信息编号11342751至11342800间共50条。
☉ 11342751:Angiotensin-Converting–Enzyme Inhibitors for Impaired Glucose Tolerance — Is There Still Hope?
In our increasingly sedentary society, the rates of overnutrition, the metabolic syndrome, and frank diabetes mellitus are rising at an alarming rate.1 Once diabetes has developed, the risks of cardiovascular and renal disease are markedly increased.2 Given the immense costs of diabetes — both personal and economic — considerable attention has been focused on identifying effective strategies to prevent or delay its onset in people at high risk. The benefits of diet and exercise in reducing the risk of diabetes are well accepted. Numerous observational studies have documented that both adiposity and physical inactivity are strong independent risk factors for diabetes. The Diabetes Prevention Program, a randomized trial of lifestyle changes and metformin in people with impaired glucose tolerance, showed a 58% reduction in the progression to diabetes in the group that underwent caloric reduction and regular exercise3; the group receiving metformin also had a reduced risk of diabetes (by 31%), but this reduction was significantly less than that resulting from the lifestyle changes. Another multicenter randomized trial, the Finnish Diabetes Prevention Study,4 reported a reduction in the incidence of diabetes as a result of lifestyle changes that was similar to the reduction in the Diabetes Prevention Program. Lifestyle changes are currently the standard recommendation for patients recognized to be at high risk for diabetes, especially obese patients. Yet maintaining adherence to these nonpharmacologic strategies is notoriously challenging. Thus, the possible use of pharmacologic interventions to prevent diabetes in people at high risk remains of keen interest. The concept that an angiotensin-converting–enzyme (ACE) inhibitor might reduce the risk of diabetes emerged from secondary findings of several trials. The Captopril Prevention Project,5 designed to assess complications and death from cardiovascular disease among patients with hypertension who were randomly assigned to receive captopril or to receive diuretics or beta-blockers, showed a 14% lower incidence of diabetes in the captopril group. However, it was unclear whether this finding might be attributable to adverse metabolic effects of the non–ACE inhibitor medications. Data from the Heart Outcomes Prevention Evaluation (HOPE) trial, designed primarily to test the hypothesis that an ACE inhibitor (ramipril) or vitamin E would reduce the risk of cardiovascular events among patients at high risk, provided more persuasive support for a possibly beneficial effect of ACE inhibitors on the risk of diabetes.6 Subjects in the HOPE trial had known vascular disease or diabetes plus at least one other risk factor for cardiovascular disease. As compared with placebo, the use of ramipril significantly reduced the rates of death, myocardial infarction, and stroke; it also reduced the risk of newly diagnosed diabetes by 34%. A subsequent meta-analysis of 12 randomized trials also showed reductions in the risk of diabetes in subjects receiving ACE inhibitors, as well as in those receiving angiotensin-receptor blockers (27% and 23%, respectively),7 suggesting that this effect might be related generally to inhibition of the renin–angiotensin system. However, none of these trials were designed with a reduction in the incidence of diabetes as the primary end point, and glucose tolerance was not assessed routinely, leaving open the possibility of ascertainment bias. In this issue of the Journal, the Diabetes Reduction Assessment with Ramipril and Rosiglitazone Medication (DREAM) Trial Investigators8 report the effects of ramipril on the risk of diabetes in a randomized trial designed with diabetes as a primary outcome. Subjects had impaired plasma glucose levels after an overnight fast (at least 110 mg per deciliter but less than 126 mg per deciliter ) or impaired glucose tolerance (a 2-hour post-load plasma glucose level greater than 140 mg per deciliter but less than 200 mg per deciliter ) and did not have prior cardiovascular disease. They were randomly assigned, according to a 2-by-2 factorial design, to receive either ramipril (up to 15 mg daily) or placebo and either the thiazolidinedione rosiglitazone or placebo and were followed for a median of 3 years. Rates of the primary end point — diabetes or death — were not significantly lower in the ramipril group (18.1%) than in the placebo group (19.5%; hazard ratio, 0.91; 95% confidence interval , 0.81 to 1.03). This end point largely reflected a diagnosis of diabetes, since deaths were uncommon. However, regression to normoglycemia, a secondary outcome, was significantly more frequent in the ramipril group than in the placebo group (42.6% vs. 38.3%), although the absolute difference between the groups was small. In addition, glucose levels measured 2 hours after a glucose challenge were significantly lower in the ramipril group (although fasting glucose levels were not). There was no significant interaction between the two therapies; the results for rosiglitazone are reported separately.9 In contrast to ramipril, the use of rosiglitazone resulted in a significant and substantial reduction in diabetes or death (11.4%, vs. 26.2% in the placebo group; hazard ratio, 0.40; 95% CI, 0.35 to 0.46).9 Thiazolidinediones are peroxisome-proliferator–activated receptor (PPAR) agonists, which are known to improve insulin sensitivity, and the concept that they would prevent or delay progression from abnormal glucose metabolism to diabetes is not new. The Troglitazone in Prevention of Diabetes (TRIPOD) study,10 for example, showed that troglitazone significantly reduced the incidence of diabetes among women at high risk, although reports of hepatotoxicity led to the withdrawal of that medication. Follow-up studies will assess whether the effect of rosiglitazone on the rate of diabetes will persist after drug washout. The observations that ramipril increased the likelihood of regression to normoglycemia and significantly reduced post-challenge glucose levels suggest that an ACE inhibitor may improve glucose metabolism. How? ACE inhibitors block the conversion of angiotensin I to angiotensin II, decreasing the angiotensin II that is available both in the circulation and, locally, in various organs and tissues.11,12 Angiotensin II may adversely affect glucose metabolism by increasing reactive oxygen species and inducing inflammation, decreasing blood flow in many tissue beds, and stimulating the sympathetic nervous system. It may also impair insulin-signaling pathways and pancreatic function. Angiotensin II inhibits the differentiation of adipocytes through the angiotensin II type 1 receptor, and low adiponectin levels are associated with insulin resistance. Inhibiting the renin–angiotensin system may improve blood flow to muscles, decrease the activity of the sympathetic nervous system, enhance insulin signaling, lower levels of free fatty acids, increase plasma adiponectin levels, and improve glucose disposal. Another putative mechanism by which the inhibition of the renin–angiotensin system may improve insulin sensitivity is through effects on PPAR, which is inhibited by angiotensin II.11,12 How can the absence of a significant effect of ramipril on the incidence of diabetes in the DREAM trial be reconciled with prior findings that this drug and other inhibitors of the renin–angiotensin system reduce the risk of diabetes? As the DREAM Investigators note, previous trials included subjects with hypertension and subjects with, or at high risk for, cardiovascular disease, populations that differed from those in the DREAM trial — and did not include standard assessments of glucose levels. It is possible that observed reductions in the incidence of diabetes with the use of inhibitors of the renin–angiotensin system may, in some cases, have reflected improvement in unrecognized hyperglycemia at baseline or reduced detection of diabetes (owing to fewer hospitalizations for cardiovascular events). The 95% CI for the hazard ratio for diabetes in the present trial (0.80 to 1.03) means that the findings are compatible with the absence of an effect of ramipril on the risk of diabetes but do not exclude the possibility of some benefit. In addition, the 3-year study duration may have been insufficient to demonstrate a benefit, although the duration of the Diabetes Prevention Program (in which interventions significantly reduced the risk of diabetes) was similar. Given the primary findings of the DREAM trial, ramipril cannot be recommended for the prevention of type 2 diabetes. For patients who take ACE inhibitors for another indication (such as hypertension, congestive heart failure, or a high risk of cardiovascular events), improvement in glycemia may turn out to be yet another benefit. For now, ongoing attention to diet and exercise remains our best hope for reducing the rising rate of diabetes. Source Information This editorial was published at www.nejm.org on September 15, 2006. References World Health Organization. Obesity and overweight. (Accessed September 21, 2006, at http://www.who.int/dietphysicalactivity/publications/facts/obesity/en/.) Nelson RG, Knowler WC, Pettitt DJ, Bennett PH. Kidney disease in diabetes. In: National Diabetes Data Group. Diabetes in America. 2nd ed. Bethesda, MD: National Institutes of Diabetes and Digestive and Kidney Diseases, 1995:349-400. (NIH publication no. 95-1468.) Diabetes Prevention Program Research Group. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 2002;346:393-403. Lindstrom J, Louheranta A, Mannelin M, et al. The Finnish Diabetes Prevention Study (DPS): lifestyle intervention and 3-year results on diet and physical activity. Diabetes Care 2003;26:3230-3236. Hansson L, Lindholm LH, Niskanen L, et al. Effect of angiotensin-converting-enzyme inhibition compared with conventional therapy on cardiovascular morbidity and mortality in hypertension: the Captopril Prevention Project (CAPPP) randomised trial. Lancet 1999;353:611-616. The Heart Outcomes Prevention Evaluation Study Investigators. Effects of an angiotensin-converting-enzyme inhibitor, ramipril, on cardiovascular events in high-risk patients. N Engl J Med 2000;342:145-153. Abuissa H, Jones PG, Marso SP, O'Keefe JH Jr. Angiotensin-converting enzyme inhibitors or angiotensin receptor blockers for prevention of type 2 diabetes: a meta-analysis of randomized clinical trials. J Am Coll Cardiol 2005;46:821-826. The DREAM Trial Investigators. Effect of ramipril on the incidence of diabetes. N Engl J Med 2006;355:1551-1562. The DREAM (Diabetes REduction Assessment with ramipril and rosiglitazone Medication) Trial Investigators. Effect of rosiglitazone on the frequency of diabetes in patients with impaired glucose tolerance or impaired fasting glucose: a randomised controlled trial. Lancet 2006;368:1096-1105. Buchanan TA, Xiang AH, Peters RK, et al. Preservation of pancreatic beta-cell function and prevention of type 2 diabetes by pharmacological treatment of insulin resistance in high-risk Hispanic women. Diabetes 2002;51:2796-2803. Kurtz TW. New treatment strategies for patients with hypertension and insulin resistance. Am J Med 2006;119:Suppl 1:S24-S30. Scheen AJ. Prevention of type 2 diabetes mellitus through inhibition of the renin-angiotensin system. Drugs 2004;64:2537-2565....查看详细 (11530字节)

☉ 11342752:Rapid Detection of Tuberculosis and Drug-Resistant Tuberculosis
Tuberculosis remains one of the major unresolved global health problems, and the situation is worsening in many parts of the world, primarily because of the association between tuberculosis and the epidemic of human immunodeficiency virus (HIV) infection and AIDS and the growing prevalence of drug resistance. The highest prevalence of both tuberculosis and drug resistance is found in countries with limited resources, which cannot afford to implement modern methods of epidemic control of tuberculosis. The detection of new cases of tuberculosis in these areas is based on provisional diagnosis by means of direct acid-fast bacilli testing of sputum smears, a diagnostic tool that can provide positive results in less than 50% of patients with newly diagnosed pulmonary tuberculosis confirmed on culture. Furthermore, the sputum-smear test does not address detection of persons with drug-resistant strains of Mycobacterium tuberculosis. The most common method of detecting drug-resistant strains of tuberculosis in many countries (even those with a moderate economic level) is often limited to use in patients with no response to the initial standard treatment regimen. Therefore, detection of drug resistance is attempted only when there is a clinical suspicion of drug resistance. The loss of 9 to 12 (or more) months of the provision of appropriate tuberculosis therapy has several potentially critical consequences: patients with multidrug-resistant tuberculosis may have progressive disease or may die while receiving ineffectual treatment; the presence of amplified drug resistance, including the loss of pyrazinamide and ethambutol, may create the next level of an extensively drug-resistant tuberculosis pathogen; and, most important, ongoing transmission is likely to occur. A more efficient and cost-effective alternative to that expensive so-called strategy would be the implementation of culture isolation for more complete detection of new cases of tuberculosis and the testing of all initial culture isolates for susceptibility to isoniazid and rifampin. Such an alternative would be feasible and effective under two conditions: the method should be inexpensive and easy to implement and the turnaround time of laboratory testing should be short enough that providers could make timely adjustments to the treatment regimens when drug resistance is detected. For many years, the implementation of testing for drug susceptibility has been one of the most neglected aspects of health care, despite the large number of bacteriologic and molecular methods developed for testing.1 The situation today demands further development in this area, with a focus on methods that can be implemented in countries with limited resources. In this issue of the Journal, Moore and colleagues2 attempt to address these problems. They have evaluated a method called the microscopic-observation drug-susceptibility (MODS) assay, which is considered an inexpensive tool for the bacteriologic diagnosis of tuberculosis and the detection of drug resistance. This method, originally described in 2000,3 with additional data reported in 2004,4 is based on direct inoculation of the selective 7H9 liquid culture medium in 24-well plates with a sputum specimen subjected to the digestion–decontamination procedure with the use of a mixture of two reagents, N-acetyl-L-cysteine and sodium hydroxide, for two purposes.5 The first purpose is digestion, or liquefaction, accomplished with N-acetyl-L-cysteine and is required for the subsequent concentration of the mycobacteria by centrifugation. The second purpose is decontamination, which is performed with sodium hydroxide and is required to kill microbes that otherwise would interfere with the isolation of a pure mycobacterial culture. Concentration by centrifugation is presumed to be the final step of the procedure. Detection of the typical cord formation ("microcolonies") of M. tuberculosis in the wells on microscopical examination (under an inverted light microscope at a magnification of x40) constitutes the basis of diagnosis. Growth (or the lack thereof) in drug-containing wells, as compared with growth in drug-free wells, is the basis for reporting the results as "susceptible" or "resistant" to medication. The reported2 sensitivity of this method in the recovery of mycobacteria from sputum specimens was higher than the sensitivity of either the MB/BacT automated mycobacterial system or traditional culture on L?wenstein–Jensen medium. The results of Moore et al. complement two previous publications from a group in Peru,3,4 in one of which3 the sensitivity of the MODS assay appears to be equal to that of the mycobacteria growth-indicator tube system and showed higher sensitivity than culture on agar plates. Results of testing for drug susceptibility with four drugs (isoniazid, rifampin, ethambutol, and streptomycin) agreed well with those obtained with the use of the MB/BacT system.2 The most impressive data reported by Moore et al. are on the turnaround time of the assay: the median time was 7 days for the detection of growth on culture and testing direct-drug susceptibility, whereas the MB/BacT system required 13 and 22 days, respectively, and culture on L?wenstein–Jensen medium required 26 and 68 days, respectively. This report by Moore et al. as well as the two previous reports provide support for an affordable, rapid method of culture-based diagnosis and detection of drug resistance in countries with limited resources. We believe that, in its current form, the MODS assay will require further improvement and standardization before it can be recommended for broad application in such countries. Reports of the identification of M. tuberculosis on the basis of the "string-and-tangle appearance"2 of growth detected on microscopical examination of the wells should be considered provisional, rather than final. Some strains of M. tuberculosis may not produce this serpentine formation. Also, many isolates of M. kansasii also may produce cording in broth and have morphologic features and a timing of appearance indistinguishable from those of isolates of M. tuberculosis. An additional problem in countries where there is a significant prevalence of bovine tuberculosis is the differentiation between M. tuberculosis and M. bovis. These problems of identification can be addressed if isolation on a solid medium to obtain a pure culture is implemented along with the MODS assay, and such cultures can be used, if necessary, for subsequent confirmation of the diagnosis. Critical drug concentrations implemented for the MODS assay were adopted by the authors from those developed for the Bactec system (BACTEC 960, Becton Dickinson). Although the results obtained correlated well with results obtained with the use of two other methods, in general, each technology is known to require special calibration of critical concentrations halfway between the highest minimal inhibitory concentrations for susceptible strains and the lowest minimal inhibitory concentrations for resistant strains. The major difficulty in the implementation of the MODS assay or any other new cultivation method is that of biosafety, which is not fully addressed by Moore et al. Although it is not stated in their report, we assume that the sputum digestion–decontamination procedure was performed in conjunction with concentration of the specimens, as required by the original description of this technique.5 Concentration must be performed in aerosol-contained centrifuges. In addition, the manipulation of infected well plates is a potential hazard requiring special caution and should be performed only in biosafety cabinets. Use of these measures, along with other biosafety issues, can be properly addressed only in a well-organized tuberculosis laboratory using mandatory implementation of biosafety level 3 standards.6 Regardless of how simple and inexpensive the newly introduced technique is, there is the issue of the cost of the materials needed for the test and the total cost of the procedure, including the costs of labor, laboratory equipment (centrifuges, biosafety cabinets, inverted light microscopes, among other types of equipment), and overhead. In other words, implementation of an "inexpensive" test can be successful only if it is incorporated into the overall algorithm of the laboratory protocol. Thus, the introduction of a new procedure (such as the MODS assay) requires the existence of a basic tuberculosis laboratory. The establishment of microbiology laboratories in countries with a high prevalence of tuberculosis and growing rates of drug-resistant tuberculosis should become one of the urgent priorities in the global fight against tuberculosis epidemics, especially in countries with limited resources. The MODS technique may well move this process forward. No potential conflict of interest relevant to this article was reported. Source Information From the National Jewish Medical and Research Center, Denver. References Heifets LB, Cangelosi GA. Drug susceptibility testing of Mycobacterium tuberculosis: a neglected problem at the turn of the century. Int J Tuberc Lung Dis 1999;3:564-581. Moore DAJ, Evans CAW, Gilman RH, et al. Microscopic-observation drug-susceptibility assay for the diagnosis of TB. N Engl J Med 2006;355:1539-1550. Caviedes L, Lee TS, Gilman RH, et al. Rapid, efficient detection and drug susceptibility testing of Mycobacterium tuberculosis in sputum by microscopic observation of broth cultures. J Clin Microbiol 2000;38:1203-1208. Moore DA, Mendoza D, Gilman RH, et al. Microscopic observation drug susceptibility assay, a rapid, reliable diagnostic test for multidrug-resistant tuberculosis suitable for use in resource-poor settings. J Clin Microbiol 2004;42:4432-4437. Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory. Atlanta: Centers for Disease Control, 1985. Heifets LB, Richmond JY. Modern biological safety standards in tuberculosis diagnostic (TB) laboratories. In: Richmond JY, ed. Anthology in biosafety. IX. Exploring the performance envelope for BSL-3 and BSL-4 laboratories. Mundelein, IL: American Biological Safety Association, 2006:155-66....查看详细 (10326字节)
☉ 11342753:Alzheimer's Disease — Clinical Trials and the Logic of Clinical Purpose
Agitation, aggression, delusions, and hallucinations are among the most common and disabling symptoms of Alzheimer's disease. These problems diminish the quality of life for both the patient and caregiver.1 They are also costly.2 Treatments for these symptoms include the second-generation, or "atypical" antipsychotic medications. The Food and Drug Administration (FDA) labels for antipsychotic medications state bluntly that they are not approved for the treatment of dementia-related psychosis, and they display a "black-box" warning: "Elderly patients with dementia-related psychosis treated with atypical antipsychotic drugs are at an increased risk of death compared to placebo."3 Yet, clinicians, including me, continue to prescribe these drugs.4 Although the results of several clinical trials suggest this practice has some evidence base,5 we have done this without clear evidence of the nature and extent of the clinical value of antipsychotic medications — until now. The article by Schneider et al. in this issue of the Journal reports the results of the Clinical Antipsychotic Trials of Intervention Effectiveness–Alzheimer's Disease (CATIE-AD).6 The trial substantially informs clinicians about the clinical value of three second-generation antipsychotic drugs (olanzapine, quetiapine, and risperidone) in a community-dwelling study population with a broad spectrum of dementia severity and behavioral problems that were severe enough to disrupt their functioning. Schneider and colleagues report that the main outcomes of their study "were the time from initial treatment to the discontinuation of treatment for any reason and the number of patients with at least minimal improvement on the Clinical Global Impression of Change (CGIC) scale at 12 weeks." The balance of the results on the primary and secondary end points suggests that olanzapine and risperidone were equally effective and were superior to placebo and quetiapine in treating behavioral problems, but this benefit was limited to a subgroup of patients who either tolerated or did not have side effects such as parkinsonism and sedation. These results and the evidence that behavioral problems in Alzheimer's disease can be reduced by specialized care that stresses nonpharmacologic management7 suggest that these drugs have a limited, but at times necessary, role in the care of patients with Alzheimer's disease. They are perhaps best prescribed in systems of care that can provide the skills and expertise needed to ensure that the risks associated with the drugs are justified by their potential benefits. The study by Schneider and colleagues addresses a number of the problems with previous clinical trials of atypical antipsychotic medications. For instance, the designs of previous trials did not reflect clinical practice. Hence, their results could not directly change clinical practice. In contrast, the study by Schneider et al. adhered to the "logic of clinical purpose." This scientific and ethical model asserts that clinical trials are logically grounded in and ethically justified by the way in which they reflect and contribute to clinical practice.8 Many of the randomized and controlled trials for behavioral problems in persons with Alzheimer's disease assign patients to fixed doses of a drug and measure efficacy at prespecified time points with the use of a measure of symptom severity such as the Neuropsychiatric Inventory. This design predominates among trials of treatments for Alzheimer's disease for many reasons. These scales are generally thought to represent a valid measure of the syndrome under study. In addition, studies in which outcomes are measured at multiple time points accrue a large volume of data; this approach, in turn, increases the likelihood that any change measured will be statistically, though not necessarily clinically, significant. Clearly, these designs are particularly valuable to companies seeking an FDA label or reprints of reports to distribute to clinicians. However, these designs are not as valuable for clinicians, for many reasons. A clinical trial with a primary end point that is a measure not used in clinical practice will generate results that are difficult to interpret and, hence, not likely to lead to appropriate changes in clinical practice.9 In addition, a fixed time of end-point assessment makes less sense for clinical trials that are intended to guide clinical decisions about treatments that may be discontinued at any time because the disorder varies substantially among patients and the intervention has side effects. The study by Schneider et al. addresses these problems. Its primary end point is based on a real-world measure of clinical practice: the decision to change treatment after a reasonable time to allow titration to a therapeutic dose of the drug. In defining effectiveness, the investigators determined whether the time from the initiation of treatment to a decision to change the medication because of either lack of effectiveness or side effects would be longer for persons receiving the study drug than for persons receiving placebo. This end point also addressed another common problem of the dominant model of studies of the treatment of symptoms in Alzheimer's disease: how to use data from patients who drop out before the end-point assessment. The typical solution regarding dropouts is to impute the missing data with the use of the "the last observation carried forward" method. But these data incorporate into the analysis different durations of exposure to both the drug and the study. This is a substantial problem when the condition under study involves variable decline or change, which is characteristic of the behavioral and cognitive problems in persons with Alzheimer's disease. The method also typically overestimates the precision of the effect and introduces bias.10 The primary end point in the study by Schneider et al. is an accurate reflection of a clinical event: the decision to change treatment because the patient's condition is worsening or not improving sufficiently. This portmanteau measure nicely mimics clinical practice and it obviates the problem of missing data. Moreover, the decision regarding how to administer the drugs well reflected clinical practice. Schneider and colleagues used flexible dosing of the drugs while maintaining blinded study conditions. Each of the medications was dispensed as identically appearing small and large capsules containing the appropriate lower and higher doses. The investigators chose the starting dose and were expected to adjust the dose on the basis of the patient's response. Although the assignment of patients to a fixed dose of a drug has considerable statistical efficiency, it does not reflect how these drugs are administered in clinical practice. Flexible dosing ensures one of the core principles of geriatric pharmacology: "start low and go slow, but go," meaning that the clinician should start a drug at a dose that is on the lower end of the plausible therapeutic index but then increase the dose until there is efficacy or intolerable side effects. The CATIE-AD study is an exemplar of the clinical trial's revolutionary role in shaping therapeutics. Recent remarks by FDA officials support wider use of these kinds of adaptive designs.11 This trial, funded by the National Institutes of Health, is also a model for how to spend our taxes on research, particularly now that taxes also pay for prescriptions. No potential conflict of interest relevant to this article was reported. Source Information From the Department of Medicine, Division of Geriatric Medicine, Alzheimer's Disease Center, Institute on Aging, University of Pennsylvania, Philadelphia. References Steele C, Rovner B, Chase GA, Folstein M. Psychiatric symptoms and nursing home placement of patients with Alzheimer's disease. Am J Psychiatry 1990;147:1049-1051. Murman DL, Colenda CC. The economic impact of neuropsychiatric symptoms in Alzheimer's disease: can drugs ease the burden? Pharmacoeconomics 2005;23:227-242. FDA Public Health Advisory: death with antipsychotics in elderly patients with behavioral disturbances. Rockville, MD: Food and Drug Administration, 2005. Thomason C, Babic T, Margolin R, Schaeffer J, Riordan H. A review of atypical antipsychotic prescribing practices in a large, managed care database of elderly patients in the United States prior to and after black-box warnings. Alzheimer's and Dementia 2006;2:S4-S5. Schneider LS, Dagerman K, Insel PS. Efficacy and adverse effects of atypical antipsychotics for dementia: meta-analysis of randomized, placebo-controlled trials. Am J Geriatr Psychiatry 2006;14:191-210. Schneider LS, Tariot PN, Dagerman KS, et al. Effectiveness of atypical antipsychotic drugs in patients with Alzheimer's disease. N Engl J Med 2006;355:1525-1538. Callahan CM, Boustani MA, Unverzagt FW, et al. Effectiveness of collaborative care for older adults with Alzheimer disease in primary care: a randomized controlled trial. JAMA 2006;295:2148-2157. Freedman B. Placebo-controlled trials and the logic of clinical purpose. IRB 1990;12:1-6. Karlawish JHT. The search for a coherent language: the science and politics of drug testing and approval. In: Kapp MB, ed. Ethics, law and aging review. Vol. 8. New York: Springer, 2002:39-56. Murphy SA. An experimental design for the development of adaptive treatment strategies. Stat Med 2005;24:1455-1481. Gottlieb S. Prepared remarks. Presented at the Conference on Adaptive Trial Design, Washington, DC, July 10, 2006. (Accessed September 21, 2006, at http://www.fda.gov/oc/speeches/2006/trialdesign0710.html.)...查看详细 (9784字节)

☉ 11342754:Case 31-2006 — A 15-Year-Old Girl with Severe Obesity
Presentation of Case A 15 1/2-year-old girl was seen in the outpatient Weight Center of this hospital for the evaluation of severe obesity. She had had a normal gestation without complications and had been adopted during the first month of life. She weighed 3.9 kg at birth and 4.8 kg at 1 month of age. At the age of 1 year, her weight-to-length ratio was in the 75th percentile. At 3 years of age, her body-mass index (BMI, the weight in kilograms divided by the square of the height in meters) was above the 97th percentile. She was referred to a nutritionist. Her appetite remained steady, and she ate most foods. Although her food intake appeared to be similar to that of the other children in the family, her BMI continued to increase (Figure 1A). Figure 1. Changes in BMI over Time (Panel A) and Their Association with Specific Weight-Loss Treatments (Panel B). Panel A shows a comparison of the patient's BMI (indicated by X) with standard percentiles, according to age and sex. At all ages shown, the patient's BMI is well above the 97th percentile for her age. Over the course of 3 years, various approaches were used to help the patient lose weight (Panel B). After the discontinuation of sertraline, sibutramine was added, which was associated with a modest weight loss and no side effects. Over a period of 15 months, in conjunction with moderate-to-high levels of physical activity, the patient lost 3.2 kg. As is typical with pharmacotherapy for obesity, she regained the weight after stopping the drug. Metformin was begun for the treatment of diabetes, in conjunction with dietary counseling. Treatment with orlistat was attempted but was discontinued after only a few doses because of steatorrhea. At the age of 17 years, she participated in a group-based program at the Weight Center, consisting of behavioral and nutritional interventions with a group of adolescents, with the parents meeting separately. The patient lost 3.2 kg during the 3-month course of the program and maintained the weight loss in the subsequent 6 months. Snoring and restless sleep began at approximately 5 to 6 years of age. At the age of 7 years, she was enrolled in a monthly weight-control program, and a year later, she was evaluated by a nutrition specialist. Physical examination revealed an overweight child with mild acanthosis nigricans of the neck with no other abnormalities. The next year, her parents noted that she was eating secretly; hyperpigmentation of the thighs was noted on physical examination. Between the ages of 10 and 11 years, her weight increased approximately 15 kg, and she began a program for weight control at her pediatrician's practice. At 12 years of age, she was seen by a psychiatrist, who noted dysthymia and poor motivation and prescribed sertraline and psychotherapy. When the patient was 13 years old, her parents noticed nocturnal somnambulation and an increased intake of food. At 13 years 3 months, the patient was referred to an endocrinologist because she had not lost weight and had chronic daytime fatigue. Her height was 162.4 cm, her weight 106.9 kg, her blood pressure 132/73 mm Hg, and her pulse 76 beats per minute. Breast development was Tanner stage 3, and pubertal development was Tanner stage 5 (with 1 representing immature development and 5 maturity); acanthosis nigricans was present around the neck and groin. The remainder of the examination was normal; there was no hirsutism. Results of laboratory tests are shown in Table 1. Table 1. Laboratory Values. Snoring and insomnia worsened during adolescence. Menarche occurred at the age of 14 years, and her menstrual cycles were irregular. Daytime sleepiness worsened, including falling asleep at school, and morning headaches developed. A combination of dextroamphetamine sulfate and amphetamine aspartate was prescribed to enhance alertness. During an evaluation by a sleep specialist at the age of 14 years, physical examination revealed a blood pressure of 120/90 mm Hg; the tonsils were enlarged, but there was no marked crowding. A series of overnight polysomnograms obtained between the ages of 13 and 15 years showed progressive worsening of obstructive sleep apnea, including intermittent oxygen desaturation, hypercapnia, periodic leg movements, and sleep disruption. Bilevel ventilation therapy was started when the patient was 13 years old, but compliance with the therapy was poor. Pulmonary-function testing revealed normal spirometric values and lung volumes. The results of electrocardiography, chest radiography, and echocardiography were normal. At the age of 14 years (17 months before this evaluation), the patient attended a summer camp and lost approximately 20 kg; she promptly regained the weight after returning home and gained an additional 12 kg during the subsequent year. Three months before presentation, she participated in a home-administered weight-loss plan on the basis of a point system, but her weight continued to increase. At the time of the evaluation in the Weight Center, she had daytime somnolence but no headaches. She drank low-calorie soft drinks and two glasses of juice daily. She snacked twice during the night on sandwiches or other carbohydrate-containing foods. She exercised with a personal trainer three to five times per week and watched television approximately 6 hours per day on weekends. She typically slept 7 hours on school nights and 12 hours per night on the weekends. She had no difficulty initiating sleep, but she was hard to arouse in the morning. The patient's early development had been normal; she walked at 12 months of age and spoke in short sentences at 15 months. Her depression had improved after a change in schools during the year preceding her presentation at the Weight Center, and the psychotherapy was discontinued. She was a good student in the 10th grade. A grandmother in her birth family was known to have been overweight; no other biologic-family history was known. Members of her adoptive family, including her parents and two younger siblings, were of normal weight. Her only medication was sertraline, and she had no known allergies. Her height was 164.5 cm, her weight 126.6 kg, and her BMI 46.7. The blood pressure was 124/95 mm Hg. Severe acanthosis nigricans was present on the neck and axillae, and there was moderate acne on the face; a slightly android pattern of hair growth was evident on the abdomen, and there were moderate striae on the lower abdomen. There was no hair growth on the face, no rash in the skin folds, and no edema. The remainder of the physical examination was normal. Additional laboratory testing was performed (Table 1), and a plan for management was developed. Differential Diagnosis Dr. Alison G. Hoppin: This patient presented with uncommon manifestations of a common disease. Obesity is common: 17.4% of adolescents in the United States are considered overweight by the standards of the Centers for Disease Control and Prevention.1 However, this patient's degree of obesity was very unusual: with a BMI of 46.7, she had adiposity levels that constituted class 3 obesity (on a scale of 1 to 3, with class 1 indicating a BMI of 30.0 to 34.9, class 2 a BMI of 35.0 to 39.9, and class 3 a BMI of 40.0 or more) in an adult. In addition, she had most of the important medical complications of obesity in children and adolescents (Table 2). Table 2. Important Health Effects of Obesity in Children and Adolescents. This patient's most acute health issues at presentation were symptoms suggestive of sleep apnea and diabetes mellitus. Because the symptoms of sleep apnea are somewhat subjective and there are no clear screening criteria, the problem is probably underdiagnosed in children and adolescents with obesity.2,3,4,5 This patient was referred to Dr. Eliot Katz, a specialist in sleep disorders in children, who will discuss the evaluation and management of her sleep apnea. Obstructive Sleep Apnea Dr. Eliot S. Katz: Testing of this patient by overnight polysomnography at 15 years of age (Figure 2) indicated that her sleep latency was less than 4 minutes (normal, 9 to 33), suggesting objective sleepiness. She had recurrent episodes of partial or complete upper-airway obstruction associated with intermittent hypoxemia (minimum oxygen saturation, 86%; normal value, 92 to 96), hypercapnia (awake, 52 mm Hg, and asleep, 64 mm Hg; normal carbon dioxide peak during sleep, 53 mm Hg), and electroencephalographic arousal (Figure 2). Her apnea–hypopnea index was markedly elevated at 21 events per hour (normal value, 1). Despite these findings, she had normal sleep architecture and sleep efficiency. Children with severe obstructive sleep apnea often have normal distribution of sleep states, despite frequent episodes of obstruction and brief electrocortical arousal. Figure 2. Sleep Study at 15 Years of Age. A 90-second portion of an overnight polysomnogram obtained while the patient was in stage 2 sleep shows a series of episodes of obstructive apnea and hypopnea (arrows), characterized by a marked reduction in airflow despite ongoing respiratory efforts. Accompanying the obstructive events are cyclic reductions in oxygen saturation of 5 to 10%. At the end of each obstruction is an arousal consisting of augmentation of electromyographic tone and a frequency shift in the electroencephalogram. EOG denotes electrooculogram, and O2-A1 right occipital lead referenced to the left mastoid lead. Obesity poses both an obstructive load to the upper airway and an elastic load to the entire pulmonary system. Although pulmonary function, as measured by spirometry, is often normal in obese children during wakefulness at rest, as it was in this child, there are often measurable deficits during exercise and sleep. Obese children are 4.5 times as likely to have obstructive sleep apnea as are children who are not obese.19 The severity of the condition is related to the degree of visceral adiposity, rather than to the amount of total body fat. This patient had a central pattern of obesity, which has the strongest correlation with the metabolic syndrome.20 More than 90% of children with both obesity and habitual snoring have obstructive sleep apnea.21 Thus, this patient probably had obstructive sleep apnea during her 8-to-10-year history of snoring before her initial polysomnography. Androgens affect ventilatory control and increase visceral fat; thus, obstructive sleep apnea is more commonly seen in boys after puberty (rather than before puberty) and in women who have excessive levels of androgen associated with the polycystic ovary syndrome, which was suspected in this patient.22 Sequelae The consequences of obstructive sleep apnea include cardiovascular abnormalities, neurocognitive impairment, daytime sleepiness, and metabolic disturbances — several of which were seen in this patient. Her hypertension was probably a consequence of both obstructive sleep apnea and obesity. In extreme cases, biventricular dysfunction and pulmonary hypertension may develop, and screening for pulmonary hypertension with echocardiography is indicated for children with severe obstructive sleep apnea. The 8-to-10-year history of probable obstructive sleep apnea during a vulnerable period of brain growth places this patient at risk for neurocognitive impairment, including mood disturbance, which she had. The disease has a minimal effect on intelligence but may impair executive functioning and attention span and has been associated with poor school performance in adolescents.23 In contrast to adults with sleep apnea, most children do not have clinical or objective sleepiness.24 Sleepiness in adolescents is frequently multifactorial and includes an insufficient amount of sleep, poor sleep hygiene, increased sleep requirements and circadian-rhythm disturbances, and disrupted sleep related to obstructive sleep apnea, sleepwalking, and nocturnal eating. Thus, this patient had many reasons for sleepiness. Both obstructive sleep apnea and obesity are associated with increased levels of inflammatory markers,20,25 which are believed to be involved in the normal homeostatic regulation of sleep. Thus, increased levels of circulating cytokines may contribute to the sleepiness that is characteristic of both obstructive sleep apnea and obesity. Obstructive sleep apnea can increase insulin resistance and leptin levels, independent of obesity,20,25 and treatment with continuous positive airway pressure can reduce insulin resistance and levels of leptin and inflammatory markers. Treatment In contrast to children of normal weight, obese children with adenotonsillar hypertrophy typically do not have complete resolution of obstructive sleep apnea after adenotonsillectomy, although their condition typically improves.26 In this patient, nasopharyngoscopy demonstrated only mild adenotonsillar hypertrophy that was not considered to warrant surgical removal. Weight loss can lessen the severity of obstructive sleep apnea, but residual obstruction is frequently present.27 Continuous positive airway pressure and bilevel ventilation are both effective therapies for obstructive sleep apnea.28 However, 15 to 20% of patients will not comply with the use of nasal positive-pressure ventilation at all; for the remainder of patients, the average duration of use is approximately 5 hours per night. Although this patient acknowledged improvements in daytime functioning after receiving nocturnal bilevel ventilation, she complied poorly with therapy. Thus, her case illustrates both the consequences of sleep apnea and the difficulties in managing it. Dr. Hoppin: This patient had features of the metabolic syndrome, a constellation of findings associated with an increased risk of atherosclerotic cardiovascular disease and type 2 diabetes mellitus.10 Insulin Resistance and Type 2 Diabetes Mellitus This patient had evidence of insulin resistance, with severe acanthosis nigricans, an elevated insulin level after an overnight fast, and a glycated hemoglobin value of 6.7%. Subsequent testing revealed a blood glucose level of 141 mg per deciliter (7.8 mmol per liter) 2 hours after an oral glucose load (Table 1), documenting impaired glucose tolerance.29 Insulin resistance is independently associated with both obesity and puberty, so this patient is at risk for both reasons.6,7,8 In a longitudinal study of obese adolescents, progression from normal to impaired glucose tolerance occurred in 10% during a 2-year period, and 25% of these patients had progression to type 2 diabetes.30 Six months after the first visit, treatment with metformin was begun, in consultation with an endocrinologist. During the next 2 years, the patient had a slight improvement in glycemic control, with glycated hemoglobin values ranging from 6.5 to 7% and fasting blood glucose levels between 90 and 95 mg per deciliter (5.0 to 5.3 mmol per liter). However, during a period of noncompliance with the metformin, her fasting blood glucose levels rose to a range of 244 to 275 mg per deciliter (13.5 to 15.3 mmol per liter), diagnostic of diabetes mellitus. Hypertension and Dyslipidemia The patient had mild hypertension at her first visit, which gradually worsened during the next 18 months. Pharmacologic intervention is recommended for hypertension that persists despite a modification in diet and for patients with diabetes mellitus.9 Angiotensin-converting–enzyme inhibitors are recommended preferentially in children with diabetes. Treatment with lisinopril was initiated 18 months after her first visit. Her fasting total cholesterol and triglyceride levels were high, whereas the level of low-density lipoprotein cholesterol was in the borderline range. Our dietary counseling included recommendations for a reduction in dietary fat.31 Polycystic Ovary Syndrome The patient also had findings that suggested the polycystic ovary syndrome and nonalcoholic fatty liver disease, both of which are associated with the metabolic syndrome phenotype. The classic clinical features of the polycystic ovary syndrome include menstrual disturbance, hirsutism, and polycystic ovaries. However, on the basis of broader diagnostic criteria, the disorder is thought to affect 5 to 10% of women of reproductive age.11 Although the patient had menarche and menstrual patterns that could be considered normal and she did not have marked hirsutism, she had an elevated level of free testosterone at 13 years of age, suggesting the presence of hyperandrogenism. Two years after her first visit here, she presented with acute right ovarian torsion due to a cyst. During surgery to remove the cyst, the contralateral ovary was polycystic on gross examination, confirming the diagnosis of the polycystic ovary syndrome. After the operation, treatment with ethinyl estradiol and drospirenone was begun. About 30% of adolescent girls with the polycystic ovary syndrome have glucose intolerance or diabetes mellitus.32 Hyperinsulinemia seems to be the common mechanism: insulin acts synergistically with luteinizing hormone to increase the production of androgen by the ovarian theca cells while also decreasing the level of sex hormone–binding globulin.12,33 Treatment with metformin can lead to clinical improvement, even in adolescents without overt diabetes mellitus.34 Nonalcoholic Fatty Liver Disease On initial evaluation, mild elevations of serum aminotransferase levels were present, without hyperbilirubinemia. Nonalcoholic fatty liver disease is the most common cause of mild aminotransferase elevations in children and is strongly associated with obesity and the metabolic syndrome.13,14 It is important to exclude other causes of liver disease, so when the finding persisted, we did laboratory testing to rule out viral hepatitis, autoimmune hepatitis, and Wilson's disease; all the test results were negative. No specific treatments have been established for fatty liver disease in children or adults, but weight loss is almost certainly helpful. Causes of Obesity We have discussed the consequences of the patient's obesity, but what can be said about the causes of it? Because she was adopted in early infancy, her case illustrates better than most how biologic determinants of obesity can dominate over lifestyle or environmental exposures, as Dr. Kaplan will discuss. Dr. Lee M. Kaplan: In obesity, a combination of genetic, developmental, and environmental determinants alters the body's normal system for the regulation of weight. The prevalence of obesity has increased in the past 50 years, with a disproportionate increase in severe obesity. Between 1986 and 2000, the prevalence of obesity (BMI >30) increased by a factor of 2, the prevalence of class 3 obesity (BMI >40) increased by a factor of 4, and the prevalence of the most severe forms of obesity (BMI >50) increased by a factor of 6.35,36 Most people are genetically susceptible to abnormal weight gain under the right conditions. The availability of highly processed, calorie-dense foods and a decreasing level of physical activity are important environmental contributors to obesity, but they are not the only ones. Disrupted meal patterns, disordered and inadequate sleep, disturbances in normal circadian rhythms, high levels of stress, social isolation, and the use of medications that promote weight gain may be equally important. Several of these factors may have affected the patient, including sleep deprivation and the stresses of adolescence. In contrast, the contribution of her diet or pattern of eating to the obesity appears to be limited. The early onset of obesity and the striking difference in weight between the patient and her adoptive siblings despite similar eating habits suggest an important contribution of genetics to her obesity.37,38 There is strong evidence that genetic background plays an important role in determining the predisposition to obesity. Obesity often runs in families, and the correlation of BMI among siblings is little affected by whether they were raised together or apart and is often independent of the type or pattern of food intake or physical activity. Studies of twins suggest that genetic factors determine about 50 to 70% of the predisposition to the development of obesity.39 We have little information about the biologic relatives of the patient, other than that her grandmother was overweight. Although the onset of her obesity was very early, most of the known monogenetic or oligogenetic causes of obesity are unlikely.40 These rare disorders reflect alterations in genes that encode central nervous system regulators of body weight, such as leptin, the leptin receptor, melanocyte-stimulating hormone, and the melanocortin 4 receptor (MC4R). Genetic testing through a research protocol revealed no evidence of an abnormal MC4R in the patient. The plasma leptin level was 47 ng per milliliter (normal range, 3.3 to 18.3); the elevated level was appropriate to her obesity, thereby excluding genetic deficiency in leptin or its receptor. Symptoms of the most common, well-defined obesity syndromes — including the Prader–Willi syndrome, the Bardet–Biedl syndrome, and a deficiency in single-minded homologue 1 (SIM-1) — were absent. Many genes contribute to the regulation of body weight, and a genetic predisposition to obesity, which the patient and other children with early-onset obesity almost certainly have, probably results from the influence of multiple genes, which combine to support an energy-thrifty phenotype.41 Discussion of Management Weight-Loss Strategies Dr. Hoppin: Despite the likelihood of a strong biologic basis for the patient's obesity, there is no specific physiological target that we can address to facilitate her weight management. The patient had lost approximately 20 kg in weight while attending a summer weight-loss camp 17 months before presentation to the Weight Center; she rapidly regained this weight after returning home. Such rebounds after acute weight loss from dietary restriction are very common and probably speak to the resilience of weight-regulatory mechanisms rather than to bad habits or a failure of willpower. Thus, we believed that the lifestyle habits of the patient and family were not the primary cause of her obesity. Nonetheless, our first approach was to work with her and her family to optimize these habits. This patient embarked on a new series of weight-control attempts, with limited success (Figure 1B). She continued to work with a personal trainer, engaging in aerobic and strength training 3 days a week, and received dietary supervision from the trainer, supplemented by individual nutritional counseling from a registered dietitian. She was able to stabilize her eating patterns to some degree, and the frequency of nocturnal eating decreased. After her first visit, sertraline was tapered and then discontinued, and a trial of sibutramine was begun 2 months later. Metformin was added 4 months later for glycemic control. With these combined therapies, her weight gain stopped, but she lost only 3.2 kg during the first year, and sibutramine was ultimately withdrawn. When she was 17 years old, she participated in a group-based program at our center with weekly meetings for 3 months. The group consisted of adolescent girls with obesity and was led by a dietitian. Specific goals were to improve food choices and planning and included both nutritional education and behavioral techniques. Dietary guidelines included modest caloric restriction and a relatively low intake of simple carbohydrates. The parents of the girls met concurrently with a psychologist to address family dynamics related to weight control. The patient participated actively and enthusiastically in the group program; she reduced her weight by another 3.2 kg and maintained the weight loss during the subsequent 6 months. Three years after her first evaluation, the patient had maintained a 6.8-kg weight loss for about a year. However, her BMI of 44 remained in the range of severe obesity, and she continued to have sleep apnea, diabetes mellitus, hypertension, and dyslipidemia. Bariatric Surgery When the patient was 18 years old, we began to consider the possibility of weight-reduction surgery. Gastric bypass is clearly the most consistently effective treatment for severe obesity in adults42,43; although less is known about outcomes in adolescents, a few published case series suggest that they are similar to those in adults.44 An expert panel45 has recommended that weight-reduction surgery be considered for adolescents with a BMI of more than 40 who have severe medical complications of obesity (such as sleep apnea or diabetes, as in this patient) or with a BMI of more than 50 who have any medical complications and in whom efforts to control weight through other measures have been unsuccessful. In adolescents, medical follow-up to monitor for and treat potential micronutrient deficiencies is essential.46 The patient was an appropriate candidate for bariatric surgery. Her weight and coexisting illnesses were appropriate indications according to criteria for both adults and adolescents. She had made a variety of sustained efforts to control her weight by other measures, and she had an excellent record of compliance with long-term medical follow-up. At the age of 19 years, the patient underwent a laparoscopic Roux-en-Y gastric bypass. Her initial postoperative recovery was uncomplicated. A wedge biopsy of the liver was performed during the operation. Dr. Gregory Y. Lauwers: The liver biopsy showed histologic evidence of nonalcoholic steatohepatitis (Figure 3). The condition is characterized by inflammation and fibrosis, indicating damage to hepatocytes, and is considered to be a progressive form of nonalcoholic fatty liver disease. It has the potential to progress to cirrhosis and hepatocellular carcinoma, but the magnitude of the risk is not known.47 Figure 3. Liver-Biopsy Specimen Obtained during Gastric Bypass Procedure. Panel A shows marked diffuse macrovesicular steatosis with inflammation (arrows) and ballooning degeneration of hepatocytes (inset, arrow) (hematoxylin and eosin). Panel B (trichrome stain) shows extensive fibrosis with bridging between portal areas; fine pericellular fibrosis is seen at a higher magnification (inset). In the absence of a history of alcohol use or other liver disease, the presence of fat is characteristic of nonalcoholic fatty liver disease. The presence of inflammation and fibrosis indicates progression to nonalcoholic steatohepatitis. Dr. Lynne L. Levitsky (Pediatric Endocrinology): At her most recent follow-up, 1 month after surgery, the patient's weight had decreased from 122.5 kg to 109.6 kg, with a BMI of 39.8. Metformin and lisinopril had been discontinued at the time of the surgery. The blood pressure was 138/79 mm Hg, and her morning blood glucose levels by finger-stick measurement had been normal on all but two occasions. On physical examination, acanthosis nigricans that had developed on the wrists and ankles had resolved but persisted on the neck. Anatomical Diagnosis Severe childhood obesity with obstructive sleep apnea, hypertension, impaired glucose tolerance progressing to type 2 diabetes mellitus, the polycystic ovary syndrome, and nonalcoholic steatohepatitis. Dr. Hoppin reports having received grant support from the Massachusetts Vitamin Litigation Fund. No other potential conflict of interest relevant to this article was reported. Source Information From the Department of Pediatrics (A.G.H., E.S.K.), the Gastrointestinal Unit (L.M.K.), and the Department of Pathology (G.Y.L.), Massachusetts General Hospital; and the Departments of Pediatrics (A.G.H., E.S.K.), Medicine (L.M.K.), and Pathology (G.Y.L.), Harvard Medical School. References Ogden CL, Carroll MD, Curtin LR, et al. Prevalence of overweight and obesity in the United States, 1999-2004. JAMA 2006;295:1549-1555. Gislason T, Benediktsdottir B. Snoring, apneic episodes, and nocturnal hypoxemia among children 6 months to 6 years old: an epidemiologic study of lower limit of prevalence. Chest 1995;107:963-966. Ali NJ, Pitson DJ, Stradling JR. Snoring, sleep disturbance, and behaviour in 4-5 year olds. Arch Dis Child 1993;68:360-366. Marcus CL, Curtis S, Koerner CB, Joffe A, Serwint JR, Loughlin GM. Evaluation of pulmonary function and polysomnography in obese children and adolescents. Pediatr Pulmonol 1996;21:176-183. Chay OM, Goh A, Abisheganaden J, et al. Obstructive sleep apnea syndrome in obese Singapore children. Pediatr Pulmonol 2000;29:284-290. Dolan LM, Bean J, D'Alessio D, et al. Frequency of abnormal carbohydrate metabolism and diabetes in a population-based screening of adolescents. J Pediatr 2005;146:751-758. Sinha R, Fisch G, Teague B, et al. Prevalence of impaired glucose tolerance among children and adolescents with marked obesity. N Engl J Med 2002;346:802-810. Williams DE, Cadwell BL, Cheng YJ, et al. Prevalence of impaired fasting glucose and its relationship with cardiovascular disease risk factors in US adolescents, 1999-2000. Pediatrics 2005;116:1122-1126. National High Blood Pressure Education Program Working Group on High Blood Pressure in Children and Adolescents. The fourth report on the diagnosis, evaluation, and treatment of high blood pressure in children and adolescents. Pediatrics 2004;114:Suppl:555-576. Cook S, Weitzman M, Auinger P, Nguyen M, Dietz WH. Prevalence of a metabolic syndrome phenotype in adolescents: findings from the Third National Health and Nutrition Examination Survey, 1988-1994. Arch Pediatr Adolesc Med 2003;157:821-827. Dunaif A. Insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis. Endocr Rev 1997;18:774-800. Ehrmann DAA. Polycystic ovary syndrome. N Engl J Med 2005;352:1223-1236. Franzese A, Vajro P, Argenziano A, et al. Liver involvement in obese children: ultrasonography and liver enzyme levels at diagnosis and during follow-up in an Italian population. Dig Dis Sci 1997;42:1428-1432. Strauss RS, Barlow SE, Dietz WH. Prevalence of abnormal serum aminotransferase values in overweight and obese adolescents. J Pediatr 2000;136:727-733. Jerre R, Karlsson J, Henrikson B. The incidence of physiolysis of the hip: a population-based study of 175 patients. Acta Orthop Scand 1996;67:53-56. Cook SD, Lavernia CJ, Burke SW, Skinner HB, Haddad RJ Jr. A biomechanical analysis of the etiology of tibia vara. J Pediatr Orthop 1983;3:449-454. von Mutius E, Schwartz J, Neas LM, Dockery D, Weiss ST. Relation of body mass index to asthma and atopy in children: the National Health and Nutrition Examination Study III. Thorax 2001;56:835-838. Balcer LJ, Liu GT, Forman S, et al. Idiopathic intracranial hypertension: relation of age and obesity in children. Neurology 1999;52:870-872. Redline S, Tishler PV, Schluchter M, Aylor J, Clark K, Graham G. Risk factors for sleep-disordered breathing in children: associations with obesity, race, and respiratory problems. Am J Respir Crit Care Med 1999;159:1527-1532. Vgontzas AN, Papanicolaou DA, Bixler EO, et al. Sleep apnea and daytime sleepiness and fatigue: relation to visceral obesity, insulin resistance, and hypercytokinemia. J Clin Endocrinol Metab 2000;85:1151-1158. Silvestri JM, Weese-Meyer DE, Bass MT, Kenny AS, Hauptman SA, Pearsall SM. Polysomnography in obese children with a history of sleep-associated breathing disorders. Pediatr Pulmonol 1993;16:124-129. Vgontzas AN, Legro RS, Bixler EO, Grayev A, Kales A, Chrousos GP. Polycystic ovary syndrome is associated with obstructive sleep apnea and daytime sleepiness: role of insulin resistance. J Clin Endocrinol Metab 2001;86:517-520. Gozal D, Pope DW Jr. Snoring during early childhood and academic performance at ages thirteen to fourteen years. Pediatrics 2001;107:1394-1399. Gozal D, Wang M, Pope DW Jr. Objective sleepiness measures in pediatric obstructive sleep apnea. Pediatrics 2001;108:693-697. Weiss R, Dziura J, Burgert TS, et al. Obesity and the metabolic syndrome in children and adolescents. N Engl J Med 2004;350:2362-2374. Wiet GJ, Bower C, Seibert R, Griebel M. Surgical correction of obstructive sleep apnea in the complicated pediatric patient documented by polysomnography. Int J Pediatr Otorhinolaryngol 1997;41:133-113. Dixon JB, Schachter LM, O'Brien PE. Polysomnography before and after weight loss in obese patients with severe sleep apnea. Int J Obes (Lond) 2005;29:1048-1054. Marcus CL, Ward SL, Mallory GB, et al. Use of nasal continuous positive airway pressure as treatment of childhood obstructive sleep apnea. J Pediatr 2002;127:88-94. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2004;27:Suppl 1:S5-S10. Weiss R, Taksali SE, Tamborlane WV, Burgert TS, Savoye M, Caprio S. Predictors of changes in glucose tolerance status in obese youth. Diabetes Care 2005;28:902-909. American Academy of Pediatrics, Committee on Nutrition. Cholesterol in childhood. Pediatrics 1998;101:141-147. Palmert MR, Gordon CM, Kartashov AI, Legro RS, Emans SJ, Dunaif A. Screening for abnormal glucose tolerance in adolescents with polycystic ovary syndrome. J Clin Endocrinol Metab 2002;87:1017-1023. Dunaif A. Hyperandrogenic anovulation (PCOS): a unique disorder of insulin action associated with an increased risk of non-insulin-dependent diabetes mellitus. Am J Med 1995;98:Suppl 1A:33S-39S. Ibanez L, Ferrer A, Ong K, Amin R, Dunger D, de Zegher F. Insulin sensitization early after menarche prevents progression from precocious pubarche to polycystic ovary syndrome. J Pediatr 2004;144:23-29. Flegal KM, Carroll MD, Ogden CL, Johnson CL. Prevalence and trends in obesity among US adults, 1999-2000. JAMA 2002;288:1723-1727. Ogden CL, Carroll MD, Curtin LR, McDowell MA, Tabak CJ, Flegal KM. Prevalence of overweight and obesity in the United States, 1999-2004. JAMA 2006;295:1549-1555. Bell CG, Walley AJ, Froguel P. The genetics of human obesity. Nat Rev Genet 2005;6:221-234. Lyon HN, Hirschhorn JN. Genetics of common forms of obesity: a brief overview. Am J Clin Nutr 2005;82:Suppl:215S-217S. Bouchard C, Tremblay A, Després J-P, et al. The response to long-term overfeeding in identical twins. N Engl J Med 1990;322:1477-1482. Rosenbaum M, Leibel RL, Hirsch J. Obesity. N Engl J Med 1997;337:396-407. Prentice AM. Early influences on human energy regulation: thrifty genotypes and thrifty phenotypes. Physiol Behav 2005;86:640-645. Buchwald H, Avidor Y, Braunwald E, et al. Bariatric surgery: a systematic review and meta-analysis. JAMA 2004;292:1724-1737. Case Records of the Massachusetts General Hospital (Case 25-2004). N Engl J Med 2004;351:696-705. Inge TH, Zeller MH, Lawson ML, Daniels SR. A critical appraisal of evidence supporting a bariatric surgical approach to weight management for adolescents. J Pediatr 2005;147:10-19. Apovian CM, Baker C, Ludwig DS, et al. Best practice guidelines in pediatric/adolescent weight loss surgery. Obes Res 2005;13:274-282. Towbin A, Inge TH, Garcia VF, et al. Beriberi after gastric bypass surgery in adolescence. J Pediatr 2004;145:263-267. Kleiner DE, Brunt EM, Van Natta M, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41:1313-1321....查看详细 (35830字节)
☉ 11342755:Medical Mystery — Visual-Field Defect
A 31-year-old woman presented with a sudden onset of an inferonasal visual-field deficit in the left eye 2 days after she had traveled 16 hours by air. Physical examination on the day after the onset of her visual symptoms showed the findings in Panels A, B, and C. Auscultation revealed a systolic ejection murmur over the right upper sternal border. The hematocrit was 57.4%. What is the diagnosis? Editor's note: We invite our readers to submit their answers at www.nejm.org/mystery. We will publish the diagnosis in the Correspondence section of the December 7 issue and e-mail it to everyone who submits an answer. All answers must be received by October 25. Jeevan R. Mathura, Jr., M.D. Lee M. Jampol, M.D. Northwestern University Chicago, IL 60611 j-mathura@northwestern.edu...查看详细 (815字节)

☉ 11342756:External-Beam Radiotherapy for Localized Prostate Cancer
This Journal feature begins with a case vignette that includes a therapeutic recommendation. A discussion of the clinical problem and the mechanism of benefit of this form of therapy follows. Major clinical studies, the clinical use of this therapy, and potential adverse effects are reviewed. Relevant formal guidelines, if they exist, are presented. The article ends with the author's clinical recommendations. A 69-year-old man undergoes a follow-up evaluation after testing showed an elevated serum prostate-specific antigen (PSA) level. One year previously, he had requested serum PSA testing after receiving counseling regarding its advantages and disadvantages. His serum PSA level at that time was 8.0 ng per milliliter, and prostatic intraepithelial neoplasia was detected on biopsy. His serum PSA level is now 11.0 ng per milliliter, and the apical prostate is indurated (clinical tumor stage, T2a). Transrectal prostatic ultrasonography shows a prostate gland 70 ml in size (twice normal size), needle biopsy reveals adenocarcinoma with a Gleason score of 7, and cancer staging shows no sign of spread beyond the prostate. A specialist recommends high-dose, image-guided external-beam radiotherapy. The Clinical Problem One in six American men receives a diagnosis of prostate cancer during his lifetime, usually after 60 years of age.1 With approximately 234,000 new cases expected in 2006, prostate cancer is the most common noncutaneous malignant disease and is the third leading cause of cancer-related death in men.1 The established risk factors for the disease include race, age, and family history.2 The prognosis for patients with prostate cancer is variable and depends on the tumor-related characteristics at diagnosis. In practice, the clinical tumor stage at presentation (according to the classification of the American Joint Committee on Cancer),3 the histologic appearance (according to the Gleason score; scores range from 2 to 10, with higher scores indicating a poorer prognosis), and serum PSA values are used to assess the risk of spread of microscopic tumor beyond the prostate,4,5,6 determine the risk of recurrence (Table 1),4 and estimate the likelihood of therapeutic success.5 The interaction among these factors can be assessed with the use of a predictive instrument, such as a nomogram,7 that quantifies the risk for the individual patient.6,7,8 Patients with higher Gleason scores, higher PSA levels, and rapidly rising PSA values (i.e., a PSA velocity of >2 ng per milliliter per year) have an increased risk of disease progression and cancer-related death.5,8,9 Table 1. Risk of Recurrence and Options for Initial Management of Clinically Localized Prostate Cancer, According to the National Comprehensive Cancer Network (NCCN). Pathophysiology and Effect of Therapy Etiologic studies have identified several genes that are associated with susceptibility to prostate cancer and that may serve as a substrate for carcinogenesis.10,11 Environmental factors such as dietary carcinogens and environmental agents may also promote somatic mutations that accumulate over a period of several decades (Figure 1).2,12 Progression from multifocal neoplasia originating in the prostate to metastatic dissemination occurs in several steps over time. The main pathways of spread to the lymph nodes and bone are lymphatic and hematogenous. Figure 1. Genomic Damage Resulting in Prostate Cancer. Genomic damage is induced by dietary carcinogens and inflammatory oxidants that give rise to proliferative epithelial cells that fail to mature into normal columnar epithelium. Proliferative inflammatory atrophy is thought to progress to prostatic intraepithelial neoplasia and prostate cancer through a multistep process. Radiotherapy interacts with the DNA helix of cancer cells and other intracellular targets, resulting in cell death. Radiotherapy can take the form of externally generated electromagnetic (x-ray) and particle (most commonly, proton) beams directed into the patient or radionuclides placed in or near cancerous tissue (brachytherapy). The biologic effects of radiotherapy result from ionization within the DNA helix, the interaction between radiation-induced chemical radicals and DNA, and the modification of other intracellular targets that are responsible for apoptosis and DNA repair (Figure 1).13 The entire prostate is the target of radiotherapy, because prostate cancer is often multifocal and is not always fully identified even with the use of extensive sampling on biopsy. In addition, the seminal vesicles, the pelvic lymph nodes, or both, may be included in the therapy for a portion of the total dose when the estimated risk of the spread of microscopic cancer to these structures exceeds approximately 15%.6,7 Doses of radiation are measured in Gray (Gy) units. One Gray is equivalent to 1 joule of radiation energy absorbed per kilogram of body weight. The probability of the eradication of the cancer improves with higher doses,14,15,16,17,18,19,20,21,22,23,24,25,26,27 but so too may the risk and severity of adverse effects. The risk of adverse effects is related to the proximity of the prostate to adjacent organs (especially the bladder and rectum) as well as variations in the patient's position during radiotherapy and random movement of the prostate between treatment sessions. The use of standard radiotherapeutic techniques reduces the effect of radiation on adjacent organs by directing multiple radiation beams at the prostate from several angles. The intersection of the beams, thus the region of the highest intensity of the radiation, is centered on the prostate (Figure 2). Figure 2. Image-Guided Radiotherapy. Image-guided radiotherapy begins with the placement of intraprostatic markers (Panel A) and a planning session at which computed tomography is used to identify the markers, the prostate, and adjacent organs. The imaging device, which is integrated into the linear accelerator, is used to locate the markers. The x-ray beams are adjusted to ensure that the prescribed dose encompasses the prostate and that the dose to the adjacent organs is limited (Panel B). Two developments have improved the precision with which external-beam radiotherapy can be directed specifically to the prostate, limiting the potential for injury to other organs and permitting the use of higher doses of radiation. In the first development, known as three-dimensional conformal radiotherapy, image-guided techniques are used to ensure that the alignment of the beams conforms tightly to the target.28 In the second development, known as intensity-modulated radiotherapy,16,17,18 further enhancements allow modulation of the intensity of the dose in each of many minute pixels, or "beamlets," within each beam, resulting in steep gradients of intensity within the prostate and between the prostate and adjacent organs. Clinical Evidence The two standard approaches to the management of early prostate cancer with a curative intent are radiotherapy (external-beam radiation, brachytherapy, or both) and radical prostatectomy. However, data from randomized trials that directly compare these two methods are lacking. Performing such trials is complicated by the rapid evolution of treatment techniques and the long clinical follow-up necessary to show an effect on survival.29 Therefore, comparison studies of these approaches have relied primarily on retrospective analyses. Unfortunately, such studies tend to be biased, because younger and healthier patients are apt to undergo surgery whereas older and less vigorous patients are usually treated with radiotherapy. A recent comparison involved 2991 patients who underwent prostatectomy or received low-dose (74 Gy) radiotherapy.23,25,26,30,43 The symptoms include abdominal cramping, tenesmus, and urgency and frequency of defecation and are usually controlled with antidiarrheal agents or topical antiinflammatory preparations. Late gastrointestinal effects include urgency, frequency, and hematochezia. Strictures, ulceration, and perforation are rare. Moderate-to-severe late gastrointestinal effects occur in approximately 20% of patients treated with high doses of radiotherapy, as compared with approximately 10% of those treated with standard doses (approximately 70 Gy).16,19,23,25,26,43,44 However, when highly conformal beams are used and the dose to the rectum is limited,16,19,23,26,30 the rate of moderate-to-severe late gastrointestinal effects approaches that seen among patients who receive standard doses. Moderate-to-severe acute genitourinary effects occur in approximately a third of patients23,25,26,30,43 and are caused by irritability of the bladder detrusor or urothelial inflammation (cystitis, urethritis, or both) resulting in urgency, frequency, or dysuria. Prostatic inflammation may result in prolonged or incomplete voiding, especially in the setting of coincident benign hypertrophy. These adverse effects are often lessened with the short-term use of 1-adrenergic–receptor antagonist medications. Late genitourinary effects are relatively uncommon, but bladder-neck or urethral stricture may cause retention, and a reduced bladder capacity may result in urgency and frequency. Acute and late genitourinary effects do not increase significantly with the use of high-dose radiotherapy.16,19,23,25,26,30,43,44 Urinary incontinence is uncommon; approximately 1% of patients use protective pads intermittently or daily.45 Erectile dysfunction occurs in perhaps a third of patients after radiotherapy, as a result of the disruption of penile vasculature46; however, this disorder may also result from causes other than radiotherapy.47 Because high doses of radiation to the corpus spongiosum double the risk of erectile dysfunction,46 measures to reduce the corporal dose are now incorporated into the planning of radiotherapy. Patients undergoing radiotherapy for prostate cancer may have a small increase in the risk of second malignant diseases, as compared with those who undergo prostatectomy48 but not as compared with the population at large, since patients receiving radiotherapy are typically older than surgical patients and often have other medical problems. With the exception of sarcoma within the irradiated volume (absolute risk, 0.03%), a causal relationship between radiotherapy and second cancers is uncertain. Areas of Uncertainty Data comparing external-beam radiotherapy and expectant management, brachytherapy, or prostatectomy are limited, so it is uncertain which approach provides the ideal balance between cancer control and the quality of life. The comparison between external-beam radiotherapy and prostatectomy has not been the subject of a major randomized clinical trial involving current therapeutic methods. Despite evidence that high doses of radiation reduce the recurrence of prostate cancer more successfully than do "standard" doses,14,15,16,17,18,19,20,21,22,23,24,25,26,27 there is no conclusive evidence from clinical trials that cancer-related deaths are reduced and that the patient's quality of life is enhanced as a result of high-dose radiotherapy. The maximal tolerated dose that can be achieved with image guidance has not been determined. It is also not clear whether combining brachytherapy or androgen suppression with radiotherapy is preferable to radiotherapy alone when high doses of radiation are used. Increasing the dose of radiation given at each treatment session may improve the efficacy of the therapy, even when the total dose is held constant or even reduced. One approach involves increasing the dose administered at each session by approximately one third (from 1.8 Gy to 2.5 Gy) while decreasing the number of sessions (from 44 to 28) and the total dose (from 79.2 Gy to 70 Gy).49 However, this approach has not yet been adequately tested against the currently recommended approach (Table 1). Guidelines The Clinical Practice Guidelines of the National Comprehensive Cancer Network4 are often cited (Table 1) and have been adopted by other organizations (including the American Cancer Society) that advise medical professionals and the public. The initial guidelines were published in 2000 and are updated annually, and they are available at www.nccn.org/professionals/physician_gls/default.asp. Because the guidelines do not imply a preferred choice among the standard options, and because they recognize the potential limitations of current clinical evidence, the principal recommendation is care in the context of a clinical trial. The category of risk recurrence and life expectancy form the basis for the guidelines. External-beam radiotherapy is considered appropriate for patients in most categories of risk. Three-dimensional conformal radiotherapy or intensity-modulated radiotherapy should be used, and doses of 70 to 75 Gy are acceptable for the treatment of patients at low risk for recurrence, whereas doses of 75 to 80 Gy are suggested for those at intermediate or high risk. Image guidance is recommended for highly conformal radiotherapy. The addition of androgen-suppression therapy for 2 to 3 years is recommended for patients at high or very high risk. A shorter duration (6 months) of androgen suppression is optional for patients at intermediate risk who have more than one risk factor or for patients at high risk who have a single risk factor. Recommendations The patient described in the vignette has intermediate-risk prostate cancer,4 a fast-paced PSA velocity (>2 ng per milliliter per year),9 and a life expectancy exceeding 10 years.32 He is a suitable candidate for either radical prostatectomy or external-beam radiotherapy with or without brachytherapy. He should be evaluated for urinary obstruction, and consultations with specialists in radiation oncology, medical oncology, and urology should be sought to provide a comprehensive and balanced discussion of the options. It should be emphasized that his chances of a recurrence of cancer and his long-term survival appear to be nearly equal with either surgery or radiotherapy; he should therefore consider the available information about adverse effects and the quality of life. Enrollment in a clinical trial should also be considered. I recommend external-beam radiotherapy as the sole treatment for this patient, without irradiation of the seminal vesicles or pelvic lymph nodes. Image-guided intensity-modulated conformal beams should be used to deliver a dose of 75 to 80 Gy to the prostate, with the final determination of the dose to be based on the proportion of adjacent organs that can be spared. The combination of brachytherapy and external-beam radiotherapy may not be superior to high-dose external-beam radiotherapy alone,22 so I do not recommend this approach for this patient. I also do not recommend the addition of androgen suppression, since its role with high-dose image-guided delivery is not firmly established16,22 and its use will increase the possibility of adverse effects. No potential conflict of interest relevant to this article was reported. Source Information From the Department of Radiation Oncology, Mayo Clinic, Rochester, MN. Address reprint requests to Dr. Pisansky at the Department of Radiation Oncology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905. References Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2006. CA Cancer J Clin 2006;56:106-130. Bostwick DG, Burke HB, Djakiew D, et al. Human prostate cancer risk factors. Cancer 2004;101:Suppl 10:2371-2490. Greene FL, Page DL, Fleming ID, et al., eds. AJCC cancer staging manual. 6th ed. New York: Springer-Verlag, 2002. Prostate cancer. In: National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology, version 2.2005. Jenkinstown, PA: National Comprehensive Cancer Network, June 2006. (Accessed September 15, 2006, at http://www.nccn.org/professionals/physician_gls/default.asp.) Pisansky TM. External beam radiotherapy as curative treatment of prostate cancer. Mayo Clin Proc 2005;80:883-898. Partin AW, Mangold LA, Lamm DM, Walsh PC, Epstein JI, Pearson JD. Contemporary update of prostate cancer staging nomograms (Partin Tables) for the new millennium. Urology 2001;58:843-848. Prostate nomogram. New York: Memorial Sloan-Kettering Cancer Center, 2006. (Accessed September 15, 2006, at http://www.nomograms.org.) Kattan MW, Zelefsky MJ, Kupelian PA, Scardino PT, Fuks Z, Leibel SA. Pretreatment nomogram for predicting the outcome of three-dimensional conformal radiotherapy in prostate cancer. J Clin Oncol 2000;18:3352-3359. D'Amico AV, Renshaw AA, Sussman B, Chen MH. Pretreatment PSA velocity and risk of death from prostate cancer following external beam radiation therapy. JAMA 2005;294:440-447. Nelson WG, De Marzo AM, Isaacs WB. Prostate cancer. N Engl J Med 2003;349:366-381. Hughes C, Murphy A, Martin C, Sheils O, O'Leary J. Molecular pathology of prostate cancer. J Clin Pathol 2005;58:673-684. Gonzalgo ML, Isaacs WB. Molecular pathways to prostate cancer. J Urol 2003;170:2444-2452. Coleman CN. 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Teh BS, Amosson CM, Mai WY, McGary J, Grant WH III, Butler EB. Intensity modulated radiation therapy (IMRT) in the management of prostate cancer. Cancer Invest 2004;22:913-924. Pollack A, Zagars GK, Starkschall G, et al. Prostate cancer radiation dose response: results of the M.D. Anderson phase III randomized trial. Int J Radiat Oncol Biol Phys 2002;53:1097-1105. Kuban DA, Thames HD, Levy LB, et al. Long-term multi-institutional analysis of stage T1-T2 prostate cancer treated with radiotherapy in the PSA era. Int J Radiat Oncol Biol Phys 2003;57:915-928. Jacob R, Hanlon AL, Horwitz EM, Movsas B, Uzzo RG, Pollack A. The relationship of increasing radiotherapy dose to reduced distant metastases and mortality in men with prostate cancer. Cancer 2004;100:538-543. Kupelian PA, Potters L, Khuntia D, et al. Radical prostatectomy, external beam radiotherapy or =72 Gy, permanent seed implantation, or combined seeds/external beam radiotherapy for stage T1-T2 prostate cancer. Int J Radiat Oncol Biol Phys 2004;58:25-33. Dearnaley DP, Hall E, Lawrence D, et al. Phase III pilot study of dose escalation using conformal radiotherapy in prostate cancer: PSA control and side effects. Br J Cancer 2005;92:488-498. Kupelian P, Kuban D, Thames H, et al. Improved biochemical relapse-free survival with increased external radiation doses in patients with localized prostate cancer: the combined experience of nine institutions in patients treated in 1994 and 1995. Int J Radiat Oncol Biol Phys 2005;61:415-419. Zietman AL, DeSilvio ML, Slater JD, et al. Comparison of conventional-dose vs high-dose conformal radiation therapy in clinically localized adenocarcinoma of the prostate: a randomized controlled trial. JAMA 2005;294:1233-1239. Peeters STH, Heemsbergen WD, Koper PCM, et al. Dose-response in radiotherapy for localized prostate cancer: results of the Dutch multicenter randomized phase III trial comparing 68 Gy of radiotherapy with 78 Gy. J Clin Oncol 2006;24:1990-1996. Symon Z, Griffith KA, McLaughlin PW, Sullivan M, Sandler HM. Dose escalation for localized prostate cancer: substantial benefit observed with 3D conformal therapy. Int J Radiat Oncol Biol Phys 2003;57:384-390. Herman MG, Pisansky TM, Kruse JJ, Prisciandaro JI, Davis BJ, King BF. Technical aspects of daily online positioning of the prostate for three-dimensional conformal radiotherapy using an electronic portal imaging device. Int J Radiat Oncol Biol Phys 2003;57:1131-1140. Jani AB, Hellman S. Early prostate cancer: clinical decision-making. Lancet 2003;361:1045-1053. Beckendorf V, Guerif S, Le Prise E, et al. The GETUG 70 Gy vs. 80 Gy randomized trial for localized prostate cancer: feasibility and acute toxicity. Int J Radiat Oncol Biol Phys 2004;60:1056-1065. Genitourinary cancer protocols. Philadelphia: Radiation Therapy Oncology Group, 2006. (Accessed September 15, 2006, at http://www.rtog.org/members/active.html#genitourinary.) Arias E. United States life tables, 2002. National vital statistics reports. Vol. 53. No. 6. Hyattsville, MD: National Center for Health Statistics, 2004:1-38. Chon BH, Loeffler JS. The effect of nonmalignant systemic disease on tolerance to radiation therapy. Oncologist 2002;7:136-143. Horwitz EM, Hanlon AL, Pinover WH, Anderson PR, Hanks GE. Defining the optimal radiation dose with three-dimensional conformal radiation therapy for patients with nonmetastatic prostate carcinoma by using recursive partitioning techniques. Cancer 2001;92:1281-1287. Roach M III, Lu J, Pilepich MV, et al. Predicting long-term survival, and the need for hormonal therapy: a meta-analysis of RTOG prostate cancer trials. Int J Radiat Oncol Biol Phys 2000;47:617-627. D'Amico AV, Manola J, Loffredo M, Renshaw AA, DellaCroce A, Kantoff PW. 6-Month androgen suppression plus radiation therapy vs radiation therapy alone for patients with clinically localized prostate cancer: a randomized controlled trial. JAMA 2004;292:821-827. Nag S, Beyer D, Friedland J, Grimm P, Nath R. American Brachytherapy Society (ABS) recommendations for transperineal permanent brachytherapy of prostate cancer. Int J Radiat Oncol Biol Phys 1999;44:789-799. Demanes DJ, Rodriguez RR, Schour L, Brandt D, Altieri G. High-dose-rate intensity-modulated brachytherapy with external beam radiotherapy for prostate cancer: California Endocurietherapy's 10-year results. Int J Radiat Oncol Biol Phys 2005;61:1306-1316. Roach M III, Hanks G, Thames H Jr, et al. Defining biochemical failure following radiotherapy with or without hormonal therapy in men with clinically localized prostate cancer: recommendations of the RTOG-ASTRO Phoenix Consensus Conference. Int J Radiat Oncol Biol Phys 2006;65:965-974. D'Amico AV, Moul JW, Carroll PR, Sun L, Lubeck D, Chen MH. Surrogate end point for prostate cancer-specific mortality after radical prostatectomy or radiation therapy. J Natl Cancer Inst 2003;95:1376-1383. Zelefsky MJ, Ben-Porat L, Scher HI, et al. Outcome predictors for the increasing PSA state after definitive external-beam radiotherapy for prostate cancer. J Clin Oncol 2005;23:826-831. Cox JD, Stetz J, Pajak TF. Toxicity criteria of the Radiation Therapy Oncology Group (RTOG) and the European Organization for Research and Treatment of Cancer (EORTC). Int J Radiat Oncol Biol Phys 1995;31:1341-1346. Ryu JK, Winter K, Michalski JM, et al. Interim report of toxicity from 3D conformal radiation therapy (3D-CRT) for prostate cancer on 3DOG/RTOG 9406, level III (79.2 Gy). Int J Radiat Oncol Biol Phys 2002;54:1036-1046. Bey P, Carrie C, Beckendorf V, et al. Dose escalation with 3D-CRT in prostate cancer: French study of dose escalation with conformal 3D radiotherapy in prostate cancer -- preliminary results. Int J Radiat Oncol Biol Phys 2000;48:513-517. Liu M, Pickles T, Berthelet E, et al. Urinary incontinence in prostate cancer patients treated with external beam radiotherapy. Radiother Oncol 2005;74:197-201. 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☉ 11342757:Chronic Health Conditions in Adult Survivors of Childhood Cancer
ABSTRACT Background Only a few small studies have assessed the long-term morbidity that follows the treatment of childhood cancer. We determined the incidence and severity of chronic health conditions in adult survivors. Methods The Childhood Cancer Survivor Study is a retrospective cohort study that tracks the health status of adults who received a diagnosis of childhood cancer between 1970 and 1986 and compares the results with those of siblings. We calculated the frequencies of chronic conditions in 10,397 survivors and 3034 siblings. A severity score (grades 1 through 4, ranging from mild to life-threatening or disabling) was assigned to each condition. Cox proportional-hazards models were used to estimate hazard ratios, reported as relative risks and 95% confidence intervals (CIs), for a chronic condition. Results Survivors and siblings had mean ages of 26.6 years (range, 18.0 to 48.0) and 29.2 years (range, 18.0 to 56.0), respectively, at the time of the study. Among 10,397 survivors, 62.3% had at least one chronic condition; 27.5% had a severe or life-threatening condition (grade 3 or 4). The adjusted relative risk of a chronic condition in a survivor, as compared with siblings, was 3.3 (95% CI, 3.0 to 3.5); for a severe or life-threatening condition, the risk was 8.2 (95% CI, 6.9 to 9.7). Among survivors, the cumulative incidence of a chronic health condition reached 73.4% (95% CI, 69.0 to 77.9) 30 years after the cancer diagnosis, with a cumulative incidence of 42.4% (95% CI, 33.7 to 51.2) for severe, disabling, or life-threatening conditions or death due to a chronic condition. Conclusions Survivors of childhood cancer have a high rate of illness owing to chronic health conditions. As a result of advances in treatment, almost 80% of children and adolescents who receive a diagnosis of cancer become long-term survivors.1 In the United States, there are approximately 270,000 survivors of pediatric cancer, or about 1 of every 640 adults between the ages of 20 and 39 years.2 The large number of survivors has prompted studies of the long-term health consequences of treatments for childhood cancer.2,3,4 It is now clear that damage to the organ systems of children caused by chemotherapy and radiation therapy may not become clinically evident for many years. To understand fully the health risks incurred by treatment of childhood cancer, it is important to measure three types of long-term outcomes: health status, mortality, and morbidity. Of these, the first two have been fairly well characterized.5,6 In a retrospective analysis of 20,227 5-year survivors of cancer, Mertens et al.5 found statistically significant excess rates of death from subsequent cancers (standardized mortality ratio , 19.4), from cardiac causes (SMR, 8.2), and from pulmonary causes (SMR, 9.2). In a study of 9535 adults who had survived childhood cancer, 44% reported having at least one domain of health status (general health, mental health, functional status, limitations in activity, cancer-related pain, and cancer-related fear or anxiety) that was moderately or severely affected.6 However, only a few small studies have assessed the long-term morbidity that follows the treatment of childhood cancer. Four studies, with a combined sample of 1330 survivors, reported an estimate of the prevalence of "late effects," or chronic conditions that were possibly associated with cancer therapy.7,8,9,10 The small sample limited the ability of these studies to assess the risk of chronic conditions associated with a particular treatment. Moreover, there were no comparison groups to provide perspective, and the outcomes were reported for survivors who were relatively young and still involved in follow-up care. The Institute of Medicine report Childhood Cancer Survivorship: Improving Care and Quality of Life highlights the need for further research to characterize the health of long-term survivors of childhood cancer and to determine the prevalence and incidence of adverse health outcomes and chronic conditions after cancer therapy.2 The purpose of this study was to investigate the large and geographically diverse cohort that is followed in the Childhood Cancer Survivor Study (CCSS), with the goal of determining the prevalence, incidence, and severity of chronic health conditions in adult survivors of childhood cancer, and to determine the risk of chronic conditions in the survivors, as compared with their siblings. In addition, we sought to identify subpopulations of survivors at highest risk for severe, debilitating, or life-threatening chronic health conditions. In short, we sought to provide an estimate of the overall physical morbidity secondary to the type of cancer and cancer therapy. Methods Subjects CCSS is a multi-institutional, retrospectively ascertained cohort of adults who have survived for at least 5 years after treatment for childhood cancer. Our study was restricted to participants who met the following criteria: a diagnosis of leukemia, central nervous system (CNS) tumor, Hodgkin's disease, non-Hodgkin's lymphoma, Wilms' (kidney) tumor, neuroblastoma, soft-tissue sarcoma, or bone tumor; a diagnosis and initial treatment at one of 26 collaborating CCSS institutions; a date of diagnosis between January 1, 1970, and December 31, 1986; an age of less than 21 years at diagnosis; and survival for at least 5 years after the date of diagnosis. The institutional review board at each participating center reviewed and approved the CCSS protocol and documents sent to participants. All study participants provided written informed consent for participation in the study and for the release of medical-record information. A detailed description of the study design and characteristics of the cohort was reported previously.11 The questionnaire that was administered to all participants at baseline and the treatment abstraction form are available at www.cancer.umn.edu/research/ltfu/ccssquestionnaires.html and in Section A of the Supplementary Appendix, available with the full text of this article at www.nejm.org. Of the 20,720 survivors of childhood cancer in the cohort, 3017 (14.6%) were lost to follow-up after extensive efforts failed to locate them. Among the remaining 17,703 subjects, 14,372 (81.2%), including 10,397 who were 18 years of age or older at the time they were interviewed, completed the baseline questionnaire. To determine the potential for introducing bias into the studied cohort, we previously compared demographic and cancer-related characteristics among participants, nonparticipants, and those who were lost to follow-up. We found that these three groups were similar with regard to sex, the type of cancer, the age at diagnosis, the age at which they were asked to participate in the study (or for those lost to follow-up, the age at which the cohort was assembled), and the type of cancer treatment.11,12 To allow for comparisons with a population that had not been treated for cancer, we asked a random sample of participating survivors to identify a nearest-age living sibling. Of 4782 eligible siblings, 3846 (80.4%) participated; of these, 3034 were 18 years of age or older. Cancer Treatment Information We obtained information on the original cancer diagnosis from the treating institution. For the 12,752 participants who returned a signed medical release, the information on cancer therapy included their initial treatment, treatment for any relapse, and preparatory regimens for bone marrow transplantation (if applicable). Data regarding exposure to 42 chemotherapeutic agents (either yes or no) were abstracted from the medical record; cumulative doses were abstracted for 22 of these agents. Data were also obtained on surgical procedures performed for cancer treatment at any time from the date of diagnosis onward, as well as on the site of the tumor and on fields and doses of radiation therapy. (See Sections B through D in the Supplementary Appendix for details concerning these treatments.) Health Condition Outcomes At enrollment, survivors and siblings completed a 289-item questionnaire that included questions regarding their physical health conditions, including their age at the onset of the condition. All second cancers that were ascertained from a participant or a physician's report were verified by a review of pathology reports performed by Dr. S. Hammond at the CCSS Pathology Center in Columbus, OH. To determine the severity of the conditions, scoring was based on the Common Terminology Criteria for Adverse Events (version 3), a scoring system developed through the National Cancer Institute by a multidisciplinary group and intended for use in scoring both acute and chronic conditions in patients with cancer and survivors of all ages.13 The system grades conditions as mild (grade 1), moderate (grade 2), severe (grade 3), life-threatening or disabling (grade 4), or fatal (grade 5). For our study, a total of 137 health conditions were scored. As specified in the scoring system, when a particular condition was not listed, it was entered in the "Other, specify" category according to the organ system affected. If there was not enough information to distinguish between grades, the lower score was selected. Adverse psychosocial outcomes, including depression and anxiety disorder, were not included in this analysis. Before data analysis, all the authors agreed on all scores. The same scoring system was used to grade the conditions of siblings. However, because the design of the study included only living siblings at the time of study entry, there were no siblings with a grade 5 (fatal) condition. Statistical Analysis We determined the prevalence of chronic conditions among survivors and siblings. Three primary outcomes were assessed: any condition (grades 1 through 4), severe or life-threatening conditions (grade 3 or 4), and multiple conditions (2). For participants who had more than one condition, the maximum grade was used. For deceased survivors, the maximum grade of a condition reported before death was used for comparisons with siblings. For example, if a survivor died of a myocardial infarction, the grade for myocardial infarction (grade 4) was used rather than the grade for a fatal condition (grade 5). Hazard ratios were estimated with the use of Cox regression and are reported as relative risks with 95% confidence intervals (CIs). Comparisons between survivors and siblings were adjusted for the age at enrollment, sex, and race or ethnic group. The analysis accounted for within-family correlations with the use of sandwich standard-error estimates.14 The risk associated with treatment with radiation and chemotherapy agents was analyzed with the use of specific classes of chemotherapeutic agents and according to specific anatomical sites of radiation therapy. Cumulative incidence rates for each of the primary outcomes were calculated for the survivors, with death treated as a competing risk according to the method described by Gooley et al.15 Cumulative incidence rates for siblings were not calculated because the design of the study included only living siblings at the time of study entry. The starting point for both survivors and siblings was 5 years after the date of diagnosis of cancer. If the date of onset of an irreversible condition, such as blindness, occurred within the first 5 years after the date of diagnosis, the condition was considered to be present 5 years after diagnosis; for the purposes of analysis, the onset date was shifted forward to that time point. Data were analyzed with SAS software, version 9.0 (SAS Institute). Results Comparisons of Survivors with Siblings Table 1 shows the characteristics of the survivor and sibling cohorts. The mean age of the survivors was 26.6 years (range, 18.0 to 48.0), and the mean interval from the date of diagnosis of cancer to the date of completion of the questionnaire was 17.5 years (range, 6.0 to 31.0). Of the survivors, 46.2% were women and 16.0% were members of minority groups. The mean age of the siblings was 29.2 years (range, 18.0 to 56.0); 52.9% were women, and 8.1% were members of minority groups (Table 1 lists relevant P values). Table 1. Demographic Characteristics of Adult Survivors of Childhood Cancer and Siblings. Among the survivors, 62.3% reported having at least one chronic health condition, with 27.5% reporting a grade 3 (severe) or grade 4 (life-threatening or disabling) condition (Table 2, and Section E of the Supplementary Appendix, which lists the 137 conditions reported in the health questionnaire). Among survivors, 37.6% reported having at least two conditions listed in Supplementary Appendix E, and 23.8% reported having three or more conditions. In contrast, among siblings, 36.8% reported having a chronic health condition, of which 5.2% reported having a condition of grade 3 or 4; 13.1% reported having at least two conditions, and 5.4% reported having three or more conditions. The relative risks of 10 selected conditions of grade 3 or 4 for survivors as compared with siblings are provided in Table 3. Table 2. Cancer Survivors and Siblings with a Chronic Health Condition, According to the Severity Score. Table 3. Relative Risk of Selected Severe (Grade 3) or Life-Threatening or Disabling (Grade 4) Health Conditions among Cancer Survivors, as Compared with Siblings. After adjustment for the age at completion of the questionnaire or death, sex, and race or ethnic group, survivors were 3.3 times as likely as their siblings to have a chronic health condition of any grade (95% CI, 3.0 to 3.5) (Table 4). The relative risk of a survivor having any condition of grade 3 or 4, as compared with siblings, was 8.2 (95% CI, 6.9 to 9.7). Survivors were 4.9 times as likely to have two or more chronic health conditions (95% CI, 4.4 to 5.5). Groups at highest risk for having a condition of grade 3 or 4 were survivors of bone tumors (relative risk, 38.9; 95% CI, 31.2 to 48.5), CNS tumors (relative risk, 12.6; 95% CI, 10.3 to 15.5), and Hodgkin's disease (relative risk, 10.2; 95% CI, 8.3 to 12.5). All cancer groups were significantly more likely to have any condition, a condition of grade 3 or 4, and two or more conditions, as compared with siblings (P<0.001 for all comparisons). Table 4. Relative Risk of a Chronic Health Condition among Cancer Survivors, According to the Type of Tumor and Treatment, as Compared with Siblings. The relative risks associated with various combinations of therapy for survivors, as compared with siblings, are listed in Table 4. (Sections B, C, and D of the Supplementary Appendix list the types of chemotherapy, surgery, and radiotherapy received by the survivors.) Exposure to one of five specific combinations was associated with a risk of having a condition of grade 3 or 4 that was at least 10 times the expected risk: chest radiation plus bleomycin (relative risk, 13.6; 95% CI, 9.8 to 18.7), chest radiation plus an anthracycline (relative risk, 13.0; 95% CI, 10.4 to 16.3), chest radiation plus abdominal or pelvic irradiation (relative risk, 10.9; 95% CI, 8.9 to 13.2), an anthracycline plus an alkylating agent (relative risk, 10.9; 95% CI, 9.0 to 13.1), and abdominal or pelvic irradiation plus an alkylating agent (relative risk, 10.0; 95% CI, 8.2 to 12.1). An increase in the cumulative dose of an alkylating agent was associated with an increased risk of any condition or multiple conditions. In a similar way, an increase in the cumulative dose of an alkylating agent in combination with any type of irradiation was associated with an increased relative risk. In contrast, an increase in the cumulative dose of an anthracycline was not associated with an increased risk of any condition (grades 1 through 4) that we listed. Comparisons among Survivors The sex and age of the survivor at the time of the diagnosis of cancer modified the risk of having a condition of grade 3, 4, or 5. As compared with male survivors and with adjustment for the type of cancer, age at the time of the study, and race or ethnic group, female survivors were 1.5 times as likely to have any condition of grade 3 or higher (95% CI, 1.3 to 1.6). Female survivors were also more likely than male survivors to have any condition (grades 1 through 5; relative risk, 1.4; 95% CI, 1.3 to 1.5) and to have multiple conditions (relative risk, 1.5; 95% CI, 1.4 to 1.7). The age of the survivor at diagnosis also independently modified the risk when the analysis was adjusted for age at the time of the study, sex, and race or ethnic group. For each cancer group, survivors who received the diagnosis at an older age were significantly more likely to report any condition, conditions of grades 3 through 5, or multiple conditions (P<0.001). The race or ethnic group of the survivor did not affect the likelihood of having a condition of grade 3 or higher. However, black non-Hispanic survivors were less likely than white non-Hispanic survivors to have any condition (grades 1 through 5; relative risk, 0.8; 95% CI, 0.6 to 0.9) or multiple conditions (relative risk, 0.7; 95% CI, 0.5 to 0.8) (data not shown). Figure 1 shows the cumulative incidence of chronic health conditions reported by survivors during the interval from the time of the diagnosis to the time of the study. The cumulative incidence of a chronic health condition was 66.8% 25 years after the diagnosis, with an estimated incidence of 73.4% (95% CI, 69.0 to 77.9) at 30 years. For conditions of grades 3 through 5 (severe, disabling, life-threatening, or fatal), the cumulative incidence 25 years after the diagnosis was 33.1%, with an estimated incidence of 42.4% (95% CI, 33.7 to 51.2) at 30 years. The cumulative incidence of multiple conditions (2) was 38.5% 25 years after diagnosis, with an estimated incidence of 39.2% (95% CI, 37.7 to 40.8) at 30 years. Figure 1. Cumulative Incidence of Chronic Health Conditions among 10,397 Adult Survivors of Pediatric Cancer, According to the Original Diagnosis and the Severity of the Later Condition. Among the survivors of various types of childhood cancer, the severity of subsequent health conditions was scored according to the Common Terminology Criteria for Adverse Events (version 3) as either mild (grade 1), moderate (grade 2), severe (grade 3), life-threatening or disabling (grade 4), or fatal (grade 5). For the total survivor cohort, the curves showing the cumulative incidence of the two outcomes by grade are truncated at 28 years, even though the text provides data up to 30 years after the original cancer diagnosis. This was done for consistency with the panels showing data for groups of patients with certain types of cancer, in which smaller samples yielded data that were not as robust at 30 years as they were at 28 years. Discussion Long-term survivors of pediatric cancer are more likely to have diminished health status and to die prematurely than are adults who never had childhood cancer, but only a few small studies have assessed the frequency and severity of chronic conditions in this population. In addition to small samples and selection bias, previous studies have been limited by their focus on the prevalence of health conditions that developed during a relatively short interval after the cancer diagnosis, thus limiting the assessment of whether the risk changes with time.7,8,9,10 Our study of more than 10,000 adult survivors of childhood cancer who were treated in the 1970s and 1980s shows that the risk of chronic health conditions is high, particularly for second cancers, cardiovascular disease, renal dysfunction, severe musculoskeletal problems, and endocrinopathies. Moreover, the incidence of chronic conditions, including these five groups of serious outcomes, increases over time and does not appear to plateau. These findings underscore the necessity of continued follow-up of survivors of childhood cancer, with an emphasis on surveillance for second cancers (e.g., breast and colorectal cancer, melanoma, and nonmelanoma skin cancer), coronary artery disease, late-onset anthracycline-related cardiomyopathy, pulmonary fibrosis, and endocrinopathies (e.g., premature gonadal failure, thyroid disease, osteoporosis, and hypothalamic and pituitary dysfunction). Follow-up care of survivors should also include secondary and tertiary prevention (e.g., strategies to promote tobacco cessation or avoidance, physical activity, and proper weight management) and management of chronic disease. As a group, cancer survivors were eight times as likely as their siblings to have severe or life-threatening chronic health conditions (e.g., myocardial infarction, congestive heart failure, premature gonadal failure, second cancers, and severe cognitive dysfunction). Three groups were at highest risk: survivors of bone tumors, CNS tumors, and Hodgkin's disease. In addition to being at increased risk for particular health conditions, survivors of these tumors were also more likely to have multiple conditions. For example, bone-tumor survivors with multiple conditions more frequently had severe musculoskeletal problems, congestive heart failure, and hearing loss. CNS-tumor survivors with multiple conditions were more likely to have cognitive dysfunction, seizure disorders, and various endocrinopathies. Hodgkin's disease survivors with multiple conditions were more likely to have cardiovascular disease (coronary artery disease, cerebrovascular accident, valvular heart disease, or cardiomyopathy), second cancers (particularly breast cancer in women), lung disease, and thyroid disorders. In the 1970s, effective treatment for osteosarcoma of the limbs generally included amputation. Even with modern limb-sparing procedures, the life-altering musculoskeletal morbidity faced by bone-tumor survivors is clinically significant and will increase as weight-bearing joints age more rapidly, owing to asymmetric stress and altered function.16,17,18 Since bone-tumor survivors are the group most likely to have severe limitations in activity,18 these limitations — and the resulting physical inactivity — may further compound the risk of cardiovascular disease as the survivors age. Survivors of CNS tumors — who often have significant cognitive, visual, and auditory impairment and endocrinopathies — are the group most likely to be functionally impaired.6,19,20,21 Hodgkin's disease survivors have the highest risk of second cancers and heart disease.22,23,24,25,26,27 Five treatment combinations were associated with a risk of severe or life-threatening conditions that was increased by a factor of at least 10; four of the treatment combinations included chest, abdominal, or pelvic irradiation. Survivors who were treated with any of these combinations should be identified for the purpose of surveillance, since many of the conditions can be diagnosed at an early and treatable stage (e.g., breast cancer, cardiomyopathy, and osteoporosis).28 As compared with men, women who survive childhood cancer have been reported to have a greater risk of diminished health status,6 second cancers,22,29 anthracycline-related cardiomyopathy and congestive heart failure,30 and cranial radiotherapy–related cognitive dysfunction, growth hormone deficiency, and obesity.31,32,33,34 Our study confirms this difference in outcomes between the sexes after cancer therapy. It has been reported that less than 20% of adult survivors of childhood cancer are followed at a cancer center or by an oncologist; the likelihood of follow-up in this setting decreases even more with time.35 For this reason, it is important for general physicians and internists to be aware of the risks facing this population. There are several considerations to keep in mind when interpreting these findings. First, the conditions were self-reported without external verification, with the exception of second cancers and death. Several key chronic conditions associated with cancer therapy, such as late-onset cardiomyopathy associated with previous anthracycline exposure, may remain clinically silent for long periods before becoming clinically apparent.36 Other conditions that may be underreported are osteoporosis, hypertension, and insulin resistance. Our list of chronic conditions, although comprehensive, is not all-inclusive; a notable omission is adverse mental health outcomes, an important component of morbidity after cancer in childhood.6,37,38 In summary, adult survivors of pediatric cancer who were treated in the 1970s and 1980s are a high-risk population. Thirty years after a diagnosis of cancer, almost three fourths of survivors have a chronic health condition, more than 40% have a serious health problem, and one third have multiple conditions. The incidence of health conditions reported by this population increases with time and does not appear to plateau. The monitoring of survivors is an important part of their overall health care. Supported by a grant (U24-CA-55727, to Dr. Robison) from the Department of Health and Human Services; by the Children's Cancer Research Fund (to the University of Minnesota); and by American Lebanese Syrian Associated Charities (St. Jude Children's Research Hospital). Presented at the Proceedings of the 2005 Annual Meeting of the American Society of Clinical Oncology, Orlando, FL, May 13–17, 2005. No potential conflict of interest relevant to this article was reported. * Members of the Childhood Cancer Survivor Study are listed in the Appendix. Source Information From Memorial Sloan-Kettering Cancer Center, New York (K.C.O., C.A.S.); the University of Minnesota, Minneapolis (A.C.M.); the Fred Hutchinson Cancer Research Center, Seattle (T.K., D.L.F., W.L.); St. Jude Children's Research Hospital, Memphis, TN (M.M.H., L.L.R.); Children's Hospital of Philadelphia, Philadelphia (A.T.M., W.H.); Stanford University Medical Center, Palo Alto, CA (N.M.); Yale University School of Medicine, New Haven, CT (N.S.K.-L.); and Brown Medical School, Providence, RI (C.L.S.). Address reprint requests to Dr. Oeffinger at the Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021, or at oeffingk@mskcc.org. References Ries LAG, Eisner MP, Kosary CL, et al., eds. SEER cancer statistics review, 1975-2002. Bethesda, MD: National Cancer Institute, 2005. (Accessed September 15, 2006, at http://seer.cancer.gov/csr/1975_2002/.) Hewitt M, Weiner SL, Simone JV, eds. Childhood cancer survivorship: improving care and quality of life. Washington, DC: National Academies Press, 2003. Oeffinger KC, Hudson MM. Long-term complications following childhood and adolescent cancer: foundations for providing risk-based health care for survivors. CA Cancer J Clin 2004;54:208-236. Friedman DL, Meadows AT. Late effects of childhood cancer therapy. Pediatr Clin North Am 2002;49:1083-1106. Mertens AC, Yasui Y, Neglia JP, et al. Late mortality experience in five-year survivors of childhood and adolescent cancer: the Childhood Cancer Survivor Study. J Clin Oncol 2001;19:3163-3172. Hudson MM, Mertens AC, Yasui Y, et al. Health status of adult long-term survivors of childhood cancer: a report from the Childhood Cancer Survivor Study. JAMA 2003;290:1583-1592. Garre ML, Gandus S, Cesana B, et al. Health status of long-term survivors after cancer in childhood: results of an uniinstitutional study in Italy. Am J Pediatr Hematol Oncol 1994;16:143-152. Stevens MC, Mahler H, Parkes S. The health status of adult survivors of cancer in childhood. Eur J Cancer 1998;34:694-698. Sklar CA. Overview of the effects of cancer therapies: the nature, scale and breadth of the problem. Acta Paediatr Suppl 1999;88:1-4. Oeffinger KC, Eshelman DA, Tomlinson GE, Buchanan GR, Foster BM. Grading of late effects in young adult survivors of childhood cancer followed in an ambulatory adult setting. Cancer 2000;88:1687-1695. Robison LL, Mertens AC, Boice JD, et al. Study design and cohort characteristics of the Childhood Cancer Survivor Study: a multi-institutional collaborative project. Med Pediatr Oncol 2002;38:229-239. Mertens AC, Walls RS, Taylor L, et al. Characteristics of childhood cancer survivors predicted their successful tracing. J Clin Epidemiol 2004;57:933-944. Cancer Therapy Evaluation Program. Common terminology criteria for adverse events, version 3.0. Bethesda, MD: National Cancer Institute, 2003. (Accessed September 15, 2006, at http://ctep.cancer.gov/reporting/ctc_v30.html.) Therneau TM, Grambsch PM. Modeling survival data: extending the Cox model. New York: Springer-Verlag, 2000. Gooley TA, Leisenring W, Crowley J, Storer BE. Estimation of failure probabilities in the presence of competing risks: new representations of old estimators. Stat Med 1999;18:695-706. Eiser C, Darlington AS, Stride CB, Grimer R. Quality of life implications as a consequence of surgery: limb salvage, primary and secondary amputation. Sarcoma 2001;5:189-95. Nagarajan R, Neglia JP, Clohisy DR, Robison LL. Limb salvage and amputation in survivors of pediatric lower-extremity bone tumors: what are the long-term implications? J Clin Oncol 2002;20:4493-4501. Ness KK, Mertens AC, Hudson MM, et al. Limitations on physical performance and daily activities among long-term survivors of childhood cancer. Ann Intern Med 2005;143:639-647. Mulhern RK, Hancock J, Fairclough D, Kun L. Neuropsychological status of children treated for brain tumors: a critical review and integrative analysis. Med Pediatr Oncol 1992;20:181-191. Mulhern RK, Reddick WE, Palmer SL, et al. Neurocognitive deficits in medulloblastoma survivors and white matter loss. Ann Neurol 1999;46:834-841. Sklar CA, Constine LS. Chronic neuroendocrinological sequelae of radiation therapy. Int J Radiat Oncol Biol Phys 1995;31:1113-1121. Bhatia S, Yasui Y, Robison LL, et al. High risk of subsequent neoplasms continues with extended follow-up of childhood Hodgkin's disease: report from the Late Effects Study Group. J Clin Oncol 2003;21:4386-4394. Bisharat N, Omari H, Lavi I, Raz R. Risk of infection and death among post-splenectomy patients. J Infect 2001;43:182-186. Travis LB, Gospodarowicz M, Curtis RE, et al. Lung cancer following chemotherapy and radiotherapy for Hodgkin's disease. J Natl Cancer Inst 2002;94:182-192. Hull MC, Morris CG, Pepine CJ, Mendenhall NP. Valvular dysfunction and carotid, subclavian, and coronary artery disease in survivors of Hodgkin lymphoma treated with radiation therapy. JAMA 2003;290:2831-2837. Kenney LB, Yasui Y, Inskip PD, et al. Breast cancer after childhood cancer: a report from the Childhood Cancer Survivor Study. Ann Intern Med 2004;141:590-597. Ng AK, Mauch PM. Late complications of therapy of Hodgkin's disease: prevention and management. Curr Hematol Rep 2004;3:27-33. Oeffinger KC. Longitudinal risk-based health care for adult survivors of childhood cancer. Curr Probl Cancer 2003;27:143-167. Neglia JP, Friedman DL, Yasui Y, et al. Second malignant neoplasms in five-year survivors of childhood cancer: Childhood Cancer Survivor Study. J Natl Cancer Inst 2001;93:618-629. Lipshultz SE, Lipsitz SR, Mone SM, et al. Female sex and drug dose as risk factors for late cardiotoxic effects of doxorubicin therapy for childhood cancer. N Engl J Med 1995;332:1738-1743. Waber DP, Tarbell NJ, Fairclough D, et al. Cognitive sequelae of treatment in childhood acute lymphoblastic leukemia: cranial radiation requires an accomplice. J Clin Oncol 1995;13:2490-2496. Robison LL, Nesbit ME Jr, Sather HN, Meadows AT, Ortega JA, Hammond GD. Height of children successfully treated for acute lymphoblastic leukemia: a report from the Late Effects Study Committee of Childrens Cancer Study Group. Med Pediatr Oncol 1985;13:14-21. Melin AE, Adan L, Leverger G, Souberbielle JC, Schaison G, Brauner R. Growth hormone secretion, puberty and adult height after cranial irradiation with 18 Gy for leukaemia. Eur J Pediatr 1998;157:703-707. Oeffinger KC, Mertens AC, Sklar CA, et al. Obesity in adult survivors of childhood acute lymphoblastic leukemia: a report from the Childhood Cancer Survivor Study. J Clin Oncol 2003;21:1359-1365. Oeffinger KC, Mertens AC, Hudson MM, et al. Health care of young adult survivors of childhood cancer: a report from the Childhood Cancer Survivor Study. Ann Fam Med 2004;2:61-70. Lipshultz SE, Lipsitz SR, Sallan SE, et al. Chronic progressive cardiac dysfunction years after doxorubicin therapy for childhood acute lymphoblastic leukemia. J Clin Oncol 2005;23:2629-2636. Zebrack BJ, Gurney JG, Oeffinger K, et al. Psychological outcomes in long-term survivors of childhood brain cancer: a report from the Childhood Cancer Survivor Study. J Clin Oncol 2004;22:999-1006. Zebrack BJ, Zeltzer LK, Whitton J, et al. Psychological outcomes in long-term survivors of childhood leukemia, Hodgkin's disease, and non-Hodgkin's lymphoma: a report from the Childhood Cancer Survivor Study. Pediatrics 2002;110:42-52....查看详细 (33353字节)
☉ 11342758:A Self-Regulation Program for Maintenance of Weight Loss
ABSTRACT Background Since many successful dieters regain the weight they lose, programs that teach maintenance skills are needed. We developed a maintenance program based on self-regulation theory and tested the efficacy of delivering the program face to face or over the Internet. Methods We randomly assigned 314 participants who had lost a mean of 19.3 kg of body weight in the previous 2 years to one of three groups: a control group, which received quarterly newsletters (105 participants), a group that received face-to-face intervention (105), and a group that received Internet-based intervention (104). The content of the programs in the two intervention groups was the same, emphasizing daily self-weighing and self-regulation, as was the frequency of contact with the groups. The primary outcome was weight gain over a period of 18 months. Results The mean (±SD) weight gain was 2.5±6.7 kg in the face-to-face group, 4.7±8.6 kg in the Internet group, and 4.9±6.5 kg in the control group, with a significant difference between the face-to-face group and the control group (2.4 kg; 95% confidence interval , 0.002 to 10.8; P=0.05). The proportion of participants who regained 2.3 kg or more over the 18-month period was significantly higher in the control group (72.4%) than in the face-to-face group (45.7%; absolute difference, 27%; 95% CI, 14 to 39; P<0.001) or the Internet group (54.8%; absolute difference, 18%; 95% CI, 5 to 30; P=0.008). Daily self-weighing increased in both intervention groups and was associated with a decreased risk of regaining 2.3 kg or more (P<0.001). Conclusions As compared with receiving quarterly newsletters, a self-regulation program based on daily weighing improved maintenance of weight loss, particularly when delivered face to face. (ClinicalTrials.gov number, NCT00067145 .) The major challenge in the treatment of obesity is maintenance of weight loss. Weight-loss programs involving diet, exercise, and behavior modification produce initial weight losses of approximately 10%,1 resulting in clinically important health benefits.2 However, most dieters regain about one third of the weight lost during the next year and are typically back to baseline in 3 to 5 years.3 Maintenance sessions are included in most programs, since continued contact has been found to be helpful in improving maintenance of weight loss.4 Such maintenance sessions have had limited efficacy, perhaps owing to the heterogeneity of weight loss at the start of the maintenance phase and the fact that the sessions are basically extensions of the weight-loss program. There is a need for maintenance programs that specifically target people who have lost substantial amounts of weight, regardless of how they lost it, and that teach skills specific to the maintenance of weight loss. We report here the results of the Study to Prevent Regain (STOP Regain), a randomized clinical trial testing the efficacy of a face-to-face program and an Internet-based program, as compared with a newsletter control group, in preventing weight regain over a period of 18 months. The study focused exclusively on maintenance of weight loss and recruited participants after they had lost a minimum of 10% of their body weight. The maintenance interventions developed for this trial were based on self-regulation theory5 and emphasized comparing current weight with a goal weight (weight at the start of the maintenance phase). Then, depending on the correspondence between these two weights, the participants were taught to adjust their eating and exercise behavior or to provide self-reinforcement.6 We hypothesized that the interventions, delivered face to face or over the Internet, would decrease average weight regain and reduce the proportion of participants who regained 2.3 kg or more during a period of 18 months. Methods Participants We recruited 314 participants between February 2001 and September 2003 through newspaper advertisements, brochures, and contacts with commercial and research weight-control programs in the Rhode Island area (Figure 1). To be eligible, participants were required to have lost at least 10% of their body weight during the prior 2 years. The 10% weight-loss criterion was selected because of its relevance to clinical measures of health and the 2-year criterion because the risk of regaining weight is greatest during the first several years after weight loss.7,8 All participants were required to have a physician, friend, or weight-loss counselor complete and sign a form indicating the amount and timing of their weight loss. Exclusion criteria included serious physical disorders (e.g., heart attack or stroke), serious psychological disorders (e.g., bipolar disease, schizophrenia, or an eating disorder), pregnancy, or a planned move. Participants with medical conditions that might affect their ability to safely complete the intervention or their ability to exercise were required to obtain written permission from a physician before entering the study. Figure 1. Study Enrollment and Retention. Study Design Participants were stratified according to the amount of prior weight loss (10 to 20% of body weight vs. more than 20% of body weight) and then randomly assigned to a control group (105 participants), a face-to-face intervention group (105), or an Internet intervention group (104). The face-to-face format was tested because this approach is used in most weight-control programs and provides a high level of accountability through objective weigh-ins and intensive therapist and peer support. We tested an Internet program because this approach has been shown to be helpful in weight loss and maintenance9,10,11; we reasoned that the Internet program would be more convenient for participants and would therefore produce better attendance at intervention sessions than the face-to-face program. Participants were assessed at 6, 12, and 18 months and were paid $25 for attending the 6- and 12-month assessments and $50 for attending the 18-month assessment. The protocol was approved by the institutional review board at the Miriam Hospital, Providence; written informed consent was obtained from all participants. The authors designed the study and secured funding; they took sole responsibility for treating participants, collecting and analyzing the data, and writing the manuscript; they vouch for the accuracy and completeness of the reported data. Interventions The face-to-face and Internet interventions were identical in frequency of contact and content and were intended to teach participants to regulate their body weight. Participants in the intervention groups were given a scale and introduced to a weight-monitoring system based on color zones. They were taught to use the scale in the way they would use a thermostat or home glucose monitor — namely, to determine whether adjustments in energy-balance behaviors were needed. Since a major difference between weight loss and weight maintenance is the reinforcement a person receives from others,3 participants were asked to submit their weight weekly through an automated telephone system (face-to-face group) or a Web-based form (Internet group). Those who reported maintaining their weight, defined as a weight gain of less than 1.4 kg12 over their starting weight, were in the green zone and were provided immediate reinforcement with positive automated messages and also received small green gifts monthly (e.g., green gum, green tea, or a green dollar bill) to foster the development of self-reinforcement skills. Participants with weight gains of 1.4 to 2.2 kg were in the yellow zone and were instructed to use problem-solving skills to bring their weight back to the green zone. Participants with a weight gain of 2.3 kg or more were in the red zone and were encouraged to restart active weight-loss efforts, using either their initial approach to weight loss or a standard behavioral approach involving a low-calorie, low-fat diet and increased physical activity. Participants in the red zone were also encouraged to use a tool kit provided at the start of the program that included their own weight-loss success story, self-monitoring diaries, a book providing information on calories and fat, a pedometer, and several cans of a meal-replacement product (Slim-Fast, Unilever). Red-zone participants were also offered individual counseling by e-mail (Internet group) or by telephone or in person (face-to-face group) until they returned to their starting weight. Throughout the program, participants in both intervention groups were encouraged to practice eating and exercise behaviors (such as exercising 60 minutes a day) that have been reported to help maintain weight loss.13,14 Both groups attended weekly meetings for the first month and thereafter (for the balance of the 18-month period) attended monthly meetings. In the face-to-face group, meetings were held at a clinic affiliated with Miriam Hospital and included an individual weigh-in and group session. Internet participants were provided with a laptop computer and an Internet connection (if needed) as well as technical support. They attended an introductory session designed to teach them how to use the laptop. Participants in the Internet group had access to a STOP Regain message board and a Web site where treatment lessons and weekly tips were posted and where they reported their weekly weight and physical-activity data. Their group meetings were conducted in a chat room. The same study personnel (nutritionists, exercise physiologists, and clinical psychologists with master's or Ph.D. degrees) led the meetings for both groups; all had experience leading behavioral weight-control groups and received uniform training and supervision for this study. The control group received a quarterly newsletter with information about diet, exercise, and weight control. These participants had no interaction with intervention staff and were seen at the clinic only for assessments. End Points The primary end point was weight gain at 18 months; secondary end points, identified a priori, were the percentage of participants who regained 2.3 kg or more (chosen because it is larger than the change expected with normal daily weight fluctuation and because of its public health implication)15 and changes in diet, activity, and use of behavioral strategies. At each assessment, participants were weighed with a calibrated scale while they were wearing light street clothes, and they completed the Paffenbarger Physical Activity Questionnaire16 and the Block Food Frequency Questionnaire.17 They also reported their frequency of self-weighing and rated the importance of key behavioral strategies on a scale of 1 to 6 (in which a score of 1 indicates not at all important and a score of 6 of major importance). Height was assessed at baseline with a wall-mounted stadiometer. Statistical Analysis The sample size was selected to provide the study with a statistical power of 80% to detect a 3-kg difference in weight regain between groups, with the use of a two-tailed test and adjustment for three comparisons. Baseline characteristics were compared with the use of the chi-square test or analysis of variance. Absolute weight gain and the percentage of weight lost from the highest weight reported in the previous 2 years were determined with analysis of variance and repeated-measures analysis of covariance, respectively, with dropouts assumed to have regained 0.3 kg per month, an assumption used in prior studies18,19 and similar to the amount regained by control-group participants in this trial. Chi-square tests were used to compare groups with respect to the percentage regaining 2.3 kg or more, with dropouts assumed to have regained this amount. The three pairwise comparisons were tested and Bonferroni's adjustment was used. All analyses were performed with the use of SPSS software, version 13.0, and no interim analyses were conducted. All reported P values are two-sided and reflect Bonferroni's adjustment. Results Baseline characteristics of the 314 participants who underwent randomization are shown in Table 1. There were no significant differences among the three study groups in any of these measures. The most commonly reported approaches used to lose weight were commercial weight-loss groups (39.5%), an individual approach with no outside help (22.9%), and exercise (36.6%), with an additional approach usually accompanying exercise. The 6- and 12-month assessments were completed by 300 and 290 participants (95.5% and 92.4%), respectively, with no significant differences among the groups. The 18-month assessments were completed by 291 participants (92.7%), with significantly better attendance in the Internet group than in the face-to-face group (97.1% vs. 87.6%, P=0.03), but with attendance in neither intervention group significantly different from that of the control group (93.3%; P=0.20 and P=0.16, respectively). Table 1. Baseline Characteristics of the Study Participants. Absolute Weight Change The mean (±SD) absolute weight gain over the 18-month period was 2.5±6.7 kg in the face-to-face group as compared with 4.7±8.6 kg in the Internet group and 4.9±6.5 kg in the control group (Table 2). There was a significant difference in weight gain between the face-to-face and control groups (absolute difference, 2.4 kg; 95% confidence interval , 0.002 to 10.8; P=0.05), but not between the face-to-face group and the Internet group (2.2 kg; 95% CI, –0.50 to 10.3; P=0.09), or between the Internet group and the control group (absolute difference, 0.2 kg; 95% CI, –4.9 to 5.9; P=1.00). Repeated-measures analysis of covariance, with adjustment for baseline percent weight reduction, showed that the face-to-face group also differed significantly from both the Internet group and the control group with respect to the percentages of weight lost during the 18 months of the trial (P=0.02 and P=0.006, respectively) (Figure 2). The failure to find a significant difference between the Internet and control groups on these measures resulted, in part, from the somewhat larger amount of weight regained in the Internet group. Among participants in the red zone, those in the Internet group had regained a mean of 9.8±8.4 kg at the end of 18 months, as compared with 7.5±5.6 kg in the control group (P=0.18) and 7.4±6.4 in the face-to-face group (P=0.24). Table 2. Mean Weight Change from Baseline. Figure 2. Mean (±SE) Percent Weight Reduction at Baseline and at 6, 12, and 18 Months. Percent weight reduction was calculated according to participants' highest weight in the prior 2 years, and it was assumed that dropouts had regained 0.3 kg per month. Data were analyzed by repeated-measures analysis of covariance, adjusting for percent weight reduction at baseline. With Bonferroni's adjustments for multiple comparisons, the percent weight reduction in the face-to-face group differed significantly from that in the Internet group (P=0.02) and the control group (P=0.006). Percentage of Participants Regaining 2.3 kg or More Assuming that all dropouts regained 2.3 kg or more, we found that 72.4% of participants in the control group regained 2.3 kg or more during the 18-month period, as compared with 54.8% of participants in the Internet group (absolute difference, 18%; 95% CI, 5 to 30; P=0.008) and 45.7% of participants in the face-to-face group (absolute difference, 27%; 95% CI, 14 to 39; P<0.001) (Figure 3). There was no significant difference in the percentage who regained 2.3 kg or more between the two intervention groups (absolute difference, 9%; 95% CI, –4 to 23; P=0.19). Figure 3. Proportion of Participants Who Regained 2.3 kg or More at 6, 12, or 18 Months. At 6 months, the proportion of participants who had regained 2.3 kg or more differed significantly between the face-to-face group and the control group (27% vs. 47%; absolute difference, 20%; 95% CI, 7 to 33; P=0.003). At 18 months, the control group differed significantly from both the face-to-face group (72.4% vs. 45.7%; absolute difference, 27%; 95% CI, 14 to 39; P<0.001) and the Internet group (72.4% vs. 54.8%; absolute difference, 18%; 95% CI, 5 to 30; P=0.008). I bars are standard errors. Effect of Interventions on the Prevention of Weight Regain Of the 314 participants in the study, 34.7% remained consistently in the green or yellow zone at 6, 12, and 18 months, including 43.8% of those in the face-to-face group, 37.5% of those in the Internet group, and 22.9% of those in the control group. There was a significant difference in these percentages between the control group and the face-to-face group (absolute difference, 21%; 95% CI, 9 to 33; P=0.001) and the control group and the Internet group (absolute difference, 15%; 95% CI, 2 to 27; P=0.02), but not between the intervention groups (absolute difference, 6%; 95% CI, –7 to 20; P=0.35). In contrast, only 23 of the 166 participants (13.9%) who regained 2.3 kg or more at 6 or 12 months subsequently returned to the green or yellow zone, with no significant difference among the face-to-face, Internet, and control groups (19.2%, 16.0%, and 7.8%, respectively). The difference between the face-to-face group and the control group approached significance (P=0.07), but the Internet group did not differ significantly from the face-to-face group (P=0.99) or the control group (P=0.17). Adherence Attendance at the face-to-face and chat-room sessions decreased over time (P<0.001) but was consistently better in the face-to-face group than in the Internet group (P=0.005). The percentage of sessions attended by participants in the face-to-face group was 78.7% from baseline to 6 months, 53.5% from 7 to 12 months, and 41.5% from 13 to 18 months, as compared with 65.7%, 41.2%, and 34.2%, respectively, in the Internet group. The percentage of participants who reported their weight each week also decreased over time (P<0.001), but the difference between the two intervention groups was not significant. In the face-to-face group, the percentage of participants who reported their weight was 84.0% from baseline to 6 months, 68.6% from 7 to 12 months, and 56.1% from 13 to 18 months, and in the Internet group the respective percentages were 82.0%, 69.1%, and 55.3%. Adverse Events Medical and psychological adverse events were monitored throughout the trial. No serious adverse events were related to the interventions (Figure 1). Behavioral Changes Despite the differences in regained weight, there were no significant differences in changes in caloric intake, percentage of calories from fat, or physical activity among the groups. However, there were significant differences in the participants' ratings of the importance of key behavioral strategies. Both intervention groups gave higher ratings for self-weighing as an important aspect of their weight-control efforts than did the control group (P=0.001). The face-to-face group, as compared with the control group, also gave higher ratings for setting a weight-loss goal (P=0.005), counting calories (P=0.03), and keeping a graph or record of eating and exercise (P=0.01). Since self-weighing is a key component of the self-regulation model, we compared the proportion of participants in each group who reported weighing themselves at least daily. Differences between treatment groups were significant at 6, 12, and 18 months (P<0.001 for all pairwise comparisons); during each period, 28.9 to 40.0% of participants in the control group reported daily weighing, as compared with 65.1 to 81.4% of participants in the Internet group and 71.2 to 78.9% of participants in the face-to-face group. In addition, in the face-to-face and Internet groups, a smaller proportion of participants who weighed themselves daily regained 2.3 kg or more, as compared with those who weighed themselves less often (face-to-face group, 26.2% vs. 58.3%; absolute difference, 32%; 95% CI, 10 to 55; P=0.005; Internet group, 39.7% vs. 67.7%; absolute difference, 28%; 95% CI, 7 to 49; P=0.01). In the control group, the difference was much smaller (65.3% vs. 71.9%; absolute difference, 7%; 95% CI, –15 to 28; P=0.54) (Figure 4). Figure 4. Proportion of Participants Who Regained 2.3 kg or More among Those Who Reported Weighing Themselves Daily versus Weighing Less Often at 18 Months. Daily weighing was reported by 26, 58, and 61 participants in the control, Internet, and face-to-face groups, respectively; less frequent weighing was reported by 64, 31, and 24 participants, respectively. The percentage of participants who regained 2.3 kg or more was significantly lower among those who weighed themselves daily than among those who weighed themselves less often in the face-to-face group (26.2% vs. 58.3%; absolute difference, 32%; 95% CI, 10 to 55; P=0.005) and the Internet group (39.7% vs. 67.7%; absolute difference, 28%; 95% CI, 7 to 49; P=0.01) but not in the control group (65.3% vs. 71.9%; absolute difference, 7%; 95% CI, –15 to 28; P=0.54). Discussion A major problem in the treatment of obesity is maintenance of weight loss. Our results suggest that with minimal intervention, 72% of successful dieters will regain more than 2.3 kg over a period of 18 months. The interventions used in our study, which focused on teaching self-regulation of body weight, significantly decreased the risk of regaining 2.3 kg or more. However, only the face-to-face format reduced the amount of weight regained. We recruited participants who had recently lost a clinically important amount of weight, regardless of how they had lost the weight, and taught them strategies to prevent weight regain instead of promoting further weight loss. The efficacy of the interventions we tested suggests that this concept of a separate and distinct weight-loss maintenance program may be important for the successful treatment of obesity. In this study and a prior study,20 preventing weight regain appears to be critical because of the difficulty of reversing even small weight regains. The face-to-face format produced the best outcomes in this trial, with an average weight gain of 2.5 kg and 46% of participants regaining 2.3 kg or more. Attendance was also superior in the face-to-face group. The Internet intervention was effective in decreasing the proportion of participants who regained weight, but it was less effective in reducing the amount of weight regained. Therefore, from a public health perspective, it might be desirable to use the Internet strategy for prevention of weight regain but to add face-to-face or telephone counseling if weight is regained. The proportion of participants who reported daily self-weighing was significantly higher in the intervention groups than in the control group, and daily self-weighing was strongly associated with successful weight-loss maintenance. However, frequent self-weighing could be either a cause or a consequence of weight-loss maintenance. The fact that participants in the intervention groups had been taught to use their weight data to regulate their eating and exercise behaviors may be what made this strategy effective for these groups but not for the control group. Several recent studies have shown the importance of frequent self-weighing for both prevention of weight gain and weight loss,21 and clinical guidelines of the National Heart, Lung, and Blood Institute7 state that "regular self-monitoring of weight is critical for long-term maintenance." Although concern has been expressed about possible adverse effects of regular self-weighing, there is little evidence to support this concern22 and no evidence of adverse consequences of the interventions in our trial. The strengths of our study include the selection of a cohort of successful dieters (who had an average weight loss of more than 19 kg at baseline), the randomized design, and the fact that 93% of participants completed the 18-month follow-up. Limitations include failure to find differences in dietary intake or activity, perhaps reflecting the limitations of using self-reported measures,23 and the inability to determine whether self-regulation was more effective than other approaches to weight-loss maintenance and which aspects of the intervention were most important. In conclusion, the two self-regulation interventions were more effective than the use of newsletters in maintaining weight loss for the duration of the study. The face-to-face intervention decreased the amount of weight regained, but both the Internet and the face-to-face interventions decreased the risk of regaining 2.3 kg or more. Future studies should examine ways to refine these interventions in order to improve their efficacy. Supported by a grant (DK57413) from the National Institute of Diabetes and Digestive and Kidney Diseases. Dr. Wing reports having received consulting fees from Sanofi and Pfizer; and Dr. Tate, lecture fees from Unilever. No other potential conflict of interest relevant to this article was reported. We are indebted to Natalie Robinson, Elizabeth Jackvony, Deborah Sepinwall, Jacki Hecht, and Marta Roberts for their contributions in coordinating the study and providing the interventions; to Jason Peng and Jason Machan for data management and statistical support; to Kristen Whitehead for administrative support; and to all the STOP Regain participants. Source Information From Brown Medical School (R.R.W., A.A.G., H.A.R.) and Miriam Hospital (R.R.W., A.A.G., H.A.R., J.L.F.) — both in Providence, RI; and the University of North Carolina, Chapel Hill (D.F.T.). Address reprint requests to Dr. Wing at the Weight Control and Diabetes Research Center, 196 Richmond St., Providence, RI 02903, or at rwing@lifespan.org. References Wing RR. Behavioral approaches to the treatment of obesity. In: Bray GA, Bouchard C, eds. Handbook of obesity: clinical applications. New York: Marcel Dekker, 2004:147-67. Knowler WC, Barrett-Connor E, Fowler SE, et al. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 2002;346:393-403. Wadden TA, Phelan S. Behavioral assessment of the obese patient. In: Wadden TA, Stunkard AJ, eds. Handbook of obesity treatment. New York: Guilford Press, 2002:186-226. Perri MG, Corsica JA. Improving the maintenance of weight lost in behavioral treatment of obesity. In: Wadden TA, Stunkard AJ, eds. Handbook of obesity treatment. New York: Guilford Press, 2002:357-79. Kanfer FH, Goldstein AP, eds. Helping people change: a textbook of methods. New York: Pergamon Press, 1975. Wing RR, Epstein LH, Nowalk MP, Lamparski DM. Behavioral self-regulation in the treatment of patients with diabetes mellitus. Psychol Bull 1986;99:78-89. National Institutes of Health. Clinical guidelines on the identification, evaluation, and treatment of overweight and obesity in adults -- The Evidence Report. Obes Res 1998;6:Suppl 2:51S-209S. McGuire MT, Wing RR, Klem ML, Lang W, Hill JO. What predicts weight regain in a group of successful weight losers? J Consult Clin Psychol 1999;67:177-185. Tate DF, Jackvony EH, Wing RR. Effects of Internet behavioral counseling on weight loss in adults at risk for type 2 diabetes: a randomized trial. JAMA 2003;289:1833-1836. Tate DF, Wing RR, Winett RA. Using Internet technology to deliver a behavioral weight loss program. JAMA 2001;285:1172-1177. Harvey-Berino J, Pintauro S, Buzzell P, Gold EC. Effect of Internet support on the long-term maintenance of weight loss. Obes Res 2004;12:320-329. Baker RC, Kirschenbaum DS. Weight control during the holidays: highly consistent self-monitoring as a potentially useful coping mechanism. Health Psychol 1998;17:367-370. Klem ML, Wing RR, McGuire MT, Seagle HM, Hill JO. A descriptive study of individuals successful at long-term maintenance of substantial weight loss. Am J Clin Nutr 1997;66:239-246. Wing RR, Hill JO. Successful weight loss maintenance. Annu Rev Nutr 2001;21:323-341. Stevens J, Truesdale KP, McClain JE, Cai J. The definition of weight maintenance. Int J Obes (Lond) 2006;30:391-399. Paffenbarger RS Jr, Wing AL, Hyde RT. Physical activity as an index of heart attack risk in college alumni. Am J Epidemiol 1978;108:161-175. Block G, Hartman AM, Dresser CM, Carroll MD, Gannon J, Gardner L. A data-based approach to diet questionnaire design and testing. Am J Epidemiol 1986;124:453-469. Wadden TA, Berkowitz RI, Womble LG, et al. Randomized trial of lifestyle modification and pharmacotherapy for obesity. N Engl J Med 2005;353:2111-2120. Wadden TA, Berkowitz RA, Sarwer DB, Prus-Wisniewski R, Steinberg C. Benefits of lifestyle modification in the pharmacologic treatment of obesity: a randomized trial. Arch Intern Med 2001;161:218-227. Phelan S, Hill JO, Lang W, Dibello JR, Wing RR. Recovery from relapse among successful weight maintainers. Am J Clin Nutr 2003;78:1079-1084. Linde JA, Jeffery RW, French SA, Pronk NP, Boyle RG. Self-weighing in weight gain prevention and weight loss trials. Ann Behav Med 2005;30:210-216. O'Neil PM, Brown JD. Weighing the evidence: benefits of regular weight monitoring for weight control. J Nutr Educ Behav 2005;37:319-322. Lichtman SW, Pisarska K, Berman ER, et al. Discrepancy between self-reported and actual caloric intake and exercise in obese participants. N Engl J Med 1992;327:1893-1898....查看详细 (29699字节)

☉ 11342759:Effect of Ramipril on the Incidence of Diabetes
ABSTRACT Background Previous studies have suggested that blockade of the renin–angiotensin system may prevent diabetes in people with cardiovascular disease or hypertension. Methods In a double-blind, randomized clinical trial with a 2-by-2 factorial design, we randomly assigned 5269 participants without cardiovascular disease but with impaired fasting glucose levels (after an 8-hour fast) or impaired glucose tolerance to receive ramipril (up to 15 mg per day) or placebo (and rosiglitazone or placebo) and followed them for a median of 3 years. We studied the effects of ramipril on the development of diabetes or death, whichever came first (the primary outcome), and on secondary outcomes, including regression to normoglycemia. Results The incidence of the primary outcome did not differ significantly between the ramipril group (18.1%) and the placebo group (19.5%; hazard ratio for the ramipril group, 0.91; 95% confidence interval , 0.81 to 1.03; P=0.15). Participants receiving ramipril were more likely to have regression to normoglycemia than those receiving placebo (hazard ratio, 1.16; 95% CI, 1.07 to 1.27; P=0.001). At the end of the study, the median fasting plasma glucose level was not significantly lower in the ramipril group (102.7 mg per deciliter ) than in the placebo group (103.4 mg per deciliter , P=0.07), though plasma glucose levels 2 hours after an oral glucose load were significantly lower in the ramipril group (135.1 mg per deciliter vs. 140.5 mg per deciliter , P=0.01). Conclusions Among persons with impaired fasting glucose levels or impaired glucose tolerance, the use of ramipril for 3 years does not significantly reduce the incidence of diabetes or death but does significantly increase regression to normoglycemia. (ClinicalTrials.gov number, NCT00095654 .) Diabetes, ranked as the fifth leading cause of death worldwide, is a major risk factor for various cardiovascular and renal disorders.1 The prevalence of diabetes is increasing, which in turn increases the risk of premature cardiovascular disease and death.2 Therefore, strategies to reduce the incidence of diabetes are needed. Physical activity, weight loss,3,4 and some glucose-lowering agents4,5 reduce the incidence of diabetes in people with elevated glucose levels that are just below the diagnostic threshold for diabetes. Several trials involving people with hypertension or cardiovascular disease have suggested that agents that block or inhibit the renin–angiotensin system may also prevent diabetes.6 The Heart Outcomes Prevention Evaluation (HOPE) study showed that, in a population at high risk for cardiovascular events, the use of ramipril reduced cardiovascular events by 22% and diabetes by 34%, as compared with placebo.7 However, the presence of diabetes was ascertained by self-report in the HOPE study and was not a prespecified outcome. Other studies reported similar findings in people with cardiovascular disease or hypertension.6,8 We conducted a prospective trial, the Diabetes Reduction Assessment with Ramipril and Rosiglitazone Medication (DREAM) study, to evaluate whether ramipril reduces the risk of diabetes in people who have impaired fasting glucose levels (after an 8-hour fast) or impaired glucose tolerance but who are at low risk for cardiovascular events. Methods A detailed description of the design of the DREAM trial has been published previously.9 Briefly, between July 2001 and August 2003, we enrolled 5269 persons 30 years of age or older who had impaired fasting plasma glucose levels (at least 110 mg per deciliter but less than 126 mg per deciliter ) or impaired glucose tolerance (a plasma glucose level of at least 140 mg per deciliter but less than 200 mg per deciliter 2 hours after an oral glucose load) but who did not have a history of diabetes (not including gestational diabetes), cardiovascular disease, or intolerance of either angiotensin-converting–enzyme (ACE) inhibitors or thiazolidinediones. In early 2003, study eligibility was expanded to include persons with isolated impaired fasting glucose levels. Eligible participants entered a 17-day, single-blind, double-placebo run-in period. If they showed adherence to their study medications during that period, participants were randomly assigned to receive either ramipril (Altace, Sanofi-Aventis, King Pharmaceuticals) (5 mg daily for the first 2 months, with an increase to 10 mg at the 2-month visit and 15 mg after 1 year) or matching placebo (and rosiglitazone or matching placebo ) (4 mg once daily for the first 2 months and then 8 mg thereafter). In a 2-by-2 factorial design, patients were randomly assigned to a study group with the use of a concealed, computerized telephone randomization system, stratified according to center, with a permuted block size of 8. Detailed results for the rosiglitazone group are reported separately.10 Visits were scheduled 2 months and 6 months after randomization and then every 6 months until the common termination window between February and April 2006. Alanine aminotransferase levels were measured every 2 months during the first year. At each visit, study drugs were dispensed, and adherence was assessed and reinforced, as was a healthy diet and lifestyle. Electrocardiograms were recorded at baseline, at 2 years, and at the end of the study. At the 2-year and final visits, a glucose-tolerance test was performed 2 hours after a 75-g oral glucose load in participants in whom diabetes had not developed. At other annual visits, fasting plasma levels of glucose and glycated hemoglobin were measured locally, and an oral glucose-tolerance test was performed if the fasting plasma glucose level was 126 mg per deciliter (7.0 mmol per liter) or higher, to confirm or refute the diagnosis of diabetes, or if the fasting plasma glucose level exceeded 95 mg per deciliter (5.3 mmol per liter) and the glycated hemoglobin value exceeded 93% of the upper limit of the normal range for the assay. If diabetes was diagnosed during the study and required pharmacologic therapy, the study medications were continued and antihyperglycemic agents other than thiazolidinediones could be prescribed. Participants who had not received a diagnosis of diabetes by the end of the study entered a single-blind placebo washout period and underwent a glucose-tolerance test (2 hours after an oral glucose load) 2 to 3 months later. The study protocol and consent forms were approved by the ethics committees at all centers, and all participants provided written informed consent. Outcomes The primary outcome was newly diagnosed diabetes or death. Death was included to account for the possibility that diabetes may develop at different rates in people who die and in those who survive. Diabetes was diagnosed if a locally measured fasting plasma glucose level was at least 126 mg per deciliter (7.0 mmol per liter) or a 2-hour post-load glucose level was at least 200 mg per deciliter (11.1 mmol per liter), with confirmation by a second test on a different day. In the event that a confirmatory second result could not be obtained, diabetes was diagnosed on the basis of one abnormal result, provided there was no clinical reason to refute the diagnosis. Diabetes was also diagnosed if a physician outside of the study diagnosed diabetes on the basis of a fasting plasma glucose level of at least 126 mg per deciliter (7.0 mmol per liter) or any plasma glucose level of at least 200 mg per deciliter (11.1 mmol per liter) and prescribed an antidiabetic agent. The key secondary outcomes were a composite of cardiac and renal events, defined as either cardiovascular events (clinical or silent myocardial infarction, stroke, death from cardiovascular events, revascularization procedures, heart failure, newly diagnosed angina with objective evidence of ischemia, or ventricular arrhythmia requiring resuscitation) or renal events (on the basis of measurements in urine and blood at a central laboratory). Both the composite of the cardiac and renal events and the renal events alone have yet to be analyzed. Other secondary outcomes included glucose levels and regression to normal glucose levels (fasting plasma glucose level, less than 110 mg per deciliter ; 2-hour post-load glucose level, less than 140 mg per deciliter ). A committee that was unaware of the study-group assignments adjudicated the diagnoses of diabetes and clinical outcomes according to predefined definitions (see the Supplementary Appendix, available with the full text of this article at www.nejm.org). Statistical Analysis We calculated that we would need to enroll at least 5000 persons with impaired fasting glucose levels or impaired glucose tolerance for the study to have a statistical power of 90% to detect a risk reduction exceeding 22% in the ramipril group. The calculation was based on an estimated annual incidence of the primary outcome in the placebo group of 4.5%, a mean follow-up of 3 years, a two-sided type 1 error rate of 5%, and a combined effect of both interventions that was 10% less than that for either intervention. All data were collected and analyzed at the Population Health Research Institute, McMaster University, with the use of an intention-to-treat approach, under the supervision of the steering committee. Data for participants whose diabetes status was unavailable at the end of the study were censored at the time of the last glucose assessment. Kaplan–Meier curves for the primary outcome as well as regression to normoglycemia were constructed for the treatment and placebo groups and were compared with the use of log-rank tests. The outcome of regression was based on available values. If glucose levels were not available (e.g., because tests were not completed), we assumed that glycemic status had not changed since the last known value. Cox proportional-hazards models were used to estimate the effect of ramipril on the hazard ratio for the primary and secondary outcomes (with stratification according to whether treatment included rosiglitazone or placebo). Interaction between the ramipril and rosiglitazone treatments was assessed with the inclusion of an interaction term in the model. We assessed the effect of the study drugs on glucose levels by calculating the median fasting plasma glucose level and the 2-hour post-load plasma glucose level at each scheduled measurement time. Since an oral glucose-tolerance test was not performed after diabetes was diagnosed, and since any fasting plasma glucose level measured after diabetes was diagnosed might have been lowered as a result of diabetes management, a calculation of the medians or means with the use of every available value would have failed to assess accurately the effect of the treatment on glucose levels. Instead, we calculated the medians by assigning people with diabetes the worst rank score, in Wilcoxon rank-sum analyses, for both the 2-hour post-load and fasting plasma glucose levels.11 Analysis of variance (with adjustment for the baseline level) was used to assess the differences between groups in the mean change in alanine aminotransferase levels and in systolic and diastolic blood pressures at the end of the trial. All reported P values are two-sided, and they were not adjusted for multiple testing. An independent trial monitoring committee reviewed the data at least annually. The committee could inform the principal investigators of the study results and recommend study termination if there was a consistent reduction (i.e., a reduction that was evident after two consecutive analyses performed more than 6 months apart) in the hazard ratio for the primary outcome of 4 SD in the first half of the planned follow-up or of 3 SD in the second half, or if there was an excess of cardiovascular events of 3 SD in the first half of the trial or of 2 SD in the second half. In October 2005, after confirming the first interim analysis, the trial monitoring committee informed the principal investigators of the results of the entire study after determining that the study question regarding rosiglitazone had been clearly answered. The committee recommended an early and orderly close-out of the study. The principal investigators agreed, and the study was terminated 5 months early. The DREAM trial was funded by the Canadian Institutes of Health Research, Sanofi-Aventis, GlaxoSmithKline, and King Pharmaceuticals through the University Industry grant program. Sanofi-Aventis and King Pharmaceuticals provided ramipril and placebo, and GlaxoSmithKline provided rosiglitazone and placebo. The trial was designed, implemented, and analyzed by the steering committee, which also wrote and reviewed the paper. The data were held by the investigators at the Population Health Research Institute, McMaster University. Representatives of the study sponsors were nonvoting members of the steering committee. Results Baseline and Follow-up A total of 24,592 participants were screened at 191 centers in 21 countries. Of those screened, 5808 entered the run-in phase of the trial. The most common reasons for exclusion were ineligibility (94.2%) and refusal to participate (3.0%). Of those entering the run-in phase, 5269 participants were randomly assigned to treatment (739 had impaired fasting glucose levels alone and 4530 had impaired glucose tolerance with or without impaired fasting glucose levels). The most common reasons for exclusion during the run-in period were ineligibility (284 participants) and refusal to participate (159 participants). Table 1 summarizes the baseline characteristics of the 5269 participants, which were similar in the ramipril and placebo groups. Table 1. Baseline Characteristics of the Study Participants. Participants were followed for a median of 3.0 years. At 1 year, 86.6% of participants randomly assigned to receive ramipril and 89.9% of those randomly assigned to receive placebo were still taking the study medication. The corresponding proportions at 2 years were 81.3% and 84.8%; at 3 years, 75.4% and 80.9%; and at the end of the study, 72.7% and 78.0%. Throughout the study, the most common reasons for the discontinuation of study medications among participants in the ramipril group and those in the placebo group were the participant's decision to stop taking the medication (17.4% and 17.7%, respectively), cough (9.7% and 1.8%), advice from a physician (2.3% and 2.5%), and peripheral edema (1.0% and 1.1%) (Table 1 in the Supplementary Appendix). Angioedema occurred in three participants receiving ramipril (0.1%) and in four participants receiving placebo (0.2%). Open-label ACE inhibitors were used by 2.7% and 4.0% of participants in the ramipril and placebo groups, respectively. The mean systolic blood pressure at baseline was 136.1 mm Hg in the ramipril group and 136.0 mm Hg in the placebo group (Table 1). This value decreased by 8.2 mm Hg among those receiving ramipril, as compared with 3.9 mm Hg among those receiving placebo at 2 months (P0.11 for all interactions). The absence of a significant interaction was further demonstrated by the similar hazard ratios for the primary outcome among participants receiving ramipril, regardless of whether they also received active rosiglitazone: the hazard ratio associated with ramipril, as compared with placebo, among participants receiving active rosiglitazone was 0.92 (95% CI, 0.74 to 1.15), and that among participants receiving placebo rosiglitazone was 0.91 (95% CI, 0.78 to 1.05). The results for the regression to normoglycemia were similar. Discussion Our study suggests that the use of up to 15 mg of ramipril daily for 3 years does not significantly prevent diabetes or death in people without cardiovascular disease who have impaired fasting glucose levels or impaired glucose tolerance. However, significantly more participants receiving ramipril had normal fasting glucose levels and glucose tolerance than those receiving placebo, and the distribution of the glucose levels had shifted downward in the ramipril group by the end of the study. These significant effects suggest that a longer or larger study would be needed to detect a reduction in the rate of newly diagnosed diabetes in this population, if such a reduction exists. There may be several reasons why these results differ from the reductions in the rates of newly diagnosed diabetes reported previously with ACE inhibitors.7,12,13,14,15,16,17,18,19,20 First, our study was specifically designed to determine whether ramipril prevents diabetes, unlike previous studies7,8,12,13 in which the analysis of the development of diabetes was either a secondary or post hoc outcome and in which glucose levels were not systematically recorded. Second, fasting glucose levels and 2-hour post-load glucose levels below the diagnostic threshold for diabetes were required for entry into our study. In contrast, diabetes status was not explicitly established in several previous studies7,8,12,13; consequently, some participants in those studies who were identified as not having diabetes may actually have had undiagnosed diabetes at baseline. The fact that participants also had preexisting cardiovascular disease, which is associated with a high prevalence of dysglycemia,21,22 supports this possibility. Previous observations concerning the effects of ACE inhibition on newly diagnosed diabetes therefore arguably reflect reduced ascertainment or increased regression of preexisting diabetes in participants with undiagnosed diabetes at baseline, decreased progression to newly diagnosed diabetes, or both. Third, participants in our trial differed from those in previous studies, who primarily had known cardiovascular disease, heart failure, hypertension, or a combination thereof. In our study, patients with cardiovascular disease and heart failure were excluded, and participants were relatively younger (mean age, 55 years, vs. 65 years in other trials). The mean blood pressure of 136/83 mm Hg at baseline in our study is substantially lower than that reported in the hypertension trials. It is possible that the degree of activation of the renin–angiotensin system is higher in people who are older or have known cardiovascular disease or hypertension and that ACE inhibition may therefore have a greater effect in these people than in others. We compared ramipril with placebo, whereas the comparison in several other trials was with another antihypertensive agent, such as a beta-blocker or a diuretic.14,15,16 Both of these medications may increase the risk of diabetes,23 thereby leading to an overestimation of the effects of ACE inhibitors. The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) compared the metabolically neutral calcium-channel blocker to an ACE inhibitor and reported a 17% reduction in the incidence of diabetes in the ACE-inhibitor group.15 The combined data from three of the largest previous trials that compared ACE inhibitors with placebo in subjects with cardiovascular disease suggest a risk reduction in diabetes of 14% (95% CI, 5 to 22),8 which appears to be consistent with the 9% lower rate of newly diagnosed diabetes in our study. The duration of follow-up was shorter in our trial (median, 3 years) than in the previous studies of ACE inhibitors and angiotensin-receptor blockers (median, about 4.5 years). The Kaplan–Meier curves in our study suggest a benefit of ramipril in the prevention of diabetes after 3.5 years (Figure 1A). Whether this apparent late divergence between the ramipril group and the placebo group is real (or simply due to chance) can be reliably ascertained only by further follow-up of the participants in our trial or by other trials with longer follow-up (e.g., the NAVIGATOR trial or the ONTARGET/TRANSCEND trials24). Finally, the fact that our study detected a significant effect of ramipril on glucose metabolism but not a significant reduction in the incidence of diabetes may reflect the entry criteria, which led to the exclusion of anyone with glucose levels in the diabetic range. As a result, most participants had baseline glucose levels that were far from the diagnostic threshold for diabetes and close to the normal threshold, which is reflected in the median fasting plasma glucose level of approximately 106.3 mg per deciliter (5.90 mmol per liter) and the median 2-hour post-load glucose level of approximately 156.7 mg per deciliter (8.70 mmol per liter) among all participants at baseline. Therefore, a modest reduction in glucose levels resulting from the use of ramipril would allow more participants to cross into the normal range rather than into the more distant diabetic range, and there would be more power to detect an effect on regression to normal levels than progression to diabetic levels. This appeared to be the case, since diabetes developed in 938 participants, whereas 2128 participants had regression to normoglycemia. Thus, despite the fact that the DREAM trial did not detect a significant effect of ramipril on the incidence of diabetes, the effect with respect to regression to normoglycemia and the totality of the available data suggest that drugs that block the renin–angiotensin system may have a modest, favorable effect on glucose metabolism. A limitation of our study was that, despite extensive efforts to obtain complete follow-up data, glucose levels were available for only 92.6% of those participants who had not reached a primary outcome by the end of the study. However, information on diabetes status was obtained from the histories of 97.7% of the participants. In addition, the 3 years of follow-up in our trial and the low rates of cardiovascular events during the trial are probably inadequate to provide reliable information on the ultimate prevention of cardiovascular outcomes. Diet and lifestyle changes are typically recommended for the prevention of diabetes in people with profiles similar to those of the participants in our study.3,4 The DREAM trial did not show that ramipril prevents diabetes in this population; however, it did demonstrate an effect of ramipril on regression to normal glucose levels. These results suggest that ramipril may have favorable effects on glucose metabolism, a finding that is consonant with other reports on studies of ACE inhibitors (when used for established indications).16,23 However, not all trials have found such an association.15 Further research will be required to clarify this effect. For now, the routine use of ramipril for the express purpose of preventing diabetes is not indicated. Supported by grants from the Canadian Institutes of Health Research (MCT41548), Sanofi-Aventis, GlaxoSmithKline, and King Pharmaceuticals. Ms. Bosch and Drs. Yusuf, Gerstein, Dagenais, and Chiasson report having received consulting and lecture fees from Sanofi-Aventis; Drs. Yusuf, Gerstein, Holman, and Chiasson, consulting and lecture fees from GlaxoSmithKline; and Dr. Probstfield, consulting fees from King Pharmaceuticals. Ms. Bosch, Drs. Yusuf and Gerstein, Ms. Pogue, and Dr. Dagenais report holding a patent for the use of ramipril to prevent diabetes and assigning all rights to Sanofi-Aventis in 2003. No other potential conflict of interest relevant to this article was reported. * The DREAM Trial Investigators are listed in the Appendix. Source Information The members of the Writing Committee (Jackie Bosch, M.Sc., Salim Yusuf, D.Phil., Hertzel C. Gerstein, M.D., Janice Pogue, M.Sc., Patrick Sheridan, M.Sc., McMaster University, Hamilton; Gilles Dagenais, M.D., Laval University Heart and Lung Institute, Quebec; Jean Louis Chiasson, M.D., Research Centre–Centre Hospitalier de l'Université de Montreal–H?tel-Dieu, Montreal — all in Canada; Rafael Diaz, M.D., Instituto Cardiovascular de Rosario, Rosario, Argentina; Alvaro Avezum, Ph.D., Dante Pazzanese Institute of Cardiology, S?o Paulo, Brazil; Fernando Lanas, M.D., Universidad de la Frontera, Temuco, Chile; Jeffrey Probstfield, M.D., Fred Hutchinson Cancer Research Center, Seattle; George Fodor, Ph.D., University of Ottawa Heart Institute, Ottawa; and Rury R. Holman, F.R.C.P., Oxford Centre for Diabetes, Endocrinology, and Metabolism, Oxford, United Kingdom) of the Diabetes Reduction Assessment with Ramipril and Rosiglitazone Medication (DREAM) trial assume responsibility for the overall content and integrity of the article. This article was published at www.nejm.org on September 15, 2006. Address reprint requests to the DREAM Project Office, Population Health Research Institute, 2nd Fl., McMaster Clinic, 237 Barton St. E, Hamilton, ON L8L 2X2, Canada, or to dream@cardio.on.ca. References Roglic G, Unwin N, Bennett PH, et al. The burden of mortality attributable to diabetes: realistic estimates for the year 2000. Diabetes Care 2005;28:2130-2135. Booth GL, Kapral MK, Fung K, Tu JV. Relation between age and cardiovascular disease in men and women with diabetes compared with non-diabetic people: a population-based retrospective cohort study. Lancet 2006;368:29-36. Tuomilehto J, Lindstrom J, Eriksson JG, et al. Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance. N Engl J Med 2001;344:1343-1350. Diabetes Prevention Program Research Group. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 2002;346:393-403. Chiasson J-L, Josse RG, Gomis R, Hanefeld M, Karasik A, Laakso M. Acarbose for prevention of type 2 diabetes mellitus: the STOP-NIDDM randomised trial. Lancet 2002;359:2072-2077. Padwal R, Majumdar SR, Johnson JA, Varney J, McAlister FA. A systematic review of drug therapy to delay or prevent type 2 diabetes. Diabetes Care 2005;28:736-744. Yusuf S, Gerstein H, Hoogwerf B, et al. Ramipril and the development of diabetes. JAMA 2001;286:1882-1885. Dagenais GR, Pogue J, Fox K, Simoons ML, Yusuf S. Angiotensin-converting-enzyme inhibitors in stable vascular disease without left ventricular systolic dysfunction or heart failure: a combined analysis of three trials. Lancet 2006;368:581-588. The DREAM Trial Investigators. Rationale, design and recruitment characteristics of a large, simple international trial of diabetes prevention: the DREAM trial. Diabetologia 2004;47:1519-1527. The DREAM (Diabetes REduction Assessment with ramipril and rosiglitazone Medication) Trial Investigators. Effect of rosiglitazone on the frequency of diabetes in patients with impaired glucose tolerance or impaired fasting glucose: a randomised controlled trial. Lancet 2006;368:1096-1105. Lachin JM. Worst-rank score analysis with informatively missing observations in clinical trials. Control Clin Trials 1999;20:408-422. Vermes E, Ducharme A, Bourassa MG, et al. Enalapril reduces the incidence of diabetes in patients with chronic heart failure: insight from the Studies Of Left Ventricular Dysfunction (SOLVD). Circulation 2003;107:1291-1296. The PEACE Trial Investigators. Angiotensin-converting-enzyme inhibition in stable coronary artery disease. N Engl J Med 2004;351:2058-2068. Hansson L, Lindholm LH, Niskanen L, et al. Effect of angiotensin-converting-enzyme inhibition compared with conventional therapy on cardiovascular morbidity and mortality in hypertension: the Captopril Prevention Project (CAPPP) randomised trial. Lancet 1999;353:611-616. The ALLHAT Officers and Coordinators for the ALLHAT Collaborative Research Group. Major outcomes in high-risk hypertensive patients randomized to angiotensin-converting enzyme inhibitor or calcium channel blocker vs diuretic: the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT). JAMA 2002;288:2981-2997. Wing LMH, Reid CM, Ryan P, et al. A comparison of outcomes with angiotensin-converting-enzyme inhibitors and diuretics for hypertension in the elderly. N Engl J Med 2003;348:583-592. Yusuf S, Ostergren JB, Gerstein HC, et al. Effects of candesartan on the development of a new diagnosis of diabetes mellitus in patients with heart failure. Circulation 2005;112:48-53. Lindholm LH, Persson M, Alaupovic P, Carlberg B, Svensson A, Samuelsson O. Metabolic outcome during 1 year in newly detected hypertensives: results of the Antihypertensive Treatment and Lipid Profile in a North of Sweden Efficacy Evaluation (ALPINE study). J Hypertens 2003;21:1563-1574. Lindholm LH, Ibsen H, Borch-Johnsen K, et al. Risk of new-onset diabetes in the Losartan Intervention For Endpoint reduction in hypertension study. J Hypertens 2002;20:1879-1886. Julius S, Kjeldsen SE, Weber M, et al. Outcomes in hypertensive patients at high cardiovascular risk treated with regimens based on valsartan or amlodipine: the VALUE randomised trial. Lancet 2004;363:2022-2031. Coutinho M, Gerstein HC, Wang Y, Yusuf S. The relationship between glucose and incident cardiovascular events: a metaregression analysis of published data from 20 studies of 95,783 individuals followed for 12.4 years. Diabetes Care 1999;22:233-240. Norhammar A, Tenerz A, Nilsson G, et al. Glucose metabolism in patients with acute myocardial infarction and no previous diagnosis of diabetes mellitus: a prospective study. Lancet 2002;359:2140-2144. Taylor EN, Hu FB, Curhan GC. Antihypertensive medications and the risk of incident type 2 diabetes. Diabetes Care 2006;29:1065-1070. Teo K, Yusuf S, Sleight P, et al. Rationale, design, and baseline characteristics of 2 large, simple, randomized trials evaluating telmisartan, ramipril, and their combination in high-risk patients: the Ongoing Telmisartan Alone and in Combination with Ramipril Global Endpoint Trial/Telmisartan Randomized Assessment Study in ACE Intolerant Subjects with Cardiovascular Disease (ONTARGET/TRANSCEND) trials. Am Heart J 2004;148:52-61....查看详细 (35792字节)
☉ 11342760:Microscopic-Observation Drug-Susceptibility Assay for the Diagnosis of TB
ABSTRACT Background New diagnostic tools are urgently needed to interrupt the transmission of tuberculosis and multidrug-resistant tuberculosis. Rapid, sensitive detection of tuberculosis and multidrug-resistant tuberculosis in sputum has been demonstrated in proof-of-principle studies of the microscopic-observation drug-susceptibility (MODS) assay, in which broth cultures are examined microscopically to detect characteristic growth. Methods In an operational setting in Peru, we investigated the performance of the MODS assay for culture and drug-susceptibility testing in three target groups: unselected patients with suspected tuberculosis, prescreened patients at high risk for tuberculosis or multidrug-resistant tuberculosis, and unselected hospitalized patients infected with the human immunodeficiency virus. We compared the MODS assay head-to-head with two reference methods: automated mycobacterial culture and culture on L?wenstein–Jensen medium with the proportion method. Results Of 3760 sputum samples, 401 (10.7%) yielded cultures positive for Mycobacterium tuberculosis. Sensitivity of detection was 97.8% for MODS culture, 89.0% for automated mycobacterial culture, and 84.0% for L?wenstein–Jensen culture (P90% of excluded patients), declined to participate (<5%), or if the clinic staff were unavailable (<5%). Numbers of eligible patients were derived from data extracted from National TB Programme log entries during the recruitment period; the numbers may have been overestimated, since follow-up patients (who were ineligible) were not always clearly identified. Of the 25 hospitalized patients with HIV infection who were eligible but not recruited, more than 90% were incapable of providing informed consent, owing to their clinical condition, or were receiving ongoing treatment for tuberculosis. Most, but not all, patients submitted two sputum samples. Laboratory Methods Detection of M. tuberculosis Sputum samples were decontaminated according to the sodium hydroxide–N-acetyl-L-cysteine method.21 An aliquot was used for microscopical examination of auramine-stained sputum smears, and the remainder was used for parallel L?wenstein–Jensen culture, automated mycobacterial culture, and MODS culture (see Fig. I in the Supplementary Appendix, available with the full text of this article at www.nejm.org). L?wenstein–Jensen culture and automated mycobacterial culture with the use of the MBBacT system (bioMérieux) were selected because they are reference methods commonly used in developing and industrialized countries, respectively. After inoculation of 250 μl of decontaminant, L?wenstein–Jensen slants were incubated at 37°C and examined twice weekly from day 7 through day 60.21 MBBacT bottles were inoculated with 500 μl of decontaminant, and cultures were monitored continuously for 42 days according to the recommendations of the manufacturer. The MODS assay was performed as described previously.6,7 Briefly, broth cultures were prepared in 24-well tissue-culture plates (Becton Dickinson), each containing 720 μl of decontaminant, Middlebrook 7H9 broth (Becton Dickinson), oxalic acid, albumin, dextrose, and catalase (OADC) (Becton Dickinson), and polymyxin, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA) (Becton Dickinson). For each sample, 12 wells were used: in 4 control wells, no drug was added, and each of the remaining 8 wells contained one of four drugs at one of two concentrations tested. The cultures were examined under an inverted light microscope at a magnification of 40x every day (except Saturday and Sunday) from day 4 to day 15, on alternate days from day 16 to day 25, and twice weekly from day 26 to day 40. To minimize cross-contamination and occupational exposure, plates were permanently sealed inside plastic ziplock bags after inoculation and were subsequently examined within the bag. Positive cultures were identified by cord formation, characteristic of M. tuberculosis growth, in liquid medium in drug-free control wells, as described previously.6,7,22 Nontuberculous mycobacteria were recognized by their lack of cording or, for M. chelonae (which is the only nontuberculous mycobacteria that does form cords), by rapid overgrowth by day 5. Fungal or bacterial contamination was recognized by rapid overgrowth and clouding in wells. If contamination was detected, the original sample was cultured again after being decontaminated once more. Spacer oligonucleotide typing (spoligotyping), polymerase chain reaction with multiple primers,23 or both were applied to all isolates from each of the three types of cultures in order to confirm the presence of M. tuberculosis. Drug-Susceptibility Testing Direct drug-susceptibility testing was performed with the use of the MODS assay, as previously described.6,7 Growth in drug-free control wells but not in drug-containing wells indicated susceptibility. The drug concentrations used were as follows: isoniazid, 0.1 and 0.4 μg per milliliter; rifampin, 1 and 2 μg per milliliter; ethambutol, 2.5 and 5.0 μg per milliliter; and streptomycin, 2 and 6 μg per milliliter. Drug-sensitive control strains were tested daily. Indirect drug-susceptibility testing was performed with the use of the proportion method21 for isolates from L?wenstein–Jensen culture (by an external laboratory) and with the automated MBBacT system24,25,26,27,28,29 for isolates from the automated mycobacterial culture. For purposes of discrepant analysis, the microplate alamar blue assay30,31,32,33,34,35 was performed in parallel, both for all L?wenstein–Jensen isolates and for isolates from drug-free control wells in the MODS assay. All procedures were performed by six staff members of the mycobacteriology laboratory who were unaware of the results of the other tests. Statistical Analysis Data were analyzed with the use of Stata 7 software, with the sample as the unit of analysis, to reflect the operational performance of a routine service laboratory. The Wilcoxon signed-rank test was used to compare the times to each end point among the three methods. A P value of less than 0.05 was used to indicate statistical significance. The concordance of susceptibility results was determined with the use of the sensitivity, specificity, and positive and negative predictive values for the detection of resistance (with 95% confidence intervals ), as well as with kappa values. To address the inherent difficulty of evaluating a test that is more sensitive than the reference tests, and to minimize incorporation bias (the use of results from the investigational test as part of the reference result), we previously undertook a comprehensive microbiologic, molecular, and epidemiologic investigation of the discordant cultures.36 We identified 17 cross-contaminated cultures (12 MODS cultures, 4 automated mycobacterial cultures, and 1 L?wenstein–Jensen culture) from 14 samples. For sensitivity and specificity of detection and predictive-value calculations for each of the three methods, a positive reference result was defined as a positive culture according to at least one method for which cross-contamination had been conclusively ruled out.36 A negative reference result was defined as any sample in which all three culture methods yielded negative results or in which two were negative and the third indeterminate, owing to repeated bacterial or fungal overgrowth, or a sample for which cross-contamination was demonstrated to be the only cause of the positive culture.36 Thus, the 17 false positive cultures were defined as positive in calculations of performance characteristics for the relevant methods, but the reference result for the 14 samples was determined to be negative. McNemar's 2 test was used to compare the sensitivities of detection of the three methods. Definition of Reference Susceptibility Test Results Concordant results from automated mycobacterial culture and the proportion method were regarded as the reference result for drug susceptibility. Discordant results from automated mycobacterial culture and the proportion method were resolved by means of discrepant analysis, with the use of the two parallel results from the microplate alamar blue assay. If both results from this assay agreed, that result was used as the reference result; if not, the strain was designated indeterminate (see Table I in the Supplementary Appendix). Results Patients and Samples Recruitment of patients and culture results are shown in Figure 1. Demographic characteristics and tuberculosis diagnoses are shown in Table 1 according to study group. Figure 1. Recruitment of Patients and Culture Results. For L?wenstein–Jensen (L-J) and automated mycobacterial cultures, positive cultures were those that were positive according to either method or both; negative cultures were those that were negative according to both methods or negative according to one method and indeterminate according to the other; and indeterminate cultures were those that were indeterminate according to both methods. For patients with suspected tuberculosis, the 242 reference-standard culture-positive samples included 4 false positive samples arising from cross-contamination36 (1 in L?wenstein–Jensen culture alone, 1 in automated mycobacterial culture alone, and 2 in both automated mycobacterial culture and MODS culture), and the 32 culture-positive samples in MODS culture alone included 7 that were false positive owing to cross-contamination. For the prescreened patients at high risk for tuberculosis or multidrug-resistant tuberculosis, the 87 reference-standard culture-positive samples included 1 false positive sample in both automated mycobacterial culture and MODS culture, and the 7 samples that were culture-positive in MODS culture alone included 2 false positive cultures. The indeterminate cultures for the prescreened patients and the hospitalized patients with HIV infection were designated as such because cultures from all three methods were repeatedly contaminated by bacterial overgrowth (two with rapid-growing non-tuberculous mycobacteria). Table 1. Demographic Characteristics, Prevalence of Disease, and Sensitivity, Specificity, and Predictive Values for the Detection of Mycobacterium tuberculosis in Sputum. Sensitivity and Specificity of Detection Of the 3760 sputum samples collected, 401 (10.7%) were positive for M. tuberculosis cultures, 3356 were negative, and 3 were indeterminate (and were removed from analysis), because all three types of cultures were repeatedly contaminated by bacterial overgrowth. MODS culture had a greater overall sensitivity of detection than either automated mycobacterial culture or L?wenstein–Jensen culture (97.8%, 89.0%, and 84.0%, respectively; P<0.001); this difference was maintained in all groups. The overall specificity of detection was 99.6% for MODS culture, 99.9% for automated mycobacterial culture, and 100.0% for L?wenstein–Jensen culture. Predictive values and data according to group are shown in Table 1, and in Figure II of the Supplementary Appendix. Added Value of Second Culture for Sensitivity of Detection The incremental benefit of a second smear for acid-fast bacilli and a second sputum culture is shown in Figure 2. A second MODS culture detected an additional 8.2% of cases among patients with suspected tuberculosis but offered no added value among prescreened patients at high risk for tuberculosis or multidrug-resistant tuberculosis. Figure 2. Incremental Benefit of One and Two Sputum Cultures as Compared with Sputum-Smear Microscopy. The percentage of cases detected during examination of the first smear or culture is shown, as well as the additional (not total) percentage detected during examination of a second smear or culture. For patients with suspected tuberculosis (Panel A) and prescreened patients at high risk for tuberculosis or multidrug-resistant tuberculosis (Panel B), examination of smears stained with the Ziehl–Neelsen (Z-N) stain was performed at the local laboratory of the National TB Programme before the sample was retrieved for study purposes. For samples from the hospitalized patients with HIV infection (Panel C), microscopical examination of sputum smears stained with auramine but not Z-N stain was performed at Universidad Peruana Cayetano Heredia, because these patients were not recruited through the National TB Programme. L-J denotes L?wenstein–Jensen. Time to Culture Positivity Of the 401 sputum samples positive for M. tuberculosis, 325 were culture-positive according to all three methods and were thus included in the head-to-head analysis of time to culture positivity (Figure 3). The median time to culture positivity was significantly shorter for MODS than for the automated mycobacterial or L?wenstein–Jensen cultures (7 days vs. 13 days and 26 days , respectively; P<0.001). Smear status had a clinically unimportant, though significant, effect on time to culture positivity in MODS culture (median, 6 days for a smear-positive sample vs. 7 days for a smear-negative sample; P<0.001). Though samples that were culture-negative (69) or contaminated (7) according to at least one method were excluded from this analysis, results were unchanged by their inclusion (data not shown). Figure 3. Cumulative Percentages of the Time to Culture Positivity for 325 Culture-Positive Samples According to Culture Method (Panel A) and the Effect of the Quantitative Status of Sputum Smears for Acid-Fast Bacilli (Panels B, C, and D). The percentages of cultures that were positive at days 7, 14, and 21 were 74%, 99%, and 100%, respectively, in MODS culture; 7%, 62%, and 89% in automated mycobacterial culture; and 0%, 5%, and 28% in L?wenstein–Jensen (L-J) culture (Panel A). In Panels B, C, and D, "auramine smear –" was defined by the presence of fewer than 10 acid-fast bacilli per 100 fields, "1+" 10 to 99 acid-fast bacilli per 100 fields, "2+" 1 to 10 acid-fast bacilli per field, and "3+" more than 10 acid-fast bacilli per field. One field was equivalent to the examination of one carbol-fuchsin–stained smear at a magnification of 1000x Bacterial and Fungal Contamination of Cultures The median time from sample collection to sample processing was 3 days, and most but not all samples were refrigerated en route. At least one culture per sample was contaminated in 739 of 3760 samples (20.0%), though in only 63 samples (1.7%) were all cultures by the three methods contaminated. The proportion of initially contaminated samples was 8.1% (95% CI, 7.2 to 9.0) for MODS culture, 4.4% (95% CI, 3.8 to 5.3) for automated mycobacterial culture, and 14.2% (95% CI, 13.1 to 15.3) for L?wenstein–Jensen culture. However, ultimately contaminated (indeterminate) cultures were less frequent in MODS culture (6 cultures, 0.2%) than in either automated mycobacterial culture (11 cultures, 0.3%; P=0.01) or L?wenstein–Jensen culture (55 cultures, 1.5%; P<0.001). The median time from initial processing of samples to the results of culture testing for initially contaminated cultures was shorter for MODS culture (24 days; 95% CI, 19 to 28) than for automated mycobacterial culture (32 days; 95% CI, 25 to 39; P=0.03) or L?wenstein–Jensen culture (50 days; 95% CI, 44 to 56; P<0.001). Positive MODS cultures that were contaminated but able to be evaluated accounted for less than 2.5% of all positive cultures for the assay. Direct Drug-Susceptibility Testing Valid drug-susceptibility testing in the MODS assay depends on the observed growth of M. tuberculosis in all four control wells, as was the case for 349 of 392 positive MODS cultures (89.0%). Of the 43 samples for which drug-susceptibility testing in the MODS assay was not possible, 28 (65.1%) were culture-negative according to automated mycobacterial culture and L?wenstein–Jensen culture. Resistance to rifampin was detected in 10.7% of all samples; to isoniazid, in 19.5%; to rifampin and isoniazid (combined to test for multidrug resistance), in 10.4%; to ethambutol, in 10.1%; and to streptomycin, in 21.4% (Table 2). The proportion of samples for which susceptibility results agreed between MODS culture and the gold-standard methods was 100% for rifampin, 96.7% for isoniazid, 98.8% for rifampin and isoniazid, 95.4% for ethambutol, and 91.7% for streptomycin. Overall median times from initial sample processing to the results of drug-susceptibility testing were 7 days for MODS culture, 22 days for automated mycobacterial culture, and 68 days for L?wenstein–Jensen culture. Table 2. Susceptibility Results from the MODS Assay and Concordance with the Gold-Standard Methods. Discussion This operational study extends and provides support for the findings of earlier proof-of-principle studies6,7,22,37 and demonstrates that the MODS assay outperforms the gold-standard reference methods of developing and industrialized countries. For all three study groups, the MODS assay detected M. tuberculosis in sputum with greater sensitivity and speed and reliably identified multidrug-resistant tuberculosis strains in less time than did L?wenstein–Jensen or automated mycobacterial cultures. These data indicate that the MODS assay could be considered for use in appropriate settings. Our study was designed to address conventional pitfalls.8,15,18,20,38 Specifically, it was performed in an appropriately broad group of patients with or without disease and in pertinent patient groups (without selection bias); all tests were performed in all patients (preventing verification bias); and all results were interpreted by staff members who were unaware of the other test results, using appropriate gold-standard reference methods for comparison. The robustness of our study derives from its operational, real-world design. Meticulous resolution of discordant results is essential when an investigational diagnostic method is more sensitive than existing reference standards. Use of two established reference methods for comparison and two samples per patient facilitated the rigorous definition of true positive results, addressing the problem of incorporation bias. The high specificity and infrequent cross-contamination in the MODS assay36 relate to the containment of the plates in ziplock bags and the absence of manipulation after inoculation, which also improve biologic security. The greater sensitivity and speed of detection in MODS culture than in the gold standards were predicted on the basis of previous studies.6,7,37 The increased sensitivity of liquid medium has long been known, and a discerning human eye can scrutinize cultures better than can automated systems with their use of necessarily rigid cutoff values. It is simpler to recognize the characteristic cord formation than to read a malarial smear; within 1 week, students training in our laboratory can comfortably and accurately read one well per minute, considerably faster than the time it takes to read a smear for acid-fast bacilli. Training in the MODS assay can be completed in less than 2 weeks (similar to training in L?wenstein–Jensen and automated mycobacterial cultures; training in drug-susceptibility testing with the proportion method takes several months). Beyond standard laboratory equipment, automated mycobacterial culture requires computer-linked automated culture incubators, whereas MODS culture requires only an inverted light microscope. As purchased by us, the cost of $2 per sample for MODS culture compares favorably with the $6 cost per sample for L?wenstein–Jensen culture and the proportion method and the cost of $52 per sample for automated mycobacterial culture; however, labor costs may be higher for MODS culture. Increased sensitivity carries the risk of increased bacterial overgrowth (for MODS culture and automated mycobacterial culture), though even after repeated decontamination, the sensitivity and specificity of MODS culture were unaffected. High speed, sensitivity, and specificity, and the requirement for only one culture, all enhance tuberculosis rule-out procedures and potentially simplify tuberculosis-screening algorithms for use in patients with HIV infection before the initiation of prophylactic treatment with isoniazid. If a MODS culture is negative on day 15, there is a 99.7% chance that the sample is truly culture-negative. Thus, we believe that a negative MODS culture can be discarded after 3 weeks. In settings with a high tuberculosis burden, the only susceptibility data that are likely to effect a change in therapy at the programmatic level are those for the detection of multidrug-resistant tuberculosis, for which the performance of the MODS assay is highly reliable and rapid (median time to the results of susceptibility testing, 7 days), providing clinically important information in a meaningful time frame. Although direct drug-susceptibility testing is conventionally viewed with suspicion — indirect testing of cultured strains is preferred — our data refute that view for rifampin and isoniazid in the MODS assay. However, susceptibility testing for M. tuberculosis is complex, and concordance among even regional laboratories performing gold-standard testing is particularly variable for ethambutol and streptomycin.39 Our findings for these two drugs agreed with previous data for the MODS assay,7 demonstrating insufficient concordance of the assay (at least in its current format) with gold standards to recommend usage. Our study defines strengths and redundancies in the first-generation MODS assay and should enable the development of a streamlined, clinically useful method. The use of fewer wells per sample than we used — two wells with no drug (to ensure high specificity), one with rifampin (1 μg per milliliter), and one with isoniazid (0.4 μg per milliliter) — reduces costs by 40% but does not affect performance. The MODS assay is "laboratory freeware," not a commercial product or a kit. Any laboratory that is adequately biologically secured, has an incubator and a centrifuge, and is capable of microscopy can safely perform MODS culture. All ingredients are available from major laboratory suppliers. Downstream effects on patient care are the litmus test of the utility of a new method, and the added value will therefore depend on context and strategy for implementation. In countries where smear-negative tuberculosis is frequently diagnosed and treated empirically, the incremental benefit of MODS culture on case detection, as compared with the smear alone, would be less than that in Peru, where only 21% of treated cases are smear-negative and where MODS culture has recently been incorporated into Ministry of Health guidelines (www.minsa.gob.pe/normaslegales/2006/RM383-2006.pdf). However, the high specificity rate would save patients and society money by minimizing overtreatment, and the early detection and treatment of multidrug-resistant tuberculosis would interrupt transmission. Equity in the access to high-performance techniques to diagnose tuberculosis thus benefits both individual health40,41 and public health. Despite the low cost per sample, in many resource-limited settings with a high tuberculosis burden, testing by the MODS assay of all patients with suspected tuberculosis (<5% of whom have culture-positive disease in Peru) would be a challenge financially and operationally. In our targeted, high-risk groups, only one MODS culture (collected in one visit) is required to achieve culture-positive rates of 20%, a good return on the investment. Programmatic studies are now needed to determine the optimal implementation strategy to maximize the effect and cost-effectiveness of this tool. The MODS assay addresses two key gaps in resource-limited settings with a high tuberculosis burden: rapid, accurate detection of M. tuberculosis and simultaneous identification of multidrug-resistant tuberculosis. The use of culture-based diagnostic techniques for case detection may not be the future as envisaged by the International Union against Tuberculosis and Lung Disease42; promotion of such a strategy may conflict with the view that the scale-up of coverage and improvement of smear microscopy is currently a more important priority. However, we believe the MODS assay could now be implemented in settings in which smear microscopy is being optimally used and the augmentation of case detection is feasible and desirable. Supported by a grant from the Wellcome Trust (064672). Dr. Gilman and Ms. Caviedes were supported by a National Institutes of Health–Fogarty–Global Research Training Grant (TW006581). Presented in part at the 35th UNION World Conference on Lung Health, Paris, October 28–November 1, 2004 (abstract nos. PS-456-493 and PC-456-496). Drs. Moore and Evans report having received grant support from Sequella for clinical trials of another tuberculosis diagnostic test. Dr. Gilman reports having received consulting fees from Sequella. Dr. Vivar reports having received grant support from Merck, Chiron, and Cerexa. No other potential conflict of interest relevant to this article was reported. We are indebted to all participants in this study, for almost all of whom this was their first experience with clinical research; to the numerous medical and laboratory staff members at the community-clinic and hospital study sites who ensured that the protocol ran smoothly, particularly Yuri García, Adolfo Orellana Marin, Raul Miranda Arrostigue (Centro de Salud Carlos Cueto Fernandini), Guillermo Vera Mallqui (Centro Base de Salud Los Olivos), Luis Rivera Pérez (CS Infantas), Walter Ramos Magui?a (CS Villa Norte), Luz Vásquez Chávez (CS Primavera), Félix Pari Loayza (CS Juan Pablo II), Ruth Flores Escobar (Puesto de Salud Los Olivos de Pro), Jesús Castillo Diaz (CS-Comites Locales de Administracíon Compartida Laura Caller Iberico), Milciades Reátegui Sanchez (CS-CLAS San Martín de Porres), Alicia Vigo Alegria (PS Enrique Milla Ochoa); and to laboratory, support, and field staff members at Universidad Peruana Cayetano Heredia and Asociación Benéfica PRISMA, particularly Paula Magui?a, Fanny Garcia, Eleana Sanchez, Yrma Chuquiruna, Rosmery Gutierrez, Sonia Lopez, Christian Solis, Indira Villaverde, Pilar Navarro, and Natalie Bowman. Source Information From the Department of Infectious Diseases and Immunity and the Wellcome Centre for Clinical Tropical Medicine, Imperial College London, Faculty of Medicine (Hammersmith Campus), London (D.A.J.M., C.A.W.E., A.R.E., J.S.F.); Asociación Benéfica PRISMA, San Miguel (D.A.J.M., C.A.W.E., R.H.G., M.-G.H.-D., A.R.E.), Laboratorio de Investigación de Enfermedades Infecciosas, Universidad Peruana Cayetano Heredia, San Martín de Porras (D.A.J.M., C.A.W.E., R.H.G., L.C., J.C., A.R.E.), Hospital Nacional General Arzobispo Loayza (A.V., Y.P.), Hospital Nacional Hipolito Unanue, El Agustino (E.S.), Dirección de Salud–III Lima Norte (Ministerio de Salud), Rimac (J.C.S.), and Dirección de Salud–IV Lima Este (Ministerio de Salud), El Agustino (C.S., D.L.) — all in Lima, Peru; the Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore (D.A.J.M., C.A.W.E., R.H.G.); and the Tulane School of Public Health and Tropical Medicine, New Orleans (R.O.). Address reprint requests to Dr. Moore at the Department of Infectious Diseases and Immunity and Wellcome Centre for Clinical Tropical Medicine, Imperial College London, Faculty of Medicine (Hammersmith Campus), DuCane Rd., London W12 0NN, United Kingdom, or at davidajmoore@msn.com. References Global tuberculosis control: surveillance, planning, financing. Geneva: World Health Organization, 2006:1-242. (Accessed September 15, 2006, at http://www.who.int/tb/publications/global_report/2006/en/index.html.) The global plan to stop TB, 2006-2015 Geneva: Stop TB Partnership and World Health Organization, 2006:1-172. (Accessed September 15, 2006, at http://www.stoptb.org/globalplan.) Espinal MA, Kim SJ, Suarez PG, et al. Standard short-course chemotherapy for drug-resistant tuberculosis: treatment outcomes in 6 countries. JAMA 2000;283:2537-2545. Chavez Pachas AM, Blank R, Smith Fawzi MC, Bayona J, Becerra MC, Mitnick CD. Identifying early treatment failure on category I therapy for pulmonary tuberculosis in Lima Ciudad, Peru. Int J Tuberc Lung Dis 2004;8:52-58. Dowdy DW, Chaisson RE, Moulton LH, Dorman SE. The potential impact of enhanced diagnostic techniques for tuberculosis driven by HIV: a mathematical model. AIDS 2006;20:751-762. Caviedes L, Lee TS, Gilman RH, et al. Rapid, efficient detection and drug susceptibility testing of Mycobacterium tuberculosis in sputum by microscopic observation of broth cultures. J Clin Microbiol 2000;38:1203-1208. Moore DA, Mendoza D, Gilman RH, et al. Microscopic observation drug susceptibility assay, a rapid, reliable diagnostic test for multidrug-resistant tuberculosis suitable for use in resource-poor settings. J Clin Microbiol 2004;42:4432-4437. Small PM, Perkins MD. More rigour needed in trials of new diagnostic agents for tuberculosis. Lancet 2000;356:1048-1049. Suffys P, Palomino JC, Cardoso Leao S, et al. Evaluation of the polymerase chain reaction for the detection of Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2000;4:179-183. Tessema TA, Hamasur B, Bjun G, Svenson S, Bjorvatn B. Diagnostic evaluation of urinary lipoarabinomannan at an Ethiopian tuberculosis centre. Scand J Infect Dis 2001;33:279-284. Tsubura E, Yamanaka M, Sakatani M, Takashima T, Maekura R, Nakatani K. A cooperative clinical study on the evaluation of an antibody detection kit (MycoDot test) for mycobacterial infections. Kekkaku 1997;72:611-615. Somi GR, O'Brien RJ, Mfinanga GS, Ipuge YA. Evaluation of the MycoDot test in patients with suspected tuberculosis in a field setting in Tanzania. Int J Tuberc Lung Dis 1999;3:231-238. Cole RA, Lu HM, Shi YZ, Wang J, De-Hua T, Zhou AT. Clinical evaluation of a rapid immunochromatographic assay based on the 38 kDa antigen of Mycobacterium tuberculosis on patients with pulmonary tuberculosis in China. Tuber Lung Dis 1996;77:363-368. Reid MC, Lachs MS, Feinstein AR. Use of methodological standards in diagnostic test research: getting better but still not good. JAMA 1995;274:645-651. Ransohoff DF, Feinstein AR. Problems of spectrum and bias in evaluating the efficacy of diagnostic tests. N Engl J Med 1978;299:926-930. Bossuyt PM, Reitsma JB, Bruns DE, et al. Towards complete and accurate reporting of studies of diagnostic accuracy: the STARD Initiative. Ann Intern Med 2003;138:40-44. Bossuyt PM, Reitsma JB, Bruns DE, et al. The STARD statement for reporting studies of diagnostic accuracy: explanation and elaboration. Ann Intern Med 2003;138:W1-W12. Begg CB. Biases in the assessment of diagnostic tests. Stat Med 1987;6:411-423. Walsh A, McNerney R. Guidelines for establishing trials of new tests to diagnose tuberculosis in endemic countries. Int J Tuberc Lung Dis 2004;8:609-613. Perkins MD, Kritski AL. Diagnostic testing in the control of tuberculosis. Bull World Health Organ 2002;80:512-513. Laboratory services in TB control. Parts I, II, and III. Geneva: World Health Organization, 1998. (Publication no. WHO/tb/98.258.) Park WG, Bishai WR, Chaisson RE, Dorman SE. Performance of the microscopic observation drug susceptibility assay in drug susceptibility testing for Mycobacterium tuberculosis. J Clin Microbiol 2002;40:4750-4752. Del Portillo P, Thomas MC, Martinez E, et al. Multiprimer PCR system for differential identification of mycobacteria in clinical samples. J Clin Microbiol 1996;34:324-328. Bemer P, Bodmer T, Munzinger J, Perrin M, Vincent V, Drugeon H. Multicenter evaluation of the MB/BACT system for susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 2004;42:1030-1034. Barreto AM, Araujo JB, de Melo Medeiros RF, de Souza Caldas PC. Evaluation of indirect susceptibility testing of Mycobacterium tuberculosis to the first- and second-line, and alternative drugs by the newer MB/BacT system. Mem Inst Oswaldo Cruz 2003;98:827-830. Yew WW, Tong SC, Lui KS, Leung SK, Chau CH, Wang EP. Comparison of MB/BacT system and agar proportion method in drug susceptibility testing of Mycobacterium tuberculosis. Diagn Microbiol Infect Dis 2001;39:229-232. Tortoli E, Mattei R, Savarino A, Bartolini L, Beer J. Comparison of Mycobacterium tuberculosis susceptibility testing performed with BACTEC 460TB (Becton Dickinson) and MB/BacT (Organon Teknika) systems. Diagn Microbiol Infect Dis 2000;38:83-86. Diaz-Infantes MS, Ruiz-Serrano MJ, Martinez-Sanchez L, Ortega A, Bouza E. Evaluation of the MB/BacT mycobacterium detection system for susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 2000;38:1988-1989. Brunello F, Fontana R. Reliability of the MB/BacT system for testing susceptibility of Mycobacterium tuberculosis complex isolates to antituberculous drugs. J Clin Microbiol 2000;38:872-873. Kumar M, Khan IA, Verma V, Qazi GN. Microplate nitrate reductase assay versus Alamar Blue assay for MIC determination of Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2005;9:939-941. Reis RS, Neves I Jr, Lourenco SL, Fonseca LS, Lourenco MC. Comparison of flow cytometric and Alamar Blue tests with the proportional method for testing susceptibility of Mycobacterium tuberculosis to rifampin and isoniazid. J Clin Microbiol 2004;42:2247-2248. Sungkanuparph S, Pracharktam R, Thakkinstian A, Buabut B, Kiatatchasai W. Correlation between susceptibility of Mycobacterium tuberculosis by microtiter plate Alamar Blue assay and clinical outcomes. J Med Assoc Thai 2002;85:820-824. Pracharktam R, Angkananukool K, Vibhagool A. In vitro susceptibility testing of levofloxacin and ofloxacin by microtiter plate Alamar Blue against multidrug and non multidrug resistant Mycobacterium tuberculosis in Thailand. J Med Assoc Thai 2001;84:1241-1245. Franzblau SG, Witzig RS, McLaughlin JC, et al. Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay. J Clin Microbiol 1998;36:362-366. Collins L, Franzblau SG. Microplate Alamar Blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother 1997;41:1004-1009. Moore DA, Caviedes L, Gilman RH, et al. Infrequent MODS TB culture cross-contamination in a high-burden resource-poor setting. Diagn Microbiol Infect Dis 2006;56:35-43. Oberhelman RA, Soto-Castellares G, Caviedes L, et al. Improved recovery of Mycobacterium tuberculosis from children using the Microscopic Observation Drug Susceptibility (MODS) method. Pediatrics 2006;118:e100-e106. Perkins MD. New diagnostic tools for tuberculosis. Int J Tuberc Lung Dis 2000;4:Suppl 2:S182-S188. Madison B, Robinson-Dunn B, George I, et al. Multicenter evaluation of ethambutol susceptibility testing of Mycobacterium tuberculosis by agar proportion and radiometric methods. J Clin Microbiol 2002;40:3976-3979. Mitnick C, Bayona J, Palacios E, et al. Community-based therapy for multidrug-resistant tuberculosis in Lima, Peru. N Engl J Med 2003;348:119-128. Shin S, Furin J, Bayona J, Mate K, Kim JY, Farmer P. Community-based treatment of multidrug-resistant tuberculosis in Lima, Peru: 7 years of experience. Soc Sci Med 2004;59:1529-1539. Tuberculosis bacteriology -- priorities and indications in high prevalence countries: position of the technical staff of the Tuberculosis Division of the International Union Against Tuberculosis and Lung Disease. Int J Tuberc Lung Dis 2005;9:355-361....查看详细 (42300字节)
☉ 11342761:Effectiveness of Atypical Antipsychotic Drugs in Patients with Alzheimer's Disease
ABSTRACT Background Second-generation (atypical) antipsychotic drugs are widely used to treat psychosis, aggression, and agitation in patients with Alzheimer's disease, but their benefits are uncertain and concerns about safety have emerged. We assessed the effectiveness of atypical antipsychotic drugs in outpatients with Alzheimer's disease. Methods In this 42-site, double-blind, placebo-controlled trial, 421 outpatients with Alzheimer's disease and psychosis, aggression, or agitation were randomly assigned to receive olanzapine (mean dose, 5.5 mg per day), quetiapine (mean dose, 56.5 mg per day), risperidone (mean dose, 1.0 mg per day), or placebo. Doses were adjusted as needed, and patients were followed for up to 36 weeks. The main outcomes were the time from initial treatment to the discontinuation of treatment for any reason and the number of patients with at least minimal improvement on the Clinical Global Impression of Change (CGIC) scale at 12 weeks. Results There were no significant differences among treatments with regard to the time to the discontinuation of treatment for any reason: olanzapine (median, 8.1 weeks), quetiapine (median, 5.3 weeks), risperidone (median, 7.4 weeks), and placebo (median, 8.0 weeks) (P=0.52). The median time to the discontinuation of treatment due to a lack of efficacy favored olanzapine (22.1 weeks) and risperidone (26.7 weeks) as compared with quetiapine (9.1 weeks) and placebo (9.0 weeks) (P=0.002). The time to the discontinuation of treatment due to adverse events or intolerability favored placebo. Overall, 24% of patients who received olanzapine, 16% of patients who received quetiapine, 18% of patients who received risperidone, and 5% of patients who received placebo discontinued their assigned treatment owing to intolerability (P=0.009). No significant differences were noted among the groups with regard to improvement on the CGIC scale. Improvement was observed in 32% of patients assigned to olanzapine, 26% of patients assigned to quetiapine, 29% of patients assigned to risperidone, and 21% of patients assigned to placebo (P=0.22). Conclusions Adverse effects offset advantages in the efficacy of atypical antipsychotic drugs for the treatment of psychosis, aggression, or agitation in patients with Alzheimer's disease. (ClinicalTrials.gov number, NCT00015548 .) Delusions, hallucinations, aggression, and agitation affect more than half of patients with Alzheimer's disease and related dementias.1,2,3,4 Antipsychotic drugs are used to treat these behaviors and symptoms and are among the most frequently used psychotropic drugs in Alzheimer's disease.5,6 Second-generation (atypical) antipsychotic drugs have been considered to be at least as effective as conventional antipsychotic agents such as haloperidol, with a lower risk of most adverse effects,7 and are used as first-line pharmacologic treatments for patients with dementia.5,8 However, there is a dearth of placebo-controlled and active-drug–controlled, randomized trials and longer-term data from controlled trials regarding the effectiveness of atypical antipsychotic drugs. Moreover, the available data on efficacy have been inconsistent, rates of response to placebo have been high, and patients have been required to receive drugs for the 6-week to 12-week study periods, whether or not they benefited, an artificial situation that does not address effectiveness.9 New safety issues have emerged with respect to atypical antipsychotic drugs. Increased risks of cerebrovascular adverse events10,11,12 and death13,14 have complicated their use. Antidepressant medications such as citalopram have been suggested as alternatives to antipsychotic drugs,15 at least for aggression or agitation,16 despite the lack of data from adequate trials.17 We conducted a double-blind, placebo-controlled trial to determine the effectiveness of olanzapine, quetiapine, and risperidone as compared with placebo in outpatients with Alzheimer's disease and psychosis, aggression, or agitation. Methods Study Setting and Design The trial was part of the National Institute of Mental Health (NIMH) Clinical Antipsychotic Trials of Intervention Effectiveness. Such trials address broad outcomes in settings and with interventions that reflect usual practices. The rationale, design, and methods of this study — Clinical Antipsychotic Trials of Intervention Effectiveness–Alzheimer's Disease (CATIE-AD) — have been described previously.18 The trial was conducted between April 2001 and November 2004 at 45 sites in the United States (26 university clinics, 7 Veterans Affairs medical centers, and 12 private-practice sites ). In phase 1 of the study, patients were randomly assigned under double-blind conditions to receive olanzapine, quetiapine, risperidone, or placebo in a 2:2:2:3 ratio. Doses were adjusted as clinically indicated by study physicians. If the physicians judged that the patient's response was not adequate at any time after the first 2 weeks, then treatment could be discontinued. Patients with an adequate response continued treatment for up to 36 weeks. Patients whose initial treatment was discontinued during phase 1 could be enrolled in phase 2 and randomly assigned under double-blind conditions to receive one of the antipsychotic drugs to which they were not initially assigned or to receive citalopram.18 This report is limited to phase 1 results. Participants Eligible participants fulfilled criteria for dementia of the Alzheimer's type (according to the Diagnostic and Statistical Manual of Mental Disorders, fourth edition)19 or probable Alzheimer's disease20 on the basis of the history, physical examination, results of structural brain imaging, and the score on the Mini–Mental State Examination (MMSE)21; the MMSE score had to be between 5 and 26 (on a scale from 0 to 30, with lower scores indicating poorer performance). To be eligible, patients had to be ambulatory and living at home or in an assisted-living facility. Eligible patients had delusions, hallucinations, aggression, or agitation that developed after the onset of dementia and was severe enough to disrupt their functioning and, in the opinion of the study physicians, to justify treatment with antipsychotic drugs. Signs and symptoms of psychosis, aggression, or agitation had to have occurred nearly daily during the previous week or at least intermittently for 4 weeks. During the week before they were randomly assigned to treatment, eligible patients also had a severity rating of at least "moderate" for conceptual disorganization, suspiciousness, or hallucinatory behavior on the Brief Psychiatric Rating Scale (BPRS).22 Alternatively, a frequency rating of "often" or "more frequently" and a severity rating of at least "moderate" were required for delusions, hallucinations, agitation, or "aberrant motor behavior" in the Neuropsychiatric Inventory.23 A study partner or caregiver who had regular contact with the patient was required to participate in the assessments.18 Patients were excluded if they had received a diagnosis of a primary psychotic disorder (e.g., schizophrenia), delirium, other dementia such as vascular dementia24 or Lewy-body dementia,25 or psychosis, agitation, or aggression that could be better accounted for by another medical condition, medication, or substance abuse. Patients were also excluded if they required psychiatric admission, were suicidal, were going to receive treatment with a cholinesterase inhibitor or antidepressant medication, had previously been treated with two of the three atypical antipsychotic drugs under study, or had contraindications to any of the study drugs. The study was approved by the NIMH data and safety monitoring board and by the institutional review board at each site. Written informed consent was obtained from the patients or their legally authorized representatives and from the partners or caregivers who participated with the patients. Interventions The trial design encouraged prescribing that reflected clinical practice while maintaining the randomized and double-blind treatment assignment. The study physicians determined the starting doses and adjusted the doses on the basis of their clinical judgment and patients' responses. Medications were dispensed at each visit in the form of identically appearing small and large capsules containing lower and higher doses of olanzapine (Zyprexa, Eli Lilly; 2.5 mg or 5.0 mg), quetiapine (Seroquel, AstraZeneca; 25 mg or 50 mg), risperidone (Risperdal, Janssen Pharmaceutica; 0.5 mg or 1.0 mg), or placebo. To treat difficult behaviors during the trial, study physicians could increase the dose of the study medication or prescribe a benzodiazepine or oral or parenteral haloperidol. All patients and caregivers were given basic information about Alzheimer's disease.26 Caregivers were offered two counseling sessions during the first 18 weeks and could speak with staff members as needed. Outcomes The primary outcome measure was the time until discontinuation of treatment for any reason in phase 1. This outcome integrates the judgments of patients, caregivers, and clinicians regarding efficacy, safety, and tolerability into a global measure of effectiveness that reflects therapeutic benefits in relation to undesirable effects. The two primary hypotheses in phase 1 were, first, there would be pairwise differences between the three groups given atypical antipsychotic agents and the placebo group in the time until discontinuation of treatment for any reason, and second, that among those antipsychotic drugs that were found to be different from placebo, none would be inferior to the others. The main secondary outcome measure was the attainment of minimal or greater improvement on the Clinical Global Impression of Change (CGIC)27 scale at week 12 while the patients continued to receive the phase 1 drug. The other secondary outcomes were the time to the discontinuation of treatment in phase 1 because of lack of efficacy and the time to the discontinuation of treatment because of adverse events, intolerability, or death. Safety was assessed by eliciting information about the occurrence of adverse events. The patients' weight and prolactin, glucose, cholesterol, and triglyceride levels were measured at weeks 12, 24, and 36. Statistical Analysis Patients who underwent randomization and received at least one dose of the study medication were included in the intention-to-treat sample. Randomization was performed with the use of permuted blocks of nine per site without stratification and was implemented with the use of an interactive voice-response telephone system. A total of 421 patients underwent randomization (from a target sample of 450 patients), yielding a statistical power of 99% to identify a difference of 33% in the rates of discontinuation of treatment by 36 weeks between any one of the drugs and placebo and a power of 80% to detect a difference of 20%, assuming a 60% rate of discontinuation in the placebo group. We used Kaplan–Meier survival curves to estimate the time to the discontinuation of treatment for all patients in the intention-to-treat population. The treatment groups were compared with the use of Cox proportional-hazards regression models,28 stratified according to site. Sites with 17 or fewer patients were grouped according to the size and type of site (i.e., university clinic, private practice, or Veterans Affairs medical center) for a total of 15 sites (8 pooled and 7 with 18 or more randomly assigned patients). In the Cox model, the overall difference among the treatment groups was evaluated with the use of a test with 3 degrees of freedom. If the difference was significant at a P value of less than 0.05, then each drug was compared with placebo by means of a Hochberg adjustment for multiple comparisons.29 Any antipsychotic drug found to be significantly better than placebo was then tested for noninferiority to the other antipsychotic agents. Noninferiority was concluded when the P value was less than 0.025 from a one-sided test with a margin of 2.0 (i.e., the upper limit of the 95% confidence interval for the hazard ratio was <2.0). Two atypical antipsychotic drugs were considered to be equivalent if the CI was contained within 0.5 and 2.0 (i.e., each drug was noninferior to the other); this was a broad equivalence margin for noninferiority. Finally, any antipsychotic drug found to be noninferior to the other antipsychotic drugs was tested for superiority on the basis of a one-sided test with a P value of less than 0.025. Discontinuation of treatment for a specific reason was analyzed in the same way, except that the data for patients who discontinued treatment for a different reason were censored at the time of discontinuation. Patients who had a response to the assigned study drug were defined as those who were still in phase 1 and who had a CGIC score indicating at least minimal improvement at 12 weeks; all patients who had discontinued medications or who did not have minimal improvement on the CGIC scale were classified as not having a response. Groups were compared with the use of a nonparametric analysis of covariance,30 stratified according to the type of site and adjusted for the following baseline covariates: age, sex, MMSE score, and total BPRS score. Statistical testing followed the same strategy as for time to discontinuation of treatment, with equivalence defined as a 95% CI of –0.20 to 0.20 for the difference between response rates. The study was funded by the NIMH. The pharmaceutical companies whose drugs were included in the study donated drug supplies; they were not involved in the design of the study, analyses, or interpretation of results. The authors and a protocol committee (see the Appendix) designed the trial. The site investigators gathered the data, and one of the authors analyzed the data. The manuscript was written by the authors, who vouch for the completeness and veracity of the data and data analyses. Results Patients, Caregivers, and Medication Doses A total of 521 patients were screened and 421 underwent randomization and received at least one dose of medication (Figure 1). Eighty-two percent of patients discontinued their initially assigned medication (i.e., during the 36-week follow-up period). Figure 1. Enrollment and Outcomes. Clinical characteristics of the patients are shown in Table 1. Fifty-seven percent required a level of care equivalent to that given in assisted-living facilities, and 17% needed the equivalent of nursing home care.31 At baseline, 46% were receiving antihypertensive drugs, 24% cholesterol-lowering drugs, 16% antiinflammatory drugs, and 30% vitamin E. Table 1. Baseline Characteristics of Patients Who Underwent Randomization. Fifty-two percent of caregivers were spouses, and 33% were children or sons-in-law or daughters-in-law. The mean ages of caregivers were 73.5 years for spouses and 51.2 years for children or their spouses; 71% were women.31 They spent a mean (±SD) of 5.2±4.0 hours per day in specific caregiving activities.32 The mean initially prescribed doses were 3.2 mg of olanzapine per day, 34.1 mg of quetiapine per day, and 0.7 mg of risperidone per day. The last prescribed mean dose in phase 1 was 5.5 mg of olanzapine per day, 56.5 mg of quetiapine per day, and 1.0 mg of risperidone per day. The mean number of capsules prescribed was similar for all treatment groups: 0.7 large or 1.4 small capsules per day initially, and 1.1 large or 2.2 small capsules per day for the last prescribed dose in phase 1. Outcomes There were no significant overall differences among treatment groups with regard to the time to discontinuation of treatment for any reason (Table 2 and Figure 2A). The median time to discontinuation of treatment ranged from 5.3 weeks with quetiapine to 8.1 weeks with olanzapine. Table 2. Medication Doses and Outcomes in the Intention-to-Treat Sample. Figure 2. Discontinuation of Treatment in Phase 1 According to Study Group. The median time to discontinuation of treatment because of lack of efficacy was longer in the olanzapine group (22.1 weeks) and the risperidone group (26.7 weeks) than in the placebo group (9.0 weeks) (Table 2 and Figure 2B). The hazard ratio for the discontinuation of treatment because of lack of efficacy was 0.51 (P<0.001) for olanzapine as compared with placebo, and 0.61 (P=0.01) for risperidone. The time to discontinuation of treatment with quetiapine owing to lack of efficacy (median, 9.1 weeks) did not differ significantly from that for placebo. For the discontinuation of treatment, olanzapine and risperidone were equivalent to each other (hazard ratio, 0.84; 95% CI, 0.53 to 1.32), and olanzapine was significantly superior to quetiapine (hazard ratio, 0.63; 95% CI, 0.41 to 0.96; P=0.02). The time to discontinuation of treatment owing to intolerance of the study drug, adverse effects, or death favored placebo, with discontinuation rates of 24% for patients who received olanzapine, 16% for patients who received quetiapine, and 18% for patients who received risperidone, as compared with 5% for patients who received placebo (Table 2 and Figure 2C). All three groups of patients who received an atypical antipsychotic drug were significantly more likely to discontinue treatment than were those who received placebo (hazard ratio for olanzapine, 4.32; 95% CI, 1.84 to 10.12; for quetiapine, 3.58; 95% CI, 1.44 to 8.91; and for risperidone, 3.62; 95% CI, 1.45 to 9.04). At 12 weeks, 32% of patients in the olanzapine group had a response (i.e., a CGIC score indicating at least minimal improvement with continued use of the phase 1 study medication), as compared with 26% in the quetiapine group, 29% in the risperidone group, and 21% in the placebo group; these rates were not significantly different (P=0.22). The overall rate of discontinuation of treatment at 12 weeks was 63%. Adverse Events There were no significant differences among the groups with regard to the proportion of patients who had at least one serious adverse event and the proportion who had any adverse event (Table 3). There were higher rates of parkinsonism or extrapyramidal signs in the olanzapine and risperidone groups (12% in each) than in the quetiapine group (2%) or the placebo group (1%). Correspondingly, the proportion of patients with a score of 1 or more on the Simpson–Angus scale (on a scale of 0 to 4, with 1 indicating mild extrapyramidal signs, and 4 the most severe signs) was higher with olanzapine (14%) and risperidone (11%) than with placebo (2%). Table 3. Adverse Events and Other Safety Outcomes in Phase 1. Sedation occurred more commonly with the three drugs (reported in 15 to 24% of patients) than with placebo (5%), and confusion or changes in mental status occurred more commonly with olanzapine (18%) and risperidone (11%) than with placebo (5%). Both cognitive disturbances and psychotic symptoms were more common with olanzapine (5% and 7%, respectively) than with the other medications or placebo (0 to 2%). The body weight and body-mass index (BMI) of patients increased with antipsychotic drugs (by 0.4 to 1.0 lb per month and 0.2 BMI unit) and decreased slightly with placebo (by –0.9 lb per month and –0.2 BMI unit). Prolactin levels at week 12 were markedly elevated in the risperidone group only. Ten patients entered nursing homes during phase 1 (two patients receiving olanzapine, four patients receiving quetiapine, three patients receiving risperidone, and one patient receiving placebo). Discussion In this study, we randomly assigned patients with Alzheimer's disease and psychosis, aggression, or agitation to placebo or one of three atypical antipsychotic medications. The median time to discontinuation of treatment for any reason ranged from 5 to 8 weeks, with no significant differences among the four groups. The study physicians discontinued the study drugs within 8 weeks after the start of treatment in about half the patients. This practice was consistent with the opinions of expert clinicians who have recommended discontinuing or switching antipsychotic treatment after 2 to 4 weeks in patients who are not benefiting from it.5,8 The time to discontinuation of treatment, due to lack of efficacy, favored olanzapine and risperidone but was offset by the increased rates of discontinuation of these drugs due to adverse events. Because the study was designed to allow patients who discontinued a study medication in phase 1 to enter phase 2 and be randomly assigned to a different study drug, expectations that the phase 2 treatments might be more effective may have increased the likelihood of earlier discontinuation of treatment during phase 1. The study physicians increased the initial medication doses from an average of 1.4 small capsules to an average of 2.2, equivalent to approximately 1.0 mg of risperidone, 5.5 mg of olanzapine, and 57.0 mg of quetiapine. The risperidone and olanzapine doses were both within the ranges recommended by experts5 and used in previous trials.9 The quetiapine dose, however, was half to a quarter that used in two nursing home trials.9 The protocol committee was unwilling to use a higher minimum starting dose of quetiapine or a larger incremental dose because of the possibility of excess sedation, but the committee expected that physicians would determine dosing individually for each patient. The rates of adverse events were similar among the drugs, however, so the apparent reluctance to increase the dose may have been related to observed adverse events or concern that adverse events would occur with higher doses of risperidone or olanzapine. Nevertheless, the decision not to increase the dose in the face of an inadequate response limits some conclusions about efficacy and adverse effects. Sedation was more likely with all of the anti-psychotic drugs than with placebo and was particularly likely with olanzapine, which was also associated with increased confusion. Although worsening of cognition has been observed in other trials of antipsychotic medications for the treatment of dementia,9 MMSE scores did not worsen in our trial. Extrapyramidal signs and symptoms with both risperidone and olanzapine were common reasons for the discontinuation of treatment. The clinical significance of the expected hyperprolactinemia with risperidone is uncertain. Patients gained weight with olanzapine and risperidone and lost weight with placebo; the possibility that antipsychotic drugs cause the metabolic syndrome in the elderly requires further investigation. The key enrollment criterion — the physician's assessment that an antipsychotic drug was the appropriate pharmacologic therapy — helped to ensure clinical equipoise. The patient population was broad and representative in terms of age, race, ethnic group, level of disability, and extent of cognitive impairment. The level of psychopathology was moderate to severe, which is similar to that in studies of patients in nursing homes.Approximately 75% of the patients required a level of care at least equivalent to that given in assisted-living facilities.31 Most of the patients in our study required a level of care equivalent to that given in an assisted-living facility, and 17% needed the equivalent of nursing home care.31 Washout from previous treatment and run-in periods were not used because of the patients' acute clinical symptoms; instead, the study design allowed for rapid assignment and initiation of treatment to be consistent with clinical practice. Specifically, 14% percent of the patients were receiving antipsychotic medications within 2 weeks before randomization (5% risperidone, 4% haloperidol, 3% quetiapine, and 2% olanzapine). The use of cholinesterase inhibitors did not influence outcomes. We used the discontinuation of treatment for any reason as a pragmatic measure of outcomes for individual patients rather than means for scores on rating scales because the discontinuation of treatment incorporates the judgments of patients, caregivers, and clinicians regarding efficacy, safety, and tolerability and reflects therapeutic benefits in relation to undesirable effects. Previous trials compared mean scores on rating scales according to group at the end of 6 to 12 weeks without regard to tolerability and did not address the outcomes among individual patients. Our results are consistent with those of the few previous outpatient trials of atypical antipsychotic medications,9 which showed modest trends or no significant effects on symptom ratings. Overall, the rates of discontinuation of treatment among the four study groups ranged from 77 to 85%. Although the differences among the groups may have been significant in a larger trial, our findings suggest that there is no large clinical benefit of treatment with atypical antipsychotic medications as compared with placebo. Physicians were likely to switch medications quickly in the face of lack of efficacy or adverse effects. Although the atypical antipsychotic drugs were more effective than placebo, adverse effects limited their overall effectiveness, and their use may be restricted to patients who have few or no side effects and for whom benefits can be discerned. Clinicians, patients, and family members must consider both risks and benefits in order to optimize a patient's care. Supported by a grant (NO1 MH9001) from the NIMH. AstraZeneca Pharmaceuticals, Forest Pharmaceuticals, Janssen Pharmaceutica, and Eli Lilly provided medications for the studies. Dr. Schneider reports having received consulting fees from AstraZeneca, Forest Laboratories, Johnson & Johnson, and Pfizer, and lecture fees from AstraZeneca, Eli Lilly, and Forest Laboratories; Dr. Tariot, consulting fees from GlaxoSmithKline and Novartis, consulting fees and research support from Abbott, AstraZeneca, Bristol-Myers Squibb, Eisai, Forest Laboratories, GlaxoSmithKline, Janssen Pharmaceutica, Eli Lilly, Merck, Pfizer, and Schwabe, educational fees from AstraZeneca, Eisai, Forest Laboratories, Lundbeck, Merz, and Pfizer, and research support from Wyeth; Dr. Ismail, consulting and lecture fees from Forest Laboratories and Janssen Pharmaceutica; Dr. Lyketsos, research funding and educational fees from GlaxoSmithKline, consulting or advisory-board fees from Eisai, Forest Laboratories, and GlaxoSmithKline, and lecture fees from Pfizer, Forest Laboratories, and Novartis; Dr. Stroup, consulting fees from Janssen Pharmaceutica, Eli Lilly, and Pfizer and lecture fees from Eli Lilly and Pfizer; Dr. Sultzer, research funding from Forest Laboratories and Pfizer, consulting fees from Eli Lilly and Novartis, and lecture fees from AstraZeneca, Bristol-Myers Squibb, Pfizer, and Forest Laboratories; Dr. Weintraub, research funding from Forest Laboratories, consulting fees from Avanir Pharmaceuticals, Boehringer Ingelheim, Teva Pharmaceuticals, and GlaxoSmithKline, and lecture fees from AstraZeneca; and Dr. Lieberman, research funding from Acadia, Bristol-Myers Squibb, Eli Lilly, GlaxoSmithKline, Janssen, and Pfizer. Dr. Davis reports having received consulting fees from Eli Lilly and Pfizer and being employed by Quintiles; Dr. Hsiao reports being employed by the NIMH; Dr. Lebowitz reports being formerly employed by the NIMH. Dr. Ryan reports having received lecture fees from Abbott, Forest Laboratories, AstraZeneca, Bristol-Myers Squibb, Eisai, Janssen Pharmaceutica, Pfizer, and research contracts from Johnson & Johnson and Eli Lilly, and being employed by Merck. No other potential conflict of interest relevant to this article was reported. * Members of the Clinical Antipsychotic Trials of Intervention Effectiveness–Alzheimer's Disease (CATIE-AD) Study Group are listed in the Appendix. Source Information From the Keck School of Medicine, University of Southern California, Los Angeles (L.S.S., K.S.D.); the Banner Alzheimer's Institute, Phoenix, AZ (P.N.T.); Quintiles, Research Triangle Park, NC (S.M.D.); the National Institute of Mental Health, National Institutes of Health, Bethesda, MD (J.K.H.); the University of Rochester Medical Center, Rochester, NY (M.S.I., J.M.R.); the School of Medicine, University of California, San Diego, La Jolla (B.D.L.); the Department of Psychiatry, Johns Hopkins Bayview, Johns Hopkins University, Baltimore (C.G.L.); the School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill (T.S.S.); Veterans Affairs Greater Los Angeles Healthcare System, University of California, Los Angeles, Los Angeles (D.L.S.); the School of Medicine, University of Pennsylvania, Philadelphia (D.W.); and the College of Physicians and Surgeons, New York (J.A.L.). Address reprint requests to Dr. Schneider at the Keck School of Medicine, University of Southern California, 1510 San Pablo St., HCC 600, Los Angeles, CA 90033, or at lschneid@usc.edu. References Tariot PN, Schneider LS, Katz IR. Anticonvulsant and other non-neuroleptic treatment of agitation in dementia. J Geriatr Psychiatry Neurol 1995;8:Suppl 1:S28-S39. Devanand DP, Jacobs DM, Tang MX, et al. The course of psychopathologic features in mild to moderate Alzheimer disease. Arch Gen Psychiatry 1997;54:257-263. Jeste DV, Finkel SI. Psychosis of Alzheimer's disease and related dementias: diagnostic criteria for a distinct syndrome. Am J Geriatr Psychiatry 2000;8:29-34. Paulsen JS, Salmon DP, Thal LJ, et al. Incidence of and risk factors for hallucinations and delusions in patients with probable AD. Neurology 2000;54:1965-1971. Alexopoulos GS, Streim J, Carpenter D, Docherty JP. Using antipsychotic agents in older patients. J Clin Psychiatry 2004;65:Suppl 2:5-104. Schneider LS, Pollock VE, Lyness SA. A metaanalysis of controlled trials of neuroleptic treatment in dementia. J Am Geriatr Soc 1990;38:553-563. Jeste DV, Lacro JP, Bailey A, Rockwell E, Harris MJ, Caligiuri MP. Lower incidence of tardive dyskinesia with risperidone compared with haloperidol in older patients. J Am Geriatr Soc 1999;47:716-719. Alexopoulos GS, Silver JM, Kahn DA, Frances A, Carpenter D. Treatment of agitation in older persons with dementia. Postgrad Med 1998;Special No.:1-88. Schneider LS, Dagerman K, Insel PS. Efficacy and adverse effects of atypical antipsychotics for dementia: meta-analysis of randomized, placebo-controlled trials. Am J Geriatr Psychiatry 2006;14:191-210. Wooltorton E. Risperidone (Risperdal): increased rate of cerebrovascular events in dementia trials. CMAJ 2002;167:1269-1270. Wooltorton E. Olanzapine (Zyprexa): increased incidence of cerebrovascular events in dementia trials. CMAJ 2004;170:1395-1395. Racoosin JA. Evaluating a safety signal in the postmarking period: cerebrovascular adverse events associated with risperidone and olanzapine. Presented at the 17th Annual Meeting of the American Association for Geriatric Psychiatry, Baltimore, February 21–24, 2004 (paper). Schneider LS, Dagerman KS, Insel P. Risk of death with atypical antipsychotic drug treatment for dementia: meta-analysis of randomized placebo-controlled trials. JAMA 2005;294:1934-1943. Center for Drug Evaluation and Research. FDA public health advisory: deaths with antipsychotics in elderly patients with behavioral disturbances. April 11, 2005. (Accessed September 15, 2006, at http://www.fda.gov/cder/drug/advisory/antipsychotics.htm.) Lyketsos CG, Olin J. Depression in Alzheimer's disease: overview and treatment. Biol Psychiatry 2002;52:243-252. Pollock BG, Mulsant BH, Rosen J, et al. Comparison of citalopram, perphenazine, and placebo for the acute treatment of psychosis and behavioral disturbances in hospitalized, demented patients. Am J Psychiatry 2002;159:460-465. Olin JT, Katz IR, Meyers BS, Schneider LS, Lebowitz BD. Provisional diagnostic criteria for depression of Alzheimer disease: rationale and background. Am J Geriatr Psychiatry 2002;10:129-141. Schneider LS, Tariot PN, Lyketsos CG, et al. National Institute of Mental Health Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE): Alzheimer disease trial methodology. Am J Geriatr Psychiatry 2001;9:346-360. Dementia of the Alzheimer's type. In: Diagnostic and statistical manual of mental disorders. 4th ed. text revision: DSM-IV-TR. Washington, DC: American Psychiatric Association, 2000:154-8. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology 1984;34:939-944. Folstein MF, Folstein SE, McHugh PR. "Mini-Mental State": a practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res 1975;12:189-198. Beller SA, Overall JE. The Brief Psychiatric Rating Scale (BPRS) in geropsychiatric research. II. Representative profile patterns. J Gerontol 1984;39:194-200. Cummings JL, Mega M, Gray K, Rosenberg-Thompson S, Carusi DA, Gornbein J. The Neuropsychiatric Inventory: comprehensive assessment of psychopathology in dementia. Neurology 1994;44:2308-2314. Roman GC, Tatemichi TK, Erkinjuntti T, et al. Vascular dementia: diagnostic criteria for research studies: report of the NINDS-AIREN International Workshop. Neurology 1993;43:250-260. McKeith IG, Galasko D, Kosaka K, et al. Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the Consortium on DLB international workshop. Neurology 1996;47:1113-1124. Mittelman MS, Bergman H, Shulman E, Steinberg G, Epstein C. Guiding the Alzheimer's Caregiver: a handbook for counselors. New York: New York University School of Medicine, 2000. Schneider LS, Olin JT, Doody RS, et al. Validity and reliability of the Alzheimer's Disease Cooperative Study-Clinical Global Impression of Change: the Alzheimer's Disease Cooperative Study. Alzheimer Dis Assoc Disord 1997;11:Suppl 2:S22-S32. Cox DR. Regression models and life-tables. J R Stat Soc 1972;34:187-220. Hochberg Y. A sharper Bonferroni procedure for multiple tests of significance. Biometrika 1988;75:800-802. Koch GG, Tangen CM, Jung JW, Amara IA. Issues for covariance analysis of dichotomous and ordered categorical data from randomized clinical trials and non-parametric strategies for addressing them. Stat Med 1998;17:1863-1892. Ismail MS, Dagerman K, Tariot PN, Abbott S, Kavanaugh S, Schneider LS. National Institute of Mental Health Clinical Antipsychotic Trials of Intervention Effectiveness-Alzheimer's Disease (CATIE-AD): baseline characteristics. Curr Alzheimer Res (in press). Davis KL, Marin DB, Kane R, et al. The Caregiver Activity Survey (CAS): development and validation of a new measure for caregivers of persons with Alzheimer's disease. Int J Geriatr Psychiatry 1997;12:978-988....查看详细 (35304字节)
☉ 11342762:The Two-Edged Sword of Curing Childhood Cancer
In the June 3, 1948, issue of the Journal, Sydney Farber and his colleagues reported that they had successfully used aminopterin to induce temporary remission in children with lymphoid leukemia. This exciting article suggested that what had been a uniformly fatal disease might be amenable to treatment and, some dared to hope, cure. Then, in 1970, DeVita and his colleagues at the National Cancer Institute showed that a combination of non–cross-reactive chemotherapeutic agents, the MOPP regimen (mechlorethamine, vincristine, procarbazine, and prednisone), could induce sustained remission in advanced Hodgkin's disease. From such beginnings, an extraordinary success story unfolded, and today, more than 75% of children with cancer can be cured of their disease.1 Nearly 20,000 children under the age of 21 receive a diagnosis of cancer each year in the United States, and more than 15,000 per year enter the ranks of the cured and are able to live into adulthood. In the "war on cancer," this would appear to be a battle won. But what happens to these survivors? In this issue of the Journal, Oeffinger and colleagues (pages 1572–1582) report that the cure may be accompanied by a host of adverse events, some of which may not appear for many years after treatment has ended (see image). These findings imply that vigorous and long-term monitoring of young cancer survivors, accompanied by early intervention when problems arise, is mandatory. There is a dark side to being cured of cancer as a young person. Computed Tomographic Scan of an Osteosarcoma in the Right Ileorectal Fossa of a Teenager Who Underwent Pelvic Irradiation for Neuroblastoma in Infancy. Oeffinger et al. report the latest follow-up data from a remarkable long-term study, which does for the survival of childhood cancer what the Framingham Heart Study did for the natural history of cardiovascular disease. The Childhood Cancer Survivor Study (CCSS), established in 1994, is conducted by a consortium of 25 pediatric oncology treatment centers that have pooled data on their survivors who were treated between 1970 and 1986, when significant strides were first made in the treatment of many of the most common types of cancer in children. Although there have been a number of previous reports (from this group and others) that document the substantial incidence of specific long-term side effects of treatment that are faced by survivors, no study has approached the sheer number of patients in the cohort followed by the CCSS. This group is clearly the standard by which all future studies should be measured. Oeffinger et al. confirm the extraordinarily high incidence of late, and often permanent, complications arising from intensive treatment with combination chemotherapy and ionizing radiation. They also demonstrate that the risks are cumulative, with no evidence of a plateau so far. For physicians and nurses who are dedicated to taking care of these patients, this is worrisome news indeed. Looking at the outcomes in more than 10,000 adult survivors, the CCSS investigators found that almost two thirds of the patients reported at least one chronic health problem, one quarter had a severe condition, and (perhaps most disturbing) almost one quarter reported having three or more chronic health problems. By any criteria, these results are alarming, and they bespeak a significant level of complications in a population in which only a minority of patients receive follow-up from specialists, as the CCSS and others have previously reported. Furthermore, the types of late sequelae run the gamut of affected organ systems, hinting that even more problems may cloud the future as this population ages. Chronic illnesses that affect the elderly more generally will also emerge in survivors of childhood cancer, who already carry a heavy physiological burden, as well as a risk of psychological problems stemming from their experience with cancer.2,3 One can only speculate about how the combination of preexisting myocardial damage from anthracycline exposure or heart irradiation for Hodgkin's disease would affect cardiac risk factors, when combined with the high incidence of hyperlipidemia, hypertension, diabetes, and kidney disease in the U.S. population at large. Indeed, it is probable that survivors of childhood cancer will be especially vulnerable to many age-related chronic health conditions, their risk magnified by their previous medical treatment. There is an unavoidable and noteworthy deficiency in the data from the CCSS cohort. All the patients were treated before 1986, and there have been considerable changes in therapy for most childhood cancers during the succeeding 20 years, often with an escalating intensity of treatment paralleling an improvement in survival rates.1 At the same time, we have become increasingly aware of the potential for late effects and have attempted to incorporate this knowledge into treatment protocols whenever it is possible to do so without sacrificing efficacy. It would be expected that patients who were treated more recently might have an analogous but different array of long-term complications. This historical effect argues for establishing another cohort for future study. It would seem to be incumbent on us to ensure that survivors of childhood cancer are followed closely by physicians who understand the effects and outcomes of cancer treatment — who know what these patients have been through and what kinds of complications to look for in the future. Sadly, such follow-up is the exception rather than the rule. To whom should we look for this expertise? Historically, most of these patients, especially the adolescents and younger adults, have been cared for by pediatric oncologists. But over time most of these patients drift away from the specialty clinic. It is clear that the complications, known and unknown, that will develop in survivors can demand a level of knowledge that is beyond the range of the general internist or family doctor, or even of medical oncologists. Oeffinger, himself a family practitioner, has argued for the wide dissemination of information about late effects,4 but this approach relies on self-education by interested physicians in the community. Perhaps we need to incorporate specific training in this area into our residency programs in internal medicine, pediatrics, and family medicine. As an alternative, we could develop a postgraduate training fellowship in an adult subspecialty, analogous to the fellowships in adult congenital heart disease that are common in most major academic medical centers. Patients themselves could be empowered by being furnished with a portable document describing their treatment, its potential late complications, and any types of behavior that could diminish their risk of late effects; certainly, cancer survivors and their parents would be very receptive to such recommendations.5 These suggestions do not supplant the need for physicians and nurses who are trained to provide seamless continuity of care from diagnosis to treatment to long-term follow-up. Whatever the eventual approach, it is clear that the effects of childhood cancer — like those of sickle cell anemia, cystic fibrosis, and many other diseases that begin in childhood — do not end when the patient reaches the magic age of 18 or 21 years. Although the child with cancer may be cured, the effects of treatment are lifelong, and we need to study and treat these late effects. Source Information Dr. Rosoff is an associate professor of pediatric hematology–oncology at Duke University School of Medicine and director of the Duke University Hospital Program in Clinical Ethics, Durham, NC. References Ries LAG, Harkins D, Krapcho M, et al. SEER cancer statistics review, 1975-2003. Bethesda, MD: National Cancer Institute, 2006. (Accessed September 20, 2006, at http://seer.cancer.gov/csr/1975_2003/.) Langeveld NE, Grootenhuis MA, Voute PA, de Haan RJ. Posttraumatic stress symptoms in adult survivors of childhood cancer. Pediatr Blood Cancer 2004;42:604-610. Stam H, Grootenhuis MA, Last BF. The course of life of survivors of childhood cancer. Psychooncology 2005;14:227-238. Oeffinger KC, Eshelman DA, Tomlinson GE, Tolle M, Schneider GW. Providing primary care for long-term survivors of childhood acute lymphoblastic leukemia. J Fam Pract 2000;49:1133-1146. Bashore L. Childhood and adolescent cancer survivors' knowledge of their disease and effects of treatment. J Pediatr Oncol Nurs 2004;21:98-102....查看详细 (8616字节)

☉ 11342763:Doctors on Strike — The Crisis in German Health Care Delivery
In mid-March 2006, physicians at Germany's university clinics went on strike, protesting an increase in the hours of the official workweek that came without a corresponding increase in pay. The physicians' union, the Marburger Bund, argued that neither the old official workweek of 38.5 hours, nor the new one of 40 or 42 hours, reflected the reality known by most doctors in Germany, where 80-hour weeks are common and physicians often put in many additional, uncompensated hours. About 15,000 of the 20,000 physicians based at university hospitals joined in this unprecedented strike. After 3 months of collective action and negotiation, employers consented to pay increases of 8 to 18%, far less than had been sought. The Marburger Bund was successful in improving working conditions by reducing the actual weekly working time to a maximum of about 48 hours (with well-defined exceptions permitted under the derogations allowed by the European Union). Furthermore, the union secured bonus payments for physicians who have earned more advanced medical qualifications. Then, in late June, physicians at German public hospitals followed their university colleagues onto the picket lines, staging rolling strikes at about 700 municipal clinics that together employ approximately 70,000 doctors. Demanding better working conditions and pay increases similar to those sought at the university hospitals, the Marburger Bund rejected an agreement that had recently been negotiated between the municipalities and the service-sector trade union Ver.di on behalf of all public employees, which the Marburger Bund said neither reflected the working patterns of physicians nor compensated doctors for wage losses. (For example, a married 29-year-old physician would have lost 31,000 over a period of 10 years and 68,000 over a period of 20 years.) This was the first time a group of highly qualified "public servants" had not agreed to contracts negotiated between public employers and the general public-service union. Late in August, after an 8-week strike, the Marburger Bund and the municipalities signed a new contract, granting doctors pay raises similar to those obtained in the university hospitals. Overall working time was limited, and better reimbursement schemes for overtime and night-call duty were accepted. Physicians on strike — a phenomenon that would have been considered unthinkable — is now a reality. Yet given that many of the factors involved are the very ones that have contributed to a health care crisis in Germany, it seems likely that walkouts will become more common in the future. (Figure) Physicians on Strike in Germany. From the Marburger Bund. To comprehend why German physicians have taken to the streets rather than staying at their patients' bedsides, one must understand the state of the German health care system and the way in which hierarchies and decision making have been restructured in recent decades. With a system based on social insurance, expenditures for health care represent 10.8% of the gross domestic product of Germany — higher than the average of 8.9% among the 30 countries in the Organisation for Economic Co-operation and Development. Ambulatory health care is provided by general practitioners and specialists, and inpatient care is delivered by a mix of public, nonprofit, and private providers. With one exception, university clinics are managed by the federal states. The population enjoys equal and easy access to care, and waiting lists and explicit rationing of health care have been unknown to date. But social politics have continuously weakened white-collar professions, including those in medicine and pharmacy, detracting from the bargaining position of physicians employed by university and municipal clinics (who make up about 30% of all doctors in Germany). For example, in a trend reminiscent of that associated with managed care in the United States in the 1990s,1 during the past three to four decades the German medical profession has permitted health insurance companies to control many aspects of medical practice. This has changed the German concept of good and affordable medicine by placing ever-greater limits on doctors' latitude in making medical decisions for their patients. At the same time, medicine has been increasingly subjected to economic considerations. Increases in the costs of diagnostic and therapeutic procedures have coincided with the shrinking of the tax base. So physicians employed by university clinics and municipal hospitals have been forced to accept increases in their workload, coupled with ever-lower incomes. Between 1991 and 2004, 1 in every 10 German hospitals was closed, resulting in a 20% reduction in the number of hospital beds. The length of stay in hospitals decreased by 38%, but the number of patients being treated increased by 20%. Thus, work in hospitals was condensed and intensified, and physicians' work hours increased accordingly. Not all physicians were unhappy with long working hours. Night calls were reimbursed and constituted the only chance for many, especially junior, physicians to earn a decent income. Others welcomed the overtime because it provided them an opportunity to perform enough operations and procedures to qualify for professional specialization quickly. But the average German physician was clearly feeling overworked. The burden was growing by more than the number or patients assigned to each physician. In 2003, a survey found that physicians in German hospitals spent more than one third of their working time on documentation and other administrative duties. Traditionally, the German hospital system has made little use of coding assistants or advanced-practice nurses to perform routine nonmedical tasks. In 2004, an international survey showed that the earnings of hospital-based physicians in 2002 were lower in Germany than in any other country under review (see graph).2 As Frank Ulrich Montgomery, chairman of the Marburger Bund, pointed out, the new wage agreement reached by Ver.di would have resulted in even further pay cuts for younger doctors. Annual Average Earnings of Hospital Doctors in 2002. The highest estimate and lowest estimate (where available) are shown, connected by a vertical line. Data are from National Economic Research Associates. It is therefore not surprising that only 55% of students who have recently entered medical school will eventually take a job in a hospital. Large numbers of German physicians have left the country to work abroad, primarily in Britain and Scandinavia. In opinion polls, German physicians emphasize that what they consider a crisis of the entire health care delivery system has been caused by several factors, including the loss of professional independence in clinical work, the increasing influence of economic considerations, and the growing load of bureaucratic tasks. There is a widespread feeling that even an increase in pay may be unable to restore the job satisfaction of physicians. In the case of this year's strikes, the German Association of Municipal Administrations has argued that acceding to the Marburger Bund's demands would result in an "unprecedented avalanche of costs." And indeed, about half of all local hospitals are allegedly operating in the red. Physicians participating in collective action are accused of jeopardizing the already tenuous existence of small hospitals, contributing to a worsening of the distribution of care, especially in rural areas. These complex problems must be addressed if we are to address the factors that underlie both the dissatisfaction of physicians and the overall crisis in health care delivery. Source Information Dr. Nowak is the director of the Institute and Outpatient Clinic for Occupational and Environmental Medicine at the Ludwig Maximilians University and a deputy for Occupational and Environmental Medicine at Technical University — both in Munich, Germany. References Kassirer JP. Managed care and the morality of the marketplace. N Engl J Med 1995;333:50-52. Bramley-Harker E, Barham L. Comparing physicians' earnings: current knowledge and challenges: a final report for the Department of Health. London: NERA Economic Consulting, 2004. (Accessed September 20, 2006, at http://www.nera.com/Publication.asp?p_ID=2552.)...查看详细 (8415字节)
☉ 11342764:Body of Research — Ownership and Use of Human Tissue
For better or worse, we have irretrievably entered an age that requires examination of our understanding of the legal rights and relationships in the human body and the human cell. — Moore v. Regents of the University of California, California 2nd District Court of Appeals, 1988 Nearly 20 years after the California courts decided Moore v. Regents — a seminal case concerning a patient's interest in the profits derived from patents on a cell line generated from his spleen tissue — U.S. jurisprudence still has no coherent answer to a deceptively simple question: Do we own our own bodies? Why deceptively simple? Because the meaning of "property" is unclear. Because the question must be asked about our relationships both to our bodies and to our excised body tissue. And because it must be asked about those relationships both before and after we die. No brief article or single court opinion could address all the complexities of the legal notion of "property." But simple or not, the question of whether we "own" our bodies must be answered soon: courts now face multiple controversies concerning the use of bits and pieces of bodies — and their derivatives — that are scattered among pathology laboratories, state hygiene laboratories, museums, archives, sperm banks, fertility clinics, and forensic DNA collections. One recent controversy concerns William Catalona, a prostate cancer surgeon and researcher formerly employed by Washington University in St. Louis. Over the course of decades, and with his patients' consent, Catalona amassed more than 3500 tissue samples, developed the prostate-specific antigen test, and led clinical trials to improve testing for prostate cancer. When he left for another university, Catalona asked his patient-donors to write to Washington University requesting that their tissue samples be sent to his new place of employment. Washington University refused to send them, and a dispute arose about the patients' right to control the tissue. On the basis of the terms of the consent documents signed when the patients originally released their tissue, the court ruled that control belonged to Washington University and that although patients might ask that their samples be destroyed, they could not direct that they be delivered to Catalona or to another university. Left unclear in the court's opinion is whether the patients ever had a property interest in their tissues. One reading suggests they did but that they relinquished it upon donation; another suggests that they never had such an interest, even while the tissues were in their living bodies. The court's ruling on the narrower issue of control of the excised tissues had some basis in precedent. A smattering of other decisions, concerning state laws covering presumed consent for the donation of cadaveric corneas, management of frozen embryos whose progenitors are still alive, and postmortem control of stored semen, suggested that donors might have some property rights in their tissues. But in two other decisions, the Moore case of 1988 (concerning rights to share in commercial gain from derivatives of tissue taken without proper informed consent) and the Greenberg case of 2003 (concerning property rights in tissue and genetic information derived from patients' tissues), courts found that state law provided little basis for granting patients a property interest in their voluntarily donated, excised tissue. Those courts did not delve deeply into the possible existence of property rights to such tissue before excision. If, on the one hand, there are no such rights, then the Catalona decision is not a question of the evaporation of a property interest after donation but a finding that we have no property right to our tissues before or after they leave our bodies. According to this view, any right we may have to control the use of our tissue rests on other considerations, such as the privacy of medical information or entitlements created through the regulation of research on human subjects. If, on the other hand, such a property right does exist, then any conclusion about whether donation constitutes a relinquishment of that right depends on the fairness of the transaction and the quality of the information provided before consent is given. Such distinctions have powerful implications for medical research. At present, research regulations are built on a theory of autonomy that is independent of any property right in one's tissue. Thus, although in general patients can refuse or consent to the donation of tissue samples for research, such rights are quite limited. Common law protects people from involuntary excision of body tissue, which would be considered battery. But after the tissue has been properly excised, its use without the patient's consent may be permitted under federal research regulations, if the patient's identity is unknown or adequately obscured.1 Federal regulations are based on a notion of voluntarism, but the right to refuse participation in research is based more on ensuring that subjects can be the ones to judge how to protect their own interests than on a pure form of autonomous decision making, which would include a right to refuse for any reason or no reason at all. Hence, no consent is required for research deemed to pose minimal risk to subjects — or for research in which identifying information is obscured or from which it has been deleted (which protects privacy and minimizes social risks such as stigmatization or discrimination, but does nothing to recognize subjects' autonomy). And when patients' preferences regarding the kinds of research that may be performed on their tissue are ascertained, this is done as a courtesy, rather than as recognition of patients' rights to prohibit the use of their tissues for purposes of which they disapprove. In a battle over such rights, the Havasupai tribe is suing researchers who took blood for a diabetes-control project and later used it in research about schizophrenia within the tribal community.2 Although institutions may be disciplined by federal authorities for regulatory lapses, courts have so far refused to recognize a private legal claim based on this sort of alleged violation of the requirements of consent for research. In other settings, however, calls for increased control by patients over the use of their tissue would suggest that only subsuming the matter under property law would suffice.3 In the emerging field of regenerative medicine, for example, California's new funding regulations require researchers to honor the limits set by donors of embryos or gametes on the kinds of work that can be done even with donated tissue that has been "anonymized" — a rule consistent with a theory of property rights in tissue. In the field of biobanking (the collection and distribution of tissue and DNA samples), some have called for caution in the common practice of asking donors to waive all rights, on the assumption that property rights exist and waivers may be ineffective if they do not satisfy all the legal rules for property transfers.4 Investigators and universities have argued that recognizing property rights in excised tissue would threaten their ability to use stored tissue samples effectively5 — a use that is essential to everything from advances in pharmacogenomics and genetic epidemiology, to the development of new tests for clinically significant mutations, to the investigation of historical events and epidemics through the medical testing of cadavers. Ultimately, the debate is less about whether the management of human tissue should be governed by property laws or by a more robust regulatory scheme than about the proper balance between respect for persons and the collective interest in promoting research involving human tissue. Respect for persons and their autonomy can be promoted through many legal frameworks. In one framework, autonomy is premised on the notion that one's body is one's property and that uninvited removal or use of tissue is theft or trespass. In the other, autonomy is premised on the liberty interests of the person within the body (such as the freedom from assault), and the uninvited removal or use of tissue is an injury and a deprivation of liberty. Despite its apparent clarity, the property-based approach creates new dilemmas that we are hardly ready to confront. If I "own" my tissue, do I have all the rights usually associated with property? Calling something property does not necessarily mean that its owner has all possible rights to its exclusive use, donation, sale, alteration, and destruction, but this is a common bundle of rights associated with ownership of property. If this pattern were extended to tissues, then selling organs might become a matter of right. And cadaveric tissue could become the property of heirs, rendering unusable even old repositories such as the one used to sequence the 1918 influenza virus. Of course, labeling something as "property" does not foreclose limitations on its use: governments can sometimes take property for public purposes or forbid its sale or alteration. But generally, calling something "property" will enhance the owner's interests considerably, especially with regard to prohibiting others from using it. Alternatively, our relationship with our bodies may be viewed as a trusteeship: even if they are not our property, we nonetheless have extensive and exclusive rights with respect to them, at least while we are alive and our bodies are intact. Even with such a view, bodies and tissues may also be viewed as part of a common heritage of humanity, to be used for the collective good if such use does not unduly infringe on our liberties. This leads to arguments for a public right to use excised tissue, provided that due care is taken to protect the privacy and social interests of the person from whom it comes. Such arguments also support a policy of "presumed consent" for the use of cadavers for tissue and organ transplantation. Moving in this direction would still require attention to respecting the interests of persons who do not wish to be research subjects or who fear the invasion of privacy or social interests that examination of their tissues might entail. Regulation of the management of banked tissue could address these questions. But recharacterizing our bodies as a public resource would also suggest an ethical duty to work toward a just distribution of the benefits of such research, both financial and therapeutic. Without some symmetry between the burdens and the benefits of research, the social compact underlying such an imposition on personal interests would be undermined. Although treating our bodies and tissues as property would offer an avenue toward strong protection of personal autonomy, it is not the only way to accomplish this goal. Nor is it without costs, in terms of possible losses to the collective interest in research and organ donation. Other systems, whether based on enhanced regulations protecting human subjects or a theory of trusteeship, could serve as well. But whichever system is chosen, it is long past time for the country to choose. State laws vary, federal regulations do not apply to all privately funded research, and a patchwork of rules cover the myriad laboratories and biobanks in the United States. Our tissue may be scattered. Our laws ought not to be. Source Information Ms. Charo is a professor of law and bioethics at the School of Law and the School of Medicine, University of Wisconsin, Madison. An interview with Professor Charo can be heard at www.nejm.org. References Protection of Human Subjects, 45 C.F.R. Public Welfare, pt. 46 (2006). Tilousi v. Arizona State Univ. Bd. of Regents, No. 04-CV-1290-PCT-FJM (March 3, 2005). Andrews L. Who owns your body? A patient's perspective on Washington University v. Catalona. J Law Med Ethics 2006;34:398-414. Winickoff DE, Winickoff RN. The charitable trust as a model for genomic biobanks. N Engl J Med 2003;349:1180-1184. National Bioethics Advisory Commission. Research involving human biological materials: ethical issues and policy guidance. Washington, DC: Government Printing Office, 1999....查看详细 (12330字节)
☉ 11342765:Evaluating Detection of an Inhalational Anthrax Outbreak
McGill University, Montreal, Quebec, Canada Veterans Affairs Palo Alto Health Care System, Palo Alto, California, USA Stanford University, Stanford, California, USA University of Toronto, Toronto, Ontario, Canada Abstract Timely detection of an inhalational anthrax outbreak is critical for clinical and public health management. Syndromic surveillance has received considerable investment, but little is known about how it will perform relative to routine clinical case finding for detection of an inhalational anthrax outbreak. We conducted a simulation study to compare clinical case finding with syndromic surveillance for detection of an outbreak of inhalational anthrax. After simulated release of 1 kg of anthrax spores, the proportion of outbreaks detected first by syndromic surveillance was 0.59 at a specificity of 0.9 and 0.28 at a specificity of 0.975. The mean detection benefit of syndromic surveillance was 1.0 day at a specificity of 0.9 and 0.32 days at a specificity of 0.975. When syndromic surveillance was sufficiently sensitive to detect a substantial proportion of outbreaks before clinical case finding, it generated frequent false alarms. In the early stage of an inhalational anthrax outbreak, a 1-day delay in the initiation of chemoprophylaxis and treatment of exposed persons can result in thousands of additional deaths and millions of dollars of additional expenditures (1,2). Thus, timely detection of an inhalational anthrax outbreak is critical. Rapid detection is also important for disease outbreaks that result from other bioterrorism agents and from emerging infectious diseases, such as severe acute respiratory syndrome or avian influenza (3). To detect an epidemic such as inhalational anthrax, which is nonendemic and results in severe symptoms, public health authorities have relied traditionally on identification and rapid reporting of the sentinel clinical case. However, because the perceived likelihood of a bioterrorism attack has increased, public health authorities have sought novel approaches for rapid outbreak detection. One approach that has received considerable economic investment over the past 5 years is syndromic surveillance. This approach follows prediagnostic data sources in an attempt to detect an increase in the prevalence of nonspecific symptoms. For example, the BioSense system (4), developed by the Centers for Disease Control and Prevention (CDC) at a cost of >$75 million (5), follows records of outpatient visits, pharmaceutical prescriptions, and laboratory orders in an attempt to detect disease outbreaks rapidly. Hundreds of similar systems are maintained or are under development by various groups around the world (6). Other examples include systems operated by the Department of Homeland Security (5) and academic centers in partnership with state or county public health departments (7–9). In addition to supporting outbreak detection, these syndromic surveillance systems provide situational awareness for public health authorities and may serve other purposes. Nevertheless, a major justification for these systems is outbreak detection. Despite substantial investment in syndromic surveillance and calls for further research from groups such as the Institute of Medicine (3), little evidence exists to suggest how syndromic surveillance will perform relative to clinical case finding for detection of an inhalational anthrax outbreak (10). The reason for this lack of evidence is that data from real outbreaks are not available to evaluate the performance of syndromic surveillance alone or in comparison to clinical case finding. Moreover, even if data were available from a large-scale outbreak, those data would allow only an evaluation of performance in 1 specific setting. CDC recently endorsed the use of simulated outbreaks to address the dearth of outbreak data (11), but existing simulation studies have not compared detection through clinical case finding with syndromic surveillance (12–14). Our aim was to develop a model for simulating use of healthcare services after a large-scale exposure to aerosol anthrax spores and then to use this model to estimate the detection benefit of syndromic surveillance compared with clinical case finding. Methods Study Design We developed a model to simulate the dispersion of released anthrax spores; the infection of exposed persons; the progression of disease in infected persons; and symptomatic persons' use of the healthcare system, including blood culture testing in clinical settings. Using the simulation model, we generated outbreak signals and time until the first clinical diagnosis for 3 amounts of spores released. To incorporate into the model the uncertainty in parameter values, we used a Latin hypercube sampling design, which allows many parameter values to vary simultaneously (15). The 3,000 simulated signals generated with this sampling strategy were superimposed in turn onto baseline administrative records of ambulatory healthcare visits in the Norfolk, Virginia, area. These records are generated daily and similar types of records are used widely for syndromic surveillance (4,7,9). We assessed the usefulness of syndromic surveillance by modeling the healthcare system use that would occur after an anthrax attack and superimposing this use onto actual administrative data over 1-year period. Finally, we assessed, over a range of specificity, the sensitivity and timeliness of syndromic surveillance and the detection benefit of syndromic surveillance compared with clinical case finding for each simulated outbreak. We summarize our methods in the remainder of this section and refer readers to the online Technical Appendix for additional details. Simulation Model The simulation model builds on our previous work (16–18) and is composed of 4 components: dispersion, infection, disease, and healthcare system use. The dispersion model simulates the number of anthrax spores a person would inhale at locations throughout the region after release of aerosolized spores. We used the Hazard Prediction and Assessment Capability (HPAC) software developed by the Defense Threat Reduction Agency to simulate the dispersion of spores (19). The HPAC model accounts for factors such as atmospheric conditions and terrain. We simulated a point release of 3 amounts of anthrax spores: 1 kg, 0.1 kg, and 0.01 kg (Figure 1A). The infection model simulates the number of persons infected, according to residential address and dispersion of spores (Figure 1B). The probability of infection given exposure to an amount of spores was modeled by using a probit regression model. The disease model uses a semi-Markov process to simulate the progression of infected persons through 3 discrete states of disease. Each infected person began in the incubation state and then progressed through the prodromal state and the fulminant state. The time in each state was sampled from a log normal distribution. The healthcare use model uses a semi-Markov process to simulate the probability and timing of a symptomatic person seeking care and submission of blood for culture and culture results when care is sought. For persons in the prodromal or fulminant state of disease who sought care, the instantaneous probability of seeking care increased linearly over the duration of the state. For patients whose blood samples were cultured, the testing process was modeled as the transition through 2 discrete states: growth and isolation. The time spent in each of these states was modeled by using an exponential distribution. Data for Simulation Model The infection model used an infection function corresponding to the data reported by Glassman (20). This is a probit model with a 50% lethal dose (LD50) of 8,600 spores and a slope of 0.67. Uncertainty exists about the values for many of the parameters in the disease and healthcare use models. To incorporate this uncertainty into our estimates, we used a Latin hypercube sampling approach to sample parameter values for random variables in our simulation model (15). This approach requires specifying equal probability bins for parameter values. We specified 3 bins for each parameter value, a narrow bin around the most likely estimate, and wider bins on either side of the estimate. Table 1 shows the bins we used for each parameter value, the probability distribution that each value parameterizes, and the sources that we used to define the bins. We used previous work modeling anthrax for the distribution of time periods in each disease state (2,21,22). For the probability of seeking care while in the prodromal disease state, cross-sectional surveys indicate that 14%–30% of persons visit a physician at some point during an episode of upper respiratory tract illness (23,24). For the fulminant disease state, we estimated the probability of seeking care before death as 90%–95%, given the severity of the symptoms in that state. After a person made a healthcare visit, we simulated the syndrome assigned to the person by using probabilities that reflect the distribution of clinical presentations for inhalational anthrax reported in the literature (25,26). Because we considered only respiratory syndromes for surveillance, we varied directly only the probability of being assigned a respiratory syndrome to persons in the prodromal disease state. For visits from persons in either symptomatic disease state, the estimate of sensitivity from published studies of blood culture testing was 0.8–0.9 (27). For a visit in the prodromal state, we estimated the probability of a physician ordering a blood culture as 0.01–0.015 on the basis of data from the National Ambulatory Medical Care Survey (28). For a visit in the fulminant state of disease, we estimated the probability of a blood culture test as 0.9–0.95. After gram-positive rods grew in the blood culture, we estimated the probability of isolating the organism to be 0.8–0.9 (29). We modeled the time until growth and isolation as exponential (25,30). Baseline Data and Release Scenarios We used records of ambulatory visits in the Norfolk, Virginia, region acquired from the TRICARE health maintenance organization as a baseline onto which we superimposed simulated outbreak records. The data covered the period 2001–2003, and the simulation region included 17 clinical facilities within an ≈160-km × 200-km area that encompasses 158 ZIP codes from 2 states. Over the 3 years of available data, 427,634 persons made >5 million visits. We classified the records into syndromes by using the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) to syndrome mapping defined by the ESSENCE system (7) and used only 351,749 records for which persons were classified as having a respiratory syndrome. The Human Subjects Panel at the Stanford School of Medicine approved the use of these data for this study. We examined 3 scenarios defined by the amount of spores released: 1 kg, 0.1 kg, and 0.01 kg. For each scenario, we performed 1,000 simulations. Outbreak Detection The time to outbreak detection through clinical case finding for a simulated outbreak was calculated for each simulated outbreak as the time between exposure to spores and the first positive blood culture. To calculate time to outbreak detection through syndromic surveillance, we superimposed the simulated records for respiratory syndrome visits onto the authentic baseline data, beginning on a randomly selected date in 2003, and then applied the outbreak detection algorithm to the combined baseline and simulated data. The outbreak detection algorithm used a time-series model (31) to generate daily 1-step-ahead forecasts for the total number of respiratory syndrome visits (13) and then applied a cumulative sum (32) to the forecast residual. To vary the specificity of the detection algorithm, we varied the decision threshold of the cumulative sum. Evaluation Metrics To evaluate outbreak detection through syndromic surveillance, we calculated sensitivity, specificity, and timeliness at a range of decision thresholds. Timeliness is the duration between the release of anthrax spores and the first report of an outbreak. We also computed the detection benefit of syndromic surveillance relative to clinical case finding, and the proportion of runs with a detection benefit >0. Detection benefit is the potential time saved in detection from using syndromic surveillance compared with clinical case finding. The benefit is calculated as the difference in the timeliness between syndromic surveillance and clinical case finding in those simulations in which detection occurred first by syndromic surveillance. When an outbreak was not detected by syndromic surveillance, the detection benefit was 0. For a given release scenario, each of the 1,000 simulations integrated both randomness in the component model outputs as well as uncertainty in component model parameters. Each of the 1,000 simulations is a sample from the integrated distribution of possible outcomes. To indicate the spread of the integrated uncertainty distribution, we calculated the upper and lower deciles from the 1,000 simulations. For plots, we calculated 95% confidence intervals, which reflect finiteness of the simulation. Results Detection Performance of Clinical Case Finding Because all outbreaks were ultimately detected by clinical case finding through routine blood culture, the sensitivity of this approach was 1.0 for the scenarios considered. Clinical case finding detected outbreaks from an average of 3.7 days to 4.1 days after release, with larger amounts of spores detected before smaller amounts . Results from analyses of additional release scenarios (data not shown) suggested that the influence of amount released on time to detection was mediated, in part, through the number infected. Mean timeliness across the scenarios examined was associated with the mean number infected (Pearson's r -0.94, 95% confidence interval -0.98 to -0.79), and an increase of 10,000 infected persons resulted in a decrease in the time until detection of ≈4 hours. Detection Performance of Syndromic Surveillance The sensitivity and timeliness of syndromic surveillance were influenced by the release amount and by specificity. Table 3 shows this relationship over the release scenarios examined and 2 levels of specificity. At a specificity of 0.90, a 1-kg release was detected in 100% of our simulations (sensitivity 1.0) at a mean detection time of 3.1 days. For a release that was much smaller, 0.01 kg, sensitivity was 0.94, and the mean detection time increased to 3.6 days. Although the sensitivity of syndromic surveillance was high when we set specificity to 0.90, this specificity resulted in a false alarm (false-positive detection) ≈1 every 10 days. By increasing specificity to 0.975, we reduced the false alarm rate to ≈1 every 40 days . However, with increased specificity, the sensitivity of syndromic surveillance decreased (from 0.98 to 0.82 depending on the size of the release) and the mean time until detection lengthened to 4.3 days for a 1-kg release and to 5.1 days for a 0.01-kg release . Results from analyses of additional release scenarios (data not shown) indicated that the trends in sensitivity and timeliness across release amount were mediated to some extent by the number infected. Sensitivity was a nonlinear function of the number of persons infected, with sensitivity increasing more quickly when fewer persons were infected. At a specificity of 0.975, an increase of 10,000 infected persons resulted in a decrease in time to detection of ≈6 hours. Detection Benefit of Syndromic Surveillance Compared with Clinical Case Finding The detection benefit of syndromic surveillance compared with clinical case finding was influenced by specificity and the release amount. Table 3 shows this relationship for the release amounts examined and 2 levels of specificity. When the specificity was 0.9, syndromic surveillance detected from 51% to 59% of outbreaks before clinical case finding, and the mean detection benefit was 1.0–1.1 days, but this specificity resulted in a false alarm every 10 days. At a specificity of 0.975, which reduced false alarms to 1 every 40 days, syndromic surveillance detected 19%–28% of outbreaks before clinical case finding and the mean detection benefit was 0.32–0.33 days, or ≈8 hours. Figure 2 shows that for the 0.01-kg and 1-kg release scenarios (results for the 0.1-kg release are similar, but are not shown), the proportion of outbreaks detected first by syndromic surveillance and the mean detection benefit of surveillance each increased as specificity decreased. Figure 2 also shows that the release amount had a strong effect on the proportion of outbreaks detected first by syndromic surveillance but that it did not have a strong effect on the mean detection benefit. At a set specificity, syndromic surveillance tended to detect a higher proportion of outbreaks before clinical case finding with increasing release amount. The mean detection benefit, in contrast, tended to decrease when the amount of spores released increased. This decrease in average detection benefit occurred because even though syndromic surveillance detected more outbreaks before clinical case finding as the release amount increased, the detection benefit for the additional outbreaks was small, and the average detection benefit thus decreased. Discussion When we compared the performance of clinical case finding with that of syndromic surveillance for detecting an inhalational anthrax outbreak, we found that clinical case finding detected outbreaks on average 3.7–4.6 days after release of spores. The ability of syndromic surveillance to detect an outbreak before clinical case finding was influenced by both specificity and release size, with specificity being the predominant factor. Our results suggest that syndromic surveillance could detect an inhalational anthrax outbreak before clinical case finding. However, we regularly observed a detection benefit only when syndromic surveillance operated at a specificity in the range of 0.9, which corresponds to 1 false alarm every 10 days. When operating at this relatively low specificity with a concomitant high sensitivity, syndromic surveillance detected outbreaks, on average, 1 day before clinical case finding did. One of the most useful findings of our study was the tradeoff between sensitivity and specificity of syndromic surveillance. To reduce the false alarm rate, specificity must be high. However, as specificity increased in our study, the sensitivity of syndromic surveillance decreased, and the proportion of outbreaks that was detected first by syndromic surveillance decreased more substantially. If the response to a result from syndromic surveillance is resource intensive and includes follow-up investigations in multiple healthcare settings, then a false alarm rate of 1 every 10 days may be too high for such a system to be useful. Alternatively, if public health personnel can rule out false-positive results with minimal investment, then a higher rate of false alarms may be acceptable. The detection benefit of syndromic surveillance might be an important lead, depending on the action triggered by a surveillance alarm. Because many clinical and public health departments have defined protocols for actions after clinical confirmation of an inhalational anthrax case (33), the action after detection of a clinical case is fairly well defined in many jurisdictions. In contrast, the appropriate action after a result from syndromic surveillance system is not well-defined (34). For example, some public health departments routinely wait 1 day for a second alarm before taking action (35). This strategy could eliminate the potential detection benefit of syndromic surveillance. Another concern is the relatively low specificity at which syndromic surveillance must operate to consistently result in a detection benefit. A system producing this many false alarms may result in excessive costs, and users may minimize the importance of these results. To be useful, however, syndromic surveillance does not necessarily have to detect all outbreaks, or even most outbreaks, before a clinician detects the first case. The additional lead in detection offered by syndromic surveillance in some outbreaks may result in enough benefit to support the use of syndromic surveillance. Syndromic surveillance may also be useful for applications other than detecting an outbreak caused by bioterrorism; e.g., for detecting other types of disease outbreaks (36), for ruling out the existence of an outbreak, or for evaluating the effect of a public health intervention. Assessment of the question of the utility of syndromic surveillance in general would require consideration of a broader range of costs and benefits than we included in our study. Our methods are an advance over those used in previous studies because we were able to examine rigorously, within a single modeling framework, the ability of clinical case finding and syndromic surveillance to detect anthrax outbreaks. The nature of our model allowed us to vary some outbreak characteristics directly (e.g., release amount) and to incorporate the uncertainty in parameter values into our final estimates of detection performance and detection benefit. Although our sampling approach did allow us to vary many parameter values simultaneously, it did not clarify how the results vary in relation to changes in the value of a single parameter. Our estimate of detection performance through syndromic surveillance is comparable to estimates observed through studies that used simulation models (12,37), but those studies did not allow direct comparison of detection through syndromic surveillance with detection through clinical case finding. Our estimate of the time until detection through clinical case finding is longer than the estimate used by the authors of a study aimed at modeling response strategies to an anthrax outbreak (2), but those authors did not provide a clear rationale for the value they chose. An initial presumptive diagnosis may occur earlier than the first positive blood culture result (e.g., through clinical symptoms and chest radiographs), but a decision for large-scale intervention would likely not be made until at least after the first definitive diagnosis was made. In our study, we considered 1 approach to syndromic surveillance for an outbreak resulting from 1 type of organism, and we considered clinical case finding through 1 type of routinely applied diagnostic test. There are many different approaches to syndromic surveillance; e.g., different types of data and different detection algorithms. Although different approaches to surveillance might produce different results, the choice of the infectious organism is likely to have a greater effect on results. Anthrax is relatively unique among bioterrorism agents in that a routinely used diagnostic test (i.e., blood culture) will identify the organism definitively. The benefit of syndromic surveillance relative to clinical case finding may therefore be greater for outbreaks caused by other organisms, and an anthrax outbreak may be a worst-case scenario for syndromic surveillance. Syndromic surveillance detected an inhalation anthrax outbreak before the first clinical case was diagnosed in as many as half of simulated outbreaks. However, the potential detection benefit of syndromic surveillance compared with clinical case finding depended critically on the specificity and sensitivity at which a surveillance system operated and on the size of the outbreak. When syndromic surveillance was sufficiently sensitive to detect a substantial proportion of outbreaks, it generated frequent false alarms. Public health authorities should be aware that the potential detection benefit of syndromic surveillance compared with clinical case finding is influenced strongly by the specificity at which a surveillance system operates. To help detect outbreaks more rapidly, future research should examine the cost-effectiveness of syndromic surveillance and explore approaches to linking syndromic surveillance and clinical case finding more closely. Acknowledgments We thank Julie Pavlin for her help in obtaining the data used in this study and for comments on earlier versions of the simulation model. Part of this work was performed while David L. Buckeridge was a Department of Veterans Affairs postdoctoral informatics fellow. The research of Dr Buckeridge is supported by a Canada research chair in public health informatics. Dr Buckeridge is assistant professor in the Department of Epidemiology, Biostatistics, and Occupational Health at McGill University and a medical consultant with the Montreal Department of Public Health and the Quebec Institute of Public Health. His research interests include public health informatics with a particular focus on the informatics of public health surveillance. References Kaufmann AF, Meltzer MI, Schmid GP. The economic impact of a bioterrorist attack: are prevention and postattack intervention programs justifiable Emerg Infect Dis. 1997;3:83–94. Wein LM, Craft DL, Kaplan EH. Emergency response to an anthrax attack. Proc Natl Acad Sci U S A. 2003;100:4346–51. Smolinski M, Hamburg ML. Lederberg J, editors. Microbial threats to health: emergence, detection, and response. Washington: National Academy Press; 2003. Loonsk JW. BioSense: a national initiative for early detection and quantification of public health emergencies. MMWR Morb Mortal Wkly Rep. 2004;53(Suppl):53–5. United States Government Accountability Office. Information technology. Federal agencies face challenges in implementing initiatives to improve public health infrastructure. Volume GAO-05–308. Washington: The Office; 2005. Buehler JW, Berkelman RL, Hartley DM, Peters CJ. Syndromic surveillance and bioterrorism-related epidemics. Emerg Infect Dis. 2003;9:1197–204. Lombardo J, Burkom H, Elbert E, Magruder S, Lewis SH, Loschen W, et al. A systems overview of the electronic surveillance system for the early notification of community-based epidemics (ESSENCE II). J Urban Health. 2003;80(Suppl 1):i32–42. Wagner MM, Robinson JM, Tsui F-C, Espino JU, Hogan WR. Design of a national retail data monitor for public health surveillance. J Am Med Inform Assoc. 2003;10:409–18. Yih WK, Caldwell B, Harmon R, Kleinman K, Lazarus R, Nelson A, et al. National Bioterrorism Syndromic Surveillance Demonstration Program. MMWR Morb Mortal Wkly Rep. 2004;53(Suppl):43–9. Bravata DM, McDonald KM, Smith WM, Rydzak C, Szeto H, Buckeridge DL, et al. Systematic review: surveillance systems for early detection of bioterrorism-related diseases. Ann Intern Med. 2004;140:910–22. Buehler JW, Hopkins RS, Overhage JM, Sosin DM, Tong V. Framework for evaluating public health surveillance systems for early detection of outbreaks: recommendations from the CDC Working Group. MMWR Recomm Rep. 2004;53:1–11. Nordin JD, Goodman M, Kuldorff M, Ritzwoller DP, Abrams AM, Kleinman K, et al. Simulated anthrax attacks and syndromic surveillance. Emerg Infect Dis. 2005;11:1394–8. Reis BY, Pagano M, Mandl KD. Using temporal context to improve biosurveillance. Proc Natl Acad Sci U S A. 2003;100:1961–5. Stoto M, Schonlau M, Mariano L. Syndromic surveillance: is it worth the effort Chance. 2004;17:19–24. McKay M, Beckman R, Conover W. A comparison of three methods for selecting values of input variables in the analysis of output from a computer code. Technometrics. 1979;21:239–45. Buckeridge DL. A method for evaluating outbreak detection in public health surveillance systems that use adminstrative data. [Doctoral dissertation]. Stanford (CA): Stanford University; 2005. Buckeridge DL, Burkom H, Moore A, Pavlin J, Cutchis P, Hogan W. Evaluation of syndromic surveillance systems: design of an epidemic simulation model. MMWR Morb Mortal Wkly Rep. 2004;53(Suppl):137–43. Buckeridge DL, Switzer P, Owens D, Siegrist D, Pavlin J, Musen M. An evaluation model for syndromic surveillance: assessing the performance of a temporal algorithm. MMWR Morb Mortal Wkly Rep. 2005;54(Suppl):109–15. Defense Threat Reduction Agency. The HPAC user's guide. Hazard prediction and assessment capability, version 4.0. Fort Belvior (VA): The Agency; 2001. Glassman HN. Industrial inhalation anthrax. Discussion. Bacteriol Rev. 1965;30:657–9. PMID 16350199 Brookmeyer R, Blades N, Hugh-Jones M, Henderson DA. The statistical analysis of truncated data: application to the Sverdlovsk anthrax outbreak. Biostatistics. 2001;2:233–47. Webb GF, Blaser MJ. Mailborne transmission of anthrax: modeling and implications. Proc Natl Acad Sci U S A. 2002;99:7027–32. McIsaac WJ, Levine N, Goel V. Visits by adults to family physicians for the common cold. J Fam Pract. 1998;47:366–9. Metzger KB, Hajat A, Crawford M, Mostashari F. How many illnesses does one emergency department visit represent Using a population-based telephone survey to estimate the syndromic multiplier. MMWR Morb Mortal Wkly Rep. 2004;53(Suppl):106–11. Inglesby TV, O'Toole T, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, et al. Anthrax as a biological weapon, 2002: updated recommendations for management. JAMA. 2002;287:2236–52. Jernigan JA, Stephens DS, Ashford DA, Omenaca C, Topiel MS, Galbraith M, et al. Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States. Emerg Infect Dis. 2001;7:933–44. Reimer LG, Wilson ML, Weinstein MP. Update on detection of bacteremia and fungemia. Clin Microbiol Rev. 1997;10:444–65. National Center for Health Statistics. Public use data tapes: National Ambulatory Medical Care Survey 2003. Hyattsville (MD): The Center; 2003. Begier EM, Barrett N, Mshar P, Johnson D, Hadler J; Connecticut Bioterrorism Field Epidemiology Response Team. Gram-positive rod surveillance for early anthrax detection. Emerg Infect Dis. 2005;11:1483–6. Beekmann SE, Diekema DJ, Chapin KC, Doern GV. Effects of rapid detection of bloodstream infections on length of hospitalization and hospital charges. J Clin Microbiol. 2003;41:3119–25. Box GEP, Jenkins GM. Time series analysis, forecasting and control. Englewood Cliffs (NJ): Prentice Hall; 1976. Page E. Continuous inspection schemes. Biometrika. 1954;41:100–15. Tengelsen L, Hudson R, Barnes S, Hahn C. Coordinated response to reports of possible anthrax contamination, Idaho, 2001. Emerg Infect Dis. 2002;8:1093–5. Pavlin JA. Investigation of disease outbreaks detected by "syndromic" surveillance systems. J Urban Health. 2003;80(Suppl 1):i107–14. Heffernan R, Mostashari F, Das D, Karpati A, Kuldorff M, Weiss D. Syndromic surveillance in public health practice, New York City. Emerg Infect Dis. 2004;10:858–64. Marx MA, Rodriguez CV, Greenko J, Das D, Heffernan R, Karpati AM, et al. Diarrheal illness detected through syndromic surveillance after a massive power outage: New York City, August 2003. Am J Public Health. 2006;96:547–53. Miller B, Kassenborg H, Dunsmuir W, Griffith J, Hadidi M, Nordin JD, et al. Syndromic surveillance for influenzalike illness in ambulatory care network. Emerg Infect Dis. 2004;10:1806–11....查看详细 (31694字节)
☉ 11342766:Influenza, Campylobacter and Mycoplasma Infections, and Hospital Admissions for Guillain-Barre Syndrome, England
London School of Hygiene and Tropical Medicine, London, United Kingdom Health Protection Agency, London, United Kingdom University of Manchester, Manchester, United Kingdom Abstract Guillain-Barre syndrome (GBS) is the most common cause of acute flaccid paralysis in polio-free regions. Considerable evidence links Campylobacter infection with GBS, but evidence that implicates other pathogens as triggers remains scarce. We conducted a time-series analysis to investigate short-term correlations between weekly laboratory-confirmed reports of putative triggering pathogens and weekly hospitalizations for GBS in England from 1993 through 2002. We found a positive association between the numbers of reports of laboratory-confirmed influenza A in any given week and GBS hospitalizations in the same week. Different pathogens may trigger GBS in persons of different ages; among those 35 years, positive associations were with influenza. Further studies should estimate the relative contribution of different pathogens to GBS incidence, overall and by age group, and determine whether influenza is a real trigger for GBS or a marker for influenza vaccination. Guillain-Barre syndrome (GBS) is the most common cause of acute flaccid paralysis in polio-free regions. Estimated incidence in high-income countries is 0.4–4.0 cases per 100,000 population (1). Campylobacter jejuni is the most commonly identified infectious trigger for GBS. Several studies have demonstrated evidence of recent C. jejuni infection in a higher proportion of GBS case-patients than in controls (2–10). Other pathogens, including cytomegalovirus (7), Epstein-Barr virus (7), Haemophilus influenzae (11–14), and Mycoplasma pneumoniae (7,15,16), have been suggested as possible GBS triggers, as was influenza vaccination in the United States during 1976–1977 (17). However, epidemiologic evidence that implicates these latter agents remains scarce. We conducted a time-series analysis to investigate temporal associations between weekly variations in reports of microbiologically confirmed infections and hospital admissions for GBS. Methods Reports of Microbiologically Confirmed Infections Positive microbiologic diagnoses ascertained through voluntary laboratory reporting in England and Wales are recorded in the national infections database (LabBase2) (18). We obtained weekly reports of infections suspected of causing GBS, namely, Campylobacter spp., cytomegalovirus, Epstein-Barr virus, Haemophilus influenzae (B and non-B), Mycoplasma pneumoniae, and influenza (A, B, and all influenza) from 1993 through 2002. Influenza vaccination figures are available only quarterly and do not provide sufficient temporal resolution for this analysis. We used the specimen date for all analyses because onset dates were rarely available. For Campylobacter, the median delay between patients' onset date and the specimen date was 4 days (interquartile range 3–7 days); for 90% of cases, the delay was 2 months, assuming that GBS risk is increased for 65 years, according to the age distribution of GBS patient admissions . Age group–specific models were fit similarly to those for all ages. All lags at which positive associations were found are presented. However, because of the large number of statistical tests performed (13 lags per pathogen per age group), a positive association at a given lag was considered potentially relevant only if it occurred within a cluster. A cluster was defined as 2 or more consecutive lags, each associated with the outcome at the 0.05 significance level. This approach reduces the probability of observing chance associations due to multiple testing. Within clusters, lags significant at the 0.01 level were considered important. The adjusted coefficient from these models was used to calculate the expected increase in GBS admissions per 10% increase in the range of laboratory reports at a given lag. All statistical analyses were performed in Stata 8.0 (Stata Corp., College Station, TX, USA). Results In the 10-year study period, 11,019 primary admissions for GBS occurred: 2,929 (26.6%) patients were 65 years. Summary statistics for the weekly number of GBS admissions and laboratory reports for the different pathogens are found in Table 1. Different pathogens are associated with GBS admission in different age groups. In those 65 years, associations were found between the number of GBS admissions and the number of all influenza and influenza A reports in the current week and 1 week before hospitalization. The results were robust to varying degrees of seasonal adjustment; we repeated the analysis and adjusted for seasonal wavelengths of up to 4 months (3 harmonics) and 1 month (12 harmonics) and used indicator variables for month as well, all with similar results. Table 5 shows those lags that consistently appeared in clusters at all levels of seasonal adjustment. Results for influenza and M. pneumoniae were not sensitive to the degree of seasonal adjustment. For Campylobacter, clusters of lags were seen with all Fourier models, but not with models that used month indicators. Discussion We found associations between the weekly number of laboratory reports of various pathogens and incidence of GBS hospitalizations. Different organisms may be responsible for triggering GBS in different age groups. In particular, Campylobacter and M. pneumoniae appear to be associated with GBS in those 35 years. Differences in the pathogens responsible for triggering GBS in different age groups have not previously been reported. No clusters of significant lags were found for cytomegalovirus, Epstein-Barr virus, and H. influenzae infections. This could be due to low statistical power (on average, 65 years (30). For elderly persons at high risk, uptake increased from 40% to 65% from 1993 through 2002 (31). These data support the hypothesis that persons in older age groups have a greater vaccine-induced risk of GBS, although a true association with the disease of influenza is still possible. Primary care–based studies investigating the influenza and influenza vaccination status of GBS patients could help resolve this issue. Acknowledgments We gratefully acknowledge Kate Byram and Susan Alpay for providing the data on GBS hospitalizations and Sallyanne Meakins for providing the data on laboratory reports. We thank Shakoor Hajat for helpful comments on the manuscript. This study was funded by the Department of Epidemiology and Population Health, Health Protection Agency Centre for Infections, and the Infectious Disease Epidemiology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine. Dr Tam is an epidemiologist at the Department of Epidemiology and Population Health, London School of Hygiene and Tropical Medicine. His main research interests include the epidemiology of infectious intestinal disease, long-term sequelae of diarrheal infections, and epidemiologic methods. References Allos BM. Association between Campylobacter infection and Guillain-Barre syndrome. J Infect Dis. 1997;176(Suppl 2):S125–8. Rees JH, Soudain SE, Gregson NA, Hughes RA. Campylobacter jejuni infection and Guillain-Barre syndrome. N Engl J Med. 1995;333:1374–9. Enders U, Karch H, Toyka KV, Michels M, Zielasek J, Pette M, et al. The spectrum of immune responses to Campylobacter jejuni and glycoconjugates in Guillain-Barre syndrome and in other neuroimmunological disorders. Ann Neurol. 1993;34:136–44. Guarino M, Casmiro M, D'Alessandro R. Campylobacter jejuni infection and Guillain-Barre syndrome: a case-control study. Emilia-Romagna Study Group on Clinical and Epidemiological Problems in Neurology. Neuroepidemiology. 1998;17:296–302. Hao Q, Saida T, Kuroki S, Nishimura M, Nukina M, Obayashi H, et al. Antibodies to gangliosides and galactocerebroside in patients with Guillain-Barre syndrome with preceding Campylobacter jejuni and other identified infections. J Neuroimmunol. 1998;81:116–26. Ho TW, Mishu B, Li CY, Gao CY, Cornblath DR, Griffin JW, et al. Guillain-Barre syndrome in northern China. Relationship to Campylobacter jejuni infection and anti-glycolipid antibodies. Brain. 1995;118:597–605. Jacobs BC, Rothbarth PH, van der Meche FG, Herbrink P, Schmitz PI, de Klerk MA, et al. The spectrum of antecedent infections in Guillain-Barre syndrome: a case-control study. Neurology. 1998;51:1110–5. Kaldor J, Speed BR. Guillain-Barre syndrome and Campylobacter jejuni: a serological study. Br Med J (Clin Res Ed). 1984;288:1867–70. Koga M, Yuki N, Takahashi M, Saito K, Hirata K. Close association of IgA anti-ganglioside antibodies with antecedent Campylobacter jejuni infection in Guillain-Barre and Fisher's syndromes. J Neuroimmunol. 1998;81:138–43. Kuroki S, Haruta T, Yoshioka M, Kobayashi Y, Nukina M, Nakanishi H. Guillain-Barre syndrome associated with Campylobacter infection. Pediatr Infect Dis J. 1991;10:149–51. Ju YY, Womersley H, Pritchard J, Gray I, Hughes RA, Gregson NA. Haemophilus influenzae as a possible cause of Guillain-Barre syndrome. J Neuroimmunol. 2004;149:160–6. Koga M, Gilbert M, Li J, Koike S, Takahashi M, Furukawa K, et al. Antecedent infections in Fisher syndrome: a common pathogenesis of molecular mimicry. Neurology. 2005;64:1605–11. Mori M, Kuwabara S, Miyake M, Dezawa M, Adachi-Usami E, Kuroki H, et al. Haemophilus influenzae has a GM1 ganglioside-like structure and elicits Guillain-Barre syndrome. Neurology. 1999;52:1282–4. Mori M, Kuwabara S, Miyake M, Noda M, Kuroki H, Kanno H, et al. Haemophilus influenzae infection and Guillain-Barre syndrome. Brain. 2000;123:2171–8. Ang CW, Tio-Gillen AP, Groen J, Herbrink P, Jacobs BC, Van Koningsveld R, et al. Cross-reactive anti-galactocerebroside antibodies and Mycoplasma pneumoniae infections in Guillain-Barre syndrome. J Neuroimmunol. 2002;130:179–83. Ginestal RC, Plaza JF, Callejo JM, Rodriguez-Espinosa N, Fernandez-Ruiz LC, Masjuan J. Bilateral optic neuritis and Guillain-Barre syndrome following an acute Mycoplasma pneumoniae infection. J Neurol. 2004;251:767–8. Safranek TJ, Lawrence DN, Kurland LT, Culver DH, Wiederholt WC, Hayner NS, et al. Reassessment of the association between Guillain-Barre syndrome and receipt of swine influenza vaccine in 1976–1977: results of a two-state study. Expert Neurology Group. Am J Epidemiol. 1991;133:940–51. Wall PG, de Louvois J, Gilbert RJ, Rowe B. Food poisoning: notifications, laboratory reports, and outbreaks–where do the statistics come from and what do they mean Commun Dis Rep CDR Rev. 1996;6:R93–100. Gillespie IA, O'Brien SJ, Frost JA, Adak GK, Horby P, Swan AV, et al. A case-case comparison of Campylobacter coli and Campylobacter jejuni infection: a tool for generating hypotheses. Emerg Infect Dis. 2002;8:937–42. Department of Health (England). Hospital episode statistics [cited 2005 Jan 12]. Available from http://www.hesonline.nhs.uk Schwartz J, Spix C, Touloumi G, Bacharova L, Barumamdzadeh T, le Tertre A, et al. Methodological issues in studies of air pollution and daily counts of deaths or hospital admissions. J Epidemiol Community Health. 1996;50(Suppl 1):S3–11. Schwartz J, Levin R, Goldstein R. Drinking water turbidity and gastrointestinal illness in the elderly of Philadelphia. J Epidemiol Community Health. 2000;54:45–51. Schwartz J, Levin R, Hodge K. Drinking water turbidity and pediatric hospital use for gastrointestinal illness in Philadelphia. Epidemiology. 1997;8:615–20. Vellinga A, Van Loock F. The dioxin crisis as experiment to determine poultry-related campylobacter enteritis. Emerg Infect Dis. 2002;8:19–22. Chatfield C. The analysis of time series: an introduction, 5th ed. London: Chapman and Hall; 1996. Hastie TJ, Tibshirani RJ. Generalized additive models. New York: Chapman and Hall; 1990. Goddard NL, Joseph CA, Zambon M, Nunn M, Fleming D, Watson JM. Influenza surveillance in the United Kingdom: October 2000 to May 2001. [serial online]. Commun Dis Rep CDR. 2001;2005:S1–7. Schonberger LB, Hurwitz ES, Katona P, Holman RC, Bregman DJ. Guillain-Barre syndrome: its epidemiology and associations with influenza vaccination. Ann Neurol. 1981;9(Suppl):31–8. Hurwitz ES, Schonberger LB, Nelson DB, Holman RC. Guillain-Barre syndrome and the 1978–1979 influenza vaccine. N Engl J Med. 1981;304:1557–61. Lasky T, Terracciano GJ, Magder L, Koski CK, Ballesteros M, Nash D, et al. The Guillain-Barre syndrome and the 1992–1993 and 1993–1994 influenza vaccines. N Engl J Med. 1998;339:1797–802. Joseph C, Goddard N, Gelb D. Influenza vaccine uptake and distribution in England and Wales using data from the General Practice Research Database, 1989/90–2003/04. J Public Health (Oxf). 2005;27:371–7....查看详细 (28894字节)

☉ 11342767:Nonpharmaceutical Influenza Mitigation Strategies, US Communities, 1918–1920 Pandemic1
The University of Michigan Medical School, Ann Arbor, Michigan, USA, The University of Michigan School of Public Health, Ann Arbor, Michigan, USA US Department of Defense, Fort Belvoir, Virginia, USA Abstract We studied nonpharmaceutical interventions used to mitigate the second, and most deadly, wave of the 1918–1920 influenza pandemic in the United States. We conclude that several small communities implemented potentially successful attempts at preventing the introduction of influenza. The 1918–1920 influenza pandemic was the deadliest pandemic in human history (1–6). We undertook a historical evaluation of nonpharmaceutical interventions (NPIs) during that pandemic (7), with an emphasis on American communities during the second wave (September–December 1918). The full report and a digital archive of primary sources for this study can be accessed online (available from http://www.med.umich.edu/medschool/chm/influenza). The Study We selected 6 US communities that reported relatively few, if any, cases of influenza and no more than 1 influenza-related death while NPIs were enforced during the second wave of the 1918 pandemic: San Francisco Naval Training Station, Yerba Buena Island, California; Gunnison, Colorado; Princeton University, Princeton, New Jersey; Western Pennsylvania Institution for the Blind, Pittsburgh, Pennsylvania; Trudeau Tuberculosis Sanatorium, Saranac Lake, New York; and Fletcher, Vermont (Table). We also studied the college community of Bryn Mawr College, Bryn Mawr, Pennsylvania, which took several intensive NPI measures and experienced no deaths during the second wave but did encounter a high case rate (25% of its student body). We identified these sites first by consulting Jordan's 1927 text, Epidemic Influenza (1). We then verified and modified this list by reviewing 240 federal, 92 state (from 40 states), and 25 special local reports and documents. We conducted in situ archival research at 34 locations and examined >1,400 newspaper and contemporary medical and scientific journal articles for the 1918–1920 period. The communities we identified were diverse and had unique characteristics. Fletcher, Vermont (population 737), was simply too small to suggest that its success resulted from anything more than remote location, good fortune, or the ways in which the virus skipped some communities altogether for unknown reasons (8–10). The Trudeau Tuberculosis Sanitarium (9) and the Western Pennsylvania Institution for the Blind (10) were already de facto quarantine islands because of the era's prevailing views toward confinement of the contagious and the disabled. Princeton University provided a good example of how a social institution with some measure of control over its population might implement NPIs to protect itself (11). The US Naval Base at Yerba Buena Island in San Francisco Bay (12) and the mining town of Gunnison, Colorado (13), also offer potential lessons for contemporary pandemic influenza preparedness planning. Under the direction of public health officers, the still-healthy island and mountain town essentially cut off all contact with the outside world to shield themselves from the incursion of influenza. The 2 sites saw almost no cases of infection and thus experienced no deaths, for 2 and 4 months, respectively. Most important, these communities enacted a policy we have termed protective sequestration, or the measures taken by the authorities to protect a defined and still-healthy population from infection before it reaches that population. These measures include the following: 1) prohibitions on members of the community from leaving the site; 2) prohibitions against visitors from entering a circumscribed perimeter; 3) typically placing in quarantine those visitors who are allowed to enter for a period of time before admission; and 4) if available, taking advantage of geographic barriers, such as an island or remote location. Several themes emerged from our historical research. First, coordination among public agencies is essential to any effective public health response. Despite some tension among city, county, and state officials in Gunnison, their relatively smooth cooperation may have played a role in their implementing and maintaining strict public health measures. Second, neither Gunnison nor Yerba Buena could have escaped the flu without full cooperation from the local population. Gunnison's low population density and self-sufficient ranching lifestyle made it easier for residents to bide their time . At Yerba Buena, the military chain of command mandated the cooperation of sailors and allowed the island's commander to close off the base from the outside world with little interference . Finally, these communities had the advantage of early warnings to prepare their populations. Both tracked influenza's westward movement from August to September and, unlike communities along the East Coast, could implement protective health strategies before cases appeared at their doorsteps. One would like to think that the 6 communities we identified fared better than others because of the NPIs they enacted. While we cannot prove this for any of them, the case is perhaps strongest for Yerba Buena and possibly Gunnison. Further complicating our task, in addition to the uneven quality and quantity of information available for study, is that some of these communities were sparsely populated and geographically isolated, and all of them were subject to the vagaries of how the influenza virus affected populations. Indeed, these communities represent the exception rather than the rule in terms of how most American communities experienced the influenza pandemic of 1918–1920 (14,15). This leads to several intriguing questions regarding what these escape communities can teach us about pandemic preparations today, let alone the question of whether such measures can even be replicated. Conclusions First, protective sequestration, if enacted early enough in the pandemic, crafted so as to encourage the compliance of the population involved, and continued for the lengthy time period in which the area is at risk, stands the best chance of guarding against infection. Second, available data from the second wave of the 1918–1920 influenza pandemic fail to show that any other NPI (apart from protective sequestration) was, or was not, effective in preventing the spread of the virus. Despite implementing several NPIs, most communities sustained considerable illness and death. We could not assess how the timing of NPI implementation across the nation affected disease mitigation efforts nor whether these NPIs lessened what might have been even higher rates had these measures not been in place in various locations. Moreover, we could not locate any consistent, reliable data supporting the conclusion that face masks, as available and as worn during the 1918–1920 influenza pandemic, conferred any protection to the populations that wore them (16). In fact, evidence suggests that in most American communities NPIs did not prevent the spread of virus in 1918. What remains unclear is the extent to which they may have been partially effective in reducing spread or mitigating community impact. However inconclusive the data from 1918 are, the collective experiences of American communities from the pandemic are noteworthy, especially in light of the fact that, if faced with a pandemic today, we would likely rely on many of these same NPIs to attempt to mitigate the spread of the infection until pharmacological supplies of vaccine and antiviral agents were available (17–19). It is true that the United States of today is a much different nation than it was in 1918, with a larger, more mobile, and more complex society. It is equally true that the communities we examined were all small and relatively isolated (or isolatable). Nevertheless, in the event of another influenza pandemic, many specific subcommunities (e.g., military installations, college and university campuses, nursing homes) may wish to consider protective sequestration measures as potential means to prevent or delay the onset of epidemic influenza in their populations. This study was funded by a contract from the Defense Threat Reduction Agency, US Department of Defense, DTRA01-03-D-0017. Dr Markel is the George E. Wantz Professor of the History of Medicine, Professor of Pediatrics and Communicable Diseases, and Director, Center for the History of Medicine, University of Michigan Medical School at Ann Arbor. References Jordan EO. Epidemic influenza: a survey. Chicago: American Medical Association; 1927. Crosby AW. America's forgotten pandemic: the influenza of 1918. New York: Cambridge University Press; 2003. Byerly CR. Fever of war: the influenza epidemic in the U.S. Army during World War I. First ed. New York: New York University Press; 2005. Hoehling AA. The great epidemic. Boston: Little, Brown and Company; 1961. Kolata G. Flu: the story of the great influenza pandemic of 1918 and the search for the virus that caused it. New York: Farrar, Straus and Giroux; 1999. Barry JM. The great influenza: the epic story of the deadliest plague in history. New York: Viking; 2004. World Health Organization Writing Group. Nonpharmaceutical interventions for pandemic influenza, international measures. Emerg Infect Dis. 2006;12:81–94. Vermont state board of health. Twenty-second [twelfth biennial] report of the state board of health of the state of Vermont from January 1, 1918, to December 31, 1919. Rutland (VT): The Turtle Company, Marble City Press Publishers; 1920. Taylor R. Saranac: America's magic mountain. Boston: Houghton-Mifflin; 1986. Johnston JI. History and epidemiology of influenza. In: University of Pittsburgh School of Medicine. Studies on epidemic influenza. Pittsburgh: University of Pittsburgh School of Medicine; 1919. Taylor JM. An exploit in control of influenza. Boston Med Surg J. 1920;182:601. Annual reports of the Navy Department for the fiscal year 1919. Washington: Government Printing Office, 1920; 2482–6. Leonard SJ. The 1918 influenza epidemic in Denver and Colorado. Essays in Colorado History. 1989;9:9. Sydenstricker E. Preliminary statistics of the influenza epidemic. Public Health Rep. 1918;33:2305–21. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–36. Kellogg WH. Influenza: a study of measures adopted for the control of the epidemic. California State Board of Health, Special bulletin no. 31. Sacramento (CA): California State Printing Office; 1919. Ferguson NM, Cummings DA, Fraser C, Cajka CJ, Cooley PC, Burke DS. Strategies for mitigating an influenza pandemic. Nature. 2006;442:448–52. Ferguson NM, Fraser C, Donnelly CA, Ghani AC, Anderson RM. Public health risk from the avian H5N1 influenza epidemic. Science. 2004;304:968–9. Longini IM, Nizam A, Xu S, Ungchusak K, Hanshaoworakul W, Cummings DA, et al. Containing pandemic influenza at the source. Science. 2005;309:1083–7....查看详细 (11174字节)
☉ 11342768:Fatal Human Infection with Rabies-related Duvenhage Virus, South Africa
National Institute for Communicable Diseases, Sandringham, South Africa Durbanville Mediclinic, Cape Town, South Africa University of Stellenbosch, Tygerberg, South Africa University of Pretoria, Pretoria, South Africa Abstract Duvenhage virus was isolated from a patient who died of a rabieslike disease after being scratched by a bat early in 2006. This occurred ≈80 km from the site where the only other known human infection with the virus had occurred 36 years earlier. The genus Lyssavirus within the family Rhabdoviridae currently includes rabies virus (RABV) (genotype 1) and 6 rabies-related viruses: 3 from Africa, Lagos bat virus (LBV) (genotype 2), Mokola virus (MOKV) (genotype 3), and Duvenhage virus (DUVV) (genotype 4); European bat lyssaviruses 1 and 2 (EBLV1 and 2) (genotypes 5 and 6); and Australian bat lyssavirus (ABLV) (genotype 7) (1). Strains of RABV (genotype 1) undergo genetic adaptation to particular animal hosts so that within specific areas the disease is manifested and transmitted predominantly by 1 host species. The canid, or dog, biotype of RABV is the most widely distributed in the world. In South Africa, RABV is transmitted by dogs and jackals in the northern region of the country, by dogs in the eastern region where most cases of human rabies occur, and by bat-eared foxes in the western region. In addition, an indigenous herpestid biotype of RABV (genotype 1) is transmitted by mongooses (Herpestidae) on the interior plateau of South Africa. This biotype does not spread readily to dogs but causes occasional cases of rabies in dogs, cats, humans, and more frequently, cattle and sheep (2). RABV (genotype 1) has never been isolated from bats outside North and South America, but rabies-related viruses have been isolated from bats elsewhere. In Africa, LBV and DUVV are associated with bats, but MOKV is uniquely associated with shrews and rodents, not bats. Fifteen isolations of LBV have been reported, including 8 from fruit bats and a cat in KwaZulu-Natal Province of South Africa, but the virus has never been associated with human disease (2,3). MOKV has been isolated from shrews, rodents, cats, and a dog in Africa and from 7 cats with rabies-like disease in KwaZulu-Natal and Eastern Cape provinces of South Africa (2). The virus is believed to have caused rabieslike disease in 2 persons in Nigeria in 1969 and 1971, shortly after its initial discovery in shrews in 1968, but no cases of human infection have subsequently been recognized (4,5). DUVV was discovered in 1970 when it caused fatal rabieslike disease in a person bitten by an unidentified insectivorous bat ≈150 km northwest of Johannesburg, South Africa (6). In 1981, the virus was isolated from what is believed to have been a Miniopterus schreibersi insectivorous bat caught in daylight by a cat in Makhado town (formerly Louis Trichardt) in Limpopo Province, South Africa, and in 1986 the virus was recovered from an insectivorous bat, Nycteris thebaica, trapped in a survey in Zimbabwe (7,8). The Study DUVV infection was recently confirmed in a 77-year-old man with type 2 diabetes who was scratched on the face by what appears to have been an insectivorous bat in February 2006 in North West Province, South Africa, ≈80 km from the location where the first DUVV infection occurred 36 years earlier. The bat flew into a room at night, landed on the man's spectacles while he was attempting to chase it out, and scratched his face as he brushed it off. The bat did not appear to have bitten him, and it escaped after the incident. He did not seek medical care, and thus no postexposure treatment was given. He became ill at home in Cape Town 27 days later and received treatment for influenzalike illness. He slept most of the following day, had hallucinations that night, and was admitted to a hospital on the third day of illness. On admission, he had a fever (40°C), tachycardia, neck and general limb rigidity, hyperreflexia, facial fasciculation, and involuntary grimacing. Within 24 hours generalized tonic-clonic seizures had developed with status epilepticus supervening. These necessitated intubation, sedation, and mechanical ventilation. He died on day 14 of his illness. Heminested reverse transcription–PCR was performed as previously described (9) with modified forward primer JW12 (10). This procedure detected lyssavirus nucleic acid in saliva taken on day 10 of illness and in brain tissue collected postmortem. Nucleotide sequencing of the PCR products and phylogenetic analysis performed as previously described (11) confirmed the identity of the agent as DUVV (Figure), and live virus was isolated from saliva and brain tissue by mouse inoculation. Immunofluorescence tests with antirabies conjugate prepared to be cross-reactive with the African rabies–related viruses (Onderstepoort Veterinary Institute, Pretoria, South Africa) showed small and sparse inclusion bodies in impression smears of the cortex, hippocampus, thalamus, medulla, and cerebellum. Histopathologic examination of sections from the cortex, hippocampus, thalamus, hypothalamus, midbrain, pons, medulla, and cerebellum showed polioencephalitis affecting predominantly the diencephalon and brainstem and involving varying degrees of neuronopathy, neuronal loss, astrocytosis, parenchymal and perivascular lymphocytic infiltration with CD45 immunopositivity, sparse macrophage activation, and axonal spheroid formation. No nuclear or cytoplasmic inclusions were observed. Conclusions The ability to distinguish between various lyssaviruses and monitor their relative distribution and prevalence has important implications for implementation of control measures. It was recognized in 1932 that mongoose-associated rabies in South Africa differs from classic dog rabies (2). Although an inadequately characterized lyssavirus was isolated from a bat trapped in a survey in 1963, before the existence of rabies-related viruses was known, awareness of lyssaviruses other than rabies viruses dates from the identification of DUVV in 1970 and was followed by detection of LBV and MOKV in South Africa (6,7,12). Routine differentiation of diagnostic isolates became feasible with the availability of monoclonal antibodies during the 1980s and the subsequent introduction of molecular epidemiology techniques (7,13–15). Although instances of persons seeking rabies prophylaxis after exposure to bats have been reported, the recent case of DUVV infection constitutes only the second known instance of a person in South Africa with lyssavirus infection after such an encounter. Nevertheless, it is clear that rabies-related viruses are widely endemic in South Africa and that active investigation of the bat-associated lyssaviruses is warranted. Dr Paweska is head of the Special Pathogens Unit, National Institute for Communicable Diseases, South Africa. His research interests include viral hemorrhagic fevers, rabies, and rabies-related viruses. References Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA. Virus taxonomy: the classification and nomenclature of viruses. The eighth report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press; 2004. p. 623–31. Swanepoel R. Rabies. In: Coetzer JAW, Tustin RC, editors. Infectious diseases of livestock. 2nd ed. Cape Town (South Africa): Oxford University Press; 2004. p. 1123–82. Markotter W, Randles J, Rupprecht CE, Sabeta CT, Wandeler AI, Taylor PJ, et al. Recent Lagos bat virus isolations from bats (suborder Megachiroptera) in South Africa. Emerg Infect Dis. 2006;12:504–6. Familusi JB, Moore DL. Isolation of a rabies related virus from the cerebrospinal fluid of a child with 'aseptic meningitis. Afr J Med Sci. 1972;3:93–6. Familusi JB, Osunkoya BO, Moore DL, Kemp GE, Fabiyi A. A fatal human infection with Mokola virus. Am J Trop Med Hyg. 1972;21:959–63. Tignor GH, Murphy FA, Clark HF, Shope RE, Madore P, Bauer SP, et al. Duvenhage virus: morphological, biochemical, histopathological and antigenic relationships to the rabies serogroup. J Gen Virol. 1977;37:595–611. Schneider LG, Barnard BJH, Schneider HP. Application of monoclonal antibodies for epidemiological investigations and oral vaccination studies: I. African virus. In: Kuwert E, Merieux C, Koprowski H, Bgel K, editors. Rabies in the tropics. Berlin: Springer-Verlag; 1985. p. 47–59. Foggin CM. Rabies and rabies-related viruses in Zimbabwe: historical, virological and ecological aspects. [Doctoral dissertation]. Harare (Zimbabwe): University of Zimbabwe; 1988. Whitby JE, Heaton PR, Whitby HE, O'Sullivan E, Johnstone P. Rapid detection of rabies and rabies-related viruses by RT-PCR and enzyme-linked immunosorbent assay. J Virol Methods. 1997;69:63–72. Black EM, McElhinney LM, Lowings JP, Smith J, Johnstone P, Heaton PR. Molecular methods to distinguish between classical and the rabies-related European bat lyssaviruses. J Virol Methods. 2000;87:123–31. Venter M, Smit S, Leman P, Swanepoel R. Phylogenetic evidence of widespread distribution of genotype 3 JC virus in Africa and identification of a type 7 isolate in an AIDS patient. J Gen Virol. 2004;85:2215–9. Mansvelt PR. The role of wild life in the epizootiology of some infectious and parasitic animal diseases in South Africa. Bull Off Int Epizoot. 1965;64:825–35. Smith JS, Yager PA, Orciari LA. Rabies in wild and domestic carnivores of Africa: epidemiological and historical associations determined by limited sequence analysis. Onderstepoort J Vet Res. 1993;60:307–14. Nel LH, Bingham J, Jacobs JA, Jaftha JB. A nucleotide-specific polymerase chain reaction assay to differentiate rabies virus biotypes in South Africa. Onderstepoort J Vet Res. 1998;65:297–303. Nel L, Jacobs JA, Jaftha JB, von Teichman BF, Bingham J. New cases of Mokola virus infection in South Africa: a genotypic comparison of southern African isolates. Virus Genes. 2000;20:103–6....查看详细 (10061字节)
☉ 11342769:West Nile Virus in Horses, sub-Saharan Africa
ecole du Val-de-Grace, Paris, France Institut de Medecine Tropicale du Service de Sante des Armees, Marseille, France Secteur Veterinaire de Marseille, Marseille, France Service Veterinaire du Regiment de Cavalerie de la Garde Republicaine, Paris, France Secteur Veterinaire de Lyon, Lyon, France Centre International de Recherches Medicales, Franceville, Gabon Direction Regionale du Service de Sante des Armees de Toulon, Toulon, France Abstract To evaluate the presence and extension of West Nile virus where French soldiers are stationed in Africa, specific antibody prevalence was determined by using ELISA and Western blot. Among 245 horses living in close proximity to the soldiers, seroprevalence was particularly high in Chad (97%) and Senegal (92%). West Nile virus (WNV), a mosquito-transmitted flavivirus, was first isolated in Africa, West Nile district of Uganda, in 1937 (1). It has been shown to infect humans and a wide spectrum of animal species, including birds and horses. WNV infection is often inapparent or mild in humans but may cause severe and even fatal encephalitis in horses (2). Since 1999, dissemination of the virus through North America has reinforced interest in WNV epidemiology and evolution. Before 1999, outbreaks have been reported in North Africa, Israel, Romania, Russia, and France, where the virus may have been imported by migratory birds (3–5). However, few data are available on the current circulation of WNV in sub-Saharan Africa because of lack of surveillance and diagnostic tools in those countries. Assessing and preventing human and zoonotic infectious diseases in tropical areas, particularly Africa, are essential missions of the French Defense Medical Service. To evaluate the presence and extension of WNV in the sub-Saharan African areas where French soldiers are stationed, serologic surveillance of horses living in close proximity was initiated in 2002. The Study From December 2002 through August 2005, blood samples were collected from 245 horses in 13 riding stables located in Senegal (Dakar, n = 25), Cte d'Ivoire (Abidjan, n = 95), Chad (N'Djamena, n = 30), Democratic Republic of the Congo (Kinshasa, n = 20), Gabon (Libreville, Port Gentil, and Moanda, n = 64), and Djibouti (Djibouti, n = 11) . Some horses were sampled twice in Chad (n = 18) and in Cte d'Ivoire (n = 18) during a period of 11–13 months. Origin, travel history, and how long the tested horses lived in the studied areas were not well known, but the horses were generally born and bred in the countries from which they were sampled (some of them in neighboring countries such as Burkina Faso, Mali, Niger, and Ethiopia), and none had a history of WNV vaccination. Blood was centrifuged within 24 hours after collection. Serum was separated, frozen at –20°C, and sent to the virology laboratory of the Institut de Medecine Tropicale du Service de Sante des Armees in Marseille, France. Each sample was systematically tested for WNV-specific immunoglobulin G (IgG) by using an ELISA made in house. Antigen was prepared from a crude supernatant of Vero cells collected after 4 days of infection with WNV reference strain Eg 101 (viral titer >107 ID/mL) and treated with 1% Triton 100 and β-propiolactone (1:1,000). IgG was detected by using commercial peroxidase anti-horse IgG and tetramethylbenzidine as the substrate and standard procedures of ELISA capture. Serum specimens were considered positive for IgG when the optical density (OD) in antigen-positive wells was >0.3 and the ratio between the OD in corresponding antigen-positive wells and the mean OD in antigen-negative wells was >3.5. Because of the antigenic cross-reactivity among viruses of the Flavivirus genus, validation of ELISA IgG–positive samples was necessary. The plaque reduction neutralization test (PRNT) is the serologic reference method. All the IgG-positive sera collected during 2002–2003 were tested as described (11). In a 96-well plate, 4 dilutions of each serum sample (1:10, 1:40, 1:160, 1:640; 4 wells for each dilution) were incubated at 37°C for 1 hour in a viral suspension of 10–50 PFU in 50 μL before the addition of 100 μL of a Vero cell suspension (4×104/well). Four days later, the cell layer was fixed in formol and stained with crystal violet. A test result was considered positive for a dilution if the plaque reduction was >90% compared with the negative control. Because this method is fastidious and slow, we have used an alternative Western blot (WB) approach as described in Figure 2 (12). Complete (100%) correlation between WB and PRNT and high specificity of WB were observed for a panel of 79 serum samples. Thus, only WB was used for validation of ELISA IgG-positive sera for the 2004–2005 samples. All serum samples that were positive for WNV IgG were further investigated using immunocapture IgM ELISA to evaluate the time of infection. Conclusions Except in Gabon (3%), high seroprevalence (28%–97%) for WNV was detected in horses in West Africa and Central Africa, especially in N'Djamena (97%) and Dakar (92%) . Seroprevalence of 9% was detected in East Africa (Djibouti). All horses positive for IgG were negative for IgM, which indicates relatively old infection. Estimating the date of onset of WNV infection is difficult because of a lack of published data relative to WNV IgM and IgG response in naturally infected horses; only persistence of IgG several years after infection has been described (4). Because histories of tested horses are not well known, determining precisely when and where horses became infected is difficult. However, infections likely occurred in sampling countries or neighboring sub-Saharan African countries. Seroconversion from negative to positive was found in 2 horses in Chad from 2003 through 2004, while 5 of 15 seropositive horses became seronegative, which suggests maintenance of an enzootic cycle in this area but at a low level. During the same period in Cte d'Ivoire, 9 of 10 previously seropositive horses were seronegative, while none of seronegative horses became seropositive. The most probable explanation is a decrease in IgG titer under the retained threshold of positivity compatible with the decrease of WNV IgG response in horses, which suggests the presence of an older epizootic in this area. The immunoblotting method is a fast and specific confirmation assay for validation of ELISA WNV IgG–positive sera. Once validated by further studies, WB could be used as an alternative to PRNT. Serologic data from our study should be considered as evidence of WNV activity in sub-Saharan Africa, which has a potential risk for populations and foreigners, including French soldiers. Previously, WNV was known to circulate in mosquitoes and some bird species without having any clear pathogenicity; outbreaks have been reported only in South Africa and in the Democratic Republic of the Congo (5,6). Before our study, no data relative to WNV circulation in horses in sub-Saharan Africa were documented, and WNV activity had never been reported in Chad or Gabon. Highest (92%–97%) seroprevalence was found in the western and central parts of the Sahelian area (Dakar and N'Djamena). This area, characterized by a semiarid climate and vegetation of steppe and brush grass, is the most frequently involved area for WNV isolations in birds and mosquitoes (7,13). The seroprevalence was lower in the east of the Sahelian area (Djibouti, 9%), where the climate is arid and the vegetation is semidesert, and in the sub-Sahelian area (3%–30%), where the vegetation is tropical rain forest or woodland savanna in a humid or semihumid climate. That forest favors the sedentariness of birds has been documented (14). The migration of birds may certainly be enhanced in the Sahelian area; the introduction of WNV by migratory birds during their flight between Senegal and Europe has been suspected as a cause of the 1996 outbreak in Morocco (10). To estimate possibilities of incursions of WNV, especially in Eurasia, effects of environmental factors such as climate and vegetation on reservoir and vector populations in sub-Saharan Africa should be precisely studied. Acknowledgments We thank all who contributed to these studies, especially Jose Gomez and Laurent Maurizi, for equine serum sampling in Africa and Olivier Merle, Yannick Sanson, and Fabienne Tock, who managed the technical samples. We also thank the French Defense Medical Service for financial and technical support. Dr Cabre is a veterinarian in the French Defense Medical Service. He was on duty in Chad during the end of 2003, where he developed an interest in known and emerging infectious diseases, especially zoonoses, that occur in areas of French military operations. References Smithburn KC, Hughes TP, Burke AW, Paul JH. A neurotropic virus isolated from the blood of a native of Uganda. Am J Trop Med Hyg. 1940;20:471–92. Campbell GL, Marfin AA, Lanciotti RS, Gubler DJ. West Nile virus. Lancet Infect Dis. 2002;2:519–29. Hubalek Z, Halouzka J. West Nile fever: a reemerging mosquito-borne viral disease in Europe. Emerg Infect Dis. 1999;5:643–50. Murgue B, Murri S, Zientara S, Durand B, Durand JP, Zeller HG. West Nile outbreak in horses in southern France, 2000: the return after 35 years later. Emerg Infect Dis. 2001;7:692–6. Zeller HG, Schuffenecker I. West Nile virus: an overview of its spread in Europe and the Mediterranean basin in contrast to its spread in the Americas. Eur J Clin Microbiol Infect Dis. 2004;23:147–56. Nur YA, Groen J, Heuvelmans H, Tuynman W, Copra C, Osterhaus AD. An outbreak of West Nile fever among migrants in Kisangani, Democratic Republic of Congo. Am J Trop Med Hyg. 1999;61:885–8. Centre Collaborateur OMS de Reference et de Recherche sur les Arbovirus. Flavivirus, West Nile: 282 souches identifiees. 1962 [updated 2005 Jul; cited 2006 Aug 17]. Available from http://www.pasteur.fr/recherche/banques/CRORA/virus/v010100.htm Murgue B, Murri S, Triki H, Deubel V, Zeller HG. West Nile in the Mediterranean basin: 1950–2000. Ann N Y Acad Sci. 2001;951:117–26. Murgue B, Zeller H, Deubel V. The ecology and epidemiology of West Nile virus in Africa, Europe and Asia. Curr Top Microbiol Immunol. 2002;267:195–221. Schuffenecker I, Peyrefitte CN, El Harrak M, Murri S, Leblond A, Zeller HG. West Nile virus in Morocco, 2003. Emerg Infect Dis. 2005;11:306–9. De Madrid AT, Porterfield JS. A simple micro-culture method for the study of group B arboviruses. Bull World Health Organ 1969;40:113–21. Prange A, Coussinier-Paris P, Davoust B, Cabre O, Gravier P, Sanson Y, et al. Diagnostic differentiel de l'infection par le virus West Nile: interêt de l'immunoblot. Proceedings of the 11th Actualites du Pharo Conference; 2004 Sep 9–11; Marseilles, France. Med Trop (Mars). 2004;64:293. Traore-Lamizana M, Zeller HG, Mondo M. Isolations of West Nile and Bagaza viruses from mosquitoes (Diptera: Culicidae) in center Senegal (Ferlo). J Med Entomol. 1994;31:934–8. Brown LH, Urban EK, Newman K, editors. The birds of Africa. Vol.1. 1st ed. New York: Academic Press; 1982....查看详细 (11295字节)
☉ 11342771:Rabies Virus Maintained by Dogs in Humans and Terrestrial Wildlife, Ceará State, Brazil
Instituto Pasteur de So Paulo, So Paulo, Brazil Universidade de So Paulo, So Paulo, Brazil Centers for Disease Control and Prevention, Atlanta, Georgia, USA Secretaria Estadual de Saúde do Ceará, Fortaleza, Brazil Abstract Rabies viruses circulating in Ceará, Brazil, were identified by molecular analysis to be related to variants maintained by dogs, bats, and other wildlife. Most of these viruses are associated with human rabies cases. We document the emergence of a rabies virus variant responsible for an independent epidemic cycle in the crab-eating fox (Cerdocyon thous). After dog rabies control programs were implemented in Ceará State, Brazil, a the number of human cases decreased (1,2). Thereafter, the epidemiologic importance of rabies in wildlife became evident. From 1990 through 2005, a total of 173 cases of rabies were reported in Cerdocyon thous (crab-eating fox), 25 in Callithrix. j. jacchus (common marmoset) and 6 in Procyon cancrivorous (crab-eating raccoon). During this period, in 13 of 40 human cases reported in Ceará, wildlife was the source of infection (2). In 1996, because of this new epidemiologic situation, public health authorities launched an educational program, and no human cases due to wildlife were recorded in 1999, despite 84 cases in wildlife registered that year (2). The objective of this study was to elucidate some of the epidemiologic events involved in rabies emergence among wildlife in Ceará. The Study We studied 22 samples, from dogs, cattle, wildlife, and humans in Ceará, obtained from 1997 to 2003 (Table). Samples were antigenically characterized by using a monoclonal antibody (MAb) panel against the viral nucleoprotein (3–5). Isolates were injected into the brains of suckling mice, and brain impressions were made for MAb typing (3,5). Characterization of the samples identified 4 antigenic variants. Antigenic variant-2 (AgV2), maintained by dogs, was found in all C. thous, P. crancrivourous, and human cases and in all dog isolates with the exception of brdg5360, which was positive with all the MAbs. Antigenic variant-3 (AgV3), epidemiologically associated with vampire bats, Desmodus rotundus, was identified in 3 bovine samples. A previously reported profile, representing an AgV that circulates in marmosets in Ceará (5), was detected in sample brsg5696. The Ceará viruses were analyzed genetically through a comparative phylogenetic study based on a 320-bp fragment of the nucleoprotein gene, from position 1157 to 1476, as compared with SADB19 (5–8). These isolates were also compared with rabies virus variants circulating among domestic animals and wildlife from the Americas. The viral RNA was extracted from infected tissues, and the cDNA was obtained by reverse transcription–PCR techniques, using primers 21 g and 304, and was sequenced with primer 304 (7,9). The phylogenetic analyses were made by using the PileUp program of the Wisconsin Package Version 10.1 (10) and the programs DNADIST, NEIGHBOR, SEQBOOT, and CONSENSE of the PHYLIP package (11). The expressed percentages of identity refer to the nucleotide sequences. The trees were obtained with the TREEVIEW program (12). The phylogenetic analyses showed segregation in 5 lineages, A–E , which was statistically supported by high bootstrap values. Lineage A was represented by a sample from a dog from Maranguape, which was obtained in 2001. This virus showed its closest genetic relationship with lineage B (identity 92.4%–94.2%). Lineage B was formed by all the C. thous isolates, a sample from a human bitten by a P. cancrivorous raccoon in Fortaleza in 1997, and a virus from a P. crancrivorous raccoon collected in Maranguape during 2001 (intrinsic identity 96.5%–100%). This lineage showed its highest percentage of identity with lineage C (intrinsic identity 90.6%–92.8%). Lineage C consisted of 9 human samples collected in 5 different counties from 2000 to 2003 and an isolate obtained from a dog in 2000. The samples were highly homologous (intrinsic identity 99.1%–100%). Lineage D included 3 bovines collected in 3 geographically distant counties during 2000 and 2001 (intrinsic identity 97.5%–98.4%). Lineage E was represented by the only sample collected from a C. j jacchus marmoset, These last 2 lineages were related distantly to all the others. When compared with representatives of rabies variants maintained by terrestrial and bat species in the Americas , lineages A, B, and C continue to segregate as independent lineages with high statistical support. Ceará bovine samples representing lineage D clustered with D. rotundus and D. rotundus–related cases from Latin America (intrinsic identity 94%–97.8%). The only sample representative of lineage E segregated with 2 isolates from humans bitten by C. j. jacchus and a sample collected from a marmoset kept as a pet (5). The isolates were highly homologous to each other (intrinsic identity 98.7%–100%). Conclusions A thorough description of rabies epidemiology depends on a comprehensive surveillance program and application of accurate molecular methods to discriminate among different variants and the emergence of new foci. Antigenic and limited sequencing analyses were used to better understand the emergent epidemiologic events in wildlife in Ceará, Brazil. These analyses allowed identification of 5 potential cycles in this region, despite antigenic homogeneity. Lack of antigenic and genetic relationships of sample brdg5693, representing lineage A, with the rest of the isolates from Ceará and the known terrestrial rabies vectors from the Americas shows that this virus is a variant not previously described. This virus was geographically and temporarily associated with samples brpcr5698 and brcth5692, obtained in Maranguape during 2001. These circumstances demonstrated the existence of at least 2 overlapping endemic cycles in this area. Lineage B was formed mainly by isolates from C. thous, which indicates the existence of an emerging rabies cycle in this species. The epidemiologic situation in Ceará was complicated because of overlapping distributions of dog and C. thous rabies cases . Tree topology and genetic relationships between dog and C. thous variants suggested that the canine virus was introduced in C. thous populations because of spillover events, which gave rise to an emergent cycle. A similar event was described between domestic dogs and Canis adustus (jackal) in Zimbawe. In this case, the variant circulating in dogs was introduced into the C. adustus population by spillover events, with the consequent emergence of an independent cycle (13). Recently, the hoary fox has been identified as a rabies reservoir in Brazil (14). Inclusion in lineage B of an isolate obtained from a human bitten by a P. cancrivorous raccoon and another sample collected from this species suggested the risk of establishing C. thous variant in P. cancrivorous. The niches of these 2 species overlap, which facilitates their encounters. Additional surveillance is necessary to clarify this situation. Epidemiologic data which indicates that humans had been exposed to dog bites, results of molecular characterization, and inclusion of a dog isolate in the C lineage strongly incriminate the dog as the reservoir of this variant. Identification of the source of infection by using classic surveillance alone is complicated by the presence of multiple cycles of transmission. Genetic comparison of samples from lineage D with viruses representing bats viruses from the Americas helped to identify D. rotundus as the source of livestock infection. The close genetic relationship of sample brsg5696 with rabies isolates obtained from C. j. jacchus and human cases bitten by marmosets further supported C. j. jaccuss as the most important vector of this variant. This finding indicates that this species plays an important role for disease maintenance in nature. Methods for antigenic and genetic identification of rabies samples isolated in the Americas have contributed effectively to the development of health programs, as well as recognition of possible wild reservoirs of urban rabies. The emergence of new cycles in Latin American wildlife indicates the need to strengthen surveillance programs in these species and research development for the evaluation of the feasibility of oral vaccination interventions. Acknowledgments We thank Debora R. V. Sacramento for her technical collaboration, Fabiana L. A. da Silveira Lebrun for structural support, Maria das Graas Silva for help with the bibliography and illustrations, and all staff at Instituto Pasteur of So Paulo, Brazil, and Laboratory of Rabies Diagnosis of Ceará, Brazil, for collaboration and incalculable incentives. Dr Favoretto is a scientific researcher and specialist in public health at the Instituto Pasteur of So Paulo. Her research interests include diagnosis and molecular epidemiology of rabies virus. References Pan-American Health Organization/World Health Organization. Prevention and control of diseases. Veterinary Public Health Unit. Elimination of the human rabies transmitted by dogs in Latin America: analysis of the situation, 2004. Washington: Pan American Health Organization; 2005. vol. 37, p. 10–29. Núcleo de Controle das Endemias Transmissíveis por vetores–NUEND, Coordenadoria de Apoio ao Desenvolvimento da Ateno a Saúde–CODAS, 1998–2003. Boletim da Secretaria da Saúde do Estado do Ceará, Brasil, 2004. Favoretto SR, Carrieri ML, Cunha EMS, Aguiar EAC, Silva LHQ, Sodre M, et al. Antigenic typing of Brazilian rabies virus samples isolated from animals and humans, 1989–2000. Rev Inst Med Trop Sao Paulo. 2002;44:91–5. Pan-American Health Organization/World Health Organization. Los anticuerpos monoclonales en la caracterizacion y vigilancia de los virus de la rábia en America Latina y el Caribe. Pan American Journal of Public Health. 2000;8:214–7. Favoretto SR, Mattos CC, Morais NB, Araújo FAA, Mattos CA. Rabies in marmosets (Callithrix jacchus) from the state of Ceará, Brazil. Emerg Infect Dis. 2001;7:1062–5. Conzelmann KK, Cox JK, Schneider LG, Thiel HJ. Molecular cloning and complete sequence of the attenuated rabies virus SABB19. Virology. 1990;175:485–99. Smith JS. Rabies virus. In:.Murray PR, Baron EJ,.Pfaller MA, Tenover FC, Yolken R, editors. Manual of clinical microbiology, 6th ed. Washington: American Society for Microbiology Press; 1995. p. 997–1003. de Mattos CC, de Mattos CA, Loza-Rubio E, Aguilar-Setien A, Orciari LA, Smith JS. Molecular characterization of rabies virus isolates from Mexico: implications for transmission dynamics and human risk. Am J Trop Med Hyg. 1999;61:587–97. de Mattos CA, de Mattos CC, Smith JS, Miller E, Papo S, Utrera A, et al. Genetic characterization of rabies field isolates from Venezuela. J Clin Microbiol. 1996;34:1553–8. Version WP. 10.1 Madison (WI): Genetics Computer Group; 2000. Felsenstein, J. Phylip: Phylogeny inference pakage. Version 3,5c. Seattle (WA): University of Washington;, 1993. Page, Ram. D. TREEVIEW: An application to display phylogenetic trees on personal computers. Computer Applied Biocience. 1996;12:357–8. Bingham J, Foggim CM, Wandeler AI, Hill FW. The epidemiology of rabies in Zimbabwe. 2. Rabies in jackals (Canis adustus and Canis mesomelas). Onderstepoort J Vet Res. 1999;1:11–23. Bernardi F, Nadin Davis SA, Wandeler AI, Armstrong J, Gomes A, Lima FS, et al. Antigenic and genetic characterization of rabies viruses isolated from domestic and wild animals of Brazil identifies the hoary fox as a rabies reservoir. J Gen Virol. 2005;86:3153–62....查看详细 (11810字节)

☉ 11342772:West Nile Virus Antibody Prevalence in Wild Mammals, Southern Wisconsin
US Geological Survey, Madison, Wisconsin, USA: and University of Wisconsin, Madison, Wisconsin, USA Abstract Twenty percent prevalence of West Nile virus antibody was found in free-ranging medium-sized Wisconsin mammals. No significant differences were noted in antibody prevalence with regard to sex, age, month of collection, or species. Our results suggest a similar route of infection in these mammals. In 1999, West Nile virus (WNV) was detected for the first time in the United States in dead American crows (Corvus brachyrhynchos), and a disease surveillance system that used dead crows was established (1,2). Serologic surveys to determine the prevalence of WNV exposure in free-ranging mammals (3–6) are relatively rare. Although WNV can infect a wide range of vertebrates, mammals are assumed to be dead-end hosts (7). We report the results of a 2003–2004 WNV serosurvey in medium-sized mammals from south-central Wisconsin. The Study We obtained samples from a part of south-central Wisconsin (Dane and Iowa Counties) recently identified as an area where white-tailed deer (Odocoileus virginianus) had chronic wasting disease infection (8). Medium-sized free-ranging mammals were collected as part of a larger study to evaluate the potential for transmission of chronic wasting disease from infected white-tailed deer carcasses to scavenging mammals. A total of 228 medium-sized mammal carcasses, consisting of 78 raccoons (Procyon lotor), 71 Virginia opossums (Didelphis virginiana), 59 coyotes (Canis latrans), 7 red foxes (Vulpes vulpes), 6 striped skunks (Mephitis mephitis), 5 feral cats (Felis catus), and 2 badgers (Taxidea taxus), were obtained by trapping, shooting, or collecting fresh road kills during October 2003 through April 2004. These animals were collected in rural areas consisting of small woodlots, agricultural fields, and roadsides. Blood samples from the carcasses were collected by absorbtion into Nobuto strips (Toyo Roshi Kaisha, Ltd, Tokyo, Japan), labeled, air dried, and stored at ambient temperature until submitted to the National Wildlife Health Center (NWHC). A 1:20 serum dilution was prepared in the laboratory by following the manufacturer's instructions for extraction from the Nobuto strip. The dilution was stored at 0°C until it was tested. Before testing, serum samples were heat inactivated (56°C for 30 min) to eliminate any nonspecific virus inhibitors. Serum controls were included for each sample to determine whether any individual serum sample was toxic to the cell culture used. The samples were screened for WNV antibody against 100 PFU by using the plaque reduction neutralization test (PRNT) (9). The WNV used was isolated by NWHC in September 1999 from the spinal cord, sciatic nerve, and brain pool of an American crow found dead in the state of New York (strain NY99–35261–11). Serum samples were considered to be positive for flavivirus antibody if they neutralized >50% of the WNV test dose at a serum dilution >1:40. Positive serum samples were subsequently titered by PRNT (9) against both WNV and Saint Louis encephalitis virus (SLEV) to determine antibody titer and specificity. The SLEV strain (TBH-28 ASFL) was obtained from the Centers for Disease Control and Prevention, Atlanta, Georgia. Serum antibody titers were determined by attempting to neutralize WNV and SLEV using 2-fold serial dilutions ranging from 1:20 to 1:2,560. The serum titer endpoint was considered to be that dilution >1:40 still capable of neutralizing >90% of the virus test dose. The antibody titer of each serum against the 2 viruses was compared. Serum samples were considered positive for WNV antibody if the titer was >4-fold more than the serum titer against SLEV. If a 75%) and opossums (>60%). Our data indicate that the WNV antibody prevalence is similar for raccoons, opossums, and coyotes; however; food preferences differ in these 3 species (11–13). Raccoons are omnivorous, consuming mostly plant material, invertebrates, and small vertebrates; acorns and other plant materials are important fall food. Opossums are also omnivorous, consuming almost any available animal or plant material; their summer and fall diets consist primarily of invertebrates, small animals, and plant material. Coyotes are primarily predators on small vertebrates and scavengers on carcasses such as deer, livestock, and poultry. Because of the similarities in WNV antibody prevalence and differences in primary food choices, we suspect a common route of WNV transmission, most likely arthropodborne. A relatively high proportion of medium-sized mammals appear to have been infected with WNV. Whether these species play a role in maintenance and transmission of WNV needs to be determined. Whether raccoons, opossums, and coyotes can be indicators of WNV transmission or potential WNV reservoirs for subsequent transmission to avian, domestic animal, or human hosts is not known. Further research is needed to understand the role these species play in the epidemiology and epizootiology of WNV and the effect of the virus infection on specific populations of free-ranging mammals. Acknowledgments We thank J. Hann and E. Berkley for assistance with sample collection and preparation. Support was provided by the Wisconsin Trappers Association, Wisconsin Department of Natural Resources, Wisconsin Veterinary Diagnostics Laboratory, and the US Geological Survey NWHC. Dr Docherty is diagnostic virologist emeritus for the US Department of the Interior, US Geologic Survey, NWHC. His primary duties consist of preparing manuscripts for publication and consulting on wildlife diagnostics and research. References Roehrig JT, Layton M, Smith P, Campbell GL, Nasci R, Lanciotti RS. The emergence of West Nile virus in North America: ecology, epidemiology, and surveillance. Curr Top Microbiol Immunol. 2002;267:223–40. Eidson M, Komar N, Sorhage F, Nelson R, Talbot T, Mostashari F, et al. Crow deaths as a sentinel surveillance system for West Nile virus in the northeastern United States, 1999. Emerg Infect Dis. 2001;7:615–20. Dietrich G, Montenieri JA, Panella NA, Langevin S, Lasater SE, Klenk K, et al. Serologic evidence of West Nile virus infection in free-ranging mammals, Slidell, LA, 2002. Vector Borne Zoonotic Dis. 2005;5:288–92. Farajollahi A, Panella N, Carr P, Crans W, Burguess K, Komar N, et al. Serologic evidence of West Nile virus infection in black bears (Ursus americanus) from New Jersey. J Wildl Dis. 2003;39:894–6. Santaella J, McLean R, Hall JS, Gill JS, Bowen RA, Hadow HH, et al. West Nile virus serosurveillance in Iowa white-tailed deer (1999–2003). Am J Trop Med Hyg. 2005;73:1038–42. Root JJ, Hall J, McLean R, Marlenee N, Beaty B, Gansowski J, et al. Serologic evidence of exposure of wild mammals to flavivirus in the central and eastern United States. Am J Trop Med Hyg. 2005;72:622–30. Blitvich BJ, Bowen RA, Marlenee NL, Hall RA, Bunning ML, Beaty BJ. Epitope-blocking enzyme-linked immunosorbent assays for detection of West Nile virus antibodies in domestic mammals. J Clin Microbiol. 2003;41:2676–9. Joly DO, Ribic CA, Langenberg JA, Beheler K, Batha CA, Dhuey BJ, et al. Chronic wasting disease in free-ranging Wisconsin white-tailed deer. Emerg Infect Dis. 2003;9:599–601. Beaty B, Calisher C, Shope R. Arboviruses. In: Lennette EH, Lennette DA, Lennette ET, editors. Diagnostic procedures for viral, rickettsial, and chlamydial infections: 7th ed. Washington: American Public Health Association;1995. p. 204–5. Austgen LE, Bowen RA, Bunning ML, Davis BS, Mitchell CJ, Chang G-JJ. Experimental infection of cats and dogs with West Nile virus. Emerg Infect Dis. 2004;10:82–6. Kaufmann JH. Raccoon and allies. In: Chapman JA, Feldhamer GA, editors. Wild mammals of North America: biology, management, and economics. Baltimore (MD): John Hopkins University Press; 1982. p. 573–4. Gardner AL. Virginia opossum. In: Chapman JA, Feldhamer GA, editors. Wild mammals of North America: biology, management, and economics. Baltimore (MD): The John Hopkins University Press; 1982. p. 22–6. Bekoff M. Coyote. In: Chapman JA, Feldhamer GA, editors. Wild mammals of North America: biology, management, and economics. Baltimore (MD): John Hopkins University Press; 1982. p. 452–3...查看详细 (11109字节)
☉ 11342773:Spongiform Encephalopathy in a Miniature Zebu
University of Berne, Berne, Switzerland Zoo Basel, Basel, Switzerland Federal Veterinary Office, Berne, Switzerland Abstract The first case of spongiform encephalopathy in a zebu (Bos indicus) was identified in a zoo in Switzerland. Although histopathologic and immunohistochemical analyses of the central nervous system indicated a diagnosis of bovine spongiform encephalopathy (BSE), molecular typing showed some features different from those of BSE in cattle (B. taurus). Spongiform encephalopathies (SEs) are transmissible neurodegenerative diseases characterized by spongiform lesions and deposition of partially proteinase K–resistant prion protein (PrPsc), a misfolded isoform of the normal host-encoded cellular prion protein (PrPc), in the central nervous system (CNS). The oldest known SE is scrapie, which occurs naturally in sheep and goats. Since the onset of the bovine spongiform encephalopathy (BSE) epidemic in British cattle (Bos taurus) in 1986, novel SEs emerged in other animal species including domestic cats (1), a goat (2), primates (3), and several members of the families Bovidae and Felidae in zoos (4,5). Experimental and epidemiologic evidence indicate that these animals were infected by ingesting BSE-infected carcasses or meat and bone meal. Zebus (B. indicus) belong to the family Bovidae. In Asia they are raised mainly as productive livestock, but in Europe they live primarily in zoos. We describe clinical, pathologic, and molecular features of the first case of SE in a zebu and address the question whether this animal was infected with the BSE agent. The Study In 2004, a 19-year-old miniature zebu in a zoo in Basel, Switzerland, fell during mating, after which it had abnormal gait and posture. After 6 weeks it started to bump into obstacles and showed anxiety and loss of proprioceptive control. Because of its old age and the progressive course of the disease, the animal was euthanized, and multiple organs were examined postmortem . Histopathologic examination showed severe spongiform changes and a moderate gliosis in the brainstem (Figure 1A, nucleus of the solitary tract), and many other CNS structures. Immunohistochemical analysis (6), which used the monoclonal antibodies (MAbs) F99/97.6.1 (VMRD, Pullman, WA, USA) and P4 (R-biopharm, Darmstadt, Germany), identified a marked deposition of PrPsc in the neuropil (granular type) and the neurons (Figure 1B and 1C). The cerebral cortex contained a moderately increased number of Alzheimer type II cells. Numerous nonnervous tissues, including the lymphoreticular system (Figure 1H), were analyzed by immunohistochemical techniques for the presence of PrPsc, but none was found. Taken together, these findings led to the diagnosis of a severe SE in combination with a mild metabolic encephalopathy. To assess the possibility that this animal was infected with the BSE agent, we compared the distribution of the SE-related histopathologic lesions and the PrPsc deposits in different brain structures of the zebu to those in the brain of a Swiss BSE-affected cow. In both animals, spongiform lesions were similarly distributed throughout the brain, but overall the lesions in the zebu were more severe than those in the cow . The depositions of PrPsc in these structures, as determined by immunohistochemical analysis with MAb F99/97.6.1 and different commercial BSE screening tests (Check Western, Prionics, Zurich, Switzerland; TeSeE, Bio-Rad, Marnes-la-Coquette, France), were well associated with the histopathologic lesions in both animals (data not shown). In comparative Western immunoblot (WB) analysis that used MAb 6H4 (Prionics), the zebu CNS samples (Figure 2, lanes 3 and 5) showed a characteristic 3-band pattern representing un-, mono- and diglycosylated moieties of the proteinase K–resistant PrPsc fragment. In the zebu these 3 bands clearly showed a migration pattern at a higher molecular mass than that of BSE in the cow (Figure 2, lanes 4 and 6) but similar to a sample from a sheep with scrapie (Figure 2, lane 7). When samples of the same animals were analyzed by WB and immunohistochemical analysis (Figure 1C–E) with P4, an MAb used to discriminate between BSE and scrapie in sheep (7), PrPsc was detectable in samples from the sheep with scrapie and the zebu under investigation but not in the cow with BSE. Sequencing of the open reading frame of the Prnp gene of the zebu confirmed that the encoded PrP protein was identical to the B. taurus PrP amino acid sequence (as translated from GenBank accession no. AJ298878). Conclusions In 1990, the first case of BSE in cattle in Switzerland was diagnosed; since then, authorities have banned meat and bone meal in ruminant feed in Switzerland. The zebu was born in 1985 and until 1990 ate commercial pellets containing meat and bone meal. Consequently, it might have been exposed to the BSE agent at <5 years of age. The clinical signs of the zebu were specific for an SE but could have been explained partially by other pathologic findings, e.g., the degenerative lesions of the spine and the metabolic encephalopathy . However, prominent spongiform changes and marked depositions of PrPsc in the brain confirmed the clinical suspicion of an SE. The distribution and type of the lesions and PrPsc deposits in the brain of the zebu were very similar to those in the brain of the Swiss BSE-affected cow and to findings that have been described previously for BSE in cattle in Switzerland (8,9) and elsewhere (10–12). In contrast, molecular analysis of PrPsc clearly showed a difference between the zebu and the BSE cow regarding 1) the apparent molecular mass of the PK-resistant fragment of PrPsc on WB analysis and 2) the immunoreactivity with MAb P4 on WB and immunohistochemical analyses. Both observations can be explained by extended proteinase K cleavage at the N terminus of PrPsc in cattle compared with the zebu, resulting in removal of the P4 epitope (7). Recently, very similar molecular findings were reported from France (13) in 3 exceptionally old (8, 10, and 11 years) cattle. These animals had an atypical PrPsc WB profile, different from that traditionally seen in cattle with BSE but indistinguishable from those in sheep with natural scrapie and cattle with experimental scrapie. This molecular phenotype was retained after transmission of the disease to C57BL/6 mice (14). The authors speculated that their findings may reflect either an infection with another type of infectious agent distinct from BSE, e.g., scrapie, or a sporadic form of SE in cattle. For the zebu, the latter hypothesis is supported by the observation that the molecular features of PrPsc were similar to the ones observed in type 1 sporadic Creutzfeldt-Jakob disease (15), an SE in humans. On the other hand, consistent with the findings on WB, MAb P4 readily detected PrPsc by immunohistochemical analyses of the CNS of the zebu and in sheep with scrapie but not in bovine BSE under the conditions used. Extracellular and intracellular PrPsc was detected by MAb P4 in the zebu and the sheep with scrapie. By contrast, in BSE-affected sheep, PrPsc was detected by MAb P4 in extracellular but not intracellular space (16). However, further investigations that use comparative pathology and biologic strain typing would be required to characterize the phenotype of SE in this zebu and the infectious agent in more detail. Whatever the origin of the disease, this case indicates that zebus are not naturally resistant to SE and, therefore, that B. indicus should be included in programs that monitor transmissible spongiform encephalopathies (TSEs) and in risk assessments in countries where these animals are part of the domestic livestock. Although the potential for this disease to cross the species barrier to other animals and humans is not known, zoos and veterinary services should be aware of the possibility of SEs in such animals so they can subsequently minimize the risk for foodborne SE infections in other animal species (especially Felidae) and humans by removing specified risk materials. Acknowledgments We thank Doris Ambühl, Valerie Juillerat, and Christoph Prisi for excellent technical support. This work was financed with resources provided by the Swiss Federal Veterinary Office. Dr Seuberlich is a veterinarian and head of the TSE rapid test division in the Swiss reference laboratory for TSE in animals. His major research interests include the diagnosis and molecular characterization of emerging TSE agents. References Wyatt JM, Pearson GR, Smerdon TN, Gruffydd-Jones TJ, Wells GA. Spongiform encephalopathy in a cat [letter]. Vet Rec. 1990;126:513. Eliot M, Adjou KT, Coulpier M, Fontaine JJ, Hamel R, Lilin T, et al. BSE agent signatures in a goat [letter]. Vet Rec. 2005;156:523–4. Erratum in Vet Rec. 2005;156:620. Bons N, Mestre-Frances N, Belli P, Cathala F, Gajdusek DC, Brown P. Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents. Proc Natl Acad Sci U S A. 1999;96:4046–51. Heim D, Geiser F, Perler L, Wyss R. Beyond BSE: Transmissible spongiform encephalopathies in other animal species [Article in German]. Schweiz Arch Tierheilkd. 2002;144:664–73. Kirkwood JK, Cunningham AA. Epidemiological observations on spongiform encephalopathies in captive wild animals in the British Isles. Vet Rec. 1994;135:296–303. Organisation Mondiale de la Sante Animale. Manual of diagnostic tests and vaccines for terrestrial animals. 5th ed. 2004. [cited 2006 Oct 11]. Available from http://www.oie.int Stack MJ, Chaplin MJ, Clark J. Differentiation of prion protein glycoforms from naturally occurring sheep scrapie, sheep-passaged scrapie strains (CH1641 and SSBP1), bovine spongiform encephalopathy (BSE) cases and Romney and Cheviot breed sheep experimentally inoculated with BSE using two monoclonal antibodies. Acta Neuropathol (Berl). 2002;104:279–86. Fatzer R, Graber HU, Meyer RK, Cardozo C, Vandevelde M, Zurbriggen A. Neuronal degeneration in brain stem nuclei in bovine spongiform encephalopathy. Zentralbl Veterinarmed A. 1996;43:23–9. Graber HU, Meyer RK, Fatzer R, Vandevelde M, Zurbriggen A. In situ hybridization and immunohistochemistry for prion protein (PrP) in bovine spongiform encephalopathy (BSE). Zentralbl Veterinarmed A. 1995;42:453–9. Casalone C, Caramelli M, Crescio MI, Spencer YI, Simmons MM. BSE immunohistochemical patterns in the brainstem: a comparison between UK and Italian cases. Acta Neuropathol (Berl). 2006;111:444–9. Simmons MM, Harris P, Jeffrey M, Meek SC, Blamire IW, Wells GA. BSE in Great Britain: consistency of the neurohistopathological findings in two random annual samples of clinically suspect cases. Vet Rec. 1996;138:175–7. Wells GA, Wilesmith JW. The neuropathology and epidemiology of bovine spongiform encephalopathy. Brain Pathol. 1995;5:91–103. Biacabe AG, Laplanche JL, Ryder S, Baron T. Distinct molecular phenotypes in bovine prion diseases. EMBO Rep. 2004;5:110–5. Baron TG, Biacabe AG, Bencsik A, Langeveld JP. Transmission of new bovine prion to mice. Emerg Infect Dis. 2006;12:1125–8. Parchi P, Castellani R, Capellari S, Ghetti B, Young K, Chen SG, et al. Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease. Ann Neurol. 1996;39:767–78. Jeffrey M, Martin S, Gonzalez L, Ryder SJ, Bellworthy SJ, Jackman R. Differential diagnosis of infections with the bovine spongiform encephalopathy (BSE) and scrapie agents in sheep. J Comp Pathol. 2001;125:271–84....查看详细 (11685字节)
☉ 11342774:Febrile Illness Associated with Rickettsia conorii Infection in Dogs from Sicily
Clinica e Laboratorio Veterinario Privato "San Marco," Padova, Italy North Carolina State University, Raleigh, North Carolina, USA Clinica Veterinaria, Catania, Italy Abstract We report serologic and molecular evidence of acute, febrile illness associated with Rickettsia conorii in 3 male Yorkshire terriers from Sicily (Italy). Rickettsia conorii, transmitted by Ripicephalus sanguineus, causes Mediterranean spotted fever (MSF) in humans in Mediterranean countries, Sub Saharan Africa and Asia (1). Rickettsia spp. seroprevalence in dogs is high (26%–60%) in disease-endemic regions, and proximity to seroreactive dogs is a risk factor for MSF in humans (2,3). Recent studies reported the detection of Rickettsia DNA in the blood of European dogs (4,5). However, evidence that R. conorii infection causes illness in dogs is lacking (2,3,6). Illness has been associated with R. conorii natural infection in only 2 dogs since human MSF was described in 1932 (6). Moreover, the only clinical signs observed in experimentally infected dogs were pain, erythema, and edema at the injection site; and regional lymphadenopathy (6). We report infection with R. conorii ssp. conorii in 3 acutely ill, febrile Yorkshire terrier dogs, supported by PCR, DNA sequencing, and seroconversion. The Study Between May and September 2005, three unrelated intact male Yorkshire terriers with a mean age of 4.3 years from Catania, Sicily, were brought to a local veterinarian; the dogs had the following histories: anorexia and lethargy of 2 days' duration (dog 1); anorexia, lethargy, and intermittent lameness of a few days' duration (dog 2); and intermittent vomiting, anorexia, and lethargy of a few days' duration (dog 3). Despite living mostly indoors, all 3 dogs had a recent history of tick exposure. All dogs had received current vaccination histories and had no history of serious illness. Results of the physical examination and hematologic, biochemical, and serum electrophoresis abnormalities at the time of onset of clinical signs and after 1 month (dogs 2 and 3) and 2 months (dog 1) of follow-up are provided in Table 1. Treatments instituted for all 3 dogs at onset of illness are described in Table 1. EDTA-blood and serum samples were obtained by the attending veterinarian at the time of clinical assessment (before treatment), then 1 week later and 1 month (dogs 2 and 3) or 2 months later (dog 1). DNA extraction was performed from whole blood samples (5,7). A quantitative PCR (qPCR) for detection of Rickettsia spp., Anaplasma phagocytophilum, Ehrlichia canis, and Leishmania infantum in DNA samples was performed by using a Light Cycler (Roche, Mannheim, Germany). PCR amplification was carried out with Rickettsia (Rr-prim3 5′-GAAACCGAAAGAGAATCTTCCGAT-3′ and Rr-prim4 5′-TCCTAGTGTAGAGGTGAAATTCTTA-3′ [8]), E. canis, A. phagocytophilum (fragment of 16S rRNA gene), and L. infantum LCSet primers and probes following manufacturer's instructions (TIB Molbiol, Centro Biotecnologie Avanzate, Genova, Italy) (5,7). Conventional Babesia genus PCR was performed (9). Borrelia burgdorferi sensu lato qPCR was performed by a commercial laboratory (http://www.scanelis.com). PCR results for all infectious agents listed above, with the exception of Rickettsia, were negative in all dogs. PCRs for Rickettsia that use the outer membrane protein A (ompA) gene to amplify 632 bp (10) and 212 bp (107F 5′-GCTTTATTCACCACCTCAAC-3′ and 299R 5′-TRATCACCACCGTAAGTAAAT-3′) (7) amplicons were performed. For dog 1, a 632-bp amplicon was cloned by using the TOPO TA Cloning (Invitrogen, Carlsbad, CA, USA) and sequenced (GenBank accession no. DQ518245) (7). For dogs 2 and 3, a 212-bp amplicon was subjected to direct sequencing (accession no. DQ518246, DQ518247) (7). PCR results are summarized in Table 1. Consensus sequences were aligned [(BIOEDIT version 7.0 (ClustalW)] with known sequences in GenBank using the basic local alignment search tool (BLAST; available from http://www.ncbi.nlm.nih.gov/BLAST/). The sequence obtained from all 3 dogs was 100% homologous to a portion of the complete genome sequence corresponding to the ompA gene from R. conorii (Malish 7, accession no. AE008674). Immunofluorescent assays to detect antibodies to R. rickettsii, R. conorii, B. burgdorferi sensu stricto, E. canis, Babesia canis, A. phagocytophilum, L. infantum, Bartonella henselae, and B. vinsonii ssp. berkhoffi antigens were performed (3,7). Results are presented in Table 2. Conclusions Clinicopathologic abnormalities detected in these dogs at initial examination, including acute onset of fever, lethargy, thrombocytopenia, anemia, mildly increased liver enzyme activities and hypoalbuminemia, were very similar to abnormalities associated with spotted fever group (SFG) rickettsioses in dogs and humans (1). In addition, R. conorii DNA was amplified in all dogs during the acute illness. Further evidence for R. conorii infection as a cause of the associated clinical signs was provided by the subsequent failure to detect DNA in dogs 1 and 2, 1 week after treatment with doxycycline and the rapid resolution of clinical signs 2 days after initiating doxycycline therapy. Clinical signs in dog 3 resolved in 4 days, while the dog was receiving ceftriaxone, which has no known anti-rickettsial efficacy (1). Spontaneous immune clearance of R. conorii likely accounted for the resolution of clinical signs in dog 3 (6). The 4-fold increase in IgG antibody titers in dogs 2 and 3 supports seroconversion, which is consistent with an acute R. conorii infection (11). Additionally, the initially high IgM titer in dog 1 after the onset of illness compared with a much lower IgM titer after 65 days is also supportive of an acute infection and is consistent with observations of human serologic test results (1). IgM titers rise rapidly and then disappear by day 35 and 80 in dogs experimentally infected with R. conorii and R. rickettsii, respectively (6,11). However, high R. rickettsii IgM titers are detected in dogs that do not seroconvert, based upon IgG antibodies (11). Thus, the presence of IgM supports but does not prove acute SFG infection in dogs. Coinfection with A. phagocytophilum or B. burgdorferi could have contributed to clinical signs observed in dog 1. This dog had a low serum A. phagocytophilum titer 7 days after initial examination and also seroconverted to B. burgdorferi. A. phagocytophilum causes an acute febrile illness in dogs and humans, similar to the findings described here (12). B. burgdorferi does not cause clinical signs in dogs until 60–150 days after experimental infection (13); therefore, despite seroconversion, the acute clinical signs in dog 1 were not likely to have been related to B. burgdorferi infection. Moreover, PCR amplification of DNA from organisms other than R. conorii was not found in any dog. All dogs were intact, male, genetically unrelated Yorkshire terriers. Although an increased risk for Rocky Mountain spotted fever has not been reported in Yorkshire terriers, purebred dogs infected with R. rickettsii appear to be more prone to clinical illness (14). Notably, this breed seems to be at increased risk for Babesia canis infection (15). Male dogs and men may be at increased risk for infection and may develop more severe illness with R. rickettsii and R. conorii (1,14), and male dogs are more likely to be R. conorii seroreactive (3). It has been suggested that more severe illness may develop in English springer spaniels with suspected phosphofructokinase deficiency and persons with glucose 6-phosphate dehydrogenase deficiency when infected with R. rickettsii and R. conorii (1,14). Although inherited immunodeficiencies have not been reported in Yorkshire terriers, and all dogs were previously healthy, an inherited metabolic or immunologic defect cannot be ruled out because specific testing was not performed. Although a metabolic or immunologic defect may be necessary for illness to develop in dogs of various breeds after R. conorii infection, other potential explanations can be made for the discrepancy between high R. conorii seroprevalence rates among healthy dogs and lack of reports of clinical illness. The high R. conorii seroprevalence in healthy dog populations suggests that exposure to SFG rickettsiae is common. However, the acute, nonspecific, and potentially self-limiting nature of R. conorii infection, combined with a low index of suspicion by regional veterinarians and a historical lack of specific diagnostic techniques, may have precluded the prior association of clinical signs with R. conorii infection in dogs. Further evidence should be gathered regarding the extent to which R. conorii causes clinical disease in dogs. Acknowledgments We thank Silvia Beccaro, Julie Bradley, and Matthew Poore for serologic testing, Claudia Zampieron for molecular testing, and Barbara Hegarty for helpful discussions. Dr Solano-Gallego is a veterinarian at the Private Veterinary Hospital and Laboratory San Marco (Padua, Italy). Her primary research interests include the study of vectorborne zoonotic diseases of dogs and cats. References Parola P, Paddock CD, Raoult D. Tick-borne rickettsioses around the world: emerging diseases challenging old concepts. Clin Microbiol Rev. 2005;18:719–56. Segura-Porta F, Diestre-Ortin G, Ortuno-Romero A, Sanfeliu-Sala I, Font-Creus B, Munoz-Espin T, et al. Prevalence of antibodies to spotted fever group rickettsiae in human beings and dogs from an endemic area of Mediterranean spotted fever in Catalonia, Spain. Eur J Epidemiol. 1998;14:395–8. Solano-Gallego LJ, Osso M, Hegarty B, Breitschwerdt E. A serological study of exposure to arthropod-borne pathogens in dogs from northeastern Spain. Vet Res. 2006;37:231–44. Estrada-Pena A, Venzal Bianchi J. Efficacy of four anti-tick chemicals to break the transmission of Rickettsia conorii to dogs. In: Abstracts of the Fourth International Conference on Rickettsiae and Rickettsial Diseases, Logroo, Spain; 2005 Jun 18–21. P-100. Solano-Gallego L, Razia L, Trotta T, Furlanello T, Caldin M. Molecular survey of Ehrlichia canis, Anaplasma phagocytophilum and Rickettsia spp. from blood of dogs living in Italy. In: Abstracts of the Fourth International Conference on Rickettsiae and Rickettsial Diseases, Logroo, Spain; 2005 Jun 18–21. P-107. Kelly PJ, Matthewman LA, Mason PR, Courtney S, Katsande C, Rukwava J. Experimental infection of dogs with a Zimbabwean strain of Rickettsia conorii. J Trop Med Hyg. 1992;95:322–6. Kidd LB. Evaluation of a PCR assay for detection of spotted fever group Rickettsia in dog blood. North Carolina State University; 2006 [PhD dssertation]. Breitschwerdt EB, Papich MG, Hegarty BC, Gilger B, Hancock SI, Davidson MG. Efficacy of doxycycline, azithromycin, or trovafloxacin for treatment of experimental Rocky Mountain spotted fever in dogs. Antimicrob Agents Chemother. 1999;43:813–21. Carret C, Walas F, Carcy B, Grande N, Precigout E, Moubri K, et al. Babesia canis canis, Babesia canis vogeli, Babesia canis rossi: differentiation of the three subspecies by a restriction fragment length polymorphism analysis on amplified small subunit ribosomal RNA genes. J Eukaryot Microbiol. 1999;46:298–303. Roux V, Fournier PE, Raoult D. Differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCR-amplified DNA of the gene encoding the protein rOmpA. J Clin Microbiol. 1996;34:2058–65. Breitschwerdt EB, Levy MG, Davidson MG, Walker DH, Burgdorfer W, Curtis BC, et al. Kinetics of IgM and IgG responses to experimental and naturally acquired Rickettsia rickettsii infection in dogs. Am J Vet Res. 1990;51:1312–6. Neer TM, Breitschwerdt EB, Greene RT, Lappin MR. Consensus statement on ehrlichial disease of small animals from the infectious disease study group of the ACVIM. American College of Veterinary Internal Medicine. J Vet Intern Med. 2002;16:309–15. Greene CE, Straubinger RK. Borreliosis. In: Green C, ed. Infectious diseases of the dog and cat. 3rd ed. Philadelphia: Saunders, Elsevier; 2006. p. 417–34. Greene CE, Breitschwerdt EB. Rocky Mountain spotted fever, murine typhuslike disease, rickettsialpox, typhus, and Q fever. In: Greene C, editor. Infectious diseases of the dog and cat. 3rd ed. Philadelphia: Saunders, Elsevier; 2006. p. 232–45. Martinod S, Laurent N, Moreau Y. Resistance and immunity of dogs against Babesia canis in an endemic area. Vet Parasitol. 1986;19:245–54....查看详细 (12673字节)
☉ 11342775:Genotyping of Chlamydophila psittaci in Human Samples
University of Amsterdam, Amsterdam, the Netherlands Saint Antonius Hospital, Nieuwegein, the Netherlands VU University Medical Center, Amsterdam, the Netherlands Abstract Chlamydophila (formerly Chlamydia) psittaci genotypes A, B, C, and a new genotype most similar to the 6BC type strain were found in 10 humans with psittacosis by outer membrane protein A gene sequencing. Genotypes B (n = 3) and C (n = 1) are endemic in nonpsittacine European birds. These birds may represent an important part of the zoonotic reservoir. Psittacosis is a zoonosis caused by infection with Chlamydophila (formerly Chlamydia) psittaci, an obligate intracellular bacterium. C. psittaci is divided into 8 serovars (A–F, M56, and WC) and at least 9 genotypes. Sequence analysis of the outer membrane protein A (ompA) gene is the most accurate method for identifying all known genotypes (1). All genotypes are associated with specific bird groups from which they are predominantly isolated (2,3). The prevalence of different genotypes of C. psittaci that cause infection in humans is unknown. In this study, we genotyped all C. psittaci PCR-positive human samples available in our laboratory. The Study Ten human samples positive for C. psittaci DNA in our previously described real-time PCR assay were characterized by ompA gene sequencing (4). These samples were collected from 2002 through 2005 and included 4 sputum, 4 bronchoalveolar lavage, 1 throat swab, and 1 serum. All samples were obtained from symptomatic psittacosis patients admitted to hospitals in the Netherlands. All patients had pneumonia, and 6 required treatment in an intensive care unit. The DNA of 1 outbreak strain, which infected as many as 10 people, was tested only once in this study. One of the samples was obtained from a patient who has been previously described (5). DNA purification was performed by using the guanidinium thiocyanate-silica–extraction procedure (6). Genotyping was performed essentially as previously described (7). Briefly, a part of the ompA gene was amplified with primers CPsittGenoFor (5′-GCT ACG GGT TCC GCT CT-3′) and CPsittGenoRev (5′-TTT GTT GAT YTG AAT CGA AGC-3′). These primers are located in the conserved regions of the ompA gene that contains the 4 variable domains (VDs). On the basis of published ompA sequence of the C. psittaci 6BC type strain (GenBank accession no. X56980), the resulting amplicon should have a size of 1,041 bp. PCR products were analyzed by agarose gel electrophoresis, and the expected 1,041-bp amplicon was excised from the gel. DNA was extracted from the gel and reamplified for 20 cycles, and amplicons were analyzed for size by agarose gel electrophoresis. C. psittaci ORNI (genotype A) strain and a C. abortus strain were used as positive controls. Calf thymus DNA was used as a negative control. If the ompA gene could not be amplified with this procedure, a nested PCR with primers CPsittFinner (5′-CGC TCT CTC CTT ACA AGC C-3′) and CPsittRinner (5′-GAT CTG AAT CGA AGC AAT TTG-3′) was used. The amplified product (n = 8) or the nested PCR product (n = 2) were subjected to sequence analysis (BigDye Terminator, version 1.1, cycle sequencing kit, Applied Biosystems, Foster City, CA, USA). Overlapping sequences were obtained with 6 sequencing primers: CPsittGenoFor and CPsittGenoRev, inner primers CPsittFinner and CPsittRinner, and primers CPsittHFor (5′-TCT TGG AGC GTR GGT GC-3′) and CPsittHRev (5′-GCA CCY ACG CTC CAA GA-3′). The resulting sequences were aligned, and a similarity index based on the translation of the 984-bp gene fragment was calculated. Similarity (1 – distance) was calculated by using the pairwise distance method generated by MEGA3 (8). Reference ompA genotype sequences A–F and the ompA sequence of the C. psittaci 6BC type strain available in GenBank (accession nos. AY762608–AY762612, X56980, and AF269261) were included in this analysis (1,9). All 10 isolates could be genotyped. The ompA sequence of 5 isolates was identical to the sequence of reference genotype A, 3 isolates were identical to genotype B, and the ompA sequence of 1 isolate was identical to genotype C. One isolate had a novel ompA sequence type that was 99.4% similar to the genotype A reference but more similar to the C. psittaci 6BC type strain (99.7%). Two nonsynonymous mutations were present in this sequence compared with reference genotype A. A substitution of thymine for adenine in VD 1 resulted in Ser instead of Thr at amino acid position 92 of the ompA amino acid sequence, which is identical to that found in genotype C. A substitution of cytosine for guanine, also located in VD 1, resulted in Gln instead of Glu at amino acid position 117, as found in genotype B and strain 6BC (numbering according to the ompA amino acid sequence of the C. psittaci 6BC strain, GenBank accession no. X56980). We designated this new variant C. psittaci 05/02 and deposited the sequence in GenBank (accession no DQ324426). Two genotype B strains, 3 genotype A strains, and the novel genotype 05/02 strain were obtained from patients admitted to an intensive care unit. Conclusions To our knowledge, ours is the first report of a series of genotyped C. psittaci strains isolated from symptomatic, hospitalized patients. These 10 samples reflect approximately one third of all cases reported each year in the Netherlands (10). From the genotypes that we identified, we may infer the zoonotic reservoirs of C. psittaci in the Netherlands. The different genotypes of C. psittaci are associated, although not exclusively, with different birds from which they are mostly isolated. Genotype A is mainly found in psittacine birds and was the most prevalent genotype in our samples (1,3). C. psittaci 05/02 was most related to C. psittaci 6BC and the reference genotype A (strain VS1). Both reference strains have been classified as serovar A strains. On the basis of 2 restriction fragment length polymorphism patterns, Sayada et al. suggested that serovar A should be divided into 2 genogroups (2). Our isolate 05/02 is a new ompA gene sequence variant within this probably heterogeneous group. Genotype B has been isolated mainly from feral pigeons and several other bird species; this genotype is considered endemic in European nonpsittacine birds (3,11). Genotype C has been isolated mainly from ducks, and we detected this genotype in 1 of our human samples. We did not find genotype D, which is prevalent among poultry, especially turkeys, or genotypes E and F. These last 2 genotypes are rare and found occasionally in birds (1,11). Imported psittacine birds, which carry mainly genotype A, have been proposed as the major source for human psittacosis infections (12). In our study, 4 of 10 isolates were genotypes B and C, which are rarely found in psittacine birds. This finding suggests that nonpsittacine birds may represent an underestimated source for human psittacosis cases. We detected isolates of genotypes A, B, C, and a new genotype similar to the C. psittaci 6BC strain in a series of 10 C. psittaci–positive samples. Genotypes B and C are endemic in European nonpsittacine birds, which may represent an important part of the zoonotic reservoir for human psittacosis cases. Acknowledgments The research was conducted at the Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands. Dr Heddema is a medical microbiologist at the Academic Medical Center, University of Amsterdam. His research interests are psittacosis and development of new diagnostic tools for detecting fastidious organisms. References Geens T, Desplanques A, van Loock M, Bonner BM, Kaleta EF, Magnino S, et al. Sequencing of the Chlamydophila psittaci ompA gene reveals a new genotype, E/B, and the need for a rapid discriminatory genotyping method. J Clin Microbiol. 2005;43:2456–61. Sayada C, Andersen AA, Storey C, Milon A, Eb F, Hashimoto N, et al. Usefulness of omp1 restriction mapping for avian Chlamydia psittaci isolate differentiation. Res Microbiol. 1995;146:155–65. Vanrompay D, Andersen AA, Ducatelle R, Haesebrouck F. Serotyping of European isolates of Chlamydia psittaci from poultry and other birds. J Clin Microbiol. 1993;31:134–7. Heddema ER, Beld MG, de Wever B, Langerak AA, Pannekoek Y, Duim B. Development of an internally controlled real-time PCR assay for detection of Chlamydophila psittaci in the LightCycler 2.0 system. Clin Microbiol Infect. 2006;12:571–5. Heddema ER, Kraan MC, Buys-Bergen HE, Smith HE, Wertheim-Van Dillen PM. A woman with a lobar infiltrate due to psittacosis detected by polymerase chain reaction. Scand J Infect Dis. 2003;35:422–4. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-Van Dillen PM, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990;28:495–503. Heddema ER. ter Sluis S, Buijs JA, Vandenbroucke-Grauls CM, van Wijnen JH, Visser CE. Prevalence of Chlamydophila psittaci in fecal droppings from feral pigeons in Amsterdam, The Netherlands. Appl Environ Microbiol. 2006;72:4423–5. Kumar S, Tamura K, Nei M. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform. 2004;5:150–63. Bush RM, Everett KD. Molecular evolution of the Chlamydiaceae. Int J Syst Evol Microbiol. 2001;51:203–20. National Institute of Public Health and the Environment (RIVM). Notified cases of infectious diseases in the Netherlands. Dutch Infectious Diseases Bulletin. 2004;15:30. Vanrompay D, Butaye P, Sayada C, Ducatelle R, Haesebrouck F. Characterization of avian Chlamydia psittaci strains using omp1 restriction mapping and serovar-specific monoclonal antibodies. Res Microbiol. 1997;148:327–33. Wreghitt TG, Taylor CE. Incidence of respiratory tract chlamydial infections and importation of psittacine birds. Lancet. 1988;1:582....查看详细 (10029字节)
☉ 11342776:Community-associated Methicillin-resistant Staphylococcus aureus and Healthcare Risk Factors
Centers for Disease Control and Prevention, Atlanta, Georgia, USA University of California, Berkeley, California, USA Connecticut Department of Health, Hartford, Connecticut, USA Colorado Emerging Infections Program, Denver, Colorado, USA Grady Memorial Hospital, Atlanta, Georgia, USA Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA Minnesota Department of Health, Minneapolis, Minnesota, USA University of Rochester, Rochester, New York, USA Oregon Health Science University, Portland, Oregon, USA Tennessee Department of Health, Nashville, Tennessee, USA Abstract To determine frequency of methicillin-resistant Staphylococcus aureus infections caused by strains typically associated with community-acquired infections (USA300) among persons with healthcare-related risk factors (HRFs), we evaluated surveillance data. Of patients with HRFs, 18%–28% had a "community-associated" strain, primarily USA300; of patients without HRFs, 26% had a "healthcare-associated" strain, typically USA100. In the United States, initial reports of methicillin-resistant Staphylococcus aureus (MRSA) infections among injection drug users in Detroit in 1981 were followed by reports of MRSA associated with the deaths of 4 children in Minnesota and North Dakota in 1997 (1). For the next few years, public health personnel in several states investigated outbreaks of MRSA infections of skin and soft tissue among diverse populations who typically had little or no previous contact with the healthcare system, such as Native Americans (2), sports teams (3), prison inmates (4), and child-care facility attendees (5). These outbreaks were initially associated with a novel MRSA strain known as MW2, or pulsed-field gel electrophoresis (PFGE) type USA400, but were soon replaced by a strain of MRSA belonging to PFGE type USA300 (6). Through 2002, the clinical appearance of cases and the microbiologic characteristics of USA300 and USA400 differed substantially from those associated with strains of MRSA acquired in healthcare settings (7). Increasingly, MRSA strains of community origin are causing healthcare-associated disease (8,9). We evaluated surveillance data from a multisite project to determine the frequency with which infections among patients with healthcare-related risk factors (HRFs) were caused by USA300 or other strains of community origin. The Study Active, population-based surveillance for invasive MRSA infections is ongoing in 9 US states (California, Colorado, Connecticut, Georgia, Maryland, Minnesota, New York, Oregon, and Tennessee) through the Active Bacterial Core Surveillance system in the Emerging Infections Program at the Centers for Disease Control and Prevention (CDC). Personnel in each state actively collect laboratory reports of positive MRSA cultures from normally sterile sites (e.g., blood; cerebrospinal, joint, or pleural fluid) of residents in their catchment areas to identify cases. In 2005, the estimated combined population under surveillance was 16.3 million, according to data from the US Bureau of the Census. To report a case, personnel must link a laboratory report to the patient's medical record. During record reviews, personnel abstract information about the following HRFs: culture obtained >48 hours after admission; presence of an invasive device (e.g., vascular catheter, G-tube); and history of MRSA infection or colonization, surgery, hospitalization, dialysis, or residence in a long-term care facility in the 12 months preceding the culture. Case-patients may have >1 HRF. For this analysis, we used information from the record review to classify cases into 3 mutually exclusive groups: 1) case-patients with classic healthcare-associated infections (HA) whose culture was obtained >48 hours after admission with or without other HRFs; 2) case-patients with HRFs but with community onset (i.e., whose cultures were obtained <48 hours after admission) (HACO); and 3) case-patients with community-associated (CA) infections without HRFs, according to medical record review. A subset of isolates from case-patients was collected from laboratories that voluntarily submitted them for microbiologic characterization. Of the isolates received at CDC by October 2005, a sample of 100 was selected for testing as follows. First, isolates were stratified by Emerging Infections Program site; none were available from Maryland. Second, all isolates from tissues other than blood were selected from each Emerging Infections Program site. To ensure 12–13 isolates per site, we selected blood isolates from case-patients classified as CA and obtained the remainder from samples from HA and HACO case-patients. Isolates were tested by PFGE; patterns were analyzed by using BioNumerics (Applied Maths, Austin, TX, USA). Isolates were grouped into PFGE types using Dice coefficients and 80% relatedness (10). We considered isolates with PFGE types USA300, 400, or 1000 to be of community origin and those with types USA100, 200, and 500 to be of healthcare origin as previously described (10). Statistical analysis consisted of comparisons of proportions between CA and HA and between CA and HACO cases using χ2 pairwise comparisons. Differences in median age were tested by using Wilcoxon rank sum test. Of 9,147 cases of invasive MRSA infection investigated from January 2004 through February 2006, 2,535 (28%) were HA, 5,353 (59%) were HACO, and 1,259 (14%) were CA. The median age of case-patients with HA and HACO was significantly higher than that of case-patients with CA . CA case-patients were 1) more likely to have pneumonia than HACO but not HA case-patients; 2) more likely to have endocarditis than either HA or HACO case-patients; and 3) less likely to die during this hospital stay than were HA or HACO case-patients. Of the 100 isolates selected for initial testing, 29 were from HA case-patients, 44 were from HACO case-patients (including 1 isolate of a unique PFGE type), and 27 were from CA case-patients (including 1 isolate that could not be typed) . Of the HA isolates, 8 (28%) were USA300. Of the HACO isolates, 6 (14%) were USA300, 1 (2%) was USA400, and 1 (2%) was USA1000. Thus, 18%–28% of isolates in patients with HRFs (HA and HACO) had PFGE patterns typical of community strains. Of the 27 isolates from CA case-patients, 5 (19%) were USA100 and 2 (7%) were USA500; thus, 7 (26%) of isolates among CA case-patients were strains typically considered to be of healthcare origin. Conclusions MRSA strains such as USA300, which were initially a cause of MRSA infections in the community, have migrated into healthcare settings. The results from this multisite project are consistent with observations from individual facilities, where USA300 isolates caused illness in patients whose infection was healthcare associated (11,12). Although age and frequency of endocarditis still differed between case-patients with HRFs (HA and HACO) and those without HRFs (CA), PFGE testing indicated that 18%–28% of patients with HRFs were infected with a "community-associated" strain of MRSA, primarily USA300. Furthermore, 26% of patients without HRFs had a "healthcare-associated" strain, typically USA100. Thus, the distinction between healthcare- and community-associated MRSA is rapidly blurring. Acknowledgments We are indebted to Rachel Gorwitz and Jeff Hageman for their review of the report and to Roberta Carey, Jean Patel, and Sigrid McAllister for their guidance and contributions to laboratory testing of isolates. Dr Klevens is a medical epidemiologist at CDC. She is the CDC principal investigator in a multistate project that measures methicillin-resistant Staphylococcus aureus in the population, and she provides epidemiologic support to the National Healthcare Safety Network. References Centers for Disease Control and Prevention. Four pediatric deaths from community-acquired methicillin-resistant Staphylococcus aureus—Minnesota and North Dakota, 1997–1999. JAMA. 1999;282:1123–5. Baggett HC, Hennessy TW, Rudolph K, Bruden D, Reasonover A, Parkinson A, et al. Community-onset methicillin-resistant Staphylococcus aureus associated with antibiotic use and the cytotoxin Panton-Valentine leukocidin during a furunculosis outbreak in rural Alaska. J Infect Dis. 2004;189:1565–73. Centers for Disease Control and Prevention. Methicillin-resistant Staphylococcus aureus infections among competitive sports participants—Colorado, Indiana, Pennsylvania, and Los Angeles County, 2000–2003. MMWR Morb Mortal Wkly Rep. 2003;52:793–5. Centers for Disease Control and Prevention. Public health dispatch: outbreaks of community-associated methicillin-resistant Staphylococcus aureus skin infections—Los Angeles County, California, 2002–2003. JAMA. 2003;289:1377. Adcock PM, Pastor P, Medley F, Patterson JE, Murphy TV. Methicillin-resistant Staphylococcus aureus in two child care centers. J Infect Dis. 1998;178:577–80. Tenover FC, McDougal LK, Goering RV, Killgore G, Projan SJ, Patel JB, et al. Characterization of a strain of community-associated methicillin-resistant Staphylococcus aureus widely disseminated in the United States. J Clin Microbiol. 2006;44:108–18. Fridkin SK, Hageman JC, Morrison M, Sanza LT, Como-Sabetti K, Jernigan JA, et al. Methicillin-resistant Staphylococcus aureus disease in three communities. N Engl J Med. 2005;352:1436–44. Erratum in: N Engl J Med. 2005;352:2362. Healy CM, Hulten KG, Palazzi DL, Campbell JR, Baker CJ. Emergence of new strains of methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit. Clin Infect Dis. 2004;39:1460–6. Saiman L, O'Keefe M, Graham PL III, Wu F, Said-Salim B, Kreiswirth B, et al. Hospital transmission of community-acquired methicillin-resistant Staphylococcus aureus among postpartum women. Clin Infect Dis. 2003;37:1313–9. McDougal LK, Steward CD, Killgore GE, Chaitram JM, McAllister SK, Tenover FC. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J Clin Microbiol. 2003;41:5113–20. Seybold U, Kourbatova EV, Johnson JG, Halvosa SJ, Wang YF, King MD, et al. Emergence of community-associated methicillin-resistant Staphylococcus aureus USA300 genotype as a major cause of health care-associated blood stream infections. Clin Infect Dis. 2006;42:647–56. Huang H, Flynn NM, King JH, Monchaud C, Morita M, Cohen SH. Comparisons of community-associated methicillin-resistant Staphylococcus aureus (MRSA) and hospital-associated MSRA infections in Sacramento, California. J Clin Microbiol. 2006;44:2423–7....查看详细 (10794字节)
☉ 11342777:Drug Trafficking Routes and Hepatitis B in Injection Drug Users, Manipur, India
ICMR Virus Unit, Kolkata, India Jadavpur University, Kolkata, India National Institute of Cholera and Enteric Diseases, Kolkata, India Abstract Prevalence of hepatitis B genotype C in injection drug users in the northeastern Indian state of Manipur, neighboring the "Golden Triangle," correlates well with overland drug-trafficking routes, the injection drug use epidemic, and the spread of HIV. Further spread to other regions of India through mobile populations is possible. Injection drug use is common in countries neighboring the "Golden Triangle" (Myanmar, Laos, and Thailand), known for heroin export to other countries. HIV and injection drug use outbreaks in countries neighboring the Golden Triangle, including the northeastern Indian state of Manipur, have been associated with drug-trafficking routes (1). Manipur shares a 358-km porous border with Myanmar. According to the National AIDS Control Organization, India (http://www.nacoonline.org), HIV infection rates among injection drug users in Manipur increased from 2%–3% in 1989 to >50% in 1991 and ≈64% in 2000. In addition, exposure rates of 100% for hepatitis B virus (HBV) and 92% for hepatitis C virus (HCV) have been detected among injection drug users in Manipur, and 95% of wives of injection drug users had HBV exposure (2). However, among both the users and their wives, prevalence of HBV genotypes and occult HBV infection (3), a known risk factor for hepatocellular carcinoma (HCC) (4), remained unknown. HBV is classified into 8 genotypes, HBV/A through HBV/H, and is further divided into subgenotypes (5) that have a distinct geographic distribution and are associated with different disease outcomes. The geographic distribution of HBV is known to correlate with the anthropologic history of migration (5) and to the origin and routes of spread of HBV infection. In addition, behavioral patterns are known to change HBV genotype distribution in a region (6). Manipur is an important location, where mainland India (prevalent genotypes HBV/A, HBV/D) geographically meets China and Southeast Asia (prevalent genotypes HBV/B, HBV/C). Our study was designed to detect HBV DNA among injection drug users in Manipur and to analyze HBV genotypes for correlation with injection drug use and the HIV epidemic. The Study We examined HIV-positive injection drug users from Manipur who had been identified as anti-HBc–positive during previous serosurveys conducted by the National Institute of Cholera and Enteric Diseases (2). Serum samples (stored at –80°C) taken from 63 men 18–25 years of age were available for the study. HBsAg detection was repeated with a monoclonal antibody–based Hepanostika hepatitis B surface antigen (HBsAg) kit (bioMerieux, Marcy l'Etoile, France). Anti-HCV antibody was detected using the Ortho HCV 3.0 test (Ortho-Clinical Diagnostics, Raritan, NJ, USA). We completed HBV DNA isolation, PCR amplification, genotype/subgenotype/subtype identification, recombination detection, and HBV DNA quantification with methods described earlier (7–9). We also compared nucleotide (Appendix Table) and deduced amino acid sequences (Table) with consensus sequence of amino acids of corresponding genotypes to detect substitutions (GenBank accession nos. DQ356432–DQ356441). All those tested were HBsAg negative, and only 10 (15.9%) had detectable HBV DNA. Anti-HCV was detected in all but 1 sample (no. 4). Serum HBV DNA level was detectable in 5 of 10 samples (Table); the rest were below the detection limit of our assay. HBV/C (all subtype adr except 1 adw2) was the predominant genotype, with 4 subgenotype C1 (HBV/C1) and 3 subgenotype C2 (HBV/C2) isolates. HBV/A1 adw2 was found in 1 of the isolates. Two other isolates (nos. 1 and 3, subtype adw2) indicated a possibility of intergenotypic recombination , but subgenotype could not be assigned for them. BLASTN search (http://www.incogen.com/public_documents/vibe/details/NcbiBlastn.html) of sequence from sample no. 1 showed similarity to HBV/D as well as to HBV/A sequences from European countries. On the other hand, sequences from sample no. 3 showed similarity to HBV/C and HBV/A sequences from Southeast Asian countries and India. Simplot analysis confirmed recombination in these 2 isolates . Apart from the genotype-specific substitutions, deduced amino acid sequences did not have any remarkable escape mutant other than G145R in 2 isolates (Table). Conclusions The data from this study showed occult HBV infection in 15.9% of the injection drug users tested. The rate of HBV DNA detection (10%–45%) was considerably different in studies reported from different cohorts of injection drug users in different countries, a finding that has been attributed to coinfection with HCV or low HBV DNA levels (3). Apart from HIV, our study group had a high frequency of HCV infection and low HBV DNA levels. Undetectable HBsAg, except in 2 cases with G145R substitution, may also be a result of the above-mentioned factors. Although the importance of occult HBV infection is not well understood, a recent study reported occult HBV to be a significant risk factor for HCC, especially among persons who were anti-HBc–positive (4). Another recent study among HIV-infected patients documented death due to liver disease in 22% who were HBV coinfected, 44% who were HCV/HBV coinfected, and 15% who were HBV coinfected and had HCC (10). Thus, the clinical relevance in our study group also needs to be followed. Although we detected a 100% prevalence of anti-HBc in our serosurveys (2), only 1 was HBsAg-positive. Therefore, the distribution of genotypes among those who were HBsAg positive could not be determined. Findings of HBV/C1 (prevalent in China) appear to support the history of human migration from China to northeastern India. However, we did not detect HBV/B, also prevalent in China. Further, we did detect HBV/C2, which has close similarity to strains from Southeast Asian countries. The presence of HBV/C correlated well with drug-trafficking routes and the injection drug use epidemic. The geographic proximity of Manipur to the Golden Triangle, needle sharing among injection drug users, and drug traders thus contributed to the spread of HBV through drug-trafficking routes, similar to HIV (1). In Manipur, HIV subtypes C and Thai B are prevalent (11); these are also prevalent in the India-Myanmar and China-Myanmar border regions. The presence of similar HIV subtypes among injection drug users in Manipur supports the presence of similar HBV strains (e.g., HBV/C1, HBV/C2) and their cotransmission through drug-trafficking routes. In addition, circulation of recombinant HBV is common among injection drug users because of repeated exposure (12). The intergenotypic HBV recombinants found in this study are thus expected. HBV/C has been associated with advanced liver disease and poses a higher risk for HCC in Asians (13) than does HBV/B. However, the clinical relevance of HBV recombinants and their pathogenesis is not well understood and needs further investigation. Poor, unemployed youths in the northeastern states of India are being recruited for drug trafficking to other regions (http://www.ipcs.org). Furthermore, national highways are associated with the prevalence of injection drug use in rural Manipur (14). As these highways connect Manipur with other parts of India, HBV/C may spread from Manipur to other parts of the country through persons who travel regularly, such as truck drivers and drug traffickers. Presence of HBV genotypes A and D among patients from northern and western India is well documented. Recent research reported HBV/C with close similarity to Southeast Asian strains only from eastern India (9,15) and suggested injection drug users as a possible route of introduction (15). In addition, persons from northeastern India frequent Kolkata for education, employment, medical treatment, and other purposes. Studies on these mobile populations might provide further important information on the route, population at risk for infection, and changing epidemiology of these viral infections in other regions. In conclusion, HBV/C, associated with severe liver disease in Southeastern Asia, may be emerging in the Manipur state of India through the trafficking routes of injection drugs. This genotype could spread to the general population through different modes. In light of growing information on the severity of liver disease in HBV-infected HIV/HCV patients, injection drug users should be the focus of additional education and healthcare efforts. The possibility of further spread of HIV/HBV/HCV through mobile populations to other regions of India warrants attention and further investigation. Acknowledgments We thank Tapan Kumar Chakrabarti for technical assistance. The University Grants Commission, New Delhi, India, and Indian Council of Medical Research, New Delhi, provided research fellowships. Mr Datta is senior research fellow at the ICMR Virus Unit, Kolkata, India. His research interests include the molecular epidemiology of HBV, its transmission among family members, genetic variability in the HBs/HBx gene regions, and studies on HBV DNA in extrahepatic sites in persons with HIV/HCV coinfection. References Beyrer C, Razak MH, Lisam K, Chen J, Lui W, Yu XF. Overland heroin trafficking routes and HIV-1 spread in south and south-east Asia. AIDS. 2000;14:75–83. Saha MK, Chakrabarti S, Panda S, Naik TN, Manna B, Chatterjee R, et al. Prevalence of HCV and HBV infection amongst HIV seropositive intravenous drug users and their non-injecting wives in Manipur, India. Indian J Med Res. 2000;111:37–9. Torbenson M, Kannangai R, Astemborski J, Strathdee SA, Vlahov D, Thomas DL. High prevalence of occult hepatitis B in Baltimore injection drug users. Hepatology. 2004;39:51–7. Pollicino T, Squadrito G, Cerenzia G, Cacciola I, Raffa G, Crax A, et al. Hepatitis B virus maintains its pro-oncogenic properties in the case of occult HBV infection. Gastroenterology. 2004;126:102–10. Norder H, Courouce AM, Coursaget JME, Lee SD, Mushahwar IK, Robertson BH, et al. Genetic diversity of hepatitis B virus strains derived worldwide: genotypes, subgenotypes and HBsAg subtypes. Intervirology. 2004;47:289–309. Kidd-Ljunggren K, Miyakawa Y, Kidd AH. Genetic variability in hepatitis B viruses. J Gen Virol. 2002;83:1267–80. Datta S, Banerjee A, Chandra PK, Chowdhury A, Chakravarty R. Genotype, phylogenetic analysis, and transmission pattern of occult hepatitis B virus (HBV) infection in families of asymptomatic HBsAg carriers. J Med Virol. 2006;78:53–9. Lole KS, Bollinger RC, Paranjape RS, Gadkari D, Kulkarni SS, Novak NG, et al. Full length human immunodefficiency virus type 1 genomes from subtype C infected seroconverters in India, with evidence of intersubtype recombination. J Virol. 1999;73:152–60. Banerjee A, Kurbanov F, Datta S, Chandra PK, Tanaka Y, Mizokami M, et al. Phylogenetic relatedness and genetic diversity of hepatitis B virus isolates in Eastern India. J Med Virol. 2006;78:1164–74. Salmon-Ceron D, Lewden C, Morlat P, Bevilacqua S, Jougla E, Bonnet F, et al. The Mortality 2000 Study Group. Liver disease as a major cause of death among HIV infected patients: role of hepatitis C and B viruses and alcohol. J Hepatol. 2005;42:799–805. Mandal D, Jana S, Bhattacharya SK, Chakrabarti S. HIV type 1 subtypes circulating in eastern and northeastern regions of India. AIDS Res Hum Retroviruses. 2002;18:1219–27. Chen BF, Kao JH, Liu CJ, Chen DS, Chen PJ. Genotypic dominance and novel recombinations in HBV genotype B and C coinfected intravenous drug users. J Med Virol. 2004;73:13–22. Chan HLY, Hui AY, Wong ML, Tse AML, Hung LCT, Wong VWS, et al. Genotype C hepatitis B virus infection is associated with an increased risk of hepatocellular carcinoma. Gut. 2004;53:1494–8. Sarkar K, Panda S, Das N, Sarkar S. Relationship of national highway with injecting drug abuse and HIV in rural Manipur, India. Indian J Public Health. 1997;41:49–51. Vivekanandan P, Abraham P, Sridharan G, Chandy G, Daniel D, Raghuraman S, et al. Distribution of hepatitis B virus genotypes in blood donors and chronically infected patients in a tertiary care hospital in southern India. Clin Infect Dis. 2004;38:e81–6....查看详细 (12461字节)
☉ 11342778:Chikungunya Disease Outbreak, Reunion Island
Institut de Veille Sanitaire, Saint Maurice, France Cire de la Reunion, Saint Denis, France Chikungunya is a self-limiting febrile viral disease characterized by arthralgia or arthritis. Symptoms may last for several months, but recovery was, until now, considered universal (3). However, in January 2006, the health authorities on this island started receiving death certificates mentioning chikungunya as a cause of death, either direct or indirect. By the end of April, 213 death certificates with this finding had been received. To assess the affect of chikungunya disease, we compared the crude death rate (CDR) observed during the outbreak period with an expected death rate computed from the 2002–2004 historical data. The study included the period January 1, 2005, through April 30, 2006. The expected number of deaths (all causes) for 2005 and 2006 was the number of deaths by sex and age observed during 2002–2004 modified by an estimation of the population size for 2005–2006. The details of this method, which was used during the heat wave in France in 2003, have been reported (4). The number of deaths in Reunion was obtained daily from 13 of 24 computerized registry offices throughout the island and represented 87% of the deaths on the island. During 2005, the monthly CDR remained within expected range of statistical variation. From January through April 2006, respectively, monthly CDRs were 7.1%, 34.4%, 25.2%, and 8.3% higher than expected rates (p75 years of age. CDRs began to exceed the expected range during the last week of January 2006 and remained elevated until the end of the study period. This situation closely matched the kinetics of the epidemic curve of chikungunya disease. CDR is a stable variable in time for a defined population. Only a massive phenomenon can have an effect on it, and no other abnormal health event affected the island at this time. Thus, the outbreak of chikungunya disease was likely responsible for most of the excess deaths observed in Reunion during the first 4 months of 2006. Deaths associated with chikungunya disease have been rarely reported. This outbreak in Reunion is the first with such a high incidence in a setting where real-time death reporting is a standard procedure. In such settings, CDR monitoring should be considered syndromic surveillance and should be implemented when an abnormal health phenomenon affects large populations. This work was supported by the French National Institute for Public Health Surveillance. References Enserink M. Infectious diseases. Massive outbreak draws fresh attention to little-known virus. Science. 2006;311:1085. Paganin F, Borgherini G, Staikowsky F, Arvin-Berod C, Poubeau P. Chikungunya on Reunion Island: chronicle of an epidemic foretold. Presse Med. 2006;35:641–6. Mackenzie JS, Chua KB, Daniels PW, Eaton BT, Field HE, Hall RA, et al. Emerging viral diseases of Southeast Asia and the Western Pacific. Emerg Infect Dis. 2001;7(3 Suppl):497–504. Pirard P, Vandentorren S, Pascal M, Laaidi K, Le Tertre A, Cassadou S, et al. Summary of the mortality impact assessment of the 2003 heat wave in France. Euro Surveill. 2005;10:153–6....查看详细 (3710字节)
☉ 11342779:Assay to Detect H5N1 Oseltamivir Resistance
Chulalongkorn University, Bangkok, Thailand National Institute of Animal Health, Bangkok National Institute of Hygiene and Epidemiology, Hanoi, Vietnam Although real-time PCR or pyrosequencing is more rapid than high-throughput assays for mutation detection (4,5), the conventional PCR technique can be applied to detect drug-resistant mutation (6) in areas lacking real-time PCR or pyrosequencing capabilities. Therefore, to discriminate between oseltamivir-sensitive and oseltamivir-resistant strains, we developed a simple method, based on PCR, which takes advantage of the H274Y substitution. The forward primer was designed from the conserved region common to both wild-type and mutant strains; the reverse primers were designed specifically for wild-type and mutant strains, respectively, derived from the 3′ terminal base of each primer. The primers consisted of a forward primer N1f (nt 517-534: 5′-GGGGCTGTGGCTGTATTG-3′) and reverse primer H274r (nt 759-784: 5′-GGATAACAGGAGCAYTCCTCATAGTG -3′) for wild-type strain detection or Y274r (nt 759-784: 5′-GGATAACAGGAGCAYTCCTCATAGTA-3′) for mutant strain detection. Both strains yielded products of ≈267 bp; hence, the assay consisted of 2 separate reactions for detecting wild-type and mutant strains, respectively. For each reaction, 1.0 μL cDNA was combined with a reaction mixture that contained 10 μL 2.5× MasterMix (Eppendorf, Hamburg, Germany), forward and reverse primers at a final concentration of 0.15 μM, and nuclease-free water to a final volume of 20 μL. Thermocycling conditions comprised initial denaturation at 94°C for 3 min and 35 cycles of amplification including denaturation (94°C, 30 s), annealing (65°C, 50 s), extension (72°C, 45 s), and final extension (72°C, 7 min). Subsequently, 10 μL of the amplified products was analyzed by using 2% agarose gel electrophoresis. To optimize the assay, we performed PCR-based H274Y mutagenesis of the N1 fragment of the H5N1 virus (primers on request). The resulting mutagenic and wild-type products were cloned into the pGEM-T Easy Vectors (Promega, Madison, WI, USA), confirmed by direct sequencing, and then used as positive controls. Preliminary results showed that the wild-type primer was specific for the oseltamivir-sensitive strain, whereas the mutant primer can be used to detect the oseltamivir-resistant strain exclusively because no significant cross-amplification had been observed. To establish sensitivity, serial 10-fold dilutions of the standard N1 plasmids (wild-type and mutant) ranging from 109 to 101 copies/μL were used as a template. The threshold concentrations for detection of wild-types and mutants were 103 copies/μL. To provide semiquantitative data to detect subpopulations of the resistant variants, the 2 control plasmids were mixed at wild-type:variant ratios of 108:102, 107:103, 106:104, 105:105, 104:106, 103:107, and 102:108. The result showed that the density of the expected bands depended on the amount of DNA templates (Figure, B). However, the mixing experiments indicated that the predominant mixtures of wild-type:resistant variant were 80:20, which is the lowest ratio of resistant variants that the assay can reliably detect (data not shown). To assess specificity, human DNA and viral cDNA extracted from other subtypes of influenza A virus (N2–N9) were subjected to this assay. No cross-reaction occurred with human DNA or other subtypes of influenza A virus. We further validated the assay by testing 3 specimens from hosts treated with oseltamivir and 17 specimens from untreated hosts; infection with H5N1 was detected by using multiplex real-time PCR (7). The specimens from oseltamivir recipients were isolated from a Vietnamese patient on the third day after he received a prophylactic dose (1) and from 2 tigers (CU-T7 and KU-11) (8). The specimens from untreated hosts were isolated from the plasma of an H5N1-infected human (9) and from virus-containing allantoic fluid isolated from infected chickens, ducks, and cats (n = 16) during a 2005 outbreak in Thailand. The specimen isolated from the Vietnamese patient yielded detectable bands after amplification by wild-type and mutant primer sets, which indicates that this specimen contains mixed populations of wild-type and resistant strains (Figure, A). The result was confirmed by cloning the amplicon of the Vietnamese strain into the pGEM-T EASY vector (Promega). Ten colonies were randomly picked and sequenced; 9 clones were mutant, and 1 was wild type. The other specimens produced a visible positive band only on amplification using the wild-type primer set, which indicates that samples containing these strains were oseltamivir sensitive. The assay described here provides an accurate, cost-effective, and highly efficient approach to detecting oseltamivir-sensitive and oseltamivir-resistant H5N1 strains; it is based on conventional PCR and takes advantage of the H274Y substitution within the neuraminidase gene. This simple technique can be applied in all laboratories that lack more advanced and expensive instruments; thus, it provides a valuable tool for managing and preventing influenza A H5N1 outbreaks. Concerning the spread of mutant viruses, however, the fitness of the viruses needs further investigation. Acknowledgments We thank Surangrat Srisuratanon for her assistance, Petra Hirsch for critically reviewing the manuscript, and Roche Diagnostics, Thailand, for providing mutagenesis primers and a plasmid purification kit. This study was supported by the Thailand Research Fund (Senior Research Scholar), Royal Golden Jubilee PhD Program, Center of Excellence in Viral Hepatitis Research, and Chulalongkorn University. References Le QM, Kiso M, Someya K, Sakai YT, Nguyen TH, Nguyen KH, et al. Avian flu: isolation of drug-resistant H5N1 virus. Nature. 2005;437:1108. de Jong MD, Tran TT, Truong HK, Vo MH, Smith GJ, Nguyen VC, et al. Oseltamivir resistance during treatment of influenza A (H5N1) infection. N Engl J Med. 2005;353:2667–72. Ward P, Small I, Smith J, Suter P, Dutkowski R. Oseltamivir (Tamiflu) and its potential for use in the event of an influenza pandemic. [Review]. J Antimicrob Chemother. 2005;55(Suppl 1):i5–21. Brown IH. Advances in molecular diagnostics for avian influenza. Dev Biol (Basel). 2006;124:93–7. Bright RA, Shay DK, Shu B, Cox NJ, Klimov AI. Adamantane resistance among influenza A viruses isolated early during the 2005–2006 influenza season in the United States. JAMA. 2006;295:891–4. Klimov AI, Rocha E, Hayden FG, Shult PA, Roumillat LF, Cox NJ. Prolonged shedding of amantadine-resistant influenza A viruses by immunodeficient patients: detection by polymerase chain reaction-restriction analysis. J Infect Dis. 1995;172:1352–5. Payungporn S, Chutinimitkul S, Chaisingh A, Damrongwantanapokin S, Buranathai C, Amonsin A, et al. Single step multiplex real-time RT-PCR for H5N1 influenza A virus detection. J Virol Methods. 2006;131:143–7. Amonsin A, Payungporn P, Theamboonlers A, Thanawongnuwech R, Suradhat S, Pariyothorn N, et al. Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand. Virology. 2006;344:480–91. Chutinimitkul S, Bhattarakosol P, Srisuratanon S, Eiamudomkan A, Kongsomboon K, Damrongwatanapokin S, et al. H5N1 influenza A virus and infected human plasma. Emerg Infect Dis. 2006;12:1041–3....查看详细 (7477字节)
☉ 11342780:Laboratory Exposure to Influenza A H2N2, Germany, 2004–2005
Robert Koch Institute, Berlin, Germany We used a standardized questionnaire to obtain from the laboratories information on when the CAP samples had been received, which of the 3 quality assurance specimens they contained, and how many employees had been involved in their handling. A second questionnaire was distributed to personnel in microbiology and virology departments. This elicited information regarding routine laboratory activities, contact with CAP samples, tasks performed in conjunction with handling of the samples, and any influenzalike symptoms (sudden onset of fever, cough, headache, and muscle pain) within the respective time frame. Persons who had worked in a receiving laboratory from September 1, 2004, to April 15, 2005, and had performed routine procedures in virology (defined as transport of samples, preparation of samples for diagnostic testing, antigen testing, nucleic acid amplification testing, and virus isolation) were eligible for the study. From May 4 to May 19, 2005, we visited the laboratories to interview supervisory personnel regarding routine work-up of samples and to collect blood from study participants for serologic testing. The national reference laboratory for influenza at Robert Koch Institute performed serologic testing for antibodies against A/Singapore/1/57(H2N2) virus by hemagglutination inhibition. We compared antibody titers of laboratory workers who worked with a CAP sample with those who did not. However, this comparison might not show a difference if (silent) virus transmission had occurred among laboratory staff. To exclude this possibility, we also compared titers of workers born before 1969 with those in a group of volunteers from Robert Koch Institute also born before 1969. Titers 3 symptoms typical for influenzalike illness in the 4 days after having worked with a CAP sample. Specific influenza A H2N2 antibody titers were determined in 25 study participants. None had a titer >80, two (8%) had a titer of 80, three (12%) had titers of 40, two (8%) had titers of 20, and the remaining 18 (72%) had titers 20. All 7 were born before 1969. The geometric mean of titers of all participating workers born before 1969 did not differ significantly from that of the Robert Koch Institute employees (p = 0.28; Figure). In summary, no evidence was found for laboratory infections with the influenza A H2N2 virus. The risk for laboratory-acquired influenza infections is unknown. Severe acute respiratory syndrome coronavirus and Mycobacterium tuberculosis are infectious agents whose transmission characteristics are similar to those of influenza. For severe acute respiratory syndrome coronavirus, laboratory-acquired infections are well documented (3,4). For M. tuberculosis, there are strong indications that they occur (5–7). From a public health perspective and in view of the current importance given to influenza and a possible pandemic, accurate characterization of the risk for influenza infections in laboratory settings is needed. The small number of persons included in this study limits the conclusions that can be drawn. Potentially exposed workers were presumably tested in other laboratories involved, but we are not aware of any publications to this end. The lack of evidence for laboratory-acquired infection with A H2N2 in our study suggests that the risk was low under controlled laboratory conditions. However, only a large-scale serologic study (which might still feasibly be undertaken) could further substantiate this finding. Acknowledgments We thank all assisting public health professionals and study participants for their support. References Eurosurveillance editorial team, Meijer A, Brown C, Paget J. Worldwide laboratory distribution of influenza A/H2N2 virus similar to 1957–58 pandemic strain, labs asked to destroy all samples immediately. Eurosurveillance. 2005;10. [cited 2006 Sep 15]. Available from http://www.eurosurveillance.org/ew/2005/050414.asp World Health Organization. International response to the distribution of a H2N2 influenza virus for laboratory testing: risk considered low for laboratory workers and the public. 2005 [cited 2006 May 22]. Available from http://www.who.int/csr/disease/influenza/h2n2_2005_04_12/en/ Normile D. Mounting lab accidents raise SARS fears. Science. 2004;304:659–61. Lim PL, Kurup A, Gopalakrishna G, Chan KP, Wong CW, Ng LC, et al. Laboratory-acquired severe respiratory syndrome. N Engl J Med. 2004;350:1740–5. Seidler A, Nienhaus A. Epidemiologie der berufsbedingten tuberkulose: ein literaturreview zu konventionellen studien. In: Nienhaus A, Brandenburg S, Teschler H, editors. Tuberkulose als berufskrankheit: ein leitfaden zur begutachtung. Landsberg (Germany): Ecomed Verlag; 2003. p. 142–69. Jacobson JT, Orlob RB, Clayton JL. Infections acquired in clinical laboratories in Utah. J Clin Microbiol. 1985;21:486–9. Kao AS, Ashford DA, McNeil MM, Warren NG, Good RC. Descriptive profile of tuberculin skin testing programs and laboratory-acquired tuberculosis infections in public health laboratories. J Clin Microbiol. 1997;35:1847–51....查看详细 (6242字节)
☉ 11342781:Methicillin-resistant Staphylococcus aureus in Cat and Owner
San Francisco Veterinary Specialists, Inc., San Francisco, California, USA California Dermatopathology Service and IDEXX Veterinary Services, West Sacramento, California, USA University of Guelph, Guelph, Ontario, Canada Cytologic evaluation of exudate from the cat's lesions identified neutrophils and eosinophils with engulfed cocci. Leukocytosis with eosinophilia was found on a complete blood cell count. No notable abnormalities were present on thoracic radiograph, abdominal ultrasonograph, urinalysis, and tests for feline leukemia and immunodeficiency virus. Skin biopsy specimens were collected for histologic examination, and swabs of the exudates were submitted for bacterial culture. Histopathologic findings demonstrated ulcers and dermal granulation tissue with linearly arranged eosinophils, mast cells, neutrophils, and plasma cells between dense, homogeneous collagen bundles (sclerosing dermatitis). This pattern of inflammation is distinct from most staphylococcal infections of the skin, and it has been suggested that this uncommon histologic finding in cats is associated with methicillin-resistant staphylococcal infection (1). Methicillin-resistant Staphylococcus aureus (MRSA) was isolated from the skin lesions. Identification was confirmed by detection of penicillin-binding protein 2a (PBP2a) by latex agglutination test (PBP2′ Test Kit, Oxoid, Hants, UK). Typing was performed by SmaI pulsed-field gel electrophoresis as previously described (2), and the isolate was classified as the USA300 clone. Genes encoding production of the Panton-Valentine leukocidin (PVL) were identified by real-time PCR (3). The isolate was susceptible to chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole, and vancomycin, but resistant to β-lactams, enrofloxacin, and erythromycin. After identification of MRSA in the cat, swabs of the anterior nares were collected from the owner and the cat, and MRSA was identified in specimens from both. All isolates were indistinguishable. This is the first report of isolation of USA300 MRSA from a household pet. USA300 is a community-associated clone that has disseminated widely throughout North America and Europe (4,5) and is reaching epidemic proportions in many regions. MRSA is becoming an important cause of skin and soft tissue infection in persons in the community (4,5) and has also been implicated in invasive infections such as necrotizing pneumonia (6). This clone possesses genes for PVL production, which may be an important factor in its apparent virulence (4,5). Additional characterization of the isolates from this study was not performed; however, USA300 has previously been reported to carry staphylococcal cassette chromosome mec (SCCmec) type IVa and classified as sequence type 8 (ST8) by multilocus sequence typing (4,5). Reports of MRSA infection and colonization in pets have increased dramatically in the past few years (3,7–9). Although this rise may be partially the result of increased testing and reporting, MRSA is definitely emerging in pet populations throughout the world. The role of pets in transmission of MRSA is still unclear; however, recent evidence suggests that MRSA can be transmitted between persons and their pets, in both directions (9,10). Reports of MRSA infection and colonization in pets have indicated that pets tend to be infected with isolates that are consistent with clones that are predominant in the human population in their area (7–9). Accordingly, USA100 accounted for initial isolations of MRSA in pets in North America (9). The similarity between pet and human isolates has led to speculation that pet MRSA is closely linked to human MRSA and that the source of MRSA in pets may often be colonized humans. If this is the case, it is not surprising that USA300 would emerge as a cause of disease in pets as it increases in prevalence in the human population. Considering the rapid dissemination of USA300 in humans in the United States, particularly in California, where it is the predominant community-associated clone, finding USA300 in a household pet in that state is not unexpected. Because indistinguishable isolates were collected from the owner and the infected cat, MRSA likely was transmitted between species in the household. However, while it is tempting to assume that the owner was the source of infection because of the owner's previous history of a soft tissue infection, this cannot be definitively determined on the basis of the timing of sampling in this case. MRSA appears to be emerging as an important veterinary and zoonotic pathogen, and the epidemiology of MRSA in household pets may take a parallel course to that in humans. Ongoing MRSA surveillance in animals is required, including proper testing of specimens from clinically affected animals and surveillance for colonization. The potential for transmission of this clone between humans and pets should also be evaluated to clarify its epidemiology and to facilitate development of measures to reduce household transmission. References Ozaki K, Yamagami T, Nomura K, Haritani M, Tsutsumi Y, Narama I. Abscess-forming inflammatory granulation tissue with gram-positive cocci and prominent eosinophil infiltration in cats: possible infection of methicillin-resistant Staphylococcus. Vet Pathol. 2003;40:283–7. Mulvey MR, Chui L, Ismail J, Louie L, Murphy C, Chang N, et al. Development of a Canadian standardized protocol for subtyping methicillin-resistant Staphylococcus aureus isolates using pulsed-field gel electrophoresis. J Clin Microbiol. 2001;39:3481–5. Rankin S, Roberts S, O'Shea K, Maloney D, Lorenzo M, Benson CE. Panton Valentine leukocidin (PVL) toxin positive MRSA strains isolated from companion animals. Vet Microbiol. 2005;108:145–8. Roberts JC, Krueger RL, Peak KK, Veguilla W, Cannons AC, Amuso PT, et al. Community-associated methicillin-resistant Staphylococcus aureus epidemic clone USA300 in isolates from Florida and Washington. J Clin Microbiol. 2006;44:225–6. Tenover FC, McDougal LK, Goering RV, Killgore G, Projan SJ, Patel JB, et al. Characterization of a strain of community-associated methicillin-resistant Staphylococcus aureus widely disseminated in the United States. J Clin Microbiol. 2006;44:108–18. Frazee BW, Salz TO, Lambert L, Perdreau-Remington F. Fatal community-associated methicillin-resistant Staphylococcus aureus pneumonia in an immunocompetent young adult. Ann Emerg Med. 2005;46:401–4. Loeffler A, Boag AK, Sung J, Lindsay JA, Guardabassi L, Dalsgaard A, et al. Prevalence of methicillin-resistant Staphylococcus aureus among staff and pets in a small animal referral hospital in the UK. J Antimicrob Chemother. 2005;56:692–7. Rich M, Roberts L. Methicillin-resistant Staphylococcus aureus isolates from companion animals. Vet Rec. 2004;154:310. Weese JS, Dick H, Willey BM, McGeer A, Kreiswirth BN, Innis B, et al. Suspected transmission of methicillin-resistant Staphylococcus aureus between domestic pets and humans in veterinary clinics and in the household. Vet Microbiol. 2006;115:148–55. van Duijkeren E, Wolfhagen MJHM, Box ATA, Heck MEOC, Wannet JB, Fluit AC. Human-to-dog transmission of methicillin-resistant Staphylococcus aureus. Emerg Infect Dis. 2004;10:2235–6....查看详细 (7343字节)

☉ 11342782:Community-associated Methicillin-resistant Staphylococcus aureus, Colombia
Hospital Simon Bolívar, Bogotá D.C., Colombia Universidad Nacional, Bogotá D.C., Colombia Universidad El Bosque, Bogotá D.C., Colombia University of Texas Medical School at Houston, Houston, Texas, USA The first case was in a 19-year-old man with a history of trauma to the left side of his body 1 week before admission after a fall. On admission, he complained of 2 days of fever, malaise, erythema and induration in the left hemithorax extending to the left thigh, and purulent secretion from an excoriation on the anterior aspect of the left thigh. He had no previous medical history. No previous hospitalizations or antimicrobial drug prescriptions were documented, nor did he report relatives with history of recent hospitalizations. Vital signs at admission were normal except for fever (39°C), and physical examination showed induration and erythema in the region of left hemithorax extending to the thigh, with an area of excoriation in the same thigh with purulent discharge. Laboratory evaluation showed a leukocyte count of 23.1×109/L (86% neutrophils with 2% band forms) and elevated C-reactive protein levels. The patient was hospitalized. Because necrotizing fascitis was suspected, intravenous ampicillin-sulbactam (12 g per day) was started, and surgical evaluation was requested. The patient underwent surgical debridement of the left thigh, left hemiabdomen, and hemithorax, which confirmed the diagnosis of necrotizing fascitis. Intravenous vancomycin (1 g every 12 h) was added to the regimen, and the patient was transferred to the intensive care unit. After several surgical debridements, the patient underwent placement of cutaneous-muscle grafts. He was discharged from the hospital without complications after completing 14 days of antimicrobial agents. The second case involved a 53-year-old man with no history of previous hospitalizations who reported to the emergency department with a 3-day history of fever, pain, swelling, and warm sensation on the posterior aspect of the left thigh. A diagnosis of cellulitis was made, and cephalexin (500 mg every 6 h) and gentamicin (160 mg intramuscularly every 24 h) were administered for 7 days without improvement. He returned to the hospital with worsening symptoms, an area of induration of 4×4 cm in the thigh, and purulent discharge. Drainage of the lesion was performed, and oral trimethoprim and sulfamethoxazole (160 and 800 mg, respectively, every 12 h) was started. His clinical signs and symptoms completely resolved after 7 days of therapy. Tissue culture from secretions from both patients showed gram-positive cocci in clusters on the Gram stain, and subsequent cultures yielded MRSA. Species identification and presence of the mecA gene were confirmed by PCR, as described previously (6). MICs were determined by using the agar diffusion test, according to Clinical and Laboratory Standards Institute recommendations (7). Both organisms were susceptible to vancomycin, teicoplanin, chloramphenicol, linezolid, ciprofloxacin, gentamicin, and rifampin. The isolate from the second patient was resistant to erythromycin and susceptible to clindamycin, exhibited the M phenotype on the double-disk diffusion assay (D test), and harbored the msrA gene, encoding an efflux pump (8). In contrast, the first isolate was susceptible to both erythromycin and clindamycin and resistant to tetracycline (MIC >64 μg/mL). Because infections caused by CA-MRSA isolated elsewhere are associated with the presence of the lukF gene encoding the Panton-Valentine leukocidin toxin and the staphylococcal chromosome cassette mec (SCCmec) type IV, the presence of both was evaluated by PCR, as described previously (9). Both isolates were positive for lukF and harbored the SCCmec type IV. The molecular epidemiology of healthcare-related MRSA in Colombia has changed during the past 3 years (10), but no reports of CA-MRSA had emerged. We believe these to be the first reports of CA-MRSA in Colombia with similar characteristics to those reported elsewhere. No risk factors associated with healthcare-associated MRSA were found in either of these patients, and the patients were not epidemiologically related. The first case involved a severe soft-tissue infection associated with CA-MRSA. Clinicians should be aware of the circulation of CA-MRSA in Colombia. References Vandenesch F, Naimi T, Enrigth MC, Lina G, Nimmo GR, Herffernan H, et al. Community-acquired methicillin-resistant Staphylococcus aureus carrying Panton-Valentin leuckocidin genes: worldwide emergence. Emerg Infect Dis. 2003;9:978–84. Maltezou HC, Giamarellou H. Community-acquired methicillin-resistant Staphylococcus aureus infections. Int J Antimicrob Agents. 2006;27:87–96. Zetola N, Francis JS, Nuermberger EL, Bishai WR. Community-acquired methicillin-resistant Staphylococcus aureus: an emerging threat. Lancet Infect Dis. 2005;5:275–86. Ma XX, Galiana A, Pedreira W, Mowszovicz M, Christophersen I, Machiavello S, et al. Community-acquired methicillin-resistant Staphylococcus aureus, Uruguay. Emerg Infect Dis. 2005;11:973–6. Ribeiro A, Días C, Silva-Carvalho MC, Berquo L, Ferreira FA, Santos RN, et al. First report of infection with community-acquired methicillin-resistant Staphylococcus aureus in South America. J Clin Microbiol. 2005;43:1985–8. Martineau F, Francois J, Picarda LG, Paul H, Roya MO, Michel G; ESPRIT Trial. Multiplex PCR assays for the detection of clinically relevant antibiotic resistance genes in staphylococci isolated from patients infected after cardiac surgery. J Antimicrob Chemother. 2000;46:527–34. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Document M100-S15. Wayne (PA): The Institute; 2005. Leclercq R. Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications. Clin Infect Dis. 2002;34:482–92. Oliveira DC, de Lencastre H. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2002;46:2155–61. Cruz C, Moreno J, Renzoni A, Hidalgo M, Reyes J, Schienzel J. el al. Tracking methicillin-resistant Staphylococcus aureus clones in Colombian hospitals over 7 years (1996–2003): emergence of a new dominant clone. Int J Antimicrob Agents. 2005;26:457–62....查看详细 (6501字节)
☉ 11342783:Human African Trypanosomiasis Transmission, Kinshasa, Democratic Republic of Congo
Institute of Medical Research and Study of Medicinal Plants, Yaounde, Cameroon University of Yaounde I, Yaounde, Cameroon Programme National de Lutte contre la Trypanosomiase Humaine Africaine, Kinshasa, Democratic Republic of Congo Institut National de Recherches Biomedicales, Kinshasa, Democratic Republic of Congo Service de Cooperation et d'Action Culturelle de Kinshasa, Kinshasa, Democratic Republic of Congo Institut de Recherche pour le Developpement, Montpellier, France Abstract To investigate the epidemiology of human African trypanosomiasis (sleeping sickness) in Kinshasa, Democratic Republic of Congo, 2 entomologic surveys were conducted in 2005. Trypanosoma brucei gambiense and human-blood meals were found in tsetse fly midguts, which suggested active disease transmission. Vector control should be used to improve human African trypanosomiasis control efforts. Human African trypanosomiasis (HAT) (sleeping sickness) is a parasitic disease caused by a protozoan parasite belonging to the genus Trypanosoma. Approximately 60 million persons are exposed to the disease, and 500,000 are currently infected (1). HAT has been described as a disease affecting rural areas (2). During the recent increase in HAT in historic foci, emergence of foci with new epidemiologic features in urban areas was reported (3,4). Investigations of these new features showed that development of contiguous relationships between urban areas and surrounding HAT-endemic villages can create conditions favorable for HAT in urban areas (3–5). Few studies have suggested urban transmission of HAT despite potential epidemiologic consequences of such transmission (4). In Kinshasa, Democratic Republic of Congo, the epidemiologic situation for HAT is complex. In 1903, Dutton-Todd reported a HAT prevalence of 2.4% in apparently healthy inhabitants of Leopoldville (6). In 1960, the Kinshasa focus was considered extinct, and no tsetse flies were found in the city. Until 1995, an average of 50 new cases of HAT were reported annually. However, >200 new cases have been reported annually since 1996 (e.g., 443 of 6,205 persons examined in 1998 and 912 of 42,746 persons examined in 1999) (7). Ebeja et al. reported that 39% of new cases were urban residents; 60% of them in the first stage of the disease (3). To understand the epidemiology of HAT in this context, several investigations have been undertaken (3,5,8). On the basis of epidemiologic data, some investigators (3,5) have suggested that urban or periurban transmission of HAT occurs in Kinshasa. However, in a case-control study, Robays et al. concluded that HAT in urban residents of Kinshasa was linked to disease transmission in Bandundu and rural Kinshasa (8). To investigate the epidemiology of HAT transmission in Kinshasa, we identified and evaluated contact between humans and flies. The prevalence of Trypanosoma brucei gambiense in tsetse fly midguts was determined to identify circulation of this trypanosome between humans and tsetse flies. The Study Two entomologic surveys were conducted in 2005 (during the rainy season in February and March and the dry season in June and July) at 8 sites (Ndjili Cecomaf, Ndjili Brasserie, Kimwenza, Mambre, Funa, Buma, Kimbanseke, and Kinkole) in Kinshasa. These sites were selected on the basis of HAT prevalence and entomologic data previously reported (3). Rural, periurban, and urban areas (Figure) were defined according to recent mapping of Kinshasa (5). During each survey, tsetse flies were collected in pyramidal traps (9), and sex and species were identified. Midguts and blood meals were collected on filter paper, dried, and stored in microtubes. DNA was extracted from midguts or blood meals with 1 mL of 5% Chelex (10). Microtubes were incubated for 1 h at 56°C, 30 min at 100°C, and centrifuged for 10 min at 14,000 rpm. The supernatant was collected and used as DNA template for PCR. Blood meals were analyzed according to a method previously described (11). Trypanosomes were identified by PCR with specific primers for T. brucei s.l. (12). and T. b. gambiense (13). Because no tsetse flies were found in Buma and Kinkole during the rainy season, these 2 sites were excluded from the second survey (Figure). Entomologic data were analyzed for 610 traps from which data were obtained during the 2 surveys. A total of 897 flies of both sexes were caught; Glossina fuscipes quanzensis was the only tsetse fly species found (Table). Fresh midguts containing blood meals or trypanosomes were obtained from 570 living flies. In rural and periurban areas, 54 (9.5%, 95% confidence interval [CI] 3.7%–15.2%) teneral flies (young flies that have never taken a blood meal) were identified. Of 570 flies dissected, 117 (20.97%) had sampled blood meals and 110 were successfully identified (Table): 78 meals (67.7%, 95% CI 57.5%–75.9%) were taken from humans and 32 (27.3%, 95% CI 19%–36%) from pigs. PCR identified T. brucei s.l. in midguts of 54 (9.5%, 95% CI 7.5%– 11.5%) flies; of these, 54 flies, T. b. gambiense was found in 13. The prevalence of T. b. gambiense in tsetse midguts was 2.3% (95% CI 1.2%–3.3%). One tsetse fly with a blood meal from a pig was positive for T. b. gambiense. Conclusions This study confirmed, as reported in previous studies, the presence of G. f. quanzensis in Kinshasa (5). The most favorable biotopes for tsetse flies are located along the Ndjili, Lukaya, and Boye River valleys. The apparent density per fly was low and similar to the value previously reported (5). Pigsties and rivers were the most favorable biotopes for tsetse flies in Kinshasa. Variations in apparent density per trap between biotopes and capture sites are probably linked to climatic factors, the environment surrounding each trap, and urbanization. Increases in human population density attract tsetse flies to rural and periurban areas and concentrate tsetse flies in regions where contact between humans and flies is possible (14). Identification of tsetse flies infected with T. b. gambiense confirms contact between flies and patients. The infection rate (2.3%) in our study is comparable to the rates of 1.4% reported in Uganda and 1.9% in Brazzaville, Republic of Congo (15). However, T. b. gambiense midgut infection is not proof of mature infection, although it shows direct circulation of T. b. gambiense between humans and tsetse flies. Infection occurs frequently, as reflected by the feeding preference of G. f. quanzensis for humans. This finding shows that urbanization can increase transmission risk by creating conditions that may increase contact between humans and flies, as probably occurred in Kinshasa where we identified a high percentage of human-blood meals (67.7%) and tsetse flies infected with T. b. gambiense. Our results provide evidence for local transmission of HAT in Kinshasa because we detected T. b. gambiense midgut infections, human-blood meals, and most urban resident patients in the first stage of the disease. Identification of T. b. gambiense infections in flies from different sites indicates transmission in rural and periurban areas. Some patients identified in Kinshasa could have been infected during their movement through areas outside the city for subsistence activities and economic purposes. Local transmission has likely contributed to the increase in HAT in the past decade. The sites of Ndjili Brasserie, Kimwenza, Kimbanseke, and Mambre showed higher risk for HAT transmission. We suggest that vector control be integrated into improved HAT control efforts in urban areas. In Kinshasa, focused vector control activities around pigsties and places with water-related activities can reduce fly density, contact between humans and flies, and disease transmission. Contact between tsetse flies and pigs should encourage investigations of the animal reservoir of HAT in Kinshasa. This recommendation is strengthened by the finding of T. b. gambiense in a blood meal taken from 1 pig. Acknowledgments We thank T. Baldet for critically reading the manuscript. This work was supported by Institut de Recherche pour le Developpement (Unite de Recherche 177) and Service de Cooperation et d'Action Culturelle de Kinshasa. Dr Simo is senior researcher at the Medical Research Center of the Institute of Medical Research and Study of Medicinal Plants of the Ministry of Scientific and Innovation Research of Cameroon. His research interests include the genetic and molecular epidemiology and diagnosis of sleeping sickness. References Control and surveillance of African trypanosomiasis. Report of a WHO Expert Committee. World Health Organ Tech Rep Ser. 1998;881:1–114. Laveissiere C, Sane B, Meda HA. Measurement of risk in endemic areas of human African trypanosomiasis in Cte d'Ivoire. Trans R Soc Trop Med Hyg. 1994;88:645–8. Ebeja AK, Lutumba P, Molisho D, Kegels G. Miaka mia Belinge C, Boelaert M. Sleeping sickness in the region of the town of Kinshasa: a retrospective analysis during the surveillance period 1996–2000. Trop Med Int Health. 2003;8:949–55. Courtin F, Dupont S, Zeze DG, Jamonneau V, Sane B, Coulibaly B, et al. Human African trypanosomiasis: urban transmission in the focus of Bonon (Cte d'Ivoire). Trop Med Int Health. 2005;10:340–6. de Deken R, Sumbu J, Mpiana S, Mansinsa P, Wat'senga F, Lutumba P, et al. Trypanosomiasis in Kinshasa: distribution of the vector, Glossina fuscipes quanzensis, and risk of transmission in the peri-urban area. Med Vet Entomol. 2005;19:353–9. Fournet F, Traore S, Cadot E, Hervouet JP. Impact of development of agricultural land on the transmission of sleeping sickness in Daloa, Cte d'Ivoire. Ann Trop Med Parasitol. 2000;94:113–21. Louis FJ, Mia Bilengue CN, Simarro PP. Meso VK, Lucas P, Jannin J. Trypanosomose Humaine Africaine en milieu urbain: une problematique emergente Bull Soc Pathol Exot. 2003;96:205–8. Robays J, Ebeja AK, Lutumba P, Miaka Mia Belinge C, Kande Betu Ku Mesu V, de Deken R, et al. Human African trypanosomiasis amongst urban residents in Kinshasa: a case-control study. Trop Med Int Health. 2004;9:869–75. Gouteux JP, Lancien J. The pyramidal trap for collecting and controlling tsetse flies (Diptera: Glossinidae). Comparative trials and description of new collecting techniques. Trop Med Parasitol. 1986;37:61–6. Walsh PS, Metzger DA, Higuchi R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques. 1991;10:506–13. Njiokou F, Simo G, Mbida Mbida JA, Truc P, Cuny G, Herder S. A study of host preference in tsetse fly using a modified heteroduplex PCR based method. Acta Trop. 2004;91:117–20. Moser DR, Cook GA, Ochs DE, Bailey CP, McKane MR, Donelson JE. Detection of Trypanosoma congolense and Trypanosoma brucei subspecies by DNA amplification using polymerase chain reaction. Parasitology. 1989;99:57–66. Herder S, Simo G, Nkinin S, Njiokou F. Identification of trypanosomes in wild animals from southern Cameroon using the polymerase chain reaction (PCR). Parasite. 2002;9:345–9. Fournet F, Traore S, Hervouet JP. Effects of urbanization on the transmission of human African trypanosomiasis in a sub-urban relict forest area of Daloa, Cte d'Ivoire. Trans R Soc Trop Med Hyg. 1999;93:130–2. Rogers A, Kenyabjui EN, Wiggwah AK. A high infection rate of Trypanosoma brucei subgroup in Glossina fuscipes. Parasitology. 1972;65:143–6....查看详细 (11567字节)
☉ 11342784:Live Nativity and Brucellosis, Sicily
Dipartimento di Patologia Umana, Universita di Messina, Messina, Italy Dipartimento di Prevenzione Azienda Unita Sanitaria Locale 5, Messina, Italy Institute for Continuing Medical Education of Ioannina, Ioannina, Greece In 2003, health authorities in the Messina province were notified of 29 cases of brucellosis; 18 of the patients were members of 9 different families. All patients had observed a Nativity pantomime that used live animals and was organized by the local population. Nativities in Sicily last ≈1 month, during which the sheep are milked, cheese and ricotta are produced, and these products are sold or offered fresh to tourists. All 29 patients had consumed dairy products: tuma cheese by 29 (100%) and tuma and ricotta by 16 (55%). No other risk factors for brucellosis were reported. Symptoms appeared after a median of 45 days (range 30–70). Eight patients were children (3 male), and 21 were adults (10 male). The median age of the children was 10.5 years (range 6–13) and of the adults, 42 years (range 16–67). Hospitalization was required for 5 patients. For 2 adults, brucellosis was complicated by spondylitis. The real extent of the outbreak was likely large because in Sicily ≈60% of cases may go unreported. Furthermore, we report only the cases that occurred in the villages of Messina province and that were reported to health authorities; but tourists from many other areas in Italy and some from outside Italy generally attend such events. Southern Italy has commonly been implicated as a venue for travel-associated brucellosis (2). In Italy, the overall incidence of brucellosis has gradually declined in the past 30 years, especially in northern Italy, where the disease is now reported only sporadically. This trend, however, has led to an increase in the percentage of total cases in Italians reported from the southern provinces of Calabria, Campagna, Puglia, and Sicily; of the 520 cases reported in 2003, 488 (93.8%) were reported from 4 southern regions, compared with 63.7% in 1994. Sicily alone reported 57.6% of the 2003 cases and for the past decade has had an average annual incidence of >100 cases per million (1,3). The disease is almost always caused by Brucella melitensis (4). The southern localization of the disease in Italy is obviously related to the relative high prevalence of infections in sheep and goats (5). Ovine and caprine population density is higher in the southern regions of Italy than in the rest of the country (6). The Italian brucellosis eradication plan consists of a test-and-slaughter practice. However, in Sicily a vaccination campaign with B. melitensis Rev.1 strain has been started. In 2003, ≈99% of the stock farms were tested, and ≈18% of them were infected (3). The major problems reside with small flocks that undergo frequent transhumance (seasonal movement of herds between regions with different climates) in isolated regions where testing by veterinarians is difficult or avoided by the owners (a typical drawback of test-and-slaughter practices). Technically, ricotta is not a cheese, but rather is a cheese by-product. The name "ricotta" means cooked again, referencing the production method. Ricotta is made from whey drained from tuma, provolone, and other cheeses. Heat is then used to separate, by precipitation, the remaining albumin from the whey left after making lactic acid/rennet–precipitated cheeses. It is eaten as is or used for food seasoning (e.g., classic Italian lasagna and ravioli). A cream made of sieved ricotta and sugar is used to prepare many desserts, like cannoli and cassata cake. Being cooked 2 times, ricotta should not contain viable Brucella organisms; however, shepherds sprinkle fresh milk on wicker baskets to refresh the ricotta they contain, thereby contaminating the product. Tuma is a typical Sicilian fresh cheese made from sheep's milk. It has a cylindrical appearance and is sold fresh, no more than 2 days old. It has no crust, and the dough is white or ivory-white without holes. The texture is very soft, tender, and wet. It is generally served with ham, wines, and fruits as a table cheese. Tuma cheese should be considered as the major vehicle of B. melitensis infection in Sicily. Although most similar dairy products produced in Sicily are derived from organized dairy companies and have been pasteurized, traditional delicacies from small villages may still cause brucellosis outbreaks. References Pappas G, Papadimitriou P, Akritidis N, Christou L, Tsianos EV. The new global map of human brucellosis. Lancet Infect Dis. 2006;6:91–9. Chanet V, Gourdon F, Baud O, Beytout J, Romazko JP. Brucella melitensis in a married couple after a trip through Sicily. South Med J. 2005;98:843–4. Ministry of Health, Italy. Results of epidemiological research. [cited 2006 Jul 18]. Available from http://www.ministerosalute.it/promozione/malattie/datidefcons.jsp Caporale V, Nannini D, Giovannini A, Morelli D, Ramasco M. Prophylaxis and control of brucellosis due to Brucella melitensis in Italy: acquired and expected results. In: Prevention of brucellosis in the Mediterranean countries. Proceedings of the International Seminar of the International Center for Advanced Mediterranean Agronomic Studies (CIHEAM), the Commission of the European Communities, and the Ministry of Agriculture and Fisheries. Valletta (Malta): CIHEAM, 1992. p. 127–45. De Massis F, Di Girolamo A, Petrini A, Pizzigallo E, Giovannini A. Correlation between animal and human brucellosis in Italy during the period 1997–2002. Clin Microbiol Infect. 2005;11:632–6. Caracappa S. Livestock production and animal health in Sicily, Italy. Parassitologia. 1999;41(Suppl 1):17–23....查看详细 (5774字节)
☉ 11342785:On the Question of Sporadic or Atypical Bovine Spongiform Encephalopathy and Creutzfeldt-Jakob Disease
Bethesda, Maryland, USA National Institutes of Health, Bethesda, Maryland, USA University of Verona, Verona, Italy Virginia-Maryland Regional College of Veterinary Medicine, College Park, Maryland, USA Abstract Strategies to investigate the possible existence of sporadic bovine spongiform encephalopathy (BSE) require systematic testing programs to identify cases in countries considered to have little or no risk of orally acquired disease or to detect a stable occurrence of atypical cases in countries in which orally acquired disease is disappearing. To achieve 95% statistical confidence that the prevalence for sporadic BSE is no greater than 1 per million (i.e., the annual incidence of sporadic Creutzfeldt-Jakob disease [CJD] in humans) would require negative tests in 3 million randomly selected older cattle. A link between BSE and sporadic CJD has been suggested on the basis of laboratory studies but is unsupported by epidemiologic observation. Such a link might yet be established by the discovery of a specific molecular marker or of particular combinations of trends over time of typical and atypical BSE and various subtypes of sporadic CJD, as their numbers are influenced by a continuation of current public health measures that exclude high-risk bovine tissues from the animal and human food chains. Bovine spongiform encephalopathy (BSE) was first recognized in 1986 in the United Kingdom and quickly reached epidemic proportions, affecting >30,000 cattle per year by 1992. Because of continuing exportation of both live cattle and meat and bone meal rendered from the carcasses of slaughtered cattle, the disease spread throughout most of Europe and a few non-European countries. By 2006, 20 years after its first appearance in the United Kingdom, the disease had been reported in an additional 24 countries (1). Beginning toward the end of the 1980s in the United Kingdom, and in the 1990s in other countries, numerous regulations were enacted to minimize the entry of contaminated tissues into both the animal and human food chains and to eliminate the international spread of disease. These measures have been extraordinarily successful, to the extent that no new countries have been added to the list during the past year and the number of new cases has dramatically diminished in most countries in which BSE has appeared (the situation in some countries with insufficient surveillance remains unclear). Although the origin of the epidemic is thought to have been caused by a species-crossing contamination by sheep scrapie during the course of rendering and recycling carcass meat and bone meal as cattle feed, an alternative hypothesis suggested an origin in a similarly recycled case of spontaneously occurring disease in cattle. The pros and cons of these competing ideas have been argued elsewhere (2,3), and neither will ever be convincingly proved or disproved. Thus, the phenomenon of spontaneous disease remained in limbo until the recent discovery of "atypical" strains of BSE reopened the question. In this article we consider the importance of atypical BSE within the overall concept of sporadic (spontaneous) disease and whether such cases, if they exist, could account for at least some cases of apparently sporadic Creutzfeldt-Jakob (CJD) in humans. Sporadic BSE Obviously, the ideal country in which to examine the question of sporadic BSE would have a large national herd that was guaranteed never to have been exposed to environmental sources of infection. Such an ideal will never be realized. Until recently, the United States appeared to have at least approached the ideal by having a large national herd, an adequate testing program, and an apparently small risk for contamination by imported cattle or cattle feed. That position was made vulnerable in late 2003 by the discovery of a case of BSE imported from Canada and was eliminated altogether by the subsequent discovery of 2 indigenously infected animals in widely separate regions of the country. Although the 2 indigenous cases might represent sporadic disease, the continuing identification of cases in western Canada, coupled with a history of substantial numbers of cattle imported from Canada into the United States (both indigenous US animals had the same molecular "signature" as the most recent Canadian case), makes it difficult to ignore the possibility of undetected instances of feed contamination from imported cattle and recycled infectious carcasses. At present, the 2 best countries in which to undertake testing programs would be Argentina and Australia; both have large national herds (≈50 million and 30 million animals, respectively), and both are considered to be free of orally acquired BSE infections, on the basis of importation history, nutritional practices, and adequacy of surveillance (4). Even in these countries, however, the discovery of a case of BSE could not be guaranteed to be spontaneous because of the widespread global distribution of potentially infected cattle and cattle feed and the vagaries of international trade: imperfect record keeping, lack of compliance, and just plain deception. By way of illustration, an incident occurred many years ago that involved a particularly bulky shipment labeled as a pesticide. The large quantity seemed unusual to the customs inspector, who opened it and discovered that the shipment contained meat and bone meal destined to be spread on fields to inhibit grazing by deer, a serious agricultural pest. Thus, a study of sporadic BSE would only be truly convincing if no cases were identified. Moreover, the criteria for answering the question of sporadic BSE are different than for orally acquired BSE. Most importantly, we do not know at what age sporadic cases of BSE might occur, but they are unlikely to be in the 3- to 5-year-old age group in which orally acquired BSE is most prevalent. If the age distribution of sporadic disease in cattle were to mimic that of sporadic CJD in humans, it would not peak until 14–20 years of age (the last third of the ≈20-year natural life span of a cow). Substantial numbers of such older cattle do not exist, and thus it may never be possible to state with assurance that spontaneous BSE does not occur. Even if we accept this practical constraint, we can still take advantage of the fact that in many countries a proportion of the total slaughter population consists of breeding stock and dairy cows that are culled at >7 years of age, and animals that go directly to rendering plants or die "on farm" further increase this number. Argentina, for example, with a national herd of ≈50 million cattle, in 2005 recorded nearly 1.4 million deaths from slaughter and natural causes in animals >7 years (L. Mascitelli, pers. comm.). Approximately 10% of cases of sporadic CJD occur in patients 25–50 years of age ; this age in humans corresponds to the middle third of a cow's normal life span, or 7–13 years of age. If the age distribution of sporadic BSE followed the same pattern, negative test results in a total of ≈3 million animals randomly selected from this group would allow us to be 95% confident that sporadic BSE is not present at a prevalence >1 per million, and ≈4.5 million negative animals would raise the level of confidence to 99%. Larger numbers of BSE-negative animals would be required to achieve these levels of confidence for a maximum prevalence 400,000 tests in 2005 . Neither of the 2 more recently indigenously infected older animals with nonspecific clinical features would have been detected without such testing, and neither would have been identified as atypical without confirmatory Western blots. Despite these facts, surveillance has now been decimated to 40,000 annual tests (USDA news release no. 0255.06, July 20, 2006) and invites the accusation that the United States will never know the true status of its involvement with BSE. In short, a great deal of further work will need to be done before the phenotypic features and prevalence of atypical BSE are understood. More than a single strain may have been present from the beginning of the epidemic, but this possibility has been overlooked by virtue of the absence of widespread Western blot confirmatory testing of positive screening test results; or these new phenotypes may be found, at least in part, to result from infections at an older age by a typical BSE agent, rather than neonatal infections with new "strains" of BSE. Neither alternative has yet been investigated. Sporadic CJD The possibility that at least some cases of apparently sporadic CJD might be due to infection by sporadic cases of BSE cannot be dismissed outright. Screening programs needed to identify sporadic BSE have yet to be implemented, and we know from already extant testing programs that at least a proportion of infected animals have no symptoms and thus would never be identified in the absence of systematic testing. Thus, sporadic BSE (or for that matter, sporadic disease in any mammalian species) might be occurring on a regular basis at perhaps the same annual frequency as sporadic CJD in humans, that is, in the range of 1 case per million animals. Whether humans might be more susceptible to atypical forms of BSE cannot be answered at this time. Experimentally transmitted BASE shows shorter incubation periods than BSE in at least 1 breed of cattle, bovinized transgenic mice, and Cynomolgus monkeys (12,13). In humanized transgenic mice, BASE transmitted, whereas typical BSE did not transmit (13). Paradoxically, the other major phenotype (H) showed an unusually long incubation period in bovinized transgenic mice (12). The limited experimental evidence bearing on a possible relationship between BSE and sporadic CJD is difficult to interpret. The original atypical BASE strain of BSE had a molecular protein signature very similar to that of 1 subtype (type 2 M/V) of sporadic CJD in humans (5). In another study, a strain of typical BSE injected into humanized mice encoding valine at codon 129 showed a glycopattern indistinguishable from the same subtype of sporadic CJD (15). In a third study, the glycopatterns of both the H and L strains of atypical BSE evidently did not resemble any of the known sporadic CJD subtypes (12). To these molecular biology observations can be added the epidemiologic data accumulated during the past 30 years. The hypothesis that at least some cases of apparently sporadic CJD are due to unrecognized BSE infections cannot be formally refuted, but if correct, we might expect by now to have some epidemiologic evidence linking BSE to at least 1 cluster of apparently sporadic cases of CJD. Although only a few clusters have been found (and still fewer published), every proposed cluster that has been investigated has failed to show any common exposure to bovines. For that matter, no common exposure has been shown to any environmental vehicles of infection, including the consumption of foodstuffs from bovine, ovine, and porcine sources, the 3 livestock species known to be susceptible to transmissible spongiform encephalopathies. Additional negative evidence comes from several large case-control studies in which no statistically significant dietary differences were observed between patients with sporadic CJD and controls (16,17). On the other hand, the difficulty of establishing a link between BSE and CJD may be compounded by our ignorance of the infectious parameters of a sporadic form of BSE (e.g., host range, tissue distribution of infectivity, route of transmission, minimum infectious dose for humans, whether single or multiple). Presumably, these parameters would resemble those of variant CJD; that is, high infectivity central nervous system and lymphoreticular tissues of an infected cow find their way into products consumed by humans. Transmissions that might have occurred in the past would be difficult to detect because meat products are generally not distributed in a way that results in detectable geographic clusters. Barring the discovery of a specific molecular signature (as in variant CJD), the most convincing clue to an association will come from the observation of trends over time of the incidence of typical and atypical BSE and of sporadic and variant CJD. With 4 diseases, each of which could have increasing, unchanging, or decreasing trends, there could be 81 (34) possible different combinations. However, it is highly likely that the trends for typical BSE and variant CJD will both decrease in parallel as feed bans continue to interrupt recycled contamination. The remaining combinations are thus reduced to 9 (32), and some of them could be highly informative. For example, if the incidence of atypical BSE declines in parallel with that of typical BSE, its candidacy as a sporadic form of disease would be eliminated (because sporadic disease would not be influenced by current measures to prevent oral infection). If, on the other hand, atypical BSE continues to occur as typical BSE disappears, this would be a strong indication that it is indeed sporadic, and if in addition at least 1 form of what is presently considered as sporadic CJD (such as the type 2 M/V subtype shown to have a Western blot signature like BASE) were to increase, this would suggest (although not prove) a causal relationship . Recognition of the different forms of BSE and CJD depends upon continuing systematic testing for both bovines and humans, but bovine testing will be vulnerable to heavy pressure from industry to dismantle the program as the commercial impact of declining BSE cases ceases to be an issue. Industry should be aware, however, of the implications of sporadic BSE. Its occurrence would necessitate the indefinite retention of all of the public health measures that exclude high-risk bovine tissues from the animal and human food chains, whereas its nonoccurrence would permit tissues that are now destroyed to be used as before, once orally acquired BSE has disappeared. Acknowledgments We thank Victoria E. Bridges and Chris Kopral for providing data about annual cattle slaughter numbers from the Food Safety and Inspection Service of the US Department of Agriculture (USDA) and for estimates of cattle dying on farms from data supplied by the National Animal Health Monitoring System, Animal and Plant Inspection Services, Veterinary Service, USDA. This study was funded in part by grant 4AN/F10 "Studio dei meccanismi patogenetici delle malattie neurodegenerative per la diagnosi e lo sviluppo di approcci terapeutici" from the Istituto Superiore di Sanita, Rome, Italy. Dr Brown recently retired from the Laboratory of CNS Studies at the National Institutes of Health. References World Organization for Animal Health. Bovine spongiform encephalopathy. Geographical distribution of countries that reported BSE confirmed cases since 1989 [cited 2006 Oct 24]. Available from http://www.oie.int/eng/info/en_esb.htm Brown P, Bradley R, Detwiler L, Dormont D, Hunter N, Wells GAH, et al. Transmissible spongiform encephalopathy as a zoonotic disease. International Life Sciences Institute (ILSI) Europe Report Series. Brussels: ILSI Press; 2003. Horn GM, Bobrow ME, Bruce M, Goedert M, McLean A, Webster J. Review of the origin of BSE 2001, London: Stationery Office; 2001. World Organization for Animal Health. Bovine spongiform encephalopathy. Recognition of the bovine spongiform encephalopathy status of member countries [cited 2006 Oct 24]. Available from http://www.oie.int/eng/info/en_statesb.htm Casalone C, Zanusso G, Acutis P, Ferrari S, Capucci L, Tagliavini F, et al. Identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic Creutzfeldt-Jakob disease. Proc Natl Acad Sci U S A. 2004;101:3065–70. Danish Institute for Food and Veterinary Research [cited 2006 Oct 24]. Available from http://www.dfvf.dk/Default.aspID=8147&M=News&PID=89507&NewsID=792 Polak M, Rozek W, Rola J, Zmudzinski JF. Prion protein glycoforms from BSE cases in Poland. Bulletin of the Veterinary Institute of Pulawy. 2004;48:201–5. De Bosschere H, Roels S, Vanopdenbosch E. Atypical case of bovine spongiform encephalopathy in an East-Flemish cow in Belgium [cited 2006 Oct 24]. Int J Appl Res Vet Med. 2004;2:52–4. Available from http://www.jarvm.com/articles/Vol2Iss1/DEBOSSCHERE.htm Yamakawa Y, Hagiwara K, Nohtomi K, Nakamurua Y, Nishijima M, Higuchi Y, et al. Atypical proteinase K–resistant prion protein (PrPrres) observed in an apparently healthy 23-month-old Holstein steer. Jpn J Infect Dis. 2003;56:221–2. Biacabe AG, Laplanche JL, Ryder S, Baron T. Distinct molecular phenotypes in bovine prion diseases. EMBO Rep. 2004;5:110–4. Canadian Food Inspection Agency. Report on the investigation of the sixth case of bovine spongiformencephalopathy (BSE) in Canada [cited 2006 Oct]. Available from http://www.inspection.gc.ca/english/anima/heasan/disemala/bseesb/mb2006/6investe.shtml Buschmann A, Gretzshel A, Biacabe AG, Schiebel K, Corona C, Hoffmann C, et al. Atypical BSE in Germany—proof of transmissibility and biochemical characterization. Vet Microbiol. 2006;117:103–16. Book of abstracts. Prion 2006, International Conference on Prion Diseases of NeuroPrion, Network of Excellence, Turin, Italy, 2006 Oct 3–6 [cited 2006 Oct]. Available from http://www.neuroprion.com/en/ev_p06_abstracts.html Seuberlich T, Botteron C, Wenker C, Cafe-Maral V, Oevermann A, Haase B, et al. Spongiform encephalopathy in a miniature zebu. Emerg Infect Dis. 2006;12:1950–3. Wadsworth JDF, Asante EA, Desbruslais M, Linehan JM, Joiner S, Gowland I, et al. Human prion protein with valine 129 prevents expression of variant CJD phenotype. Science. 2004;306:1793–6. Wientjens DP, Davanipour Z, Hofman A, Kondo K, Matthews WB, Will RG, et al. Risk factors for Creutzfeldt-Jakob disease: a reanalysis of case control studies. Neurology. 1996;46:1267–91. PMID: 8628468 Van Duijn CM, Delasnerie-Lauprêtre N, Masullo C, Zerr I, de Silva R, Wientjens DPWM, et al. Case-control study of risk factors of Creutzfeldt-Jakob disease in Europe during 1993–1995. Lancet. 1998;351:1081–5....查看详细 (22236字节)
☉ 11342786:Ecologic Niche Modeling and Spatial Patterns of Disease Transmission
University of Kansas, Lawrence, Kansas, USA Abstract Ecologic niche modeling (ENM) is a growing field with many potential applications to questions regarding the geography and ecology of disease transmission. Specifically, ENM has the potential to inform investigations concerned with the geography, or potential geography, of vectors, hosts, pathogens, or human cases, and it can achieve fine spatial resolution without the loss of information inherent in many other techniques. Potential applications and current frontiers and challenges are reviewed. The emerging and evolving field of landscape epidemiology has explored techniques for summarizing spatial patterns in disease transmission data. These techniques seek spatial patterns at some level of generalization or averaging and then summarize overall patterns and trends in the form of a smoothed surface. Techniques typically applied to these challenges include splining and kriging, as well as smoothing based on average values within coarser-grained windows across landscapes (1–3). These approaches always involve some loss of resolution to smooth the surfaces, and some degree of averaging is involved (Figure). Although these approaches provide simple summaries of spatial patterns, they do not often succeed in illustrating true levels of complexity and heterogeneity that characterize biologic landscapes. Disease transmission cycles are composite phenomena that represent interactions between sets of species: hosts, vectors, and pathogens. The complexities of spatial occurrence of disease will represent the combination of complexities of occurrence of the component species, as well as effects of chance events. Thus, broad-trend generalizations such as those produced using the smoothing techniques mentioned above are unlikely to lead to novel insights and new understanding of complex systems. The approach advocated in this report improves the pattern summary by estimating species-specific ecologic niches. In this way, the complex influences of environmental variation on species' distributions and their translation into disease transmission patterns can be appreciated in greater detail (Figure). Ecologic Niche Modeling (ENM) Joseph Grinnell originated the concept of ecologic niches and was the first to explore the connections between ecologic niches and geographic distributions of species (4). His idea, translated into more modern terminology, was that the ecologic niche of a species is the set of conditions under which the species can maintain populations without immigration of individuals from other areas. A more complete discussion of the concept of ecologic niches and their mapping onto the geographic distributions of species has been provided elsewhere (5). Use of the ENM approach has grown considerably in the biodiversity community in recent years (6–10). The idea is that known occurrences of species across landscapes can be related to raster geographic information system coverages summarizing environmental variation across those landscapes to develop a quantitative picture of the ecologic distribution of the species. ENM characterizes the distribution of the species in a space defined by environmental parameters, which are precisely those that govern the species' geographic distribution under Grinnell's definition. A particular strength of ENM is its independence from any particular landscape. ENM can be used to identify potential distributional areas on any landscape: unsampled or unstudied portions of the native landscape, areas of actual or potential invasion by a species with an expanding range, or changing potential distributional areas as a consequence of change (e.g., land use change or climate change). Thus, ENM represents a powerful tool for characterizing ecologic and geographic distributions of species across real-world landscapes. Applications to Disease Systems In recent years, the ENM approach has seen several prototype applications to disease transmission systems by public health and epidemiology specialists who have been willing to explore novel ideas and approaches. I outline what the technique has to offer to the field and provide citations of example publications for each benefit and use. Understanding Ecology of Diseases In many cases, the details of ecologic parameters associated with occurrences of diseases or of species participating in disease transmission (e.g., vectors, hosts, pathogens) may be unclear because of small sample sizes, biased reporting, or simply lack of detailed geographic or ecologic analysis. ENM encompasses a suite of tools that relate known occurrences of these species or phenomena to raster geographic information system layers that summarize variation in several environmental dimensions. The result is an objective, quantitative picture of how what is known about a species or phenomenon relates to environmental variation across a landscape. Studies using these approaches include an examination of ecologic differences among different Chagas disease vectors in Brazil (11) and a characterization of ecologic features of outbreaks of hemorrhagic fever caused by Ebola and Marburg viruses (12,13). Characterizing Distributional Areas A next step in applying ENM approaches to understanding disease systems is characterizing geographic distributions. Here, ENM (or something akin to it) is used to investigate landscapes for areas that meet the ecologic requirements of the species. The result is an interpolation between known sampling locations informed by observed associations between the species and environmental characteristics. Previous attempts to characterize geographic distributions of species in the disease realm have demonstrated the potential of the approach but have not always used the most powerful inferential techniques available (14,15). In at least 1 case (14), the methods used failed to generalize and predict into areas of sparse sampling. ENM produces statistically robust predictions of geographic distributions of species or phenomena (even in unsampled areas), greatly exceeding expectations under random (null) models. Numerous examples of applications of this functionality to disease systems have been published (11–13,16–22). Identifying Areas of Potential Invasion in Other Regions ENMs characterize general environmental regimes under which species or phenomena may occur. To the extent that the model is appropriately and correctly calibrated, it may be used to seek areas of potential distribution. Thus, ENMs can be used to identify areas that fit the ecologic bill for a species, even if the species is not present there. This approach has seen extensive experimentation and testing in the biodiversity realm (8,23), but applications to disease transmission have as yet been few. One study attempted to identify the particular species in the Anopheles gambiae complex that was responsible for the large-scale South American malaria outbreaks in the early 20th century (19), and another evaluated the geographic potential of a possible monkeypox host (Cricetomys spp.) in North America (24). Anticipating Risk Areas with Changing Climates A logical extension of using ENMs to identify potential distributional areas is to address the question of likely geographic shifts in distributional areas of species or phenomena under scenarios of climate change or changing land use (25). This approach has seen considerable attention in the biodiversity realm, with both tests and validations (26–28), and with broad applications across faunas and floras (29–32). In the disease world, applications have been few, although 1 study used likely climate change–mediated range shifts to hypothesize the identity of Lutzomyia vectors of recent leishmaniasis outbreaks in southern Brazil (21). Identifying Unknown Vectors or Hosts ENM approaches can be applied to various parts of disease transmission cycles (e.g., overall case distribution, reservoir host distribution, vector distribution) to identify unknown elements in systems. The geography of overall case distributions can provide an indication of which clades are potential reservoirs and which are not. A first application was an attempt to identify mammalian hosts of the Triatoma protracta group of Chagas disease vectors in Mexico (22), which succeeded in anticipating the mammal hosts of 5 of 5 species for which a test was possible. Further exploration of this possible application of ENM methods has focused on the mysterious long-term reservoir of the filoviruses (Ebola and Marburg viruses) by comparing African mammal distributions with those of filovirus-caused disease outbreaks (33). Discussion Current Challenges in ENM ENM, although it has old roots (4), is nonetheless a relatively new tool in distributional ecology and biogeography. Only a few recent studies have compared the performance of different methodologic approaches under the ENM rubric (34–37). As such, numerous challenges remain in terms of refining approaches toward a more powerful and synthetic methodology. One central challenge is that of choosing modeling methods appropriate to a particular question, in the sense of discerning interpolation challenges from extrapolation challenges. In a recent comparative study focused on interpolation, which inferred details of patterns of presence and absence on a densely sampled landscape, several techniques that have internal controls on overfitting were superior (34). Extrapolative challenges, such as predicting potential distribution of invasive species, anticipating species' responses to global climate change, and identifying unknown reservoirs or vectors, require different qualities of modeling algorithms; different methods therefore appear to emerge as superior, according to the particular challenge (5). This balance of ability to interpolate accurately versus ability to extrapolate effectively remains a challenge for the ENM methods. A second frontier that includes yet-to-be-resolved details for ENM is that of testing and evaluating model results. Currently accepted approaches center on the ability to predict independent test occurrence data in the smallest area predicted (34,38). However, efficient predictions can be poor descriptors of a species' geographic range. Simpler techniques that place greater emphasis on minimizing the omission of known occurrences may be more appropriate. Pairing significance tests (which demonstrate that the coincidence between a prediction and test data is better than that achieved by random or null models) with setting minimum performance criteria (which ensure that that the prediction is accurate enough to meet the needs of the study) is probably the best approach (38). However, these methods have yet to be agreed upon broadly in the ENM community. Current Challenges in Applications of ENM to Disease Systems Beyond methodologic challenges, several issues remain to be addressed for full application of ENM methods to disease systems. The first, and perhaps most important, is understanding the role of scale in space and time. Preliminary explorations suggest that proper matching of temporal and spatial scales in analyses may offer particular opportunities for precise and accurate prediction of the behavior of disease phenomena (39). Similarly, proper choice of environmental datasets requires further exploration. Climate data provide longer temporal applicability, but remotely sensed data that summarize aspects of surface reflectance can provide finer spatial resolution, and may measure aspects of ecologic landscapes that climate parameters alone may not capture (40). Such issues will be resolved only through further exploration and testing with predictive challenges for diverse disease systems. Finally, because disease transmission systems often represent complex interactions among multiple species (e.g., vectors, hosts, pathogens), options exist for how they should be analyzed and modeled. Simple focus on disease occurrences, such as human cases, treats the entire transmission system as a black box and as such gives an overall picture of the ecology of the transmission chain of that disease (12). An alternative, however, is modeling each component species in the transmission system and then assembling the component ENMs into a geographic picture of the transmission system (22). Each of these approaches has its relative advantages and disadvantages, but a best-practices method has yet to be established, pending further testing and exploration. Conclusions The emerging field of ENM applied to questions of ecologic and geographic characteristics of disease systems has considerable potential. In particular, it can solve several problems of spatial resolution of summaries of geographic risk for disease. In sharp contrast to surface-fitting approaches to the same questions, ENM does not lose resolution to generalize and produce a result. Rather, ENM can achieve fine-scale resolution of distributions limited only by the spatial precision of the input occurrence data and the input environmental datasets. This characteristic makes possible a clear improvement in the spatial resolution that is possible in representing spatial patterns in disease risk. ENM is in the early stages of being explored for its potential for illuminating unknown phenomena in the world of disease transmission. The extensive explorations of ENM in the biodiversity field, however, serve as a benchmark of quality and acceptance for the technique. It can, once tested and prototyped extensively in the disease realm, offer a much-improved representation of spatial patterns in distributions of species or other phenomena. Acknowledgments I send many thanks for years of collaboration and education in the world of diseases and their geography to Ben Beard, Janine Ramsey, Jim Mills, Darin Carroll, Karl Johnson, Mark Benedict, Bex Levine, Ken Gage, Rusty Enscore, Erin Staples, Jeffrey Shaw, and Roger Nasci, as well as numerous other colleagues whose omission here is not reflective of my appreciation. Dr Peterson is professor of ecology and evolutionary biology at the Biodiversity Institute of the University of Kansas. His research interests include many aspects of geographic distributions of species, including the geography and ecology of filoviruses and other disease systems. References Waller LA, Carlin BP, Xia H, Gelfand AE. Hierarchical spatio-temporal mapping of disease rates. J Am Stat Assoc. 1997;92:607–17. Kleinschmidt I, Bagayoko M, Clarke GPY, Craig M, Le Sueur D. A spatial statistical approach to malaria mapping. Int J Epidemiol. 2000;29:355–61. MacNab YC, Dean CB. Autoregressive spatial smoothing and temporal spline smoothing for mapping rates. Biometrics. 2001;57:949–56. Grinnell J. Field tests of theories concerning distributional control. Am Nat. 1917;51:115–28. Soberon J, Peterson AT. Interpretation of models of fundamental ecological niches and species' distributional areas. Biodiversity Informatics. 2005;2:1–10. Austin MP, Nicholls AO, Margules CR. Measurement of the realized qualitative niche: environmental niches of five Eucalyptus species. Ecol Monogr. 1990;60:161–77. Guisan A, Zimmermann NE. Predictive habitat distribution models in ecology. Ecological Modelling. 2000;135:147–86. Peterson AT. Predicting the geography of species' invasions via ecological niche modeling. Q Rev Biol. 2003;78:419–33. Wiley EO, McNyset KM, Peterson AT, Robins CR, Stewart AM. Niche modeling and geographic range predictions in the marine environment using a machine-learning algorithm. Oceanography. 2003;16:120–7. Soberon J, Peterson AT. Biodiversity informatics: managing and applying primary biodiversity data. Philos Trans R Soc Lond B Biol Sci. 2004;359:689–98. Costa J, Peterson AT, Beard CB. Ecological niche modeling and differentiation of populations of Triatoma brasiliensis Neiva, 1911, the most important Chagas disease vector in northeastern Brazil (Hemiptera, Reduviidae, Triatominae). Am J Trop Med Hyg. 2002;67:516–20. Peterson AT, Bauer JT, Mills JN. Ecologic and geographic distribution of filovirus disease. Emerg Infect Dis. 2004;10:40–7. Peterson AT, Lash RR, Carroll DS, Johnson KM. Geographic potential for outbreaks of Marburg hemorrhagic fever. Am J Trop Med Hyg. 2006;75:9–15. Rogers DJ, Randolph SE, Snow RW, Hay SI. Satellite imagery in the study and forecast of malaria. Nature. 2002;415:710–5. Thomson MC, Elnaiem DA, Ashford RW, Connor SJ. Towards a kala azar risk map for Sudan: mapping the potential distribution of Phlebotomus orientalis using digital data of environmental variables. Trop Med Int Health. 1999;4:105–13. Sánchez-Cordero V, Peterson AT, Martínez-Meyer E, Flores R. Distribucion de roedores reservorios del virus causante del sindrome pulmonar por hantavirus y regiones de posible riesgo en Mexico. Acta Zoologica Mexicana. 2005;21:79–91. Peterson AT, Martínez-Campos C, Nakazawa Y, Martínez-Meyer E. Time-specific ecological niche modeling predicts spatial dynamics of vector insects and human dengue cases. Trans R Soc Trop Med Hyg. 2005;99:647–55. Lopez-Cardenas J, González-Bravo FE, Salazar-Schettino PM, Gallaga-Solorzano JC, Ramírez-Barba E, Martínez-Mendez J, et al. Fine-scale predictions of distributions of Chagas disease vectors in the state of Guanajuato, Mexico. J Med Entomol. 2005;42:1068–81. Levine RS, Peterson AT, Benedict MQ. Geographic and ecologic distributions of the Anopheles gambiae complex predicted using a genetic algorithm. Am J Trop Med Hyg. 2004;70:105–9. Levine RS, Benedict MQ, Peterson AT. Distribution of Anopheles quadrimaculatus Say s.l. and implications for its role in malaria transmission in the US. J Med Entomol. 2004;41:607–13. Peterson AT, Shaw JJ. Lutzomyia vectors for cutaneous leishmaniasis in southern Brazil: ecological niche models, predicted geographic distributions, and climate change effects. Int J Parasitol. 2003;33:919–31. Peterson AT, Sánchez-Cordero V, Beard CB, Ramsey JM. Ecologic niche modeling and potential reservoirs for Chagas disease, Mexico. Emerg Infect Dis. 2002;8:662–7. Skov F. Potential plant distribution mapping based on climatic similarity. Taxon. 2000;49:503–15. Peterson AT, Papes M, Reynolds MG, Perry ND, Hanson B, Regnery R. Native-range ecology and invasive potential of Cricetomys in North America. Journal of Mammalogy. 2006;87:427–32. Peterson AT, Tian H, Martínez-Meyer E, Soberon J, Sánchez-Cordero V, Huntley B. Modeling distributional shifts of individual species and biomes. In: Lovejoy TE, Hannah L, editors. Climate change and biodiversity. New Haven (CT): Yale University Press; 2005. p. 211–28. Martínez-Meyer E, Peterson AT. Conservatism of ecological niche characteristics in North American plant species over the Pleistocene-to-recent transition. Journal of Biogeography. 2006;33:1779–89. Martínez-Meyer E, Peterson AT, Hargrove WW. Ecological niches as stable distributional constraints on mammal species, with implications for Pleistocene extinctions and climate change projections for biodiversity. Global Ecology and Biogeography. 2004;13:305–14. Araujo MB, Pearson RG, Thuiller W, Erhard M. Validation of species-climate impact models under climate change. Global Change Biology. 2005;11:1504–13. Thuiller W, Lavorel S, Araujo MB, Sykes MT, Prentice IC. Climate change threats to plant diversity in Europe. Proc Natl Acad Sci U S A. 2005;102:8245–50. Bakkenes M, Alkemade JR, Ihle F, Leemansand R, Latour JB. Assessing effects of forecasted climate change on the diversity and distribution of European higher plants for 2050. Global Change Biology. 2002;8:390–407. Erasmus BFN, Van Jaarsveld AS, Chown SL, Kshatriya M, Wessels KJ. Vulnerability of South African animal taxa to climate change. Global Change Biology. 2002;8:679–93. Peterson AT, Ortega-Huerta MA, Bartley J, Sánchez-Cordero V, Soberon J, Buddemeier RH, et al. Future projections for Mexican faunas under global climate change scenarios. Nature. 2002;416:626–9. Peterson AT, Carroll D, Mills JN. Potential mammalian filovirus reservoirs. Emerg Infect Dis. 2004;10:2073–81. Elith J, Graham CH, Anderson RP, Dudik M, Ferrier S, Guisan A, et al. Novel methods improve prediction of species' distributions from occurrence data. Ecography. 2006;29:129–51. Manel S, Dias JM, Ormerod SJ. Comparing discriminant analysis, neural networks, and logistic regression for predicting species distributions: a case study with a Himalayan river bird. Ecological Modelling. 1999;120:337–47. Stockwell DR, Peterson AT. Effects of sample size on accuracy of species distribution models. Ecological Modelling. 2002;148:1–13. Stockwell DR, Peterson AT. Comparison of resolution of methods used in mapping biodiversity patterns from point occurrence data. Ecological Indicators. 2003;3:213–21. Fielding AH, Bell JF. A review of methods for the assessment of prediction errors in conservation presence/absence models. Environmental Conservation. 1997;24:38–49. Peterson AT, Martínez-Campos C, Nakazawa Y, Martínez-Meyer E. Time-specific ecological niche modeling predicts spatial dynamics of vector insects and human dengue cases. Trans R Soc Trop Med Hyg. 2005;99:647–55. Roura-Pascual N, Suarez AV, McNyset K, Gomez C, Pons P, Wild TO, et al. Potential geographic distribution, ecological niche differentiation, and fine-scale regional projections for Argentine ants based on remotely-sensed data. Ecological Applications. 2006;16 (in press.)...查看详细 (21884字节)
☉ 11342787:International Attention for Zoonotic Infections
Centers for Disease Control and Prevention, Atlanta, Georgia, USA University of Minnesota, Minneapolis, Minnesota, USA University of California, Davis, Davis, California, USA Swiss Tropical Institute, Basel, Switzerland Food and Agricultural Organization of the United Nations, Rome, Italy Snakes on a Plane was poised to be the blockbuster movie of the 2006 summer season. It featured reptiles smuggled onto an airplane as an unusual bioweapon of mass destruction. In a much less dramatic fashion, this absurd scenario plays out every day, when tons of live animals and unprocessed animal products are shipped internationally around the globe, providing many opportunities for rapidly translocating zoonotic pathogens. Episodes of emerging zoonoses are being increasingly recognized around the world. From 1996 to 2004, some 21% of 10,490 reports of animal diseases from 191 countries submitted to the Program for Monitoring Emerging Diseases (ProMED) concerned humans affected by zoonotic disease (1). This zoonoses theme issue of Emerging Infectious Diseases (EID) corroborates this finding, presenting reports of zoonotic disease from all corners of the globe, including the People's Republic of China, Vietnam, Slovakia, Indonesia, the United States, Israel, Bangladesh, the Netherlands, Brazil, Algeria, India, the Democratic Republic of the Congo, and Italy. As public health and animal health organizations attempt to respond to these emerging and reemerging zoonotic diseases, their ability and skill in forming new strategic partnerships are of paramount importance. This year was highlighted by the establishment of the Centers for Disease Control and Prevention (CDC) as a World Organization for Animal Health (OIE) Collaborating Center for Emerging and Remerging Zoonoses. To initiate this collaborating center, the International Symposium on Emerging Zoonoses (ISEZ) was held in conjunction with the International Conference on Emerging Infectious Diseases and the International Conference on Women and Infectious Diseases in Atlanta in March 2006. ISEZ was cosponsored by OIE, the United Nations Food and Agriculture Organization, the US Department of Agriculture Animal and Plant Health Inspection Service, the Department of Interior US Geological Survey, National Wildlife Health Center, the World Health Organization, and CDC. ISEZ was attended by >400 veterinarians, physicians, and public health professionals from all over the world. The objective of the symposium was to share information among public health and animal health professionals so that more effective and cooperative partnerships could be developed. In turn, this would help to better understand, prevent, and control new microbial dangers to human and animal health that occur globally every day. The 44 internationally renowned ISEZ speakers and moderators recognized the spirit in which this meeting was created. Their participation reflected a true commitment to the partnership between public health and animal health needed to meet the ever-growing microbial challenges threatening human, animal, and environmental health on a seemingly continual basis. The speakers lent their talent and expertise to the following themes: the epidemiology of pathogens and diseases shared among humans and animals, the risks of wildlife and exotic pet trade to human and animal health, the effects of agricultural practices on human and ecosystem health, the lessons learned from previous experiences, and collaborative achievements. Speakers offered solutions that had been tested through innovative, interdisciplinary, and intersectoral research to deal with the problems of emerging zoonoses. A few presentations are highlighted below. In his presentation, Emerging and Remerging Zoonoses from Wildlife Reservoirs to Exotic Pets, Bruno Chomel, University of California, Davis, College of Veterinary Medicine, reminded participants that most emerging infectious diseases are zoonotic, with wildlife constituting a large and often unknown reservoir. Wildlife can also be a source for reemergence of previously eradicated zoonoses. The discovery of such zoonoses is often related to better diagnostic tools. However, human modification to natural wildlife habitats and human behavior, such as deforestation, which causes range expansion of the tick vector for Kyasanur Forest disease in India, also create opportunities for emergence of zoonotic diseases. Translocation of raccoon dogs from Asia into Europe has provided a new potential rabies reservoir. Human behavior includes sleeping with pets, reported by 30% of Americans, and keeping of exotic pets that provide opportunities for microbial transmission that have not existed until recently. More than 10,000 pet tigers are kept in the United States—more tigers than are currently living in the wild throughout the world. Thus, addressing human influences on ecosystems and behavior with regard to wild and exotic animals must be incorporated into efforts at preventing and controlling emerging diseases that involve wildlife and other valued natural resources. In her presentation, Combined Vaccination Delivery to Remote and Mobile Pastoral Families and Their Animals, Esther Schelling, Swiss Tropical Institute, focused on the potential of joining public health and veterinary services to achieve higher vaccination coverage in Africa's remote rural settings. Such sharing is aimed at reducing vaccine-preventable diseases in persons and their livestock. Her findings from Chad indicated that sharing transport logistics and cold chain equipment between the public health and veterinary sectors in the most remote areas reduced total program delivery costs, was highly valued by pastoralists, and was an important strategy to connect hard-to-reach populations with needed vaccinations such as for measles and polio for children and anthrax for livestock. By optimizing the use of limited logistic and human resources, public health and veterinary services were strengthened, especially at the district level, and, in turn, the services can become more prepared to respond to endemic and epidemic diseases. ISEZ participants learned about the impact of human behavior on emergence and subsequent spread of highly pathogenic avian influenza (HPAI) H5N1 strain in Asia from Vincent Martin of the Food and Agriculture Organization. Human behavior that supported disease emergence includes agriculture practices that allow species mixing, limited biosecurity to prevent mixing of domestic and wild animals, and a cultural preference for warm meat that has made live bird markets common in urban areas. These factors act synergistically with the intrinsic characteristics of the virus and the rapid evolution of animal and farming production systems in the region to support emergence. Martin emphasized that understanding the underlying farming practices and cultural preferences that influenced the emergence and spread of HPAI in developing countries has been instrumental in implementing effective risk reduction measures. The experience of fighting HPAI in Asia shows that control efforts should be focused on production sectors with low biosecurity standards and free-ranging chickens and ducks. Creating conditions that are not conducive to microbial selection and emergence represents a major challenge, from a disease management and cultural point of view, for reducing the risk for avian influenza occurrence and subsequent human infection. This issue of EID continues in the tradition of the 2 previous December theme issues by focusing on zoonoses, reminding readers again that we must maintain a vigilance in combatting these microbial threats. The issue highlights efforts directed at identifying disease reservoirs and finding better ways to understand, evaluate, prevent, and control disease transmission. At the same time, healthy ecosystems need to be promoted, economic livelihoods safeguarded, and cultural beliefs respected at household to national and international levels. Contained in this issue are articles based on the following ISEZ presentations: Review of Bats and SARS (2) and Ecologic Niche Modeling and Spatial Patterns of Disease Transmission (3). Also featured are articles that focus on the role of risk factors for introducing zoonotic diseases, such as monkeypox associated with domestic trade in certain animal species and human behavior as a risk factor for exposure to avian influenza in Vietnam. Many species of animals are highlighted, including cats colonized with methicillin-resistant Staphylococcus aureus, horses and wild mammals infected with West Nile virus, dogs with rickettsial infections, turkeys with human metapneumovirus, and nonhuman primates infected with malaria. We encourage our readers to continue to conduct and submit the findings of essential research on emerging zoonotic diseases to EID as we strive to share and disseminate this information to our multidisciplinary readership. References Cowen P, Garland T, Hugh-Jones ME, Shimshony A, Handysides S, Kaye D, et al. Evaluation of ProMED-mail as an electronic early warning system for emerging animal diseases: 1996 to 2004. J Am Vet Med Assoc. 2006;229:1090–9. Wang L-F, Shi Z, Zhang S, Field H, Daszak P, Eaton BT. Review of bats and SARS. Emerg Infect Dis. 2006:12:1834–40. Peterson AT. Ecologic niche modeling and spatial patterns of disease transmission. Emerg Infect Dis. 2006;12:1822–6....查看详细 (9528字节)
☉ 11342788:Qualitative Assessment of Risk for Monkeypox Associated with Domestic Trade in Certain Animal Species, United States
US Food and Drug Administration, College Park, Maryland, USA Abstract In 2003, US officials identified several human monkeypox cases and traced the virus exposure to infected captive prairie dogs. The virus was likely introduced through a shipment of imported African rodents, which were kept with other mammals, including prairie dogs, in a pet distribution facility in the Midwest. To prevent the further introduction and spread of the virus, federal agencies restricted the importation of African rodents and restricted the domestic trade or movement of prairie dogs and certain other rodents. In this qualitative assessment of the risk for monkeypox associated with the 2003 outbreak, we conclude that the probability of further human infection is low; the risk is further mitigated by rodent import restrictions. Were this zoonotic disease to become established domestically, the public health effects could be substantial. In May and June 2003, public health officials identified an outbreak of human monkeypox in the United States (1–3). This was the first instance of human monkeypox virus (MPXV) infection detected outside its endemic range in Africa (3). As of July 30, 2003, a total of 72 human cases had been reported (4,5). Thirty-seven (51%) cases were eventually laboratory confirmed, and 35 met the case definition set by the Centers for Disease Control and Prevention (CDC) (4,5). Among the 35 patients whose cases were laboratory confirmed before July 11, 2003 (1), 32 (91%) tested positive for MPXV by PCR, culture, immunohistochemical testing, or electron microscopy of skin lesions; 2 tested positive by PCR and/or culture of an oropharyngeal or nasopharyngeal swab; and 1 tested positive by PCR and culture of a lymph node aspirate (1). To date, no new animal or human cases have been reported. The outbreak was relatively large compared with most reported events in Africa, but clinical features were milder than typically seen there (3,6,7). No human deaths occurred (1,8), although 2 children required intensive care (1,8). One patient received a corneal transplant due to chronic ocular infection (8). Most patients were exposed to prairie dogs, primarily from an Illinois animal distributor (IL-1). Most of those infected had direct physical contact with infected animals; infection likely resulted from bites or scratches or through open wounds (1–3). Some patients were exposed to premises where prairie dogs were kept (1). Traceback implicated rodents from a shipment of African animals imported to Texas on April 9, 2003, as the probable source of MPXV (1,4). The shipment contained ≈800 small mammals of 9 different species, including 6 genera of African rodents (762 rodents total): rope squirrels, tree squirrels, Gambian giant rats, brushtail porcupines, dormice, and striped mice (1,2), as well as cusimanses, genets, and palm civets (9). Rodents from the shipment were housed with or in close proximity to prairie dogs at IL-1. Approximately 200 prairie dogs were at IL-1 coincident with the arrival of the imported African rodents (1). Many prairie dogs from IL-1 were distributed to other states for sale as pets (1,4). CDC traced 93 infected or potentially infected prairie dogs from IL-1 (1). An additional, unknown number of prairie dogs died or were sold at animal swap meets for which records are not available (1) . To prevent the introduction and spread of infected animals into susceptible populations, on June 11, 2003, the Food and Drug Administration (FDA) and CDC issued an order that prohibited 1) importation of all rodents from Africa and 2) transportation, sale, or any other commercial or public distribution, including release into the environment, of prairie dogs or rodents from 6 species represented in the African shipment (10). On November 4, 2003, FDA and CDC published an interim final rule (11) that imposed import restrictions on all African rodents and established or modified restrictions on the import, capture, transport, sale, barter, exchange, distribution, and release of prairie dogs, the 6 imported species, and possibly, by order, other animals with the potential to transmit MPXV. Neither CDC nor FDA exercised its statutory authority to seize and destroy animals to prevent the spread of MPXV. We prepared this qualitative risk analysis to help understand the impact of the domestic trade restrictions on the current risk for human monkeypox infections. We evaluated the data and uncertainties concerning monkeypox and its potential spread to animal and human populations and characterized the probability of harm on the basis of those data. Because of CDC's import restrictions on all African rodents, we did not estimate the risk posed by importation of animals into the United States. We focused only on monkeypox and did not consider other zoonotic agents that might be transmitted by the species discussed. This risk assessment follows a generally accepted 4-part framework (12). The hazards are MPXV and its potential for transmission and spread from animals to humans; the risk is human infection from prairie dogs and possibly imported rodents. Hazard Identification Human monkeypox is a sporadic zoonotic viral disease, caused by an orthopoxvirus that until 2003 was known to have occurred only in parts of Africa (3,7,13). The first human illness was identified in 1970 in a child (7,14). Previous cases were likely mistaken for smallpox (14). Although it was first isolated from a captive primate (3,6), rodents are its likely primary natural reservoir (7,15–17); its complete mammalian host range is unknown. The mode of transmission between infected animals and humans is not well defined (18). Direct mucocutaneous contact and respiratory routes have been implicated in epidemiologic and experimental research (15,18,19). The estimated mean human incubation period is 12 days (1,3,15,16). The disease is characterized by a rash similar to that observed with smallpox (14) or chickenpox (10,15,20). The infectious period occurs during the first week of the rash (7); symptoms include headache, fever, sweats, and severe lymphadenopathy (15,16,20). Among African patients with a history of smallpox vaccination, monkeypox is usually milder with lower numbers of deaths (3,6,16). Subclinical or very mild infection can occur in humans (16,21,22). Case-fatality rates in African outbreaks range from 4% to 33% (6,23) and are high among children (3,6,14,23). Variability in case-fatality rates may reflect incomplete assessment of the total number of cases, variations in case definition, and variability in the virulence of MPXV strains. The US outbreak has been associated with a milder strain (3,24–26). Case fatality also likely depends on differences in exposure, susceptibility, and healthcare (14). Repeated animal reintroduction of MPXV is believed necessary to endemic infections in human populations. Human cases in disease-endemic areas tend to be sporadic and isolated and primarily associated with direct animal-to-human transmission (24,27). However, clusters associated with common source and human-to-human transmission occur and may in Africa be increasing with decreased prevalence of prior smallpox vaccination (6,7,15,16,28,29). FDA has not approved a treatment for monkeypox. Suggested treatment options include cidofovir (30–32). Efficacy of vaccinia immune globulin in humans has not been established (30,31). After the onset of symptoms, supportive therapy is usually the recommended treatment (31). Preexposure and postexposure smallpox vaccine was used during the 2003 outbreak, with only relatively minor adverse events reported (1,32). Hazard Characterization Much is unknown about pathogenesis and transmission dynamics of MPXV in humans and animals. Limited research suggests that at least in some host mammals latent or inapparent infection occurs (15). In addition to serologic evidence of orthopoxvirus exposure, MPXV has been recovered from the kidneys of healthy-appearing animals (15,16). The latency period is unknown, as is whether the virus can be transmitted during such periods. The complete host range of MPXV in Africa is unknown. Animal antibody surveys in disease-endemic areas suggest infection is enzootic among squirrels, other rodents, and monkeys, although other animals may be infected (6,15,16,33–35). The number of animals exposed or infected in the United States is unknown and impossible to estimate. Approximately 800 animals were recorded in the African shipment, but disposition information is available only for rodents (1). A Gambian giant pouched rat, 3 dormice, and 2 rope squirrels from the shipment were tested and found to be infected with MPXV (1). Infected animals from the shipment were housed or transported with prairie dogs and other mammals. An unknown number of prairie dogs and animals from other species became infected. Although many prairie dogs became ill and several died, some infected animals survived. The secondary attack rate among susceptible animals is unknown and cannot be estimated with available data. CDC necropsied 249 animals involved with the outbreak, confirming infection in 33 animals with PCR (36) and in 22 animals through virus isolation from various tissues. Infection was confirmed in 14 prairie dogs, 2 Gambian giant pouched rats, 9 dormice, 3 rope squirrels, 1 ground hog, 1 hedgehog, 1 jerboa, and 2 opossums. CDC performed extensive histopathologic examination on 2 necropsied prairie dogs from IL-1 and detected MPXV DNA by using real-time PCR (18). The necropsied prairie dogs had MPXV in saliva, lesion exudates, and bronchi and lung parenchyma (18). Approximately 110 of the ≈200 prairie dogs likely exposed at IL-1 were sold after the African animals were introduced and before 15 of the prairie dogs at IL-1 became ill. Ten of the ill prairie dogs died rapidly (1,3,18). In June 2003, CDC evaluated an unspecified number of prairie dogs, dormice, hedgehogs, jerboas, opossums, and numerous other species (a total of 18 species) from IL-1; of these, 2 prairie dogs, 7 dormice, 1 African hedgehog, 1 jerboa, and 1 gray short-tailed opossum tested positive for MPXV by PCR (36). When these animals were infected or if they could transmit disease is not known. On June 19, 2003, CDC acquired 61 live animals from the original shipment. On August 20, 2003, CDC acquired from the state of Illinois 291 animals remaining at IL-1, including African and domestic species. Numerous other animals were acquired from Iowa, Wisconsin, Indiana, and Ohio. Of 172 animals tested from the various states as well as from the original shipment, 25 showed serologic evidence of infection without overt signs of disease (i.e., PCR and tissue culture negative). On June 24, 2003, an oral and ocular swab from a dormouse from IL-1 tested positive by PCR. After the dormouse died a month later, its tissues tested positive for MPXV by PCR and culture. A second dormouse from IL-1 that also tested positive in June appeared healthy; however, when it was euthanized in December 2003, swabs and necropsy samples of various tissues, urine, and feces were positive by PCR. No viral antigen was detected on pathologic examination of tissues (36). Investigations of human cases from the outbreak support the hypothesis that close direct contact with infected animals was necessary for infection. Cases occurred among persons who were bitten by infected prairie dogs or infected through open wounds (3,8). The 11 Wisconsin patients included a child and parents; a meat distributor who also distributed exotic animals; his wife; 2 employees of 2 different pet stores; 2 veterinarians from different clinics; a person who had bought prairie dogs; and that person's houseguest. All of these patients reported direct contact with an infected prairie dog (3), although human-to-human transmission could not be ruled out for the parents (3). Data on duration of infection are limited. Virus appears to be present in some animals months after infection, regardless of clinical illness. In addition to CDC's data on dormice, data derived from experimental infection of small numbers of laboratory animals documented infectious MPXV in tissues 3–6 weeks after exposure (18). Clinical and asymptomatic infections have been reported among captive primates; severity varied depending on the species and route of inoculation (16). CDC has reported elevated tissue viral loads in 2 necropsied prairie dogs (18). In another study, 10 experimentally infected North American ground squirrels died within 9 days, although no obvious signs of disease except for lethargy and anorexia developed (37). Squirrels infected intranasally had a longer incubation period and later death (36). Ten prairie dogs infected experimentally with a human MPXV isolate were highly susceptible to infection but had a lower death rate and less severe pathologic change than were seen in the squirrel study that used the same dose (19). A human adult infected during the 2003 outbreak experienced keratitis and corneal ulceration as a complication of infection and ultimately received a corneal transplant (8). Corneal ulceration has also been reported in some African patients (16). Exposure Assessment In African outbreaks, capturing, handling, and eating wild animals have been associated with infection (6,23,34). In the United States, monkeypox occurred in humans who had direct contact with infected animals and were bitten or infected through open wounds (3). These persons included pet dealers, pet owners and their children, and contacts of these people at risk of coming into direct contact with the infected animal. Although potential exposure occurred in settings that included pet stores, swap meets, and wild animal trade centers (1), no evidence exists that persons casually exposed to infected animals were infected. The magnitude and scope of this pet trade are not well quantified. In 2002, ≈30,000 prairie dogs were sold at pet dealers, swap meets, flea markets, and other venues open to the public (11). Of the 762 rodents in the African shipment, CDC traced 584 (77%) (1). The remaining 178 (≈23%) could not be traced beyond the point of entry (1). The fate of the 50 nonrodent animals on the shipment is unknown. Of the ≈200 prairie dogs that may have been exposed to MPXV at IL-1, 107 (54%) have not been accounted for. These animals will not likely be traced. A small number of animals associated with the outbreak, including some known to have been infected, are in the possession of pet dealers and private owners; their capacity to transmit infection is unknown. Animals from species other than the listed species—gerbil, hamster, chinchilla, opossum, groundhog, hedgehog, and jerboa—were discovered to be infected, although no confirmed human cases of infection were associated with contact with any animal except prairie dogs (1,9). To evaluate the potential spread of the disease beyond the initially exposed animals, the US Geological Survey's National Wildlife Health Center trapped 237 small mammals from 14 species at 9 sites in Wisconsin and Illinois where cases of monkeypox were reported. All were negative for monkeypoxvirus or monkeypoxvirus-specific antibodies (38). These small amounts of data are insufficient to establish the absence of MPXV in the wild. The federal restrictions on importation of high-risk species and trade in the listed species have likely substantially reduced the potential risk for exposure of uninfected animals or persons to MPXV. However, some residual risk for MPXV infection through illegal importation or infection in legally imported, nonlisted species may exist. Risk Characterization Several categories were established to define and qualitatively characterize the risks. Low risk denoted no direct human contact with a captive animal(s); even if animal infection status was unknown or the animal was infected, the exposure to the animal was likely insufficient for animal-to-human disease transmission to occur. Medium risk described human contact that was direct but the exposure involved type I contact with a potentially infected animal or animal(s) of unknown infectious status. High risk designated direct human contact and involved a type II contact with a captive animal(s) infected with MPXV or with unknown infection status but likely MPXV exposure. Finally, the term severity of infection and illness denoted any individual infection with MPXV that should be considered serious and potentially fatal. The risk to the persons and the risk for the spread of the disease to others made MPXV infection a potentially serious public health matter. The probability that any surviving animal directly involved in the outbreak may be infected must be considered high, given the possibility of latent infection. Making the unlikely assumption that all of these animals are still alive, the group includes the 178 African rodents, mostly dormice, lost to follow-up, 107 prairie dogs from IL-1 that were not traceable, and 50 nonrodent animals included in the African shipment. An unknown number, but clearly most, of the affected African rodents and IL-1 prairie dogs that were traced and identified as alive as of July 2003 (121 African rodents and ≈93 prairie dogs) have since died or been euthanized. Some animals from other species that were in the affected pet distribution facilities during the outbreak tested positive for MPXV. An unknown number of these exposed animals are likely to be alive and in private or commercial ownership; what proportion of these animals is infected with MPXV is unknown but is assumed to be small. No confirmed cases of human infection or further cases of animal infection have been associated with these animals. However, all animals directly associated with the 2003 outbreak should be considered to pose a continued high risk for infection. The probability of infection in rodents or other animals imported from monkeypox-endemic regions is unknown. Imported African rodents were almost certainly the source of the US outbreak. Animals imported as pets are handled by several persons as they pass from importer to owner, and they may be housed and transported in close proximity with nonimported susceptible animals. Current import restrictions on African rodents substantially reduce the risk for introduction and spread of MPXV, but a potential residual risk remains because of illegal importation as well as import of nonrestricted species that may carry the virus. Some previously imported animals from restricted species might also be infected with MPXV, although this risk is unknown and assumed to be extremely low. For domestically bred African rodents, the risk they may pose of transmitting MPXV to humans depends on the risk that the rodents will be exposed to infected animals. Absent a tracking or pedigree system that distinguishes domestically bred from imported, wild-caught animals is impossible. Trade in domestically bred African rodents could increase the risk for human infection if illegally imported infected animals are identified as captive-bred. The monkeypox risk to humans posed by prairie dogs is a function of the animals' possible contact with infected animals and their potential for viral transmission. The number of animals infected with or exposed to MPXV in the outbreak that might still be alive is likely small. However, these animals may be widely distributed geographically, and they may have spread the virus to other animals not currently known to pose a risk. The risk for MPXV infection and spread among prairie dogs are mitigated by current import and trade restrictions and the death or euthanization of most animals directly associated with the outbreak. The probability that an uninfected prairie dog will come into contact with an infected captive or released animal and that there will be sufficient exposure for infection is likely low. If such contact occurs, however, these animals are highly susceptible. Little evidence about the MPXV status of wild prairie dogs exists. Given the high rates of illness and death among captive prairie dogs exposed to MPXV in 2003, anticipating that the virus would result in a die-off that would be detected may be reasonable; however, in addition to the lack of data, uncertainties about the virus and the susceptibility of the animals in the wild preclude drawing any conclusions. The risk for new domestically acquired human cases is low with the current restrictions on import and trade in certain species in place. No new cases have been reported in humans or animals since the outbreak, despite the likelihood that some surviving infected animals have been kept alive by individual or commercial owners. Limited surveillance efforts have not identified MPXV in wild animal populations; however, the virus could possibly become enzootic here if an infected animal were released or escaped into the wild and spread the virus to susceptible mammals. Were that to occur, human cases would likely result. The risk that monkeypox could become enzootic is relevant in evaluating the risk of importing potential mammalian carriers of MPXV or in allowing contact between likely carriers and susceptible domestic mammals. Data limitations preclude quantitative, and limit accurate qualitative, estimation of the human risk for monkeypox in the United States . Research is needed on disease dynamics, range of host species, and the parameters of wild animal trade and ownership. Acknowledgments We thank Beth Karp, Philip L. Chao, and the reviewers external to FDA, who provided expert comment on an earlier draft of this work. Dr Bernard is the associate director for regulations in FDA's Center for Food Safety and Applied Nutrition. Her professional and academic focus has been on population health risks, law, and policy; projects at FDA include issues such as antimicrobial drug resistance, foodborne pathogens and toxins, communicable diseases, and obesity and nutrition-related diseases. Dr Anderson is the associate director for risk assessment in the Office of Biostatistics and Epidemiology at the FDA Center for Biologics Evaluation and Research. He uses computer modeling and risk assessment techniques to address critical scientific and policy issues pertaining to the safety of blood and blood products, vaccines and cellular, tissue, and gene therapies. References Centers for Disease Control and Prevention. Update: multistate outbreak of monkeypox—Illinois, Indiana, Kansas, Missouri, Ohio, and Wisconsin, 2003. MMWR Morb Mortal Wkly Rep. 2003;52:642–6. Centers for Disease Control and Prevention. 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☉ 11342789:Review of Bats and SARS
Australian Animal Health Laboratory, Geelong, Victoria, Australia Wuhan Institute of Virology of Chinese Academy of Sciences, Wuhan, People's Republic of China Institute of Zoology of Chinese Academy of Sciences, Beijing, People's Republic of China East China Normal University, Shanghai, People's Republic of China Department of Primary Industries and Fisheries, Brisbane, Queensland, Australia Consortium for Conservation Medicine, New York, New York, USA Abstract Bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, including henipaviruses and variants of rabies viruses. Recently, we and another group independently identified several horseshoe bat species (genus Rhinolophus) as the reservoir host for a large number of viruses that have a close genetic relationship with the coronavirus associated with severe acute respiratory syndrome (SARS). Our current research focused on the identification of the reservoir species for the progenitor virus of the SARS coronaviruses responsible for outbreaks during 2002–2003 and 2003–2004. In addition to SARS-like coronaviruses, many other novel bat coronaviruses, which belong to groups 1 and 2 of the 3 existing coronavirus groups, have been detected by PCR. The discovery of bat SARS-like coronaviruses and the great genetic diversity of coronaviruses in bats have shed new light on the origin and transmission of SARS coronaviruses. Severe acute respiratory syndrome (SARS) represents the 21st century's first pandemic of a transmissible disease with a previously unknown cause. The pandemic started in November 2002 and was brought under control in July 2003, after it had spread to 33 countries on 5 continents, resulting in >8,000 infections and >700 deaths (1). The outbreaks were caused by a newly emerged coronavirus, now known as the SARS coronavirus (SARS-CoV). In late 2003 and early 2004, sporadic outbreaks were reported in the region of the People's Republic of China where the 2002–2003 outbreaks originated (2). However, molecular epidemiologic studies showed that the viruses responsible for the 2003–2004 outbreaks were not the same as those isolated during the 2002–2003 outbreaks (3). These findings indicate independent species-crossing events. They also indicate that a SARS epidemic may recur in the future and that SARS-like coronaviruses (SARS-like–CoVs) that originate from different reservoir host populations may lead to epidemics at different times or in different regions, depending on the distribution of the reservoirs and transmitting hosts. The recent discovery of a group of diverse SARS-like–CoVs in bats supports the possibility of these events and further highlights the need to understand reservoir distribution and transmission to prevent future outbreaks. Animal Origin of SARS Coronaviruses Because of the sudden and unpredictable nature of the SARS outbreaks that started in November 2002 in southern People's Republic of China, structured and reliable epidemiologic studies to conclusively trace the origin of SARS-CoV were not conducted. However, accumulated studies from different groups, which used a variety of approaches, indicated an animal origin on the basis of the following findings. 1) Genome sequencing indicated that SARS-CoV is a new virus with no genetic relatedness to any known human coronaviruses (4,5). 2) Retrospective serologic studies found no evidence of seroprevalence to SARS-CoV or related viruses in the human population (6). 3) Serologic surveys among market traders during the 2002–2003 outbreaks showed that antibodies against SARS-CoV or related viruses were present at a higher ratio in animal traders than control populations (7–9). 4) Epidemiologic studies indicated that early case-patients were more likely than later case-patients to report living near a produce market but not near a farm, and almost half of them were food handlers with probable animal contact (7). 5) SARS-CoVs isolated from animals in markets were almost identical to human isolates (9). 6) Molecular epidemiologic analyses indicated that human SARS-CoV isolates could be divided into 3 groups from the early, middle, and late phases of the outbreaks and that early-phase isolates were more closely related to the animal isolates (10). 7) Human SARS-CoVs isolates from the 2003–2004 outbreaks had higher sequence identity to animal isolates of the same period than to human isolates from the 2002–2003 outbreaks (3). Susceptible Animals in Markets and Laboratories The first evidence of SARS-CoV infection in animals came from a study conducted in a live animal market in early 2003 (9). From the 25 animals sampled, viruses closely related to SARS-CoV were detected in 3 masked palm civets (Paguma larvata) and 1 raccoon dog (Nyctereutes procyonoides). In addition, neutralizing antibodies against SARS-CoV were detected in 2 Chinese ferret badgers (Melogale moschata). This initial study indicated that at least 3 different animal species in the Shenzhen market were infected by coronaviruses that are closely related to SARS-CoV. Given the vast number of live animals being traded in animal markets in southern People's Republic of China, knowing which other animals are also susceptible to these viruses is crucial. Unfortunately, for a variety of reasons no systematic studies were conducted on traded animals during the outbreak period. Experimental infection of different animals therefore became a component of the SARS-CoV investigation. Currently, >10 mammalian species have been proven to be susceptible to infection by SARS-CoV or related viruses . Rats were also implicated as potentially susceptible animals that may have played a role in the transmission and spread of SARS-CoV in the well-publicized SARS outbreaks in the Amoy Gardens apartment block in Hong Kong Special Administrative Region, People's Republic of China (23). In Guangdong in 2004, the first human with a confirmed case of SARS was reported to have had no contact with any animals except rats (2). Experimentally, we have obtained serologic evidence that SARS-CoV replicates asymptomatically in rats (B.T. Eaton et al., unpub. data). Further studies are needed to clarify the potential role of rats in the transmission of SARS-CoV. Studies by 2 independent groups suggested that avian species were not susceptible to SARS-CoV infection and that, hence, domestic poultry were unlikely to be the reservoir or associated with the dissemination of SARS-CoV in the animal markets of southern People's Republic of China (22,24). Role of Masked Palm Civets Although in 1 live animal market, 3 species were found to be infected by viruses related to SARS-CoV (9), all subsequent studies have focused mainly on palm civets, possibly because the rate of detection was higher in civets or because the number of civets traded in southern People's Republic of China exceeds that of other wildlife groups. The isolation of closely related SARS-CoV in civets during the 2002–2003 and 2003–2004 outbreaks and the close match of virus sequences between the human and civet isolates from each outbreak (3,9,25) strongly suggest that civets are a direct source of human infection. However, these studies did not clarify whether animals other than civets were involved in transmission of SARS-CoV to humans or whether civets were an intermediate host or the natural reservoir host of SARS-CoVs. During the 2002–2003 outbreaks, none of the animal traders surveyed in the markets, who supposedly had very close contact with live civets, displayed SARS symptoms (7–9). During the 2003–2004 outbreaks, at least 1 human SARS patient had had no contact with civets (2). These observations seem to indicate that >1 other animal species may play a role in transmission of SARS-CoV to humans. Most, if not all, civets traded in the markets are not truly wildlife animals; rather, they are farmed animals. Civet farming is relatively new in People's Republic of China and has rapidly expanded during the past 15 years or so. Tu et al. conducted the first comparative study of market and farmed civets (26). Serologic testing was performed on 103 serum samples taken from civets in an animal market in Guangdong and several civet farms in different regions of People's Republic of China in June 2003 and January 2004. No significant level of SARS-CoV antibody was detected in any of the 75 samples taken from 6 farms in 3 provinces. In contrast, of the 18 samples taken from an animal market in Guangdong Province in January 2004, 14 (79%) had neutralizing antibodies to SARS-CoV. In a parallel study conducted between January and September 2004 (27), molecular analysis was used to investigate the distribution of SARS-CoV in palm civets in markets and on farms. PCR analysis of samples from 91 palm civets and 15 raccoon dogs in 1 animal market and 1,107 civets from 25 farms in 12 provinces showed positive results for all animals from the market and negative results for all animals from the farms. Similar results were obtained in wild-trapped civets in Hong Kong; none of the 21 wild civets sampled had positive antibody or PCR results for SARS-CoV (28). Although not universally true, natural reservoir hosts tend to have coevolved with their viruses and usually do not display clinical signs of infection (29). However, when palm civets were experimentally infected with 2 strains of human SARS-CoV, all developed clinical signs of fever, lethargy, and loss of aggressiveness (11). Civits' high susceptibility to SARS-CoV infection and wide presence in markets and restaurants strongly indicates an important role for civets in the 2002–2003 and 2003–2004 SARS outbreaks. However, the lack of widespread infection in wild or farmed palm civets makes them unlikely to have been the natural reservoir host. SARS-like Coronaviruses in Bats The presence of SARS-like–CoVs in different species of horseshoe bats in the genus Rhinolophus has recently been reported. We found, in a study of horseshoe bat species in different regions of mainland People's Republic of China in 2004 (30), that each of the 4 species surveyed had evidence of infection by a SARS-like–CoV: 2 species (R. pearsoni and R. macrotis) had positive results by both serologic and PCR tests, and 2 (R. pussilus and R. ferrumequinum) had positive results by either serologic or PCR tests, respectively. Bats with positive results were detected in the provinces of Hubei and Guangxi, which are >1,000 km apart. A group in Hong Kong (31) found that, when analyzed by PCR, 23 (39%) of 59 anal swabs of wild Chinese horseshoe bats (R. sinicus) contained genetic material closely related to SARS-CoV. They also found that as many as 84% of the horseshoe bats examined contained antibodies to a recombinant N protein of SARS-CoV. A previous study indicated a certain level of antigenic cross-reactivity between SARS-CoV and some group 1 coronaviruses (6) and that several group 1 coronaviruses had recently been found in bats. Therefore, the actual seropositive proportion of R. sinicus might be 1 intermediate host before it could efficiently infect humans. The existence of at least 3 discontinuous highly variable genomic regions between SARS-CoV and SARS-like–CoV indicates that the second mechanism is more likely. In conclusion, the discovery of bat SARS-like–CoVs and the great genetic diversity of coronaviruses in bats have shed new light on the origin and transmission of SARS-CoV. Although the exact natural reservoir host for the progenitor virus of SARS-CoV is still unknown, we believe that a continued search in different bat populations in People's Republic of China and neighboring countries, combined with experimental infection of different bat species with SARS-CoV, will eventually identify the native reservoir species. A positive outcome of these investigations will greatly enhance our understanding of spillover mechanisms, which will in turn facilitate development and implementation of effective prevention strategies. The discovery of SARS-like–CoVs in bats highlights the increasingly recognized importance of bats as reservoirs of emerging viruses (36). Moreover, the recent emergence of SARS-CoVs and other bat-associated viruses such as henipaviruses (37,38), Menangle, and Tioman viruses (36), and variants of rabies viruses and bat lyssaviruses (38,39) also supports the contention that viruses, especially RNA viruses, possess more risk than other pathogens for disease emergence in human and domestic mammals because of their higher mutation rates (40). Acknowledgments The work conducted by our multination collaborative team was jointly funded by a special grant for Animal Reservoirs of SARS-CoV, State Key Program for Basic Research grant no. 2005CB523004, and State High Technology Development Program grant no. 2005AA219070 from the Ministry of Science and Technology, People's Republic of China; a special fund from the president of the Chinese Academy of Sciences (no. 1009); the Sixth Framework Program, EPISARS, from the European Commission (no. 51163); the Australian Biosecurity Cooperative Research Centre for Emerging Infectious Diseases (Project 1.007R); an National Institutes of Health-National Science Foundation Ecology of Infectious Diseases award (no. R01-TW05869) from the John E. Fogarty International Center; and an award to the Consortium for Conservation Medicine from the V. Kann Rasmussen Foundation. 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☉ 11342790:Risk Factors for Human Infection with Avian Influenza A H5N1, Vietnam, 2004
National Institute of Hygiene and Epidemiology, Hanoi, Vietnam Pasteur Institute, Ho Chi Minh City, Vietnam Abstract To evaluate risk factors for human infection with influenza A subtype H5N1, we performed a matched case-control study in Vietnam. We enrolled 28 case-patients who had laboratory-confirmed H5N1 infection during 2004 and 106 age-, sex-, and location-matched control-respondents. Data were analyzed by matched-pair analysis and multivariate conditional logistic regression. Factors that were independently associated with H5N1 infection were preparing sick or dead poultry for consumption 200 human cases, knowledge of influenza H5N1 remains inadequate. Neither how these viruses are transmitted to humans nor, consequently, the most effective way to reduce the risk for infection is fully understood. Descriptive and analytic epidemiologic studies conducted in Hong Kong Special Administrative Region, People's Republic of China, during the 1997 outbreak of influenza H5N1 (2–5) identified visiting a live bird market as a risk factor. However, the current outbreak encompasses different viruses and different sociodemographic, farming, and behavioral contexts. Several seroprevalence studies of healthcare workers and a case-control study from Thailand have been published from the current outbreak (6–9), but further work is needed to develop and test hypotheses on the mechanism of transmission of influenza H5N1 to humans. To clarify the source and mode(s) of transmission of influenza H5N1 to humans and to guide the control and prevention of influenza, we conducted a case-control study of all cases of avian influenza H5N1 identified in humans in Vietnam in 2004. Materials and Methods All persons with laboratory-confirmed influenza A H5 cases detected in Vietnam from January 1 through December 31, 2004, were eligible for enrollment as case-patients. Case-patients were identified from persons hospitalized with an acute respiratory infection considered by clinicians, on the basis of clinical and epidemiologic findings, to have a suspected case of H5N1 infection. Clinicians did not use a systematic case definition or screening protocol to identify patients eligible for testing for H5N1 infection. Throat swabs or tracheal aspirate samples were sent to the National Institute of Hygiene and Epidemiology in Hanoi or to the Pasteur Institute in Ho Chi Minh City for reverse transcription (RT) PCR and viral isolation. When possible, samples with positive results for influenza A H5 were sent to a World Health Organization (WHO) reference laboratory for confirmatory diagnosis. For each case-patient, 4 control-respondents, individually matched by gender, age (age difference 38°C) during their matched case-patient's period of illness (onset to recovery or death). If the selected control-respondent refused to participate or did not meet the inclusion criteria, the geographically closest eligible person was then selected from the list. All eligible control-respondents were asked to provide a throat swab and venous blood (5 mL) to confirm that they were not currently or had not previously been infected with influenza A H5. Participation of case-patients and control-respondents was voluntary and required written consent and, for those aged 40 was obtained by 2 independent assays. Statistical Analysis Data entry and analysis of individual explanatory variables was performed by using Epi-Info 6 (CDC, Atlanta, GA, USA). Mantel-Haenszel matched-pair analysis (McNemar test) was used to estimate the strength and statistical significance of associations between exposures and influenza A H5 infection. An association was considered statistically significant if 2-sided tests of significance had a p-value 0.1 for removal and a significance level of 7 days, or infection from a contaminated environment as discussed above. Study Limitations A major source of potential bias in this and other studies of risk factors for human H5N1 infection is the use of self-reported prior exposure to sick poultry as a screening tool for identifying potential case-patients. Use of this tool introduces a selection bias that favors finding greater exposure to sick poultry among case-patients than other groups, regardless whether the relationship is causal. In our study, clinicians were not using a systematic screening tool to identify possible H5N1 case-patients, but knowledge of poultry as the source of human H5N1 infection was ubiquitous. Possibly, H5N1 case-patients who did not have exposure to sick poultry may have been less likely to be identified than case-patients who did report this exposure. However, 5 of the 28 case-patients (18%) did not report exposure to sick poultry, indicating that exposure to sick poultry was not a prerequisite for identification as a case-patient. In light of this conflict between clinical necessity and study purity, estimates of the size of the association between exposure to sick poultry and H5N1 infection could be interpreted as maximums that are likely to have been inflated by this selection bias. The relatively small number of case-patients means that the study may be underpowered to detect factors posing only a moderate risk for infection and to detect effect modification. A standardized questionnaire and trained interviewing staff were used to try to minimize interviewer bias, but masking the interviewers as to the case or control status of the respondents was not possible. Recall bias was likely to have occurred, especially because the high case-fatality rate meant that a larger proportion of interviews in the case group (26/28) than the control group (35/106) were completed by proxies. The substantial delay between onset of illness and interviews (mean 35.7 days) is also a potential source of recall bias. The finding of a significant positive association between level of education and risk for infection was unexpected and is difficult to explain. It may be the consequence of a bias introduced by proxy respondents for deceased case-patients reporting higher levels of education than case-patients had actually achieved. Misclassification of case-patients and control-respondents was unlikely. All control-respondents were demonstrated to have no detectable antibodies to H5N1, and all case-patients had a clinically compatible illness with laboratory evidence of H5N1 infection, which was independently verified for 25 (89%) of the 28 cases. Public Health and Research Implications Preparing sick or dead poultry for consumption in an H5N1-affected area is a risky practice. Although this study cannot estimate the absolute risk, among those who prepared sick or dead poultry for consumption, a high proportion of infections could be attributed to this practice. However, as the practice was not that widespread in our study participants, stopping it would prevent only an estimated 28% of H5N1 cases. Less risky but more widespread practices probably account for a greater proportion of H5N1 cases; these practices must also be identified and tackled. Regardless whether consumption of infected poultry is itself a risk factor, preparation and consumption of sick or dead poultry in infected areas must stop. That all 106 persons selected as control-respondents from communities with at least 1 confirmed human H5N1 case were negative for H5N1 antibodies adds further evidence to the belief that widespread subclinical H5N1 infection has not yet occurred in Southeast Asia (20). The finding of an association between lack of access to an indoor water source and H5N1 infection provides an interesting basis for formulating new hypotheses, but it is not sufficiently strong evidence for concluding that H5N1 transmission is occurring by water or as a result of inadequate hygiene. Despite 2 reports of exposure to potentially contaminated water in Vietnamese H5N1 case-patients ([21]; pers. comm., Ministry of Health, Vietnam), no human cases of H5N1 infection have been directly attributed to exposure to contaminated water. Nevertheless, hygiene practices and access to safe water have collateral benefits regardless of H5N1 and should be encouraged and pursued. Environmental investigations are needed to sample water sources in and around the households of incident H5N1 case-patients and compare the findings to water sources sampled in and around unaffected households. Familial clusters of cases have been a significant feature of the epidemiology of H5N1 infection since 2004 in that numerous clusters have occurred in Vietnam, Thailand, Cambodia, Indonesia, and Turkey (22,23). Although common exposures and behavior may be one explanation for the marked clustering, most clusters have involved blood relatives such as sibling pairs or parent-child groups rather than unrelated pairs such as husbands and wives. This finding suggests that inherited biologic factors, such as sialic acid receptor phenotype or immune response, may be determinants of infection and disease. Studying intrinsic determinants of susceptibility will require pooling of data and samples from affected families across affected countries. If intrinsic susceptibility were a risk determinant, it might dilute associations between certain behavior and infection unless the analyses were undertaken within subgroups that are homogeneous with respect to their intrinsic susceptibility. In this respect, intrafamilial studies that combine measures of biologic susceptibility with data about behavioral patterns, including food consumption and hygiene practices, may be particularly enlightening. Acknowledgments The National Institute of Hygiene and Epidemiology thanks the World Health Organization office in Vietnam for identifying the financial resources and providing technical support and the influenza laboratory of US Centers for Disease Control and Prevention for conducting the microneutralization assays. We respectfully thank the Ministry of Health, Vietnam Administration of Preventive Medicine, Preventive Medicine Centers of 15 provinces where the H5N1 cases occurred, National Pediatric Hospital, National Institute for Infectious and Tropical Diseases, Pediatric Hospital No 1, Pediatric Hospital No 2, and the Hospital of Tropical Diseases-Ho Chi Minh City for providing the conditions and human resources needed to implement this study. Particularly, the authors of this study are indebted to the 28 influenza A H5N1 patients in Vietnam (2004) and their relatives. Without their great cooperation and help, this study would not have been completed. The members of the WHO/Global Outbreak Alert and Response Network Avian Influenza Investigation Team in Vietnam are Bach Huy Anh (Hanoi Medical University, Vietnam); Philippe Barboza (Institut de Veille Sanitaire, France); Niranjan Bhat, Aaron Curns, Nguyen Cong Doan, Jackie Katz, Keiji Fukuda, Amy Funk, Taronna Maines, Mark Simmerman, Terry Tumpey, Timothy Uyeki (CDC, United States); Arnold Bosman, Mirna Du Ry van Beest Holle (National Institute for Public Health and the Environment, the Netherlands); Sofia Boqvist (Smittskyddsinstitutet, Sweden); Rick Brown, Futoshi Hasabe (Asian Development Bank, the Phillipines); Pascale Brudon, Philippe Calain, Robert Dietz, Rodger Doran, Tom Grein, Peter Horby, Hitoshi Oshitani (WHO); Valerie Delpech (Health Protection Agency, United Kingdom); Patrice Gautier (Veterinaires sans Frontieres, Vietnam); Shigeyuki Itamura, Takehiko Saito (National Institute for Infectious Diseases, Japan); Donna Mak (Curtin University of Technology, Australia); Noel Miranda (Association of Southeast Asian Nations Secretariat, Indonesia); Reiko Saito (Niigata University, Japan); and Joel Francart. This study was generously supported by a donation from the Italian Development Cooperation Fund (Cooperazione Italiana) through the WHO office in Vietnam. Dr Dinh is associate professor of epidemiology and vice-director of the National Institute of Hygiene and Epidemiology in Vietnam. His research interests focus on the epidemiology, etiology, and control of communicable diseases of public health importance, especially emerging communicable diseases such as severe acute respiratory syndrome, avian influenza, and viral encephalitides. References World Health Organization. Avian influenza A (H5N1) in humans and poultry in Viet Nam, 2004 January 13 [cited 2006 May 18]. Available from http://www.who.int/csr/don/2004_01_13/en/index.html Mounts AW, Kwong H, Izurieta HS, Ho Y, Au T, Lee M, et al. Case-control study of risk factors for avian influenza A (H5N1) disease, Hong Kong, 1997. J Infect Dis. 1999;180:505–8. Buxton Bridges C, Katz JM, Seto WH, Chan PK, Tsang D, Ho W, et al. Risk of influenza A (H5N1) infection among health care workers exposed to patients with influenza A (H5N1), Hong Kong. J Infect Dis. 2000;181:344–8. Bridges CB, Lim W, Hu-Primmer J, Sims L, Fukuda K, Mak KH, et al. Risk of influenza A (H5N1) infection among poultry workers, Hong Kong, 1997–1998. J Infect Dis. 2002;185:1005–10. Katz JM, Lim W, Bridges CB, Rowe T, Hu-Primmer J, Lu X, et al. Antibody response in individuals infected with avian influenza A (H5N1) viruses and detection of anti-H5 antibody among household and social contacts. J Infect Dis. 1999;180:1763–70. Liem NT, Lim W; World Health Organization International Avian Influenza Investigation Team, Vietnam. Lack of H5N1 avian influenza transmission to hospital employees, Hanoi, 2004. Emerg Infect Dis. 2005;11:210–5. Apisarnthanarak A, Erb S, Stephenson I, Katz JM, Chittaganpitch M, Sangkitporn S, et al. Seroprevalence of anti-H5 antibody among Thai health care workers after exposure to Avian influenza (H5N1) in a tertiary care center. Clin Infect Dis. 2005;40:e16–8. Schultsz C, Dong VC, Chau NV, Le NT, Lim W, Thanh TT, et al. Avian influenza H5N1 and healthcare workers. Emerg Infect Dis. 2005;11:1158–9. Centers for Disease Control and Prevention. Investigation of avian influenza (H5N1) outbreak in humans–Thailand, 2004. MMWR Morb Mortal Wkly Rep. 2006;55(Suppl 1):3–6. Rowe T, Abernathy RA, Hu-Primmer J, Thompson WW, Lu X, Lim W, et al. Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays. J Clin Microbiol. 1999;37:937–43. Sturm-Ramirez KM, Hulse-Post DJ, Govorkova EA, Humberd J, Seiler P, Puthavathana P, et al. Are ducks contributing to the endemicity of highly pathogenic H5N1 influenza virus in Asia J Virol. 2005;79:11269–79. Ungchusak K, Auewarakul P, Dowell SF, Kitphati R, Auwanit W, Puthavathana P, et al. Probable person-to-person transmission of avian influenza A (H5N1). N Engl J Med. 2005;352:333–40. Bridges CB, Kuehnert MJ, Hall CB. Transmission of influenza: implications for control in health care settings. Clin Infect Dis. 2003;37:1094–101. Rimmelzwaan GF, van Riel D, Baars M, Bestebroer TM, van Amerongen G, Fouchier RA, et al. Influenza A virus (H5N1) infection in cats causes systemic disease with potential novel routes of virus spread within and between hosts. Am J Pathol. 2006;168:176–83. Keawcharoen J, Oraveerakul K, Kuiken T, Fouchier RA, Amonsin A, Payungporn S, et al. Avian influenza H5N1 in tigers and leopards. Emerg Infect Dis. 2004;10:2189–91. Shortridge KF, Zhou NN, Guan Y, Gao P, Ito T, Kawaoka Y, et al. Characterization of avian H5N1 influenza viruses from poultry in Hong Kong. Virology. 1998;252:331–42. Luby SP, Agboatwalla M, Feikin DR, Painter J, Billhimer W, Altaf A, et al. Effect of handwashing on child health: a randomised controlled trial. 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☉ 11342791:Salmonella Enteritidis in Broiler Chickens, United States, 2000–2005
US Department of Agriculture Food Safety and Inspection Service, Washington, DC, USA US Department of Agriculture Food Safety and Inspection Service, College Station, Texas, USA US Department of Agriculture Food Safety and Inspection Service, Omaha, Nebraska, USA Abstract US Department of Agriculture Food Safety and Inspection Service (FSIS) data on Salmonella enterica serotype Enteritidis in broiler chicken carcass rinses collected from 2000 through 2005 showed the annual number of isolates increased >4-fold and the proportion of establishments with Salmonella Enteritidis–positive rinses increased nearly 3-fold (test for trend, p20,000 chickens per year were eligible for FSIS regulatory Salmonella testing. These establishments accounted for >95% of raw poultry marketed in the United States. The sampling frame for the present study included all eligible FSIS-inspected establishments. Each month, eligible facilities were randomly selected for Salmonella testing to begin in the following month. In each broiler slaughter setting that was tested, 1 broiler chicken carcass rinse (hereafter referred to as broiler rinse) was collected per day for 51 days of operation. The 51 broiler rinses constitute a "Salmonella set." Sets were scheduled approximately once a year. When a plant did not meet the Salmonella performance standard, a follow-up set was scheduled. To limit bias, this report does not include data from follow-up sets. Carcasses were collected after they exited the chiller, downstream from the slaughter line. The chiller is designed to bring carcass temperatures down to the refrigeration range. The postchill collection site was selected as the sampling site because interventions for pathogen reduction are generally located before this point. Broiler Rinse Collection Carcasses were collected after they exited the chiller and aseptically placed in a sterile bag. A 400-mL volume of buffered peptone water was added to the carcass in the bag. Half the volume was poured into the interior cavity and the other half over the skin. The carcass was rinsed with a rocking motion for 1 minute at a rate of ≈35 cycles per minute. After the carcass was removed from the bag, the rinse was poured into a sterile container and shipped on a freezer pack by overnight mail to 1 of 3 FSIS laboratories (Athens, GA; Alameda, CA; St. Louis, MO, USA) for analysis (10). Microbiologic Testing Testing of broiler rinses for Salmonella was performed by using standard FSIS isolation methods (11). Before October 2003, an immunoassay system (Assurance polyclonal enzyme immunoassay, BioControl Systems, Inc., Bellevue, WA, USA) was used to screen enrichment broths for Salmonella. Beginning in October 2003, Salmonella gene amplification (BAX System PCR Assay, DuPont Qualicon, Wilmington, DE, USA) was performed on lysed cells after overnight incubation in buffered peptone broth (35°C). Broiler rinses that tested positive on the screening test were cultured for Salmonella with standard methods (i.e., selective enrichment, plating, serologic and biochemical confirmation). Three presumptive Salmonella colonies with the predominant colony form were selected from each plate for biochemical and serologic confirmation. One confirmed Salmonella isolate was sent to the National Veterinary Services Laboratories (NVSL, USDA-APHIS-VS, Ames, IA, USA) for Salmonella serotyping (12). Beginning in 2001, isolates of Salmonella Enteritidis were phage typed at NVSL (13). Because the predominant Salmonella Enteritidis phage types were clonal (6,14) and pulsed-field gel electrophoresis and antimicrobial susceptibility patterns were not available on all isolates during the study period, no further characterization of the isolates was performed for this report. Analysis Analysis was restricted to Salmonella sets performed in calendar years 2000–2005. A χ2 test (2-sided) was used to test trends for annual percent of Salmonella Enteritidis isolates among Salmonella-positive broiler rinses and all analyzed broiler rinses, respectively. A χ2 test for trend was also performed to assess the percent of establishments tested annually with Salmonella Enteritidis–positive broiler rinses, with subanalyses by establishment size. Approximately two thirds of establishments were large (>500 employees), one fourth were small (10 employees), and 5% were very small (4 positive broiler rinses per year (of 51 broiler rinse tests per set) increased, beginning in 2002. From 2000 to 2002, Salmonella Enteritidis was isolated from broiler rinses in 14 states, compared with 24 states from 2003 to 2005 . Phage type (PT) 13 was predominant, accounting for half of all isolates, followed by Salmonella Enteritidis PT 8, which accounted for more than one third of isolates . In 2005, the number of isolates that were PT 8 increased >3-fold compared with 2004. Discussion The principal finding of this study was a significant increase in the number of broiler chicken slaughter establishments with Salmonella Enteritidis–positive broiler rinses in the years from 2000 through 2005. The 90 slaughter establishments with positive rinses were dispersed across 24 states, reflecting the geographic distribution of the US broiler industry. During the study period, increases were seen in the proportion of both large and small establishments that had such positive broiler rinses. Some caution is warranted when interpreting our findings. The purpose of the FSIS Salmonella program is to assess performance of individual establishments. The program is not designed to estimate national prevalence of poultry contamination because it does not fully account for production volume or regional or seasonal effects. Furthermore, samples are collected after slaughter processes that are intended to reduce carcass contamination. Nonetheless, the apparent emergence of Salmonella Enteritidis in broilers is noteworthy given the increase in human Salmonella Enteritidis infection rates in the United States (8) and recent findings that eating chicken is a new and important risk factor for sporadic infection (5,6). Additional epidemiologic studies are recommended to further elucidate the role of contaminated chicken in human Salmonella Enteritidis infections and estimate the extent of illness attributable to chicken. Retail food surveillance and laboratory subtyping studies (6) may also be valuable because they enable comparisons of human and poultry strains. In this report, 2 Salmonella Enteritidis phage types, PT 8 and PT 13, accounted for most isolates from broiler rinses. In a recent FoodNet study, the association between Salmonella Enteritidis infection and eating chicken strengthened in analyses restricted to patients infected with these 2 phage types (6). The possible emergence of these phage types in broiler chickens suggests that industry should implement appropriate Salmonella Enteritidis controls for broiler chickens (17,18). The present study preceded a new FSIS policy to control Salmonella in broilers that emphasizes common serotypes of human illness (16). As part of this effort, FSIS held 2 public meetings on Salmonella in broilers: 1 in Athens, Georgia, in August 2005 on controls before slaughter (preharvest), and another in Atlanta, Georgia, in February 2006 on controls in the slaughter plant (postharvest). Information from these meeting was used to prepare guidelines to help broiler plants control salmonellae (19). The agency is also monitoring progress of meat and poultry plants in controlling this organism. If, in July 2007, most plants (e.g., 90%) that manufacture a specific product (e.g., broiler carcasses) have not reduced the percentage of Salmonella tests that are positive to at least half the FSIS performance standard, the agency will consider actions to improve control of salmonellae. One option that FSIS is considering is to post Salmonella results on the web for product classes that have not made sufficient progress, listing data by plant name. In the 1990s, successful voluntary quality assurance programs to control Salmonella Enteritidis were developed by the egg industry and state poultry health officials (20). Many of the interventions are adaptable to the control of this organism in broilers. For example, control points for the organism in broilers are likely to include monitoring and sanitation of breeding flocks, hatcheries, broiler flocks, and slaughter establishments. Serotype data that FSIS provides to plants on each isolate as part of its new Salmonella policy (16) may also assist plant officials to make informed SE risk management decisions. Acknowledgments We thank the FSIS headquarters, inspection, and laboratory personnel and APHIS laboratory personnel who made the report possible. Dr Altekruse is a veterinary epidemiologist in the Public Health Service assigned to the USDA Food Safety and Inspection Service. His research interests include characterization of Salmonella isolates from meat and poultry and reductions in indicator and pathogen counts during slaughter. References Patrick ME, Adcock PM, Gomez TM, Altekruse SF, Holland BH, Tauxe RV, et al. Salmonella Enteritidis infections, United States, 1985–1999. Emerg Infect Dis. 2004;10:1–7. St Louis ME, Morse DL, Potter ME, DeMelfi TM, Guzewich JJ, Tauxe RV, et al. The emergence of grade A eggs as a major source of Salmonella Enteritidis infections. New implications for the control of salmonellosis. JAMA. 1988;259:2103–7. Trepka MJ, Archer JR, Altekruse SF, Proctor ME, Davis JP. An increase in sporadic and outbreak- associated Salmonella Enteritidis infections in Wisconsin: the role of eggs. J Infect Dis. 1999;180:1214–9. Altekruse S, Koehler J, Hickman-Brenner F, Tauxe RV, Ferris K. A comparison of Salmonella Enteritidis phage types from egg-associated outbreaks and implicated laying flocks. Epidemiol Infect. 1993;110:17–22. Kimura AC, Reddy V, Marcus R, Cieslak PR, Mohle-Boetani JC, Kassenborg HD, et al. Chicken consumption is a newly identified risk factor for sporadic Salmonella enterica serotype Enteritidis infections in the United States: a case-control study in FoodNet sites. Clin Infect Dis. 2004;38(Suppl 3):S244–52. Marcus R, Varma JK, Medus C, Boothe EJ, Anderson BJ, Crume T, et al. Re-assessment of risk factors for sporadic Salmonella serotype Enteritidis infections: a case-control study in five FoodNet sites, 2002–2003. Epidemiol Infect. 2006;7:1–9. Cowden JM, Lynch D, Joseph CA, O'Mahony M, Mawer SL, Rowe B, et al. Case-control study of infections with Salmonella Enteritidis phage type 4 in England. BMJ. 1989;299:771–3. Centers for Disease Control and Prevention. Preliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food—10 states, United States, 2005. MMWR Morb Mortal Wkly Rep. 2006;55:392–5. Food Safety and Inspection Service. Pathogen reduction; hazard analysis and critical control point (HACCP) systems; final rule. Fed Regist. 1996;61:38805–55 [cited 2006 Oct 11]. Available from http://www.fsis.usda.gov/OPPDE/rdad/FRPubs/93-016F.htm Food Safety and Inspection Service. FSIS directive 10,230.5: Self-instruction guide for collecting raw meat and poultry product samples for Salmonella analysis. February 4, 1998. [cited 2006 Oct 11]. Available from http://www.fsis.usda.gov/OPPDE/rdad/FSISDirectives/10230-5.pdf Food Safety and Inspection Service. Microbiology laboratory guidebook. August 17, 2006 [cited 2006 Oct 11]. Available from http://www.fsis.usda.gov/Science/Microbiological_Lab_Guidebook Ewing E, editor. Edwards and Ewing's identification of Enterobacteriaceae. 5th ed. London: Elsevier; 1986. Ward LR, de Sa JD, Rowe B. A phage-typing scheme for Salmonella Enteritidis. Epidemiol Infect. 1987;99:291–4. Laconcha I, Baggesen DL, Rementeria A, Garaizar J. Genotypic characterisation by PFGE of Salmonella enterica serotype Enteritidis phage types 1, 4, 6, and 8 isolated from animal and human sources in three European countries. Vet Microbiol. 2000;75:155–65. USDA National Agricultural Statistics Service. Number of broilers and other meat-type chickens sold: 2002 [cited 2006 Oct 11]. Available from http://www.nass.usda.gov/research/atlas02/Livestock/Poultry/Number%20of%20Broilers% 20and%20Other%20Meat-Type%20Chickens%20Sold.gif Food Safety and Inspection Service. Salmonella verification sample result reporting: agency policy and use in public health protection. Fed Regist. 2006;71:9772–7. Gast RK, Holt PS. Experimental horizontal transmission of Salmonella Enteritidis strains (phage types 4, 8, and 13a) in chicks. Avian Dis. 1999;43:774–8. McIlroy SG, McCracken RM, Neill SD, O'Brien JJ. Control, prevention and eradication of Salmonella Enteritidis infection in broiler and broiler breeder flocks. Vet Rec. 1989;125:545–8. Food Safety and Inspection Service. Compliance guideline for controlling Salmonella in poultry. 1st ed. August 2006 [cited 2006 Oct 11]. Available from http://www.fsis.usda.gov/PDF/Compliance_Guideline_Controlling_Salmonella_Poultry.pdfsearch= %22Compliance%20guideline%20for%20controlling%20Salmonella%20in%20poultry%22 Mumma GA, Griffin PM, Meltzer MI, Braden CR, Tauxe RV. Egg quality assurance programs and egg-associated Salmonella Enteritidis infections, United States. Emerg Infect Dis. 2004;10:1782–9....查看详细 (17700字节)
☉ 11342793:Serologic Evidence of Widespread Everglades Virus Activity in Dogs, Florida
University of Texas Medical Branch, Galveston, Texas, USA University of Florida, Gainesville, Florida, USA Hollywood Animal Hospital, Hollywood, Florida, USA Animal and Plant Health Inspection Service, Fort Collins, Colorado, USA Abstract Everglades virus (EVEV), an alphavirus in the Venezuelan equine encephalitis complex, circulates among rodents and vector mosquitoes in Florida and occasionally infects humans. It causes febrile disease, sometimes accompanied by neurologic manifestations. Although previous surveys showed high seroprevalence in humans, EVEV infections may be underdiagnosed because the disease is not severe enough to warrant a clinic visit or the undifferentiated presentations complicate diagnosis. Documented EVEV activity, as recent as 1993, was limited to south Florida. Using dogs as sentinels, a serosurvey was conducted to evaluate whether EVEV circulated recently in Florida and whether EVEV's spatial distribution parallels that of the mosquito vector, Culex cedecei. Four percent of dog sera contained neutralizing EVEV antibodies, and many seropositive animals lived farther north than both recorded EVEV activity and the principal vector. These results indicate that EVEV is widespread in Florida and may be an important, unrecognized cause of human illness. Everglades virus (EVEV), a mosquitoborne Venezuelan equine encephalitis (VEE) complex alphavirus (Togaviridae; Alphavirus), circulates continuously in enzootic foci in Florida. EVEV infection of humans can result in a nonspecific, flulike, febrile illness that can progress to severe neurologic disease (1,2). Human EVEV serosurveys in the 1960s and 1970s indicated that people in south Florida were frequently exposed to EVEV. In 1 survey (3), >50% of Seminole Indians who resided north of Everglades National Park had antibody to EVEV, and 9% of other groups living in 3 rural communities at the periphery of the park were EVEV seropositive in 1973 (4). Despite high antibody prevalence, most seropositive persons reported no history of symptoms or signs consistent with VEE-like disease, although exceptions have been noted (1,2). Among the small number of seropositive persons who experienced illnesses consistent with EVEV infection, the most common signs and symptoms were fever, myalgia, headache, tender lymph nodes, and diarrhea (4). Although serologic data indicate that persons are frequently exposed to EVEV, disease is probably most often asymptomatic or not sufficiently severe to require a visit to a physician. In addition, if an EVEV-infected person seeks medical attention, the nonspecific clinical signs and symptoms, similar to those caused by other viral diseases, may not warrant etiologic diagnosis. Persons with undiagnosed diseases of suspected viral etiology are not routinely tested for EVEV by the Florida Department of Health (L.M. Stark, pers. comm.). However, repeated evidence of EVEV antibody in persons living at different locales in south Florida (3,4) suggests that EVEV may be an unrecognized cause of febrile illness. All recorded EVEV activity has been limited to south Florida, from Everglades National Park north to Indian River County (5) . The last EVEV isolation was reported in 1993 (6). As is the case with surveillance for many arboviruses, EVEV activity may be noted only in regions where virologists actively search. No recent serosurveys to detect current EVEV transmission have been performed, and the geographic distribution of EVEV circulation has never been defined by comprehensive surveys. Laboratory susceptibility experiments suggest that Culex (Melanoconion) cedecei may be the only important EVEV vector (7,8). This species has only been reported in 12 southern counties of Florida (9), which indicates that if the mosquito vector regulates EVEV distribution, EVEV activity is probably limited to those areas. Therefore, the goal of this study was to answer 3 questions: 1) Has EVEV recently been circulating in Florida 2) What is the geographic distribution of EVEV throughout the state and 3) Does the spatial distribution of EVEV activity, as measured by seroprevalence, parallel the recorded distribution of the principal vector, Cx. cedecei Testing human sera for EVEV antibody would be an ideal measure of human exposure, but obtaining these samples is difficult from a logistic and regulatory standpoint. Some human pathogen studies have used antibody prevalence of domestic animals to predict human disease risk (10–12). Canines are effective EVEV sentinels; hemagglutination-inhibiting (HI) and neutralizing antibodies without clinical disease developed in military sentry dogs stationed outdoors in Homestead, Florida (13). Dogs experimentally infected with VEEV (strains not reported) survived infection, and minimal HI titers of 320 developed (14). Furthermore, beagles exposed to Aedes triseriatus mosquitoes infected with the VEEV subtype IAB Trinidad Donkey strain became viremic from days 1 to 5 postinoculation, with virus titers ranging from 1 to 3.8 log10 mouse intraperitoneal median lethal doses per milliliter (MIPLD50) (15). In a reciprocal study, experimentally infected beagles with viremias of at least 3.7 log10 MIPLD50 of VEEV were capable of infecting Ae. triseriatus mosquitoes (16). Taken together, these results demonstrate that dogs become infected with EVEV or VEEV by artificial and natural inoculation routes; produce viremias of 3–4 days' duration; sustain a nonfatal, febrile infection; and develop detectable antibody. Field studies in VEEV-enzootic foci outside Florida also indicate that dogs are frequently infected during outbreaks. Domestic dogs tested after epidemics in Colombia (17), Venezuela (18), and Guatemala (19) commonly had neutralizing antibody to VEEV. In these dogs, the average neutralizing antibody titer was lower than titers in experimentally infected canines, possibly reflecting a longer window of time between exposure to VEEV and the time of blood collection, a period during which antibody levels could wane. Because pet dogs live in close proximity to humans and can serve as effective EVEV sentinels, human exposure to EVEV can be estimated on the basis of dog seroprevalence. Pet dogs are also good sentinels for human arbovirus risk because they more closely approximate the biomass of a human than a hamster or another small mammal, they are restricted to confined geographic zones such as a backyard or neighborhood, and dog owners are knowledgeable about the travel history of their pets. Therefore, we evaluated the distribution of EVEV in Florida by using pet dogs as sentinels of EVEV activity. Materials and Methods Serum Collection Whole blood was collected from pet dogs seen for treatment of various conditions at the University of Florida Veterinary Medical Center in Gainesville, Florida, USA, from July 2003 to January 2004, and at Hollywood Animal Hospital in Hollywood (Miami), Florida, from June to December 2004. Samples from animals living far from Florida, in areas not known to be enzootic for VEEV complex alphaviruses (Galveston, Texas, USA, and Munich, Germany) were kindly provided by resident veterinarians at local clinics and used as negative controls. Dogs from all locations were randomly sampled independent of the reason for the clinic visit. Serum was separated from erythrocytes after low-speed centrifugation. For dogs seen in Gainesville, each pet owner was asked whether the animal had traveled outside of their city of residence, except for the visit to the veterinary clinic. Antibody Assays Each serum sample was tested by standard 80% plaque reduction neutralization test (PRNT) (20). In brief, neutralizing antibody titers were determined by a constant-virus, serum dilution procedure that used Vero (African green monkey kidney) cell monolayers attached to 6- or 12-well plates. Serum samples were heated at 56°C for 30 min for inactivation, and a 1:10 starting serum dilution was serially 2-fold diluted and incubated with an equal volume (250 μL) of ≈800 PFU/mL of EVEV strain FE3–7c for 1 h at 37°C. The virus-serum mixture was incubated onto confluent Vero cell monolayers, overlaid after 1 h with 0.4% agar in Eagle's minimal essential medium and incubated at 37°C for 2 days. The virus was inactivated with 10% formaldehyde, and cell monolayers were stained with 0.05% crystal violet in 30% methanol to visualize plaques. Dilutions of serum that caused a >80% reduction in the number of plaques, as compared with negative controls (commercial fetal bovine serum and serum from dogs living outside alphavirus-enzootic areas [Texas and Germany]), were considered positive. Serum from an experimentally infected, EVEV-immune cotton rat was included as a positive control. The reciprocal of the highest dilution of serum (indicated as the final virus-serum dilution) that inhibited at least 80% of plaques was recorded as the antibody titer. To rule out infection with Eastern equine encephalitis virus (EEEV) and Highlands J viruses (HJV), related alphaviruses also enzootic to Florida, all EVEV-seropositive sera were screened by PRNT for antibody to these viruses by using the North American EEEV strain FL-93 (21) and HJV strain 86–31227 (22). Location Mapping To delineate the geographic distribution of antibody to EVEV in canines, the home location of each dog was mapped with ArcGIS for ArcView 9.1 (ESRI, Redlands, CA, USA). Owner street addresses were geocoded (Tele Atlas, Lebanon, NH, USA) and overlaid with land cover and water features (e.g., lakes, ponds, reservoirs, streams, wetlands) from the National Land Cover Dataset (United States Geological Survey, EROS Data Center, Sioux Falls, SD, USA). The proximity of each dog residence relative to water features and forest, agricultural, or suburban or urban land was calculated. The landcover type at the residence location was also determined. Results Serology A total of 633 serum samples from dogs living in Florida were tested for EVEV antibody by PRNT. Of these, 422 were from the Gainesville clinic, and 211 were from the Miami clinic. At least 1 serum sample was obtained from 54 of the 67 counties in Florida, and >20 samples per county were tested from 6 counties, including the greater Miami-Dade area (Broward n = 152, Miami-Dade n = 55, and Palm Beach n = 27) and Ocala region (Marion n = 63). Although the greatest number of samples came from the Miami area, the relative number of dog sera collected per human population by county was highest for Marion County (1 dog per 4,330 people), compared with ratios of 1:45,392 and 1:12,544 in Miami-Dade and Broward Counties, respectively. Of the 633 sera tested, 26 (4%) contained antibody to EVEV, with 80% PRNT endpoint titers ranging from 20 to 2,560 (Table). None of the EVEV PRNT-positive sera contained detectable neutralizing antibody against EEEV or HJV, which ruled out the possibility of cross-reactions with related, sympatric alphaviruses. None of the dogs from Texas or Germany had detectable antibody to EVEV. The proportion of 80% PRNT-seropositive dogs from Florida (26/633) was significantly greater than that for dogs from Texas and Germany (0/61),which indicated that serosurvey results from Florida dogs were not false positives (χ2 = 2.6, degrees of freedom [df] = 1, p0.05). Environmental Characteristics of Seropositive-Dog Residences Discussion The objective of this study was to determine the distribution of EVEV activity in Florida on the basis of seroprevalence in pet dogs that live in close proximity to humans. Although virus isolation from mosquitoes or vertebrates would be the most definitive measure of virus activity, attempting to isolate EVEV throughout Florida is logistically challenging and not currently conducted by the state health department. To more precisely determine locations in which canine EVEV infection occurs, seronegative dogs could be housed in specific habitat types in which mosquitoes test positive for EVEV. Other studies that would be useful to corroborate our findings include serosurveys in rodents that serve as EVEV reservoir hosts and that have very limited dispersal ranges. Cattle with known histories have also proven useful as VEEV sentinels (23). EVEV Seropositivity in Northern Florida Detection of EVEV antibody in dogs living in north and central Florida, without history of travel to south Florida, where EVEV was previously known to be endemic, indicates that EVEV distribution probably extends farther north than previously reported. Possible explanations for this unexpected finding include the following: 1) Dogs residing in the northern parts of Florida became infected during their trip to the veterinary clinic in Gainesville (a clinic visit was common to the history of all of these dogs). We believe that this explanation is highly unlikely because the clinics are indoors and probably are not infested by many mosquitoes and because even if a dog were bitten by an infected mosquito in or on the way into the clinic, dogs do not seroconvert until several days after infection. 2) Cross-reactive antibodies generated by other alphaviruses endemic to northern Florida were not responsible for EVEV neutralizing antibodies. Overall, 4% of the dog sera we tested contained neutralizing EVEV antibodies, a rate similar to the seroprevalence for the major human pathogenic arboviruses (EVEV, EEEV, Saint Louis encephalitis virus, Western equine encephalitis virus) in Florida detected in >2,500 sera from wildlife in 38 of the state's 67 counties from 1965 to 1974 (24). However, alphavirus-neutralizing antibodies generally do not cross-react, and none of the EVEV-seropositive dogs had neutralizing antibodies to EEEV or HJV, which indicates that the dogs sampled were not infected with any known, related, sympatric alphaviruses. When one considers that pet dogs live close to humans and experience similar mosquito exposure as standard arbovirus sentinels that are housed outdoors continuously in cages (e.g., hamsters, chickens), these results suggest that human infections with EVEV may also occur regularly in many areas of Florida. Therefore, implementing procedures to screen for EVEV in cases of febrile illness or encephalitis might result in recognition of undiagnosed disease. Our geographic results may have been skewed by the fact that two thirds of the dogs sampled were taken to the referral hospital in Gainesville, potentially oversampling dogs from this region of the state. Despite limitations inherent to using serum samples that were not randomly collected from all areas in Florida, detection of antibody in north and central Florida suggests that the geographic distribution of EVEV is more extensive than previously recorded and extends as far north as Tallahassee. EVEV distribution could also extend outside of Florida, although a serosurvey of raccoons in Georgia did not show EVEV activity that far north (25). Determinants of EVEV Distribution These results raise a question: What determines the spatial EVEV distribution in Florida Several hypotheses warrant consideration. First, reservoir host susceptibility and viremia limit the EVEV distribution. The distribution of different cotton rat populations with respect to the known EVEV distribution has not been delineated. However, genetically distinct cotton rats from outside Florida are highly permissive for EVEV viremia (26), which diminishes support for this theory. Second, mosquito species other than Cx. (Mel.) cedecei serve as enzootic vectors in northern Florida. The most abundant mammalophilic species in regions of Florida in which EVEV was previously detected, Aedes taeniorhynchus and Cx. nigripalpus, are not competent EVEV vectors in laboratory experiments (8). Although marginally susceptible mosquito species have been implicated as arbovirus vectors during outbreaks when their large population sizes allow for efficient transmission, the species implicated in those settings showed at least some competence in laboratory susceptibility experiments (27,28). By contrast, Ae. taeniorhynchus and Cx. nigripalpus were completely refractory to experimental laboratory infection with EVEV (8). Also, enzootic viruses in the VEEV complex, including EVEV, are typically highly specific in their use of Culex. (Melanoconion) spp. as primary vectors (29). Of the 7 vectors of enzootic VEE complex viruses identified to date, all are members of the Spissipes section in the Culex (Melanoconion) subgenus (29). Nevertheless, other species, as well as populations of Ae. taeniorhynchus and Cx. nigripalpus from northern Florida, should be evaluated in laboratory studies. Field studies should also be conducted since EVEV circulating in northern Florida could be capable of infecting Ae. taeniorhynchus or Cx. nigripalpus, unlike EVEV isolates and mosquitoes from the Everglades that were used for laboratory vector competence studies. Ticks or other ectoparasites that are widely distributed in Florida could also serve as vectors. Finally, one other possible explanation is that the distribution of EVEV is limited by that of Cx. cedecei, but the range of this vector extends beyond that previously recorded or has expanded (or both). Because this species is difficult to identify morphologically, systematic mosquito sampling throughout the state is needed to address this possibility. Location mapping showed that EVEV-seropositive dogs were not more likely than seronegative dogs to live in environments typically inhabited by Cx. cedecei. These results should be interpreted with caution because of limitations inherent to using owner reports of pet travel histories. Cx. cedecei has been trapped in hardwood hammocks in the Everglades, mangrove swamps, and hardwood forests, but little is known about its proclivity for disturbed or suburban habitats. A closely related VEEV complex virus vector that occurs in Central America, Cx. (Mel.) taeniopus (30), is found in high abundance in habitats subject to heavy human disturbance. Cx. cedecei might also inhabit suburban or urban areas, but it has not been identified in such environments because of limitations in identification practices. Clinical Data In spite of antibody detection in 4% of animals sampled, clinical signs reported for all of the seropositive dogs that were seen at the referral veterinary hospital in Gainesville (detailed data not shown; most animals were diagnosed with tumors or bacterial infections) were inconsistent with EVEV disease in humans (2) or laboratory rodents (L.L. Coffey, unpub. data). These observations indicate that EVEV was likely not the underlying cause for the manifestations seen at these hospitals. Military sentry dogs observed continuously during periods of natural infection, verified by seroconversion, did not show signs of VEE-like illness (13). Despite the low EVEV antibody titers observed in many of the EVEV-seropositive dogs, these results likely represent real infections, especially given that no serum samples from 61 dogs living in areas outside Florida tested positive. Antibody titers in sentry dogs naturally infected with EVEV waned to low levels during a 1- to 2-year period (13). The low titers reported here could result from a long interval between infection and blood collection. Alternatively, high antibody titers may never develop in dogs naturally infected with EVEV. For example, 1 young dog born in September 2003 and then sampled <1 year later had a titer of only 40 (Table). The low EVEV antibody titers in naturally infected pet dogs in this study are similar to those in sentry dogs (13) but are not as high as titers in dogs experimentally injected with 1,000 suckling mouse ICLD50 of EVEV (31) or VEE virus (14). Higher neutralizing antibody titers might have developed in experimentally infected dogs because the dose of virus administered by intramuscular injection was greater than the amount delivered by a feeding mosquito. In summary, detection of antibody in dogs throughout Florida suggests that EVEV extends as far north as Tallahassee and has been circulating as recently as 2003. Additional serosurveys involving more dogs, rodents, or both throughout the state, in addition to attempts to isolate EVEV from mosquitoes and vertebrates in regions where seropositive dogs occur, will further define the geographic distribution of EVEV. Enhanced vector surveillance could better define the range of Cx. cedecei and may help to explain the unexpected finding of EVEV activity in northern Florida. Screening for EVEV in human patients may also show wide spatial dispersion and a high rate of human infection. EVEV may be a cause of febrile illness or encephalitis in many areas of Florida and should be considered by physicians as a potential cause. Acknowledgments We thank Leah Paessler-Fistein and Martin Pfeffer for providing the negative control dog sera and Charles Calisher for critically reviewing the manuscript. L.L.C. was supported by the James W. McLaughlin Fellowship Fund. This research was supported by NIH grant AI48807. Dr Coffey is a recent graduate of the Experimental Pathology Department at the University of Texas Medical Branch, Galveston, Texas. Her primary research interests include virus ecology, pathogenesis, evolution, and transmission dynamics. References Ehrenkranz NJ, Sinclair MC, Buff E, Lyman DO. The natural occurrence of Venezuelan equine encephalitis in the United States. N Engl J Med. 1970;282:298–302. Ehrenkranz NJ, Ventura AK. Venezuelan equine encephalitis virus infection in man. Annu Rev Med. 1974;25:9–14. Work TH. Serological evidence of arbovirus infection in the Seminole Indians of southern Florida. Science. 1964;145:270–2. Ventura AK, Buff EE, Ehrenkranz NJ. Human Venezuelan equine encephalitis virus infection in Florida. Am J Trop Med Hyg. 1974;23:507–12. Day JF, Stark LM, Zhang JT, Ramsey AM, Scott TW. Antibodies to arthropod-borne encephalitis viruses in small mammals from southern Florida. J Wildl Dis. 1996;32:431–6. Mitchell CJ, Morris CD, Smith GC, Karabatsos N, Vanlandingham D, Cody E. Arboviruses associated with mosquitoes from nine Florida counties during 1993. J Am Mosq Control Assoc. 1996;12:255–62. Weaver SC, Scherer WF, Taylor CA, Castello DA, Cupp EW. Laboratory vector competence of Culex (Melanoconion) cedecei for sympatric and allopatric Venezuelan equine encephalomyelitis viruses. Am J Trop Med Hyg. 1986;35:619–23. Coffey LL, Weaver SC. Susceptibility of Ochlerotatus taeniorhynchus and Culex nigripalpus for Everglades virus. Am J Trop Med Hyg. 2005;73:11–6. Darsie RF, Morris CD. Keys to the adult females and fourth instar larvae of the mosquitoes of Florida (Diptera, Culicidae). Vol. 1. E.O. DeLeon Springs (FL): Painter Printing Company; 2000. Allela L, Bourry O, Pouillot R, Delicat A, Yaba P, Kumulungui B, et al. Ebola virus antibody prevalence in dogs and human risk. Emerg Infect Dis. 2005;11:385–90. Duncan AW, Correa MT, Levine JF, Breitschwerdt EB. The dog as a sentinel for human infection: prevalence of Borrelia burgdorferi C6 antibodies in dogs from southeastern and mid-Atlantic states. Vector Borne Zoonotic Dis. 2004;4:221–9. Mannelli A, Mandola M, Pedri P, Tripoli M, Nebbia P. Associations between dogs that were serologically positive for Rickettsia conorii relative to the residences of two human cases of Mediterranean spotted fever in Piemonte (Italy). Prev Vet Med. 2003;60:13–26. Nichols JB, Lassing EB, Bigler WJ, Hoff GL. 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The involvement of dogs in endemic cycles of Venezuelan encephalitis virus. Am J Epidemiol. 1973;98:311–4. Scherer WF, Ordonez JV, Jahrling PB, Pancake PB, Dickerman RW. Observations of equines, humans and domestic and wild vertebrates during the 1969 equine epizootic and epidemic of Venezuelan encephalitis in Guatemala. Am J Epidemiol. 1972;95:255–66. Beaty BJ, Calisher CH, Shope RE. Arboviruses. In: Emmons RW, editor. Diagnostic procedures for viral, rickettsial and chlamydial infections. 6th edition, Washington: American Public Health Association; 1989. p. 797–855. Aguilar PV, Paessler S, Carrara AS, Baron S, Poast J, Wang E, et al. Variation in interferon sensitivity and induction among strains of eastern equine encephalitis virus. J Virol. 2005;79:11300–10. Cilnis MJ, Kang W, Weaver SC. Genetic conservation of Highlands J viruses. Virology. 1996;218:343–51. Estrada-Franco JG, Navarro-Lopez R, Freier JE, Cordova D, Clements T, Moncayo A, et al. Venezuelan equine encephalitis virus, southern Mexico. Emerg Infect Dis. 2004;10:2113–21. Bigler WJ, Lassing E, Buff E, Lewis AL, Hoff GL. Arbovirus surveillance in Florida: wild vertebrate studies 1965–1974. J Wildl Dis. 1975;11:348–56. Bigler WJ. Serologic evidence of Venezuelan equine encephalitis virus infections in raccoons of south central Florida. J Wildl Dis. 1971;7:166–70. Coffey LL, Carrara A, Paessler S, Haynie M, Bradley R, Tesh R, et al. Experimental Everglades virus infection of the cotton rat (Sigmodon hispidus). Emerg Infect Dis. 2004;10:2182–8. Sudia WD, Lord RD, Newhouse VF, Miller DL, Kissling RE. Vector-host studies of an epizootic of Venezuelan equine encephalomyelitis in Guatemala, 1969. Am J Epidemiol. 1971;93:137–43. Miller BR, Monath TP, Tabachnick WJ, Ezike VI. 1989. Epidemic yellow fever caused by an incompetent mosquito vector. Trop Med Parasitol. 1989;40:396–9. Weaver SC, Ferro C, Barrera R, Boshell J, Navarro JC. Venezuelan equine encephalitis. Annu Rev Entomol. 2004;49:141–74. Cupp EW, Scherer WF, Lok JB, Brenner RJ, Dziem GM, Ordonez JV. Entomological studies at an enzootic Venezuelan equine encephalitis virus focus in Guatemala, 1977–1980. Am J Trop Med Hyg. 1986;35:851–9. Calisher CH. Silent hosts of Venezuelan equine encephalitis (VEE) virus in endemic situations: mammals. In: Proceedings of the Workshop-Symposium on Venezuelan Encephalitis Virus. Scientific pub. no. 243, Washington: Regional Office of the World Health Organization; 1972....查看详细 (29112字节)
☉ 11342794:Mastomys natalensis and Lassa Fever, West Africa
Philipps University Institute of Virology, Marburg, Germany Museum National d'Histoire Naturelle, Paris, France Washington University School of Medicine, Saint Louis, Missouri, USA Projet de Recherches sur les Fievres Hemorragiques en Guinee, Conakry, Guinea Severtsov Institute of Ecology and Evolution, Moscow, Russia Institut Pasteur d'Abidjan, Abidjan, Cte d'Ivoire Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany Leiden University Medical Center, Leiden, the Netherlands Abstract PCR screening of 1,482 murid rodents from 13 genera caught in 18 different localities of Guinea, West Africa, showed Lassa virus infection only in molecularly typed Mastomys natalensis. Distribution of this rodent and relative abundance compared with M. erythroleucus correlates geographically with Lassa virus seroprevalence in humans. Arenaviruses are emerging in the Americas and Africa and can cause hemorrhagic fevers with case fatalities of up to 15%. These viruses are mainly transmitted through contact with the excreta of their natural hosts, rodents of the family Muridae. The Old World arenavirus Lassa virus causes up to 300,000 cases of Lassa fever annually in endemic foci of 2 geographically disjunct regions of West Africa (1). Most arenaviruses have been associated with 1 specific reservoir host species (2). Knowledge of the geographic distribution of the taxonomically defined host is therefore essential to understand the epidemiology of human infections. In the 1970s, the rodent host of Lassa virus was classified as Mastomys natalensis (3); later, when hemoglobin electrophoresis was used for species determination, M. erythroleucus and possibly M. huberti were also proposed as hosts (1). In addition, Lassa virus antigen was detected in Rattus and Mus genera, raising the possibility that other rodent genera could be involved in transmission (4). The taxonomy of Mastomys is considered unresolved, and species determination remains problematic; 8 distinct species are recognized, and several coexist in Lassa fever–endemic areas (5). The uncertainty about their precise natural host relationships with Lassa virus is considered a major obstacle for a better understanding of the restricted distribution of Lassa fever in West Africa (6). In this study, we molecularly typed >1,000 specimens of Mastomys spp. from Guinea with a recently established species-specific PCR (7). The rodents were then screened for Lassa virus infection with a reverse transcription PCR (RT-PCR), which was shown to amplify Lassa virus strains from Sierra Leone, Liberia, Guinea, Cte d'Ivoire, and Nigeria, as well as the African arenaviruses Mobala and Ippy and 3 strains of the related lymphocytic choriomeningitis virus (8). The Study In a survey of rodentborne hemorrhagic fever viruses, 1,591 small mammals were trapped in Guinea from 2002 to 2005. Total RNA was extracted from rodent blood preserved in liquid nitrogen by using the Blood RNA kit (Peqlab, Erlangen, Germany). A 1-step RT-PCR targeting a highly conserved region of the RNA polymerase (L) gene was performed by using primers LVL3359A-plus (5′-AGAATTAGTGAAAGGGAGAGCAATTC), LVL3359D-plus (5′-AGAATCAGTGAAAGGGAAAGCAATTC), LVL3359G-plus (5′-AGAATTAGTGAAAGGGAGAGTAACTC), LVL3754A-minus (5′-CACATCATTGGTCCCCATTTACTATGATC), and LVL3754D-minus (5′-CACATCATTGGTCCCCATTTACTGTGATC) (Note: Underlined letters represent differences in nucleotides among plus and minus primers). Because Lassa virus antigen should be frequently detectable in the natural host, and specific antibodies are known to be negatively correlated with its presence, they were not investigated in this study (9). The animals were caught in 18 different study sites representative of the principal geographic regions of Guinea (10) . All sites were rural villages with a population <1,000. Human Lassa fever seroprevalence was previously reported to be low (0%–11%) in 6 trapping areas and high (25%–55%) in 12 trapping areas (11–13, Figure 1). Of 1,482 murid rodents belonging to 13 genera and at least 20 species, we typed 847 as M. natalensis and 202 as M. erythroleucus but none as M. huberti, by using DNA from liver biopsies. In addition, we karyotyped 12 members of the genus Mastomys in the field by using standard procedures (14). We obtained positive RT-PCR results from 98 (1.2%) of 1,482 murid rodents. Sequence analysis showed 96 Lassa virus strains with 96%–100% amino acid homology with the prototypic strain Josiah. Lassa virus–positive rodents were only captured in the prefectures of Faranah (villages of Gbetaya, Bantou, Tanganya) and Gueckedou (Denguedou), both situated along the border with Sierra Leone . We PCR-typed all Lassa virus–positive rodents unequivocally as M. natalensis, with 1 male (no. BA686) additionally confirmed by karyotyping (2n = 32, autosomal fundamental number = 53). Overall, 11.3% of M. natalensis were infected with Lassa virus (Table), with 0% in the low seroprevalence area and 5.4%–32.1% in the Lassa fever high seroprevalence area. In the coastal region, where the lowest human Lassa virus seroprevalence (0%–6%) has been reported, only M. erythroleucus was captured. In contrast, in the forest region, where the highest seroprevalence of up to 55% in selected villages has been found, only M. natalensis was trapped (11). Both species were captured in the savannah regions, where seroprevalence was 2%–42%, but M. natalensis was captured more frequently . Lassa virus was isolated in cell culture from 32 rodents, and a 631-bp fragment of the nucleoprotein gene previously used for phylogenetic analysis was sequenced (GenBank accession nos. DQ832667–DQ832699) (15). The phylogenetic tree shows that all isolates belong to the lineage IV of Lassa virus and that strains from the prefecture of Faranah cluster with strains isolated previously from human patients of the same region (15) . We detected 2 novel L-gene sequences that shared 68%–74% homology with lymphocytic choriomeningitis virus in 2 rodents of the Mus subgenus Nannomys. Conclusions Our results indicate that M. natalensis is very likely the only reservoir host of Lassa virus in Guinea. Previous studies that identified M. erythroleucus and M. huberti as reservoirs of Lassa virus in Sierra Leone may have confused the species, especially because M. natalensis and M. huberti have an identical number of chromosomes (5,7). Despite a massive trapping effort, M. huberti was not detected in the regions of Guinea bordering Sierra Leone that had high human Lassa virus seroprevalence. Therefore, M. natalensis is likely also the only reservoir in Sierra Leone. Whether this is also the case for the genetically more remote lineage I–III strains of Lassa virus remains to be evaluated because M. kollmannspergeri is also present in Nigeria (7,16). While it is reassuring that the reservoir for Lassa virus is M. natalensis, as reported in the literature since the 1970s, our study demonstrates that proper taxonomic identification of the host is necessary before drawing inferences about the ecology of Lassa virus infection. The relative abundance of Mastomys correlated with human Lassa virus seroprevalence in Guinea: M. natalensis was absent from the region with the lowest seroprevalence and was the only Mastomys species caught in the highest seroprevalence region. However, if both M. natalensis and M. erythroleucus were present, human Lassa virus prevalence was either low, intermediate, or high . This is a novel finding, which confirms and expands on 2 previous studies conducted in Guinea that reported a correlation between the absence of Mastomys spp. and a low human Lassa virus seroprevalence (9,12). The results of this study have multiple implications for explaining the patchy occurrence of Lassa virus in Guinea and neighboring countries, as well as for Lassa fever control. First, assuming that M. natalensis is the only host of Lassa virus, natural nidality may occur in a similar fashion as that described for Bolivian hemorrhagic fever caused by Machupo virus. The Machupo virus reservoir host, Callomys callosus, has been shown by genetic analysis to be paraphyletic. The populations of rodents responsible for the maintenance and transmission of Machupo virus were monophyletic lineages different from C. callosus in non-disease–endemic regions of Bolivian hemorrhagic fever and coevolving with the virus (17). M. natalensis specimens from regions with high and low Lassa virus activity are being genetically investigated to determine lineages and population structure. Second, the geographic region of potential rodent-to-human transmission of lineage IV strains of Lassa virus is most likely defined by the occurrence of M. natalensis. Mastomys collections from Sierra Leone and Liberia could be molecularly retyped to reevaluate areas of potential Lassa fever reemergence. Third, our mass screening for arenaviruses found only Lassa virus in Mastomys and a novel lymphocytic choriomeningitislike virus in Mus spp. of the subgenus Nannomys, which are closely related to Mus musculus, the Eurasian host of lymphocytic choriomeningitis virus. These findings support the hypothesis of a species-specific association of arenaviruses with their rodent hosts, resulting in cophylogeny. Acknowledgments We thank Aboubakar Camara, Mohamed Camara, and Thomas Strecker for their help with the field work and C. Akoua-Koffi (Institut Pasteur, Abidjan) for her assistance. This study was supported by the European Community (INCO-DEV grant ICA4-CT2002-10050), a Marie Curie Intra-European Fellowship within the 6th European Community Framework Programme (no. MEIF-CT2003-506686), and the Howard Hughes Medical Institute. Dr Lecompte is a postdoctoral fellow at the Institute of Virology in Marburg, Germany. Her research interests include rodent systematics and molecular evolution of rodents and arenaviruses. References McCormick JB, Webb PA, Krebs JW, Johnson KM, Smith ES. A prospective study of the epidemiology and ecology of Lassa fever. J Infect Dis. 1987;155:437–44. Salazar-Bravo J, Ruedas LA, Yates TL. Mammalian reservoirs of arenaviruses. Curr Top Microbiol Immunol. 2002;262:25–63. Monath TP, Newhouse VF, Kemp GE, Setzer HW, Cacciapuoti A. Lassa virus isolation from Mastomys natalensis rodents during an epidemic in Sierra Leone. Science. 1974;185:263–5. Wulff H, Fabiyi A, Monath TP. Recent isolations of Lassa virus from Nigerian rodents. Bull World Health Organ. 1975;52:609–12. Granjon L, Duplantier J-M, Catalan J, Britton-Davidian J. Systematics of the genus Mastomys (Thomas, 1915) (Rodentia: Muridae). A review. Belg J Zool. 1997;127(Suppl. 1):7–18. Mills JN, Childs JE. Ecologic studies of rodents reservoirs: their relevance for human health. Emerg Infect Dis. 1998;4:529–37. Lecompte E, Brouat C, Duplantier JM, Galan M, Granjon L, Loiseau A, et al. Molecular identification of four cryptic species of Mastomys (Rodentia, Murinae). Biochem Syst Ecol. 2005;33:681–9. Vieth S, Drosten C, Lenz O, Vincent M, Omilabu S, Hass M, et al. A reverse transcription PCR assay for detection of Lassa virus and related Old World arenaviruses targeting the L gene. Trans R Soc Trop Med Hyg. In press. Demby AH, Inapogui A, Kargbo K, Koninga J, Kourouma K, Kanu J, et al. Lassa fever in Guinea: II. Distribution and prevalence of Lassa virus infection in small mammals. Vector Borne Zoonotic Dis. 2001;1:283–96. White F. 1983. The vegetation of Africa; a descriptive memoir to accompany the UNESCO/AETFAT/UNSO vegetation map of Africa. Paris: UNESCO; 1983. Lukashevich LS, Clegg JC, Sidibe K. Lassa virus activity in Guinea: distribution of human antiviral antibody defined using enzyme-linked immunosorbent assay with recombinant antigen. J Med Virol. 1993;40:210–7. Bausch DG, Demby AH, Coulibaly M, Kanu J, Goba A, Bah A, et al. Lassa fever in Guinea: I. Epidemiology of human disease and clinical observations. Vector Borne Zoonotic Dis. 2001;1:269–81. ter Meulen J, Lukashevich I, Sidibe K, Inapogui A, Marx M, Dorlemann A, et al. Hunting of peridomestic rodents and consumption of their meat as possible risk factors for rodent-to-human transmission of Lassa virus in the Republic of Guinea. Am J Trop Med Hyg. 1996;55:661–6. Ford CE, Hamerton JL. A colchicine, hypotonic citrate, squash sequence for mammalian chromosomes. Stain Technol. 1956;31:247–51. Bowen MD, Rollin PE, Ksiazek TG, Hustad HL, Bausch DG, Demby AH, et al. Genetic diversity among Lassa virus strains. J Virol. 2000;74:6992–7004. Robbins CB, Van der Straeten E. Comments on the systematics of Mastomys Thomas 1915 with a description of a new west African species (Mammalia: Rodentia: Muridae). Senckenbergiania Biologica. 1989;69:1–14. Salazar-Bravo J, Dragoo JW, Bowen MD, Peters CJ, Ksiazek TG, Yates TL. Natural nidality in Bolivian hemorrhagic fever and the systematics of the reservoir species. Infect Genet Evol. 2002;1:191–9....查看详细 (13146字节)
☉ 11342795:Methicillin-resistant Staphylococcus aureus in Veterinary Doctors and Students, the Netherlands
Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands Utrecht University, Utrecht, the Netherlands Canisius Wilhelmina Hospital, Nijmegen, the Netherlands Abstract The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the Netherlands, at 1.0%, is among the lowest in Europe. In 2004, a relationship between pig farming and a high risk for MRSA carriage was found. To investigate if those in professional contact with livestock are at higher risk for MRSA carriage, we screened 80 veterinary students and 99 veterinarians and questioned them about animal contacts and known MRSA risk factors. Of these, 27 students who did not have livestock contact were excluded from further analysis. We found 7 carriers of MRSA, a prevalence of 4.6%, which is similar to that found in patients who had previously been treated at foreign hospitals. A correlation of MRSA carriage with a specific animal group could not be established. To preserve the low prevalence of MRSA in the Netherlands, persons involved in the care of livestock should be isolated and screened on admission to the hospital. In the Netherlands, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in clinical isolates of S. aureus has been 20%, which suggested that contact with pigs, at least in that region of the Netherlands, posed a substantial risk of acquiring MRSA (4). If that hypothesis were true, isolation on admission and screening of pig farmers and their family members for MRSA would be indicated. To further investigate if those in professional contact with livestock are at higher risk for MRSA carriage, we screened a selection of veterinary doctors and students. Materials and Methods We screened 80 veterinary students in the last phases of their education and 99 veterinarians attending a conference on livestock. Cultures were taken from both anterior nares and throat. All participants were asked to fill in a questionnaire about the type of animal contacts and possible exposure to known MRSA risk factors. We incubated all cultures in a salt-enriched nutrient broth and after 24 hours subcultured them on blood agar plates and MRSA-ID agars (bioMerieux, La Balme Les Grottes, France). Colony morphology and latex agglutination test (Staphaurex, Remel, Lenexa, KS, USA) initially identified staphylococci; cefoxitine-disc diffusion determined methicillin resistance, according to Clinical and Laboratory Standards Institute criteria (5). All cefoxitine-resistant isolates underwent further identification and susceptibility testing to cefoxitine, gentamicin, vancomycin, teicoplanin, clindamycin, erythromycin, rifampicin, ciprofloxacin, cotrimoxazol, and tetracycline, using the Phoenix Automated Microbiology System (Becton Dickinson, Franklin Lakes, NJ, USA). We also performed mecA gene PCR, typing by pulsed-field gel electrophoresis (PFGE) with SmaI (the standard method), and spa-typing on all cefoxitine-resistant strains. Results The main characteristics of the veterinary doctors and students are listed in Table 1. Among the 179 persons tested, 7 (3.9%) MRSA carriers were found: 2 students and 5 veterinarians . MRSA carriage varied depending on whether or not study participants had contact with livestock. MRSA carriage was 4.6% among 152 students and doctors in contact with livestock and 0% among 27 students who reported no contact with livestock. All MRSA carriers in this study had recent or regular contact with pigs and cows; only 3 veterinarians reported regular contact with sheep. Because all carriers reported contact with cows and pigs, no relative risk could be calculated . In each group, 1 person indicated a known risk factor for MRSA carriage (1 had been admitted to a foreign hospital; 1 had an MRSA-positive family member), but both tested MRSA negative. In addition to 7 MRSA isolates, S. sciuri was isolated from 1 veterinarian. This strain showed green colonies on the ID-MRSA plates and was Staphaurex positive (http://www.dcss.cs.amedd.army.mil/field/FLIP30/documents/pdfs/staphaurex_insert.pdf), which caused the risk to be wrongly identified as MRSA. All cefoxitine-resistant isolates were susceptible to vancomycin, teicoplanin, rifampicin, and ciprofloxacin, but all were resistant to tetracycline. All MRSA strains and the S. sciuri were mecA positive and were resistant to digestion with restriction endonuclease SmaI when typing by PFGE was attempted, similar to the strains described by Voss et al. (4). Overall, 3 different MRSA types were identified by spa typing; 2 students and 1 veterinarian carried spa-type t011, 3 veterinarians carried spa-type t108, and 1 veterinarian carried spa-type t034. In contrast to the study of Voss et al., t108 was not a dominant spa-type. Discussion MRSA has been found in various animals, such as horses (6) and livestock (7), including pigs (4,8). So far, only 1 study has indicated transmission from livestock to caretakers (4). The extent of this transmission and its clinical significance remain unknown, also undetermined is whether persons in professions other than farming are at increased risk of becoming MRSA carriers. The overall MRSA prevalence in veterinary students and doctors involved in farm animal health in the Netherlands was about 160× higher than that among patients at hospital admissions (4.6% vs. 0.03%) (9); this prevalence falls within the range of that found in patients from foreign hospitals (3.5%–5%) (10). At least with regard to the search and destroy policy in the Netherlands, veterinarians and veterinary students who come in contact with the healthcare system may therefore qualify as patients at high risk, warranting screening and isolation on admission to hospitals. The high frequency of MRSA carriage among veterinary doctors and students is unexpected. While protective coveralls and boots are routinely used during veterinary contact with livestock, protective masks are not. Because S. aureus colonization and transmission occur mainly through contact from the hands to the anterior nares, the standard measures are probably insufficient to prevent MRSA colonization. Therefore, masks and gloves could be considered as additional protective measures. Although low in comparison with several other countries, the quantity and intensity of antimicrobial use in livestock has increased in the Netherlands (11). Data from 1997 to 2004 show that the main antimicrobial classes used in livestock are tetracycline and trimethoprim sulfonamide combinations. All the MRSA strains in this study, and all the strains found by Voss et al., were resistant to tetracycline. We conclude that veterinary doctors and students caring for livestock have a high risk of being colonized by MRSA. The percentage of MRSA carriage in the doctors and students surveyed is such that, to preserve the low prevalence of MRSA in the Netherlands, all persons involved in the care of livestock should be isolated and screened on admission to the hospital, according to national policy. Further investigation is needed to determine the exact source of MRSA in livestock and the effect of risk factors such as the use of antimicrobial agents on MRSA carriage in livestock. This type of research should be conducted in other countries to find out if this phenomenon is limited to the Netherlands or is international. Dr Wulf is a clinical microbiologist at Radboud University Nijmegen Medical Centre. Her current research interests are the interaction of infection control measures and strategies in different healthcare settings and the community. References Tiemersma EW, Bronzwaer SL, Degener JE, Lyytikinen O, Schrijnemakers P, Bruinsma N, et al. Methicillin-resistant Staphylococcus aureus in Europe, 1999–2002. Emerg Infect Dis. 2004;10:1627–34. Voss A, Milatovic D, Wallrauch-Schwarz C, Rosdahl VT, Braveny I. Methicillin-resistant Staphylococcus aureus in Europe. Europ J Clin Microbiol Infect Dis. 1994;13:50–5. European Antimicrobal Resistance Surveillance System (EARSS). EARSS annual report 2004. [cited 2006 Oct 4]. Available from http://www.rivm.nl/earss/Images/EARSS%20annual%20report%202004%20webversie_tcm61- 25345.pdf Voss A, Loeffen F, Bakker J, Wulf M, Klaassen C. Methicillin-resistant Staphylococcus aureus in pig farming. Emerg Infect Dis. 2005;11:1965–6. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; 15th informational supplement. CLSI/NCCLS M100-S15. Wayne (PA): The Institute; 2005. Weese JS, Archambault M, Willey BM, Dick H, Hearn P, Kreiswirth BN, et al. Methicillin-resistant Staphylococcus aureus in horses and horse personnel, 2000–2002. Emerg Infect Dis. 2005;11:430–5. Lee JH. Methicillin (oxacillin)-resistant Staphylococcus aureus strains isolated from major food animals and their potential transmission to humans. Appl Environ Microbiol. 2003;69:6489–94. Armand-Lefevre L, Ruimy R, Andremont A. Clonal comparison of Staphylococcus aureus isolates from healthy pig farmers, human controls, and pigs. Emerg Infect Dis. 2005;11:711–4. Wertheim HF, Vos MC, Boelens HA, Voss A, Vandenbroucke-Grauls CM, Meester MH, et al. Low prevalence of methicillin resistant Staphylococcus aureus (MRSA) at hospital admission in the Netherlands: the value of search and destroy and restrictive antibiotic use. J Hosp Infect. 2004;56:321–5. Kaiser AM, Schultsz C, Kruithof GJ, Debets-Ossenkopp YJ, Vandenbroucke-Grauls CM. Carriage of resistant micro-organisms in repatriates from foreign hospitals to the Netherlands. Clin Microbiol Infect. 2004;110:972–9. Mevius DJ, Pellicaan C, van Pelt W. MARAN-2004: monitoring of antimicrobial resistance and antibiotic usage in animals in the Netherlands in 2004. [cited 2006 Oct 6]. Available from http://www.cidc-lelystad.wur.nl/NR/rdonlyres/7F79ACE6-0FD2-41AB-81B2- BB17FA89603C/11382/MARAN2004web1.pdf...查看详细 (10770字节)
☉ 11342796:Bartonella quintana Endocarditis in Dogs
Ross University, Basseterre, Saint Kitts, West Indies Unite des Rickettsies, Marseille, France North Carolina State University, Raleigh, North Carolina, USA Massey University, Palmerston North, New Zealand Abstract We provide the first evidence that Bartonella quintana can infect dogs and cause typical signs of endocarditis. Using PCR and sequencing, we identified B. quintana in the blood of a dog from the United States with aortic valve endocarditis and probably also in the mitral valve of a dog from New Zealand with endocarditis. Bacterial endocarditis is an uncommon, often fatal, disease of dogs (1). Although a variety of bacteria can be isolated with routine blood cultures, Bartonella spp., gram-negative bacteria with fastidious growth requirements, are the most common etiologic agents (1). While B. vinsonii subsp. berkhoffii (2), B. clarridgeiae (3), and B. washoensis (4) cause endocarditis in dogs, B. henselae and B. quintana are the most common species that cause endocarditis in humans (5). We provide the first evidence that B. quintana can also infect dogs and cause endocarditis. Case 1 A 3-year-old castrated mixed breed dog was referred to the Veterinary Teaching Hospital of North Carolina State University on December 21, 1999, with lethargy, anorexia, fever, occasional cough, and lameness in the right rear leg of 8 days' duration. The dog was maintained mostly indoors but lived on a farm and was occasionally observed chasing wild animals or fighting with the other 5 dogs in the household. It also had frequent exposure to 2 pet cats. At the time of the dog's illness, all other pets in the household were considered healthy. Physical examination showed a grade 4/6, to-and-fro murmur and changes in the right rear leg compatible with vascular occlusion secondary to thromboembolism. Laboratory abnormalities included marked neutrophilia (43,000/μL, reference range 3,000–11,000/μL) and mild lymphocytosis, eosinophilia, hypoalbuminemia, and hyperglobulinemia. Thoracic radiographs showed mild left atrial enlargement and mild pulmonary interstitial infiltrates. Results of an electrocardiogram were normal, but echocardiography showed a large vegetative lesion on the aortic valve that caused stenosis and severe insufficiency. After 1 week's treatment with oral amoxicillin-clavulanate, enrofloxacin, enalapril, atenolol, and subcutaneous heparin sodium, the lameness resolved, and the demeanor was normal. Oral aspirin was substituted for heparin, and medications continued for 6 months, at which time the owner reported that the dog was healthy. On reexamination, the murmur was softer (grade 3/6), but the aortic valve vegetation and insufficiency persisted with progressive left atrial and ventricular enlargement. Subsequently, atrial fibrillation developed, and the dog died from refractory congestive heart failure on September 25, 2002. A necropsy was not performed. Case 2 In November 2003, a 3-year-old castrated mixed breed dog weighing 48 kg was referred to the Veterinary Teaching Hospital of Massey University, Palmerston North, New Zealand, for evaluation of a heart murmur. The dog lived mainly outdoors and had a 1-week history of depression, fever (40.1°C), and swelling of the left tarsus, which resolved with administration of ampicillin and clavulanate. On examination, the dog was febrile (39.8°C) and had marked dyspnea with mild cyanosis. Crackles were heard on both sides of the chest, and a grade 4/6 pansystolic murmur was loudest over the mitral valve area. The dog had numerous fleas (Ctenocephalides felis). Laboratory abnormalities included mild nonregenerative anemia, mature neutrophilia (23,000/μL, reference range 3,600–11,500/μL), mild hypoalbuminemia, and mildly elevated urea and creatinine levels. The urine was concentrated (1.033) and contained large numbers of granular casts. The heart appeared normal in thoracic radiographs, but the pulmonary vasculature was mildly enlarged, and a marked diffuse alveolar pattern occurred throughout the lungs. The heart appeared normal on echocardiography. Despite symptomatic treatment with fluids, furosemide, and amoxicillin-clavulanate, the dog's condition deteriorated rapidly, and the animal was euthanized at the owner's request. Permission was obtained for postmortem examination. Materials and Methods Case 1 Routine blood and urine cultures were performed. Specialized blood cultures for Bartonella that used blood agar plates and liquid cell culture medium (6) were obtained. A year after the dog died, frozen (-80°C) stored aliquots of whole blood (200 μL) and the culture-negative liquid cell-culture medium (1 mL) were thawed, and DNA was extracted with the QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA, USA). PCR was performed with primers that amplify portions of the α-Proteobacteria citrate synthase gene (gltA) (5′ CAT GCA GAY CAR GAR CAR AAT GCT TCT AC 3′ and 5′ ATW CCN GAA TAA AAR TCA ACA TTN GGR TAH A 3′) and the phage-associated gene (pap31) found in several Bartonella spp. (Pap31 1(s): 5′ GAC TTC TGT TAT CGC TTT GAT TT 3′ and Pap31 688 (as): 5′ cac cac cag caa mat aag gca t 3′), as described previously (7). With both primer sets, products were amplified by using DNA from the whole blood and the liquid cell-culture medium. The amplicons were cloned with the pGEM-T Easy Vector System (Promega, Madison, WI, USA) and the sequences determined by Davis Sequencing, Inc. (Davis, CA, USA). Sequences obtained were compared with those in GenBank by using AlignX software (Vector NTI Suite 6.0, InforMax, Inc., Invitrogen Corp., Carlsbad, CA, USA). Case 2 Abnormal tissues found at postmortem examination were fixed in 10% formalin, embedded in paraffin, and sectioned and routinely stained with hematoxylin and eosin. Immunohistochemical testing was performed with rabbit anti–B. quintana antibody (1:1000) and hematoxylin counterstaining as described previously (8). DNA was extracted from the formalin-fixed mitral valve with the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany). PCR was performed with primers for gltA and the ITS fragment as described previously (3,4). Also, a 1-step LightCycler nested PCR was performed as previously described (9) with external and internal primers amplifying the fur gene (10). PCR products were purified with the QIAquick PCR Purification Kit (Qiagen) and sequenced with the dRhodamine Terminator Cycle Sequencing Ready Reaction Mix (Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 310 DNA Sequencer (Applied Biosystems). Multiple alignments were made with the sequences obtained with the Clustal W software, version 1.81 (11). Results Case 1 Routine blood and urine cultures and specialized blood cultures for Bartonella were negative. Amplicons were obtained with primers for the gltA (422 bp; GenBank accession no. DQ383817) and the pap31 (526 bp; GenBank accession no. DQ383818). These sequences had 99% (gltA) and 99.8% (pap 31) homology with B. quintana Fuller (GenBank accession no. BQCSFULLR) and B. quintana strain Toulouse (GenBank accession no. BX897700), respectively. Case 2 On postmortem examination, severe congestion and edema of the lungs with blood-tinged pleural (250 mL) and pericardial (75 mL) effusion were evident. Although the heart was of normal size and shape, multiple soft, friable, irregular red masses, the largest measuring 10 mm in diameter, were firmly attached to 3 cusps of the mitral valve. The aortic valve was normal. Histologic sections of the mitral valve showed multifocal erosions of the endothelium that contained large masses of fibrin admixed with pockets of degenerate neutrophils. While bacteria were not seen in hematoxylin and eosin or gram-stained sections, Warthin-Starry staining showed multiple clusters of rod-shaped organisms within the masses of fibrin. The organisms were also seen by immunohistochemistry with the genus-reactive polyclonal rabbit anti–B. quintana antibody and hematoxylin counterstaining (Figure). The spleen, pancreas, and left kidney had multiple areas of infarction and hemorrhage with numerous intravascular fibrin thrombi. Warthin-Starry–stained sections showed numerous organisms, similar to those found in the valvular masses, within and surrounding many of the thrombosed blood vessels. No product was obtained with primers for gltA and the ITS fragment. Nested PCR that used the fur primers, however, did provide a 202-bp amplicon (GenBank accession no. DQ666269) that had 99% homology with B. quintana strain Toulouse (GenBank accession no. BX897700) and B. koehlerae (GenBank accession no. DQ666271). It had 97% homology with B. clarridgeiae strain 94-F40 (GenBank accession no. DQ683729) and lower levels with sequences in GenBank of B. bacilliformis (GenBank accession no. AF388198) and other Bartonella spp. known to cause endocarditis in dogs and humans: B. elizabethae (GenBank accession no. DQ666270), B. henselae strain Houston-1 (GenBank accession no. BX897699), and B. vinsoni subsp. berkhoffi (GenBank accession no. DQ666272). Discussion The diagnosis of canine bacterial endocarditis is usually based on appropriate clinical and echocardiographic findings or typical pathologic lesions (1). The abnormalities we found were similar to those reported in dogs with bacterial endocarditis and endocarditis due to Bartonella spp., namely murmur (89%), fever (72%), leukocytosis (78%), hypoalbuminemia (67%), thrombocytopenia (56%), elevated liver enzymes (56%), lameness (43%), azotemia (33%), respiratory abnormalities (28%), and weakness and collapse (17%) (1). One dog had clear echocardiographic evidence of endocarditis; the other had distinct lesions at necropsy not seen with echocardiography. B. quintana was the most likely cause of endocarditis identified in our dogs. In the first dog, routine blood cultures were negative for other bacteria that cause endocarditis. PCR and sequencing, however, demonstrated DNA of B. quintana in the dog's blood at the time endocarditis was diagnosed. Although specialized blood cultures for Bartonella spp. were negative, these organisms have fastidious growth requirements, and blood cultures that use solid media have poor diagnostic sensitivity in both humans (9) and dogs (1). The most useful techniques for detecting Bartonella endocarditis are immunohistochemical analysis of affected valves and PCR (1,5,8). In case 2, the dog had typical histologic lesions of endocarditis that contained large numbers of Bartonella organisms, as shown by Warthin-Starry staining and immunohistochemical analysis. When the sequences of the fur gene were compared with those of Bartonella spp. that are known to cause endocarditis in dogs and humans, the sequencing results showed the Bartonella that infected the dog had highest homology (99%) with B. quintana and B. koehlerae. We did not have control DNA to test for B. washoensis, which has been described as an agent of endocarditis in a dog (4) and myocarditis in a human (12), but we regarded infection with this organism as unlikely because it has only been identified in the United States. Although we know of no specific reports of B. quintana in New Zealand, the organism is ubiquitous (13) and is the most likely cause of the endocarditis in the dog we studied. We decided the organism was not B. koehlerae because it has not been reported in New Zealand or found in recent studies of its natural host (domestic cat) and vector (cat flea) in New Zealand (14–16). Although the organism causes endocarditis in humans (17), it does not appear to be pathogenic in cats, the natural host (18). Our description of B. quintana causing disease in the dog is the first report of the organism's pathogenicity in vertebrates other than humans, the natural reservoirs of the organism. Also, our report adds to the growing evidence that B. quintana can infect species other than humans. In recent reports, B. quintana was identified in a cat euthanized for medical reasons not related to infectious diseases (19) and in an apparently healthy captive-bred cynomolgus monkey (Macaca fascicularis) (20). B. quintana was first described as the agent of trench fever in soldiers in World War I. The organism causes a variety of clinical signs, including endocarditis, which is seen most commonly in immunocompetent, homeless men with a history of alcohol abuse (5). Although the body louse is the traditional vector of B. quintana in humans, this parasite was not a likely source of infection for our dogs since it is host specific, and we found no evidence of infestation. Recent reports of B. quintana in persons with no history of body lice have suggested that other vectors may be involved. In France, a high percentages (17%) of C. felis contain DNA of B. quintana, which suggests that cat fleas might be vectors (21). Although the dog from New Zealand had fleas, B. quintana has not been identified in C. felis in the country (15,16). Another proposed vector is Ixodes pacificus (22), but this tick does not occur in North Carolina or New Zealand. Further, ticks are very rarely found on dogs in New Zealand, where PCR studies on the only prevalent species, Haemaphysalis longicornis, have been negative for Bartonella spp. (23). The source of the B. quintana infections in the dogs we describe is unclear. In summary, our study has shown B. quintana can infect dogs and cause endocarditis. Further studies are indicated to investigate the epidemiology of these infections. Dr Kelly teaches at Ross University Veterinary School in Basseterre, Saint Kitts. His research interest is primarily in vectorborne pathogens. References MacDonald KA, Chomel BB, Kittleson MD, Kasten RW, Thomas WP, Pesavento P. A prospective study of canine infective endocarditis in northern California (1999–2001): emergence of Bartonella as a prevalent etiologic agent. J Vet Intern Med. 2004;18:56–64. Breitschwerdt EB, Atkins CE, Brown TT, Kordick DL, Snyder PS. Bartonella vinsonii subsp. berkhoffii and related members of the alpha subdivision of the Proteobacteria in dogs with cardiac arrhythmias, endocarditis, or myocarditis. J Clin Microbiol. 1999;37:3618–26. Chomel BB, Mac Donald KA, Kasten RW, Chang CC, Wey AC, Foley JE, et al. Aortic valve endocarditis in a dog due to Bartonella clarridgeiae. J Clin Microbiol. 2001;39:3548–54. Chomel BB, Wey AC, Kasten RW. Isolation of Bartonella washoensis from a dog with mitral valve endocarditis. J Clin Microbiol. 2003;41:5327–33. Fournier PE, Lelievre H, Eykyn SJ, Mainardi JL, Marrie TJ, Bruneel F, et al. Epidemiologic and clinical characteristics of Bartonella quintana and Bartonella henselae endocarditis: a study of 48 patients. Medicine. 2001;80:245–51. Maggi RG, Duncan AW, Breitschwerdt EB. A novel chemically modified liquid medium that will support the growth of seven Bartonella species. J Clin Microbiol. 2005;43:2651–5. Maggi RG, Chomel B, Hegarty BC, Henn J, Breitschwerdt EB. A Bartonella vinsonii berkhoffii typing scheme based upon 16S-23S ITS and Pap31 sequences from dog, coyote, gray fox, and human isolates. Mol Cell Probes. 2006;20:128–34. Lepidi H, Fournier PE, Raoult D. Quantitative analysis of valvular lesions during Bartonella endocarditis. Am J Clin Pathol. 2000;114:880–9. Zeaiter Z, Fournier PE, Greub G, Raoult D. Diagnosis of Bartonella endocarditis by a real-time nested PCR assay using serum. J Clin Microbiol. 2003;41:919–25. Znazen A, Rolain JM, Hammami N, Kammoun S, Hammami A, Raoult D. High.prevalence of Bartonella quintana endocarditis in Sfax, Tunisia. Am J Trop Med Hyg. 2005;72:503–7. Dessen P, Fondrat C, Valencien C, Munier G. BISANCE: a French service for access to biomolecular databases. Comput Appl Biosci. 1990;6:355–6. Kosoy M, Murray M, Gilmore RD Jr, Bai Y, Gage KL. Bartonella strains from ground squirrels are identical to Bartonella washoensis isolated from a human patient. J Clin Microbiol. 2003;41:645–50. Maurin M, Raoult D. Bartonella (Rochalimaea) quintana infections. Clin Microbiol Rev. 1996;9:273–92. Joseph AK, Wood CW, Robson JM, Paul SL, Morris AJ. Bartonella henselae bacteremia in domestic cats from Auckland. N Z Vet J. 1997;45:185–7. Kelly PJ, Meads N, Theobald A, Raoult D. Rickettsia felis, Bartonella henselae, and Bartonella clarridgeiae, New Zealand. Emerg Infect Dis. 2004;10:967–8. Kelly P, Rolain J-M, Raoult D. Prevalence of human pathogens in cat and dog fleas from New Zealand. N Z Med J. 2005;118:U1754. Avidor B, Graidy M, Efrat G, Leibowitz C, Shapira G, Schattner A, et al. Bartonella koehlerae, a new cat-associated agent of culture-negative human endocarditis. J Clin Microbiol. 2004;42:3462–8. Yamamoto K, Chomel BB, Kasten RW, Hew CM, Weber DK, Lee WI, et al. Experimental infection of domestic cats with Bartonella koehlerae and comparison of protein and DNA profiles with those of other Bartonella species infecting felines. J Clin Microbiol. 2002;40:466–74. La VD, Tran-Hung L, Aboudharam G, Raoult D, Drancourt M. Bartonella quintana in domestic cat. Emerg Infect Dis. 2005;11:1287–9. O'Rourke LG, Pitulle C, Hegarty BC, Kraycirik S, Killary KA, Grosenstein P, et al. Bartonella quintana in cynomolgus monkey (Macaca fascicularis). Emerg Infect Dis. 2005;11:1931–4. Rolain JM, Franc M, Davoust B, Raoult D. Molecular detection of Bartonella quintana, B. koehlerae, B. henselae, B. clarridgeiae, Rickettsia felis, and Wolbachia pipientis in cat fleas, France. Emerg Infect Dis. 2003;9:338–42. Chang CC, Chomel BB, Kasten RW, Romano V, Tietze N. Molecular evidence of Bartonella spp. in questing adult Ixodes pacificus ticks in California. J Clin Microbiol. 2001;39:1221–6. Kelly PJ, Rolain J-M, Midwinter A, Raoult D. Polymerase chain reaction (PCR) survey for rickettsias and bartonellas in ticks from New Zealand. N Z Vet J. 2005;53:468–9....查看详细 (18088字节)
☉ 11342797:Borrelia lusitaniae and Green Lizards(Lacerta viridis), Karst Region, Slovakia
Parasitological Institute of Slovak Academy of Sciences, Koice, Slovakia University of P.J. afárik in Koice, Koice, Slovakia Institute of Zoology of Slovak Academy of Sciences, Koice, Slovakia Abstract In Europe, spirochetes within the Borrelia burgdorferi sensu lato complex are transmitted by Ixodes ricinus ticks. Specific associations are described between reservoir hosts and individual genospecies. We focused on green lizard (Lacerta viridis) as a host for ticks and potential host for borreliae. In 2004 and 2005, a total of 146 green lizards infested by ticks were captured, and 469 I. ricinus ticks were removed. Borrelial infection was detected in 16.6% of ticks from lizards. Of 102 skin biopsy specimens collected from lizards, 18.6% tested positive. The most frequently detected genospecies was B. lusitaniae (77.9%–94.7%). More than 19% of questing I. ricinus collected in areas where lizards were sampled tested positive for borreliae. B. garinii was the dominant species, and B. lusitaniae represented 11.1%. The presence of B. lusitaniae in skin biopsy specimens and in ticks that had fed on green lizards implicates this species in the transmission cycle of B. lusitaniae. The causative agents of Lyme borreliosis, spirochetes of the Borrelia burgdorferi sensu lato complex, are maintained in natural foci by circulation between the vector ticks in the Ixodes ricinus complex and reservoir hosts of various vertebrate taxa. The B. burgdorferi s.l. complex encompasses 12 species (1–3); 4 species have been clearly established as pathogenic to humans: B. afzelii, B. garinii, B. burgdorferi s. s., and B. spielmanii (4–6). B. valaisiana and B. lusitaniae, which were previously considered nonpathogenic, may cause disease as well (7,8). Different species are associated with distinct ecologic features, levels of pathogenicity, and clinical symptoms in patients. In Europe, I. ricinus ticks infest a wide variety of vertebrate hosts, such as mammals, birds, and lizards. The vertebrate hosts are necessary to maintain the tick population and may also serve as reservoirs for the pathogen. Therefore, the identification of reservoir host species is essential to clarify the transmission patterns of B. burgdorferi s.l. in natural foci. The importance of rodents for maintaining B. afzelii (9), and of birds for B. garinii and B. valaisiana (10), in endemic regions of Slovakia is now indisputable. The National Park Slovak Karst is within the region in which B. burgdorferi s.l. in questing ticks and birds has been reported (V. Tarageová, unpub. data). In this area, 2 lizard species occur sympatrically, the common wall lizard (Podarcis muralis) and the green lizard (Lacerta viridis). The green lizard, the dominant species, is frequently infested by immature stages of I. ricinus ticks (11). The importance of lizards in the maintenance cycles of B. burgdorferi s.l. spirochetes is still controversial. In Italy, B. lusitaniae was detected in blood and tissue samples of P. muralis (12). Furthermore, Psammodromus algirus, the most abundant lizard species in North Tunisia, was found to be the primary host for immature stages of I. ricinus. Thus, it could play a role in the circulation of borreliae (13). B. burgdorferi s.s., B. andersonii, and B. bisettii were detected in the blood of 9 lizard species in the southeastern United States (14). On the other hand, several other studies in the United States have shown that the lizards Sceloporus occidentalis and Elgaria multicarinata are reservoir-incompetent for borreliae because they possess borreliacidal factor in their blood (15,16). However, 2 lizard species, Eumeces inexpectatus and Anolis carolinensis, can sustain B. burgdorferi s. s. infection (17). In the Slovak Karst (southeastern part of Slovakia), the green lizard is the major host for immature stages of I. ricinus ticks (11). Therefore, the main aim of this study was to find out whether green lizards can participate in the maintenance cycles of B. burgdorferi s.l. in natural foci and whether an association with specific borrelial genospecies exists. Materials and Methods Study Area The study was conducted in the National Park Slovak Karst. This area represents a part of the Inner Carpathians in southeastern Slovakia (48°36′ N, 20°52′ E). The climate is warm with low humidity and average temperatures of -4°C in January and 18°C in July. The average rainfall is 700 mm/year. Tick and Lizard Collection This survey was conducted in 2004–2005, from May to September, when lizards and ticks are active. Questing I. ricinus nymphs and adults were collected by flagging the vegetation in areas where lizards were sampled. Ticks were immediately stored in 70% ethanol. Green lizards were captured along hiking paths by hand or by noosing, in which a loop made from fishing nylon was attached to the end of a wooden stick and dangled in front of a lizard, which would be captured as it walked through the loop. Animals were characterized by sex and age (adult, subadult, juvenile) and examined for ticks. Ticks were removed with forceps immediately after capture and stored in 70% ethanol. Biopsy specimens (a 2-cm distal part of the tail and a 1-mm × 1.5-mm piece of skin from collar scales) were taken from each lizard with sterile scissors and put in separate vials with 70% ethanol. Ticks were identified to the species and sex. Only I. ricinus ticks were further examined for B. burgdorferi sensu lato. DNA Isolation Immediately before extraction, ticks and tissues were dried for 30 min to evaporate the ethanol. Each sample was cut with a disposable sterile scalpel. Tissue DNA from lizards' tails and scales was extracted by using DNeasy tissue kit (Qiagen, Hilden, Germany). Extraction steps were conducted according to the manufacturer's protocol. Genomic DNA from ticks was isolated by alkaline hydrolysis (18). Incubation time was extended from 5 to 30 min. Isolated DNA was stored at -20°C. PCR PCR amplification was performed in a 25-μL reaction mixture from the MasterTaq DNA polymerase kit (Eppendorf AG, Hamburg, Germany) containing 10.4 μL deionized water, 5 μL 5× TaqMaster PCR Enhancer, 2.5 μL 10× Taq buffer (with 15 mmol/L Mg2+), 1.5 μL 25-mmol/L solution of Mg (OAc)2, 0.1 μL Taq DNA polymerase (5 U/μL), 0.5 μL deoxynucleoside triphosphate (dNTP) mix (10 mmol/L) (Fermentas, Vilnius, Lithuania), 1.25 μL of each primer (10 pmol/μL) (Invitrogen, Paisley, Scotland), and 2.5 μL DNA template. To verify that DNA had been successfully isolated from each tick, primers for the fragment of the tick's mitochondrial cytochrome b gene (620 bp) were used (19). Negative samples were excluded from the further analysis. Positive samples were examined for the presence of B. burgdorferi s. l. by amplifying a portion of the 5S (rrfA)-23S (rrlB) rDNA intergenic spacer (20). PCR products were subjected to electrophoresis on a 1% agarose gel, stained with ethidium bromide, and visualized with a UV transilluminator. RFLP Analysis The positive PCR products of the 5S-23S rDNA intergenic spacer regions were further analyzed by restriction fragment length polymorphism (RFLP). Previously extracted DNA of B. afzelii, B. garinii, B. valaisiana, and B. burgdorferi s.s. were used as positive controls. For each positive sample, 13 μL amplified DNA was digested at 65°C overnight in a solution containing 5 U of Tru1I (300 U/mL) and 1× Buffer R (Fermentas). Electrophoresis was conducted in 16% polyacrylamide gel at 150 V for 3 h. The gels were stained with SYBR gold nucleic acid gel stain (Molecular Probes, Leiden, the Netherlands) for 20 min, and bands were visualized with a UV transilluminator. RFLP profiles that differed from the known profiles of positive controls were further analyzed by sequence analysis. DNA Sequencing of PCR Products Sequencing was performed at the Department of Molecular Biology (Faculty of Natural Sciences Commenius University, Bratislava, Slovak Republic). PCR of the 5S-23S rDNA intergenic spacer was conducted according to the protocol described above. For the fla gene amplicons, DNA strands were sequenced as described previously (21). PCR products were purified by using a QIAquick PCR purification kit (Qiagen). The complementary strands of each sequenced product were manually assembled. Sequences were compared with GenBank entries by Blast N2.2.13 (22). Homologous sequences were aligned by using the CLUSTAL W Multiple Sequence Alignment Program (version 1.81) (23). Sequence similarity among the sequences were calculated by EMBOSS Align, a pairwise alignment algorithm (http://www.ebi.ac.uk/emboss/align). The accession numbers of 5S-23S rDNA intergenic spacer sequences obtained in this study are DQ539339 and DQ539340. Accession numbers of flagellin sequences obtained in this study are DQ788618, DQ788619, and DQ788620. Data Analysis and Statistics To estimate the probability of a tick's becoming infected after engorging on a green lizard and to measure the degree of infectiousness of infected animals, specific infectivity Is (24) and transmission coeficient βH-T (9) were calculated. Individual infectivity (i) is defined as the proportion of larvae derived from an individual lizard that are infected (i = li/lh, li is the number of larvae that become infected, and lh is the total number of larvae derived from that host). The specific infectivity (Is) of a reservoir host species is defined as the sum of individual infectivities and number of individual lizards sampled (Is = ΣiS/nS, n is the number of individual ticks captured). The host-to-tick transmission coefficient (βH–T) is defined as the portion of the sum of individual infectivities and the number of lizards that infected >1 larva (βH–T = ΣiS/niS (ni is the number of individual hosts that gave rise to at least 1 infected tick). Differences in the prevalence of B. burgdorferi s.l. in I. ricinus were evaluated statistically with the 2-tailed χ2 test (degrees of freedom [df] = 1). A value of p1 infected tick. Infected lizards yielded ≈2 infected larvae per host. Genotyping with PCR-RFLP identified the following species: B. lusitaniae, B. afzelii, B. garinii,B. burgdorferi s.s., and B. valaisiana. Of the B. burgdorferi–positive ticks, most (77.9%) were infected with B. lusitaniae. The presence of this species was significantly higher than that of other species (p75% of all host-feeding ticks, as well as 79.2% of all infected ticks, the specific infectivity and host-to-tick transmission coefficient were almost the same for male and female lizards. The seasonal activity of green lizards and different patterns in male and female behavior were monitored in the Slovak Karst (I. Majlath, unpub. data). Larger numbers of ticks feeding on male lizards are associated with higher male activity in spring months, when tick activity peaks as well. Male lizards end hibernation first and are active when the air temperature reaches 10°C–12°C. They need to restock the energy that was depleted during winter and to gain energy for fighting other male lizards to compete for territory and females, for seeking female lizards, and for mating. Female activity increases in summer months when they are incubating eggs. As determined by PCR, the overall prevalence of infection in our sample of questing ticks (19.3%) is consistent with 20.5% found in southern Czech Republic (20) but lower than that reported for a geographically close area in western Slovakia (40%–49%) (30). The total prevalence was higher in adults (22.5%) than in nymphs (19.6%), which is in agreement with the general pattern of increasing Borrelia prevalence through the life stages of ticks as their adults feed on a multiple hosts (31). The total prevalence of borreliae in male and female ticks was identical, but the distribution of genospecies was different. B. garinii was the predominant genospecies in this locality. B. garinii and B. valaisiana are the most commonly reported species in central Europe (32). The high prevalence of B. lusitaniae in borrelia-positive larvae and nymphs as well as skin biopsy specimens from lizards suggests that green lizards are susceptible and transmission competent for B. lusitaniae. On the other hand, a lack or low prevalence of other genospecies in ticks that had fed on lizards may suggest that these genospecies could be negatively selected against by green lizards. A similar suppressive effect of Madeiran wall lizard (Podarcis dugesii) on the transmission of spirochetes was observed (33). Borreliacidal activity against B. burgdorferi s.s. was observed in the lizards S. occidentalis and E. multicarinata in North America (15,16). These findings add to the growing support for the hypothesis that there are Borrelia species-specific associations with specific reservoir host species that result from Borrelia species–specific interactions with host serum complement (29). Significant differences were found in B. lusitaniae prevalence in fed larvae compared with questing nymphs (p<0.001, df = 1); none of 183 examined nymphs was infected by this genospecies. This finding raises the questions of whether borreliae are eliminated during molting and thus do not contribute to the transmission cycle or whether we were just unable to detect it. Significant differences were found in B. lusitaniae prevalence also in fed nymphs compared with questing adults (p<0.01, df = 1). The infection prevalence decreased from 74.5% in fed nymphs to 5% in questing adults. Reduction of infection prevalence has been observed in B. afzelii from 47% in nymphs engorged on the rodents to 7% in questing nymphs (9). The occurrence of B. lusitaniae in ticks is frequent in some areas of the Iberian Peninsula and North Africa, where the organism often represents the only species of B. burgdorferi s.l. complex (13,34). In the rest of the Europe, it has been isolated or detected less frequently, with low prevalence in ticks (30,35,36). The prevalence of B. lusitaniae is the highest in southern Europe and can be exported to other areas by hosts such as birds (37). The 5S-23S rDNA and flagellin sequences of B. lusitaniae–positive ticks and skin biopsy specimens in our study were 100% identical to the B. lusitaniae strain Tr213 from a tick in Turkey (38). The distribution of this borrelial species may be associated with the distribution range of reservoir hosts, including lizards, that inhabit drier and warmer areas. These ecosystems are less abundant in central Europe than in the Mediterranean. Thus, lizards may influence the transmission cycle of borreliae in some localities in which they are the predominant host for ticks. In our study, we found B. lusitaniae in skin biopsy specimens and ticks that fed on green lizards. These findings implicate this species of lizard in the transmission cycle of B. lusitaniae. The competence of other lizard species that feed ticks should be also investigated. 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Poupon M-A, Lommano E, Humair PF, Douet V, Rais O, Schaad M, et al. Prevalence of Borrelia burgdorferi sensu lato in ticks collected from migratory birds in Switzerland. Appl Environ Microbiol. 2006;72:976–9. Guner ES, Hashimoto N, Takada N, Kaneda K, Imai Y, Masuzawa T. First isolation and characterization of Borrelia burgdorferi sensu lato strains from Ixodes ricinus ticks in Turkey. J Med Microbiol. 2003;52:807–13....查看详细 (31763字节)
☉ 11342798:Transmission of Human and Macaque Plasmodium spp. to Ex-Captive Orangutans in Kalimantan, Indonesia
Simon Fraser University, Burnaby, British Columbia, Canada Orangutan Foundation International, Los Angeles, California, USA Orangutan Care Center and Quarantine, Desa Pasir Panjang, Kaliamantan Tengah, Indonesia Abstract Data are lacking on the specific diseases to which great apes are susceptible and the transmission dynamics and overall impact of these diseases. We examined the prevalence of Plasmodium spp. infections in semicaptive orangutans housed at the Orangutan Care Center and Quarantine, Central Kalimantan, Indonesia, by using a combination of microscopic and DNA molecular techniques to identify the Plasmodium spp. in each animal. Previous studies indicated 2 orangutan-specific Plasmodium spp., but our data show 4 Plasmodium spp. These findings provide evidence for P. vivax transmission between humans and orangutans and for P. cynomolgi transmission between macaques and orangutans. These data have potential implications for the conservation of orangutans and also for the bidirectional transmission of parasites between orangutans and humans visiting or living in the region. The following great apes are classified as endangered: the robust chimpanzees, Pan troglodytes; the gracile chimpanzee/bonobo, Pan paniscus; the gorilla, Gorilla gorilla; and the orangutans, Pongo pygmaeus and P. abelii. Habitat loss and hunting by humans are 2 direct threats to the survival of the great apes (1). Until recently, diseases have been overlooked as key threats to primate conservation efforts (2). However, recent research has emphasized the threat of disease transmission between human and nonhuman primates and the effects of these diseases on nonhuman primates (3). Malaria, caused by protozoan parasites in the genus Plasmodium, is 1 disease identified as a potential threat to the conservation of orangutans (4). Two species of Plasmodium naturally infect orangutans: P. pitheci, first isolated from the blood of a Bornean orangutan (5), and P. sylvaticum, identified from orangutans housed at the Sepilok Orangutan Rehabilitation Centre (SORC), Sabah, Malaysia (6,7). Both P. pitheci and P. silvaticum have tertian periodicities (5,7) and are distinguishable from human plasmodia (7). Three major studies of orangutan malaria at SORC (4,5,7) found prevalences of infection of >50%, which may have been influenced by the unusually high population density of orangutans at SORC, estimated at 100/km2 (4). Wolfe et al. (4) found the highest Plasmodium spp. prevalence at 93.5% (29/31) in captive animals but 11.6% (5/43) in wild orangutans. Recent reports indicate that nonhuman primate plasmodia are the source of zoonotic disease outbreaks among humans in Thailand and Malaysia (8,9). Although this finding has implications for human disease outbreaks, few studies have investigated the distribution and transmission of Plasmodium spp. among orangutans and whether these great apes serve as reservoirs for human infections. Similarly, no studies have indicated that human plasmodia might infect and cause the death of captive or feral orangutans, a finding which would have serious implications for great ape conservation efforts. We report here the identification of plasmodia found in semicaptive and recently arrived orangutans at the Orangutan Care Center and Quarantine (OCC&Q) in the province of Central Kalimantan, Indonesia. Materials and Methods Study Site and Population The OCC&Q is located in the village of Pasir Panjang, ≈5 km from the city of Pangkalan Bun in the province of Central Kalimantan, Indonesia. The OCC&Q was established by the Orangutan Foundation International in 1998 to serve as a hospital, orphanage, and rehabilitation center for sick and injured orangutans. Most orangutans are delivered by police or forestry officials after they have been confiscated from illegal pet owners; thus, the history of these animals is often unknown. All orangutans living at the OCC&Q are considered to be semicaptive; during the day, these orangutans have access to a nursery forest where they can learn the skills necessary for survival in the wild. At night these orangutans return to cages for sleep. Approximately 200 orangutans were housed at the OCC&Q during the 2003 study period. Samples were obtained from resident orangutans at OCC&Q as part of biannual health checks done by OCC&Q veterinary staff, and samples were obtained from newly confiscated animals as part of routine medical examinations. Orangutans were grouped into 4 categories on the basis of their residence history at OCC&Q: 1) OCC&Q residents (living at OCC&Q for >4 months) (n = 69), 2) newly confiscated arrivals (living at OCC&Q for 30 kg) (Table). Sample Collection and Preservation Blood samples were collected by an OCC&Q veterinarian with a 25-gauge × 1-inch PrecisionGlide needle (Becton, Dickinson, and Company, Oakville, Ontario, Canada) and a 3-mL syringe. Some animals were sampled more than once so the effectiveness of antimalarial chemotherapy could be monitored. Thin and thick blood smears were prepared, fixed with methanol, stained with 10% Giemsa stain for 30 min, and destained with water (10). Samples were examined microscopically under 1,000× magnification, and levels of parasitemia were estimated (10). Aliquots of blood (5–10 μL) were placed by pipette onto each of 4 circular areas on Whatman FTA Classic Cards (Whatman Inc., Florham Park, NJ, USA), dried overnight at room temperature, and transported to Simon Fraser University for subsequent analysis. DNA Extraction DNA was extracted from the Whatman FTA Classic Cards following the manufacturer's instructions (11,12). In subsequent PCR analyses, we used an entire punch or 2 μL of eluted DNA per reaction. PCR Analysis We used primers designed against the 18S small subunit ribosomal RNA (4,13). The DNA samples were used in a 3-step PCR process. In step 1, PCR of DNA on the disks or from elutant was amplified with primers rPLU1 (5′-TCA AAG ATT AAG CCA TGC AAG TGA-3′) and rPLU5 (5′-CCT GTT GTT GCC TTA AAC TCC-3′) in a standard 50-μL PCR with a PTC-200 Thermocycler (MJ Research; Waltham, MA, USA) under the conditions of 94°C for 4 min, and 35 cycles at 94°C (30 sec), 55°C (1 min), 72°C (1 min), with an additional extension at 72°C (4 min). In step 2, this product was used in a nested PCR with primers rPLU 3 (5′-TTT TTA TAA GGA TAA CTA CGG AAA AGC TGT-3′) and rPLU Cal 2 (5′-CGC TAT TGG AGC TGG AAT TAC C-3′) in a 25-μL reaction under the conditions of 94°C (4 min) and 35 cycles at 94°C (10 sec), 60°C (10 sec), 72°C (45 sec), with an additional extension at 72°C (4 min). PCR products were size fractionated by electrophoresis on a 1% agarose gel containing ethidium bromide and examined on a BioDoc gel documentation System (UVP, Upland, CA, USA). A ≈500-bp band confirmed Plasmodium spp. DNA in the initial PCR sample. We used the DNA from step 1 that tested positive in step 2 in a third PCR with primers rPLU 3 (5′-TTT TTA TAA GGA TAA CTA CGG AAA AGC TGT-3′) and rPLU Cal (5′-ACA CAW RGT KCC TCT AAG AAG C-3′) by using BD Sprint Advantage Single Shots (Clontech, Palo Alto, CA, USA) under the conditions of 95°C (1 min), and 35 cycles at 95°C (30 sec), and 58° (3 min), with an additional extension at 58°C (3 min). These nested primers amplified an ≈1,500-bp fragment that contains 3–4 variable regions of the ribosomal sequence that can be used for species determination. After electrophoresis, PCR products were excised from the gel, purified by using a Qiagen Gel Purification Kit (Qiagen, Valencia, CA, USA), and ligated into pGEM-T-Easy Vector (Promega, Madison WI, USA). Putative transformants were identified by using blue-white screening of XL1-Blue Cells (Stratagene, La Jolla, CA, USA), grown overnight in 5 mL LB medium with ampicillin (100 μg/μL) and purified by using Wizard Plus Miniprep DNA Purification System (Promega). Sequencing of clones was done by using BigDye Chemistry (version 3.1) (Applied Biosystems, Foster City, CA, USA) and with the plasmid primers SP6 and T7 (14). Data Analysis The nucleotide sequences obtained were compared with those Plasmodium spp. sequences available in public databases by using BLASTN (nucleotide-nucleotide) (available from http://www.ncbi.nlm.nih.gov/blast/). All available sequences of the Plasmodium spp. 18S sRNA gene, which contained complete sequences of our target region, were downloaded. Results Field Results We collected 97 blood samples from 86 animals: 19 small, 63 medium, and 4 large orangutans, of which 41 were males and 45 were females (Table). Of the 69 OCC&Q residents tested (34 males, 35 females), 14 (20.3%) tested positive for Plasmodium spp. infections (8 males and 6 females) (Table). Eight of 14 newly arrived animals tested positive for Plasmodium spp. (57%) (2 small males and 6 small females) (Table). The newly arrived animal treated previously for malaria symptoms proved negative for Plasmodium spp. Both previously released ex-captive, medium-sized male orangutans, brought to OCC&Q from a release site in Tanjung Puting National Park for treatment of amebic dysentery, tested positive for Plasmodium spp. (Table). DNA Analysis We amplified, cloned, and sequenced a ≈1,500-bp segment of the Plasmodium spp. 18S sRNA gene from 13 of the 24 orangutans whose blood had been found positive for Plasmodium spp.: 4 from OCC&Q residents (3 males, 1 female), 7 from newly confiscated arrivals (2 males, 5 females), and 2 newly arrived feral animals (2 males) (Table). We aligned the sequences with similar sequences available in the databases, and generated phylogenetic trees based on a nearest neighbor analysis at the nucleotide level. We then aligned at the nucleotide level the sequences we obtained from our 13 Plasmodium spp.–infected orangutans. Phylogenetic trees showing the nearest neighbor relationships of our sequences were created from these alignments . The 13 sequences we obtained form 4 distinct groups at the nucleotide level. On the basis of these groupings, we designated samples VS25, VS28, VM75, and VM82 as group 1; samples VS14 and VM88 as group 2; VS21, VS32, VS33, VM40, VM55, and VM71 as group 3; and VS63 as group 4. From each of these groupings, we selected 1 sequence to represent the entire group. These representative samples were then aligned at the nucleotide and translated amino acid level with available sequences downloaded from the databases to generate a phylogenetic tree . Group 1 (representative sequence VM82) consists of 1,519 bp at the nucleotide level and translates to a putative protein with a length of 506 amino acids (aa). VM82 shares the greatest sequence identity with P. cynomolgi (94%) and P. inui (95%). A phylogenetic tree shows the close association with P. inui and P. cynomolgi. We then aligned VM82 solely with its 2 closest homologs, P. inui and P. cynomolgi. These 3 sequences are highly conserved but have variable areas between bases 106–155, 633–672, 720–730, and 1,018–1,050, which indicate a closer similarity with P. cynomolgi. Because of differences between VM82 and P. cynomolgi in the region of bases 143–155, 646–673, and 1,023–1,043, we designated this group as a P. cynomolgi–like parasite. Group 2 (representative sequence VM88) consists of 1,544 bp at the nucleotide level and translates to a putative protein with a length of 514 aa. Throughout the variable regions, VM88 shares substantial similarity with P. hylobati (89%–96%), P. inui (90%–97%), P. fieldi, (89%–95%) and P. cynomolgi (90%–95%). We then aligned VM88 solely with its 2 closest homologs, P. inui and P. cynomolgi. These 3 sequences are highly conserved but show 2 large variable areas between bases 113–175 and 630–670. Despite sharing the greatest homology with P. inui, differences between VM88 and P. inui in the region of bases 110–175, 712–735, 789–887, and 1,495–1,551 indicate that this is a P. inui–like parasite. Group 3 (representative sequence VM40) consists of 1,515 bp at the nucleotide level and translates to a putative protein with a length of 505 aa. VM40 shares significant identity with 3 Plasmodium spp. at the nucleotide level: P. hylobati, (96%–97%), P. inui (95%–97%), and P. cynomolgi (96%–98%). Alignment of VM40 solely with its 2 closest sequences indicated this sequence is homologous to P. cynomolgi. Group 4, sequence VS63, consists of 1,582 bp at the nucleotide level and translates to a putative protein with a length of 526 aa. At the nucleotide level, VS63 shares the greatest identity with P. simium (97%–98%) and P. vivax (96%–98%). Discussion Our results indicate that newly arrived orangutans to OCC&Q are statistically more likely to be infected with Plasmodium spp. than resident orangutans (χ2 = 8.11, degrees of freedom [df] = 1, p98% shared identity. Areas of notable differences were found between VS63 and P. knowlesi. The BLAST pairwise comparison indicates 5 discrete regions of alignment separated by nonsimilar regions, which indicates that VS63 is not a known variant of P. knowlesi. P. vivax and P. simium are genetically indistinguishable at 13 microsatellite loci and 8 tandem repeats (27). Because P. simium does not exist in Southeast Asia, we have the first report of an orangutan being infected with human P. vivax. This orangutan was a recent arrival at OCC&Q, had been confiscated from human captivity, had extensive interactions with humans in a domestic setting, and had arrived at OCC&Q with a low-level infection that was untreated 3 months before this study began, which indicates a fully functional infection. P. vivax is one of the most widespread of the human plasmodia; it infects 70–80 million persons in the low-lying, coastal, and marshy regions of the world (5,24). Data on the human plasmodia of Central Kalimantan are not easily accessible through the available scientific databases. We assume that P. vivax is present in this region on the basis of the results of our study, the widespread nature of this parasite (5,24), and reports of chloroquine-resistant P. vivax in neighboring West Kalimantan (28). That P. vivax is infective to orangutans is not surprising. Current evidence suggests that P. vivax originated 80,000–10,000 years ago from a macaque Plasmodium sp. (22–24). Because humans are genetically closer to orangutans than to macaques, if P. vivax arose as the result of a recent host switch, then orangutans also could be infected with P. vivax. Our data indicating that orangutans can be infected with human P. vivax, and the corresponding infection of humans with macaque plasmodia (8,9) emphasize the potential importance of the bidirectional transmission of these parasites between humans and nonhuman primates living in close proximity. Increasing our understanding of potential host species and phylogenetic associations of closely related parasites may help identify the origins of human diseases (3,29,30). The data presented here suggest that Bornean orangutans (Pongo pygmaeus) may be infected by 4 species of plasmodia; 2 of these may represent the previously identified orangutan plasmodia P. pitheci and P. silvaticum. Macaque malaria in orangutans suggests cross-species transmission of a parasite between macaques and orangutans living sympatrically in Kalimantan (as had been described for human infections with macaque malaria in Thailand and Malaysia) (8,9). Orangutans also may be susceptible and may be exposed to infection from the human parasite, P. vivax, although few data are available on the symptoms of macaque or human malaria infections in orangutans. Nonetheless, these findings could have important implications for orangutan conservation and rehabilitation programs and for humans who live in close proximity to orangutans. The role of humans and great apes as reservoirs of parasites that can be shared and transmitted between both hosts has not been well-studied. Conservation and rehabilitation programs that permit visits by humans must take into consideration the exchange of parasites between humans and endangered species, the implications of human parasites on the survival of the great apes in these centers, and the potential of these animals to serve as reservoirs of human parasites. Acknowledgments We thank the OCC&Q staff for their help in this research, the Indonesian Institute of Sciences, Nello Angerelli and Chris Dagg for permission to conduct this research, and for facilitating the export of blood samples to Canada. This research was reviewed and approved by the Simon Fraser University Animal Care Committee (675B-03) under Canadian Council on Animal Care guidelines. This research was funded in part by the Canada Research Chairs program, Canadian Institutes of Health Research (no. 69558), and National Sciences and Engineering Research Council of Canada (no. RPG261940) grants to C.L. Mr Reid recently completed a master's degree at Simon Fraser University, Burnaby, British Columbia, Canada, where he studied malaria parasites in orphaned orangutans. His main research interests include cross-species transmission of pathogens among primates and issues surrounding primate conservation and reintroduction. References Butynski TM. Africa's great apes. In: Beck BB, Stoinski TS, Hutchins M, Maple TL, Norton B, Rowan A, et al., editors. Great apes and humans: the ethics of coexistence. Washington: Smithsonian; 2001. p. 3–56. Wallis J, Lee DR. Primate conservation: the prevention of disease transmission. Int J Primatol. 1999;20:803–26. Jones-Engel L, Schillaci MA, Engel G, Paputungan U, Froehlich JW. Characterizing primate pet ownership in Sulawesi: implications for disease transmission. In: Paterson JD, Wallis J, editors. Commensalism and conflict: the human primate interface. Special topics in primatology, vol. 4. 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The evolution of primate malaria parasites based on the gene encoding cytochrome b from the linear mitochondrial genome. Proc Natl Acad Sci U S A. 1998;95:8124–9. Escalante AA, Cornejo OE, Freeland DE, Poe AC, Durrego E, Collins WE, et al. A monkey's tale: The origin of Plasmodium vivax as a human malaria parasite. Proc Natl Acad Sci U S A. 2005;102:1980–5. Leclerc MC, Hugot JP, Durand P, Renaud F. Evolutionary relationships between 15 Plasmodium species from Old and New World primates (including humans): a 18S rDNA cladistic analysis. Parasitology. 2004;129:677–84. Mu J, Joy DA, Duan J, Huang Y, Carlton J, Walker J, et al. Host switch leads to emergence of Plasmodium vivax malaria in humans. Mol Biol Evol. 2005;22:1686–93. Rathore D, Wahl AM, Sullivan M, McCutchan TF. A phylogenetic comparison of gene trees constructed from plastid, mitochondrial and genomic DNA of Plasmodium species. Mol Biochem Parasitol. 2001;114:89–94. Waters AP, Higgins DG, McCutchan TF. 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☉ 11342799:Borrelia garinii in Seabird Ticks(Ixodes uriae), Atlantic Coast, North America
Maine Medical Center Research Institute, South Portland, Maine, USA Memorial University of Newfoundland, Saint John's, Newfoundland, Canada Abstract Borrelia garinii is the most neurotropic of the genospecies of B. burgdorferi sensu lato that cause Lyme disease in Europe, where it is transmitted to avian and mammalian reservoir hosts and to humans by Ixodes ricinus. B. garinii is also maintained in an enzootic cycle in seabirds by I. uriae, a tick found at high latitudes in both the Northern and Southern Hemispheres. To determine whether B. garinii is present in seabird ticks on the Atlantic Coast of North America, we examined 261 I. uriae ticks by polyclonal antiborrelial fluorescent antibody. Ten of 61 ticks from Gull Island, Newfoundland, were positive for borreliae by this screen. Amplicons of DNA obtained by PCR that targeted the B. garinii rrs-rrla intergenic spacer were sequenced and matched to GenBank sequences for B. garinii. The potential for introduction of this agent into the North American Lyme disease enzootic is unknown. In Europe, Lyme disease is caused by 3 genospecies of Borrelia burgdorferi (i.e., B. afzelii, B. garinii, B. burgdorferi sensu stricto), while in North America only B. burgdorferi sensu stricto, the genospecies present in I. scapularis ticks, has been implicated in human disease. B. garinii, the most neurotropic of these 3 genospecies, causes most neurologic Lyme disease in Europe, including cases of meningopolyneuritis and, rarely, encephalomyelitis (1–3). The presence of multiple pathogenic genospecies that cause Lyme disease in Europe complicates serodiagnostic testing (4). In Eurasia, B. garinii is transmitted to avian and rodent hosts and to humans by I. ricinus, the sheep or forest tick, and I. persulcatus, the taiga tick (5–9). I. uriae, the seabird tick, also maintains this agent in a "silent" enzootic cycle in seabirds at their nesting sites over a wide but discontinuous area (10–13). Although these 2 enzootic cycles are generally geographically and ecologically separate, interchange of B. garinii strains may occur at sites where both vectors coexist (14). The risk for seabird-associated strain types of B. garinii to cause Lyme disease, however, is not known (15). Although B. garinii is present in seabird ticks in a nearly circumpolar distribution in both the Northern and Southern Hemispheres (12,13), including Alaska, the presence of B. garinii in I. uriae ticks at sites on the North Atlantic Coast has not previously been documented. We sought to determine whether B. garinii is present in ticks obtained from colonial seabird nesting sites on the Atlantic Coast of North America. Methods Seabird researchers working at several sites on the Atlantic Coast in the United States and Canada submitted I. uriae ticks to our laboratory for analysis (16). Ticks were identified to species, stage, and degree of engorgement (17). A subset of ticks was dissected, and midguts were screened for spirochetes by fluorescent microscopy by using a polyclonal antiborrelial antibody (18). DNA was extracted from Borrelia-positive ticks by using the Qiagen DNeasy Tissue Kit (Qiagen, Valencia, CA, USA). DNA amplification was performed in a designated room with genus-specific primers that include the partial sequence of rrs-rrla intergenic spacer region as described by Bunikis et al. (19) with use of negative controls. Amplification products were viewed on a 1% agarose gel containing 0.5 μg/mL ethidium bromide. At a second laboratory, ticks positive by fluorescent antibody screen were prepared as above for DNA extraction, and PCR was performed by using primers directed at the 16S ribosomal DNA. Sequences of amplicons obtained at both laboratories were confirmed to be B. garinii by comparison with known sequences in the GenBank database. Results I. uriae ticks submitted from 6 seabird colony sites and several hosts in the United States and Canada were processed (Table). Through 2005, 880 I. uriae ticks recovered primarily from Atlantic puffin chicks or their nests, were submitted from Maine sites, and another 383 were submitted from sites on the Atlantic Coast of Canada. Over 200 ticks from Maine sites and 61 ticks from Canadian sites off the coast of Newfoundland and Labrador were screened for borreliae by fluorescent microscopy. Spirochetes were detected only in ticks from Gull Island, Newfoundland (47° 15′N, 52° 46′W), where 9 of 22 adults and 1 of 39 nymphal ticks were positive. DNA was extracted from 2 of these ticks, and PCR showed a 1,900-base amplicon of the rrs-rrls intergenic spacer region that matched with B. garinii on comparison with GenBank sequences. Two additional ticks were examined in the laboratory of Sam Telford (Tufts University School of Veterinary Medicine, Grafton, MA, USA) by means of PCR targeting of 16S ribosomal DNA and again confirmed a match for B. garinii (GenBank bankit no. 800902 DG463373). Figure 2 shows the sequence from one of the ticks shown in an alignment with sequences from B. burgdorferi strain B31 and a representative B. garinii sample from GenBank. Discussion The finding of B. garinii in I. uriae ticks from Gull Island, Newfoundland, adds to the known distribution of this agent and increases the likelihood that this agent is present in other colonial seabird nesting sites on the Atlantic Coast of North America, as is the case in Europe. The recent emergence of I. scapularis in coastal Maine and some Maritime Canadian sites (20,21) brings these 2 enzootic cycles of different genospecies of B. burgdorferi into proximate areas, although their ecologic settings differ. The public health importance of this finding depends on the probability of the introduction of B. garinii into emergent I. scapularis-vectored B. burgdorferi s.s. cycles and its potential maintenance in this cycle. The public health effects also depend on the pathogenic potential for human disease caused by seabird-associated strains of B. garinii (15). The remote geographic and, at some sites, isolated topographic location of colonial seabird colonies provide significant barriers to the introduction of B. garinii into other vector ticks and their reservoir hosts. I. scapularis is dispersed to remote coastal islands of the North American Atlantic Coast during bird migration (22,23), but its establishment at these sites requires the presence of deer (24–26). With rare exception, deer are absent from sites with large seabird colonies, which are usually limited to offshore islands. Dispersal of infected I. uriae to proximate coastal areas by seabird hosts is unlikely because most species of seabirds parasitized by I. uriae are highly philopatric and forage at sea. One exception might be gulls, which may move between coastal and island sites. Passerine birds, which may forage near seabird colonies, provide another potential mechanism for dispersal of B. garinii, either through movement of infected I. uriae ticks or by serving as reservoir hosts of this agent. However, the frequency of parasitism of passerine birds by I. uriae ticks is unknown. If B. garinii was introduced into I. scapularis ticks, its maintenance in this cycle would depend on the vector competence of I. scapularis for B. garinii, the reservoir competence of available hosts, and perhaps the population genetics and strain diversity of B. garinii (14,27). Although I. scapularis is vector-competent for transmission of B. garinii to rodents, its efficiency of transmission appears lower than for B. burgdorferi s.s. (28). In addition, the vector competence of I. scapularis for seabird-associated strains of B. garinii has not been tested. The presence of similar ribotypes of B. garinii in I. ricinus ticks on a European island suggests that interchange of different B. garinii strains in different ecologic cycles may occur (14). To determine the public health importance of B. garinii in seabird colonies along the North Atlantic coast, additional studies on the issues of dispersal, vector competence, and reservoir host competence are needed. All strain types of B. garinii may not be pathogenic for humans, and future studies should also address the potential for seabird-associated strains to cause disease. Acknowledgments We thank Anthony Diamond of the University of New Brunswick, Linda Welch of the Maine Coastal Islands National Wildlife Refuge, and Stephen Kress of the National Audubon Society, and their staffs, for the collection of I. uriae from Machias Seal Island, Petit Manan Island, Seal Island, and Matinicus Rock during the past 12 years. We are also indebted to Jonas Bunikis, Sam R. Telford III, Heidi K. Goethert, Barbara Conley, and Cal Vary for their assistance in confirming the genetic identity of B. garinii. Dr Smith is an infectious disease physician and codirector of the Vector-borne Disease Laboratory at the Maine Medical Center Research Institute. His research interests include the ecology of Lyme disease emergence and the strain diversity of B. burgdorferi. References Steere AC, Coburn J, Glickstein L. 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Differential transmission of the genospecies of Borrelia burgdorferi sensu lato by game birds and small rodents in England. Appl Environ Microbiol. 1998;64:1169–73. Humair P-F, Postic D, Wallich R, Gern L. An avian reservoir (Turdus merula) of the Lyme borreliosis spirochetes. Zentrabl. Bakteriol. 1998;287:521–38. Richter D, Endepols S, Ohlenbusch A, Eiffert H, Spielman A, Matuschka FR. Genospecies diversity of Lyme disease spirochetes in rodent reservoirs. Emerg Infect Dis. 1999;5:291–6. Olsen B, Jaenson TGT, Noppa L, Bunikis J, Bergstrom S. A Lyme borreliosis cycle in seabirds and Ixodes uriae ticks. Nature. 1993;362:340–2. Olsen B, Duffy DC, Jaenson TGT, Gylfe A. J. Bonnedahl J, Bergstrom S. Transhemispheric exchange of Lyme disease spirochetes by seabirds. J Clin Microbiol. 1995;33:3270–4. Gylfe A, Olsen B, Strasevicius D, Ras NM, Weihe P, Noppa L, et al. Isolation of Lyme disease borrelia from puffins (Fratercula arctica) and seabird ticks (Ixodes uriae) on the Faeroe Islands. 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Morshed MG, Scott JD, Banerjee SN, Banerjee M, Fitzgerald T, Fernando K, et al. First isolation of Lyme disease spirochete, Borrelia burgdorferi, from black-legged tick, Ixodes scapularis, removed from a bird in Nova Scotia, Canada. Can Com Dis Rep. 1999;25:153–5. Wilson ML, Telford SR III, Piesman J, Spielman A. Reduced abundance of immature Ixodes dammini (Acari: Ixodidae) following elimination of deer. J Med Entomol. 1988;25:224–8. Rand PW, Lubelczyk C, Holman MS, Lacombe EH, Smith RP Jr. Abundance of Ixodes scapularis (Acari:Ixodidae) after the complete removal of deer from an isolated offshore island, endemic for Lyme disease. J Med Entomol. 2004;41:779–84. Rand PW, Lubelczyk C, Lavigne GR, Elias S, Holman MS, Lacombe EH, et al. Deer density and the abundance of Ixodes scapularis (Acari:Ixodidae). J Med Entomol. 2003;40:179–84. Kurtenbach K, Schaefer SM, Sewall H, Peacey M, Hoodless A, Nuttal PA, et al. Differential survival of Lyme borreliosis in ticks that feed on birds. 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☉ 11342800:Isolation of Lagos Bat Virus from Water Mongoose
University of Pretoria, Pretoria, South Africa Centers for Disease Control and Prevention, Atlanta, Georgia, USA Allerton Veterinary Laboratory, Pietermaritzburg, South Africa Onderstepoort Veterinary Research Institute, Pretoria, South Africa Canadian Food Inspection Agency, Nepean, Ontario, Canada Abstract A genotype 2 lyssavirus, Lagos bat virus (LBV), was isolated from a terrestrial wildlife species (water mongoose) in August 2004 in the Durban area of the KwaZulu-Natal Province of South Africa. The virus isolate was confirmed as LBV by antigenic and genetic characterization, and the mongoose was identified as Atilax paludinosus by mitochondrial cytochrome b sequence analysis. Phylogenetic analysis demonstrated sequence homology with previous LBV isolates from South African bats. Studies performed in mice indicated that the peripheral pathogenicity of LBV had been underestimated in previous studies. Surveillance strategies for LBV in Africa must be improved to better understand the epidemiology of this virus and to make informed decisions on future vaccine strategies because evidence is insufficent that current rabies vaccines provide protection against LBV. Lagos bat virus (LBV) belongs to the genus Lyssavirus in the family Rhabdoviridae. The prototype lyssavirus genotype and species, rabies virus (RABV), has a single, continuous, negative-strand RNA of ≈12,000 nt that codes for 5 proteins: nucleoprotein, matrix protein, phosphoprotein, glycoprotein, and polymerase (1). The Lyssavirus genus was created after isolation of several viruses in Africa and Europe that were related to, but serologically distinct from, RABV (2). Seven genotypes (gts) or species in this genus are recognized (3), and diversity may expand with the addition of new isolates from Eurasia (4), which are tentative species in the Lyssavirus genus. RABV (gt1) is distributed worldwide, Australian bat lyssavirus (gt7) has only been identified in Australia, and European bat lyssavirus 1 (EBLV-1) (gt5) and European bat lyssavirus 2 (EBLV-2) (gt6) have been found only in Europe. Lagos bat virus (LBV) (gt2), Mokola virus (gt3), and Duvenhage virus (gt4) have been found only in Africa. Recognized lyssavirus genotypes are divided into 2 serologically, pathogenically, and genetically distinct phylogroups (5). One phylogroup consists of Mokola virus and LBV (group II), while all other genotypes are in group I. Members of phylogroup I are reported to be pathogenic for mice when introduced intramuscularly and intracerebrally. In contrast, members of phylogroup II are believed to be pathogenic in mice only when introduced by the intracerebral (i.c.) route (5). Commercial vaccine strains belong to gt1 (RABV) phylogroup 1, and these vaccines provide protection against RABV and all the other members of phylogroup I. However, laboratory data suggest that these vaccines (gt1 based) will not offer protection against lyssaviruses in the phylogroup II cluster (6,7). On the basis of criteria proposed for lyssavirus phylogroups, West Caucasian bat virus could be considered an independent phylogroup III because of genetic distance and absence of serologic cross-reactivity with phylogroup I and II viruses (7). LBV was first isolated from a fruit bat (Eidolon helvum) in 1956 on Lagos Island in Nigeria (2,8). Fourteen isolations of this virus have been reported throughout Africa, including 8 in South Africa (9). Most LBV isolates were obtained from bats; 2 were from domestic cats (10,11), and 1 was from a domestic dog in Ethiopia (12). LBV has never been isolated from any terrestrial wildlife species. Globally and throughout Africa, RABV (gt1) is the most common lyssavirus. In southern Africa, 2 biotypes of RABV are recognized (13,14): the canid biotype, which mainly circulates among dogs, jackals, and bat-eared foxes, and the mongoose biotype, which is well adapted and unique to mongooses in southern Africa (15). RABV is responsible for all mongoose rabies cases in Africa. In South Africa, the principal vector of the mongoose biotype is the yellow mongoose (Cynictis penicillata), but RABV has been reported in other mongoose species, such as slender (Galerella sanguinea), water (Atilax paludinosus), small gray (Galerella pulverulenta), banded (Mungos mungo), selous (Paracynictis selousi), dwarf (Helogale parvula), and white-tailed (Ichneumia albicauda) mongooses. Mongoose rabies in South Africa commonly occurs in the central highveld regions (15,16), whereas KwaZulu-Natal Province, which is located on the east coast of South Africa, is associated with epizootic canid rabies in domestic dogs; mongoose rabies is not reported in this province. We report the first identification of LBV in a wildlife terrestrial species, A. paludinosus, commonly known as the water or marsh mongoose. The mongoose species was identified by generation and analysis of cytochrome b sequencing data. We characterized this LBV isolate by antigenic typing with antinucleocapsid monoclonal antibodies, sequencing of the nucleoprotein gene, and peripheral pathogenicity in laboratory mice in comparison with other LBV isolates from South Africa and a bat RABV isolate from North America. Materials and Methods Sample Collection In August 2004, a brain sample from a suspected rabid mongoose was submitted to the Allerton Veterinary Institute in Pietermaritzburg, KwaZulu-Natal, South Africa. The mongoose was captured by the Society for the Prevention of Cruelty to Animals in a marshy valley in a residential area in Westville near Durban after the mongoose displayed abnormal behavior. The animal was disorientated, attacked inanimate objects, and alternated between being friendly and aggressive. Only the brain of the animal was submitted for testing; the carcass was not preserved. The mongoose species was not identified. Virus Characterization Lyssavirus antigen was detected by the standard fluorescent antibody test (FAT) (17), with modifications, by using a polyclonal fluorescein isothiocyanate–conjugated immunoglobulin (Rabies Unit, Onderstepoort Veterinary Institute, Pretoria, South Africa) that could detect all lyssavirus genotypes. Virus isolation was performed by using the i.c. mouse inoculation test in suckling mice (18). Antigenic typing was performed by using the indirect fluorescent antibody test with a panel of 16 antinucleocapsid monoclonal antibodies (N-MAbs) (Centre of Expertise for Rabies, Canadian Food Inspection Agency, Nepean, Ontario, Canada) as previously described (19). Genetic characterization was based on sequencing of the entire nucleoprotein (N) gene. Briefly, total RNA was extracted from infected brain material with Trizol (Invitrogen, Croningen, the Netherlands) according to the manufacturer's instructions. Complementary DNA was produced by a reverse transcription reaction by using an oligonucleotide primer specific for the noncoding messenger RNA of the lyssavirus genome (Lys001: 5′-ACGCTTAACGAMAAA-3′ position 1–15 according to the Pasteur virus [PV] RABV genome, GenBank accession no. M13215). Complementary DNA was amplified with a PCR by using different combinations of the oligonucleotide primers Lys001, LagNF (9), 550B (5′-GTRCTCCARTTAGCRCACAT-3′, position 647–666 according to the PV genome), and 304 (5′-TTGACAAAGATCTTGCTCAT-3′, position 1514–1533 according to the PV genome) as described elsewhere (20). The PCR products were visualized after electrophoresis on a 2% agarose gel and purified by using the Wizard PCR Preps DNA Purification System (Promega, Madison, WI, USA). The purified products were sequenced with the BigDye Termination Cycle Sequencing Ready Reaction Kit 1.1 (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's protocol, with subsequent analysis on an Applied Biosystems 377 DNA automated sequencer. Sequence Analysis DNA sequencing information was compared with nucleoprotein sequence information for other lyssavirus genotypes in GenBank, as well as with nucleoprotein sequencing data obtained during this study from previous LBV isolates in South Africa from Epomophorus whalbergi fruit bats in 1980 (21), 1982 (11), 2003 (9), and 2004 (9), by using the same method as described above. CLUSTAL W (22) was used to produce sequence alignments and generate a neighbor-joining phylogenetic tree. A graphic representation of the tree was constructed with the TREEVIEW program (23). Virus Pathogenicity Two LBV isolates from South Africa (LBVSA2004) (9) and the LBV mongoose isolate described in this report (Mongoose2004), as well as a North American bat RABV (Myotis spp. variant, isolated in Washington, USA, 2004), were injected into 4-week-old inbred ICR mice (5 mice/group) by different routes. The i.c. 50% lethal dose (LD50) was determined by titration of the virus suspension injected into 4-week-old ICR mice by the i.c. route. Thereafter, 4-week-old ICR mice were injected with 30 μL of 103 LD50 of each virus by the i.c. route and 30 μL of 105 LD50 of each virus by the intramuscular (i.m.) route. Virus inoculum was prepared by 1 i.c. passage of the original mongoose brain material in suckling mice. Species Identification of the LBV-infected Mongoose Because the mongoose carcass was destroyed, we attempted to accurately identify the animal by using DNA sequencing analyses of the mitochondrial cytochrome b region of mongoose genomic DNA obtained from the brain sample. The mitochondrial cytochrome b region has been used to characterize relationships between mongoose species (24). Genomic DNA was extracted from mongoose brain by using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany), followed by PCR conducted according to the method of Veron et al. (24). PCR products were purified by using the Wizard SV PCR and gel purification kit (Promega) and sequenced by using the BigDye Termination Cycle Sequencing Ready Reaction Kit 3.1 (Applied Biosystems) according to the manufacturer's protocol, with subsequent analysis on an Applied Biosystems 3100 DNA automated sequencer. A DNA sequence of 893 bp of the cytochrome b gene was compared with cytochrome b sequences for mongooses available in GenBank by the same method as described earlier for the analysis of LBV nucleoprotein gene sequences. Results Virus Characterization and DNA Sequence Analysis FAT performed on mongoose brain material showed a positive reaction for lyssavirus antigen. During the mouse inoculation test, suckling mice died 9 days after i.c. injections with mongoose brain suspensions. FAT of the suckling mouse brains showed a positive reaction for lyssavirus antigen. The isolate reacted with N-MAb 38HF2, which is an antibody that reacts with all lyssaviruses tested, and with the antibody N-MAb M612, which is highly specific for LBV and does not react with any other lyssaviruses tested. These findings indicate that the new isolate belongs to LBV (Table). A reverse transcription–PCR method followed by a cycle sequencing method was used to amplify and determine the nucleic acid sequence of the entire nucleoprotein-encoding gene of the new putative LBV isolate. Phylogenetic analysis indicated that the new isolate clusters together with LBV isolates from South Africa and with LBV isolates from Nigeria (2) and Ethiopia (12) . LBV isolates from South Africa, including the new mongoose isolate of LBV, showed high nucleotide sequence identity with each other (99.1%–99.7%), compared with low sequence identity (≈82%) with the LBV isolate from Nigeria. The LBV isolate from Ethiopia (isolated from a dog; GenBank accession no. AY333110) showed 99.1%–99.9% nucleotide sequence homology with the South African LBV isolates. This result warrants further investigation of the DNA sequence identity of the Ethopian LBV isolate. Virus Pathogenicity Genotypes 1 and 2 viruses were pathogenic for mice by the i.c. and i.m. routes of injection . A similar death rate was observed for both genotypes (100%) after i.c. injection of equal amounts of virus (103 LD50 dose). Although the LBV isolates were lethal to mice when 105 LD50 was injected intramuscularly, they were less efficient than the RABV isolate. Of mice injected with the LBV isolate from the mongoose, 20% died; 60% of the mice died after injection with the LBVSA2004 isolate from the fruit bat E. whalbergi. However, the RABV isolate showed 100% lethality in mice. Species Identification of the LBV-infected Mongoose Analysis of 893 bp of the cytochrome b gene obtained from mongoose brain indicated that the infected animal shared a 98% DNA nucleotide sequence homology with the African water mongoose (A. paludinosus) . Water mongooses are solitary and mainly nocturnal mammals, but they may also be active during the day. These animals live near water in areas with sufficient bush cover and have been found throughout sub-Saharan Africa (25). Discussion Isolation of LBV from terrestrial wildlife serves as further confirmation of our lack of understanding of the incidence and host range of lyssaviruses in Africa. Poor surveillance of rabies-related viruses and poor diagnostic capability in most of Africa are large contributors to our lack of information and the obscurity of the African lyssaviruses. The fluorescent antibody test used as a diagnostic test for rabies can only indicate the presence of lyssavirus antigens and cannot distinguish between lyssavirus genotypes. To identify a lyssavirus precisely, antigenic typing or genetic characterization is necessary, but these techniques are beyond the capability of most laboratories responsible for rabies diagnostics in Africa. Our phylogenetic analysis indicated a strong nucleoprotein sequence homology between LBV isolates from South Africa. Geographic partitioning is a well-known characteristic of RABV epidemiology worldwide. The strong sequence homology we observed may result from the defined geographic location from which all LBV isolates were obtained. Although cases in domestic animals have been recorded, no human cases of infection with LBV have been documented. However, cross-neutralization data obtained with human sera and in rodent models suggest that preexposure and postexposure treatments for rabies are not effective against LBV (6,7). The infected mongoose showed aggressive behavior and was captured in a populated residential area. Although the incidence of the rabies-related viruses seems to be low, human exposure to these viruses is possible. Results of pathogenicity experiments indicated that death can occur from the i.c. and i.m. routes of injection, although gt2 viruses showed lower lethality to mice when injected i.m. Our results differ from those of another study (5), which reported that a gt2 virus was not pathogenic to mice when administered by the i.m. route at the same dose (3×105 LD50) used in our experiment. What amount of virus is involved in natural infection is not known. Cumulatively, our results indicate that LBV may be a health risk for humans and other mammals, and future vaccine strategies against rabies in Africa should consider these possibilities. Although laboratory data suggest little cross-neutralization of LBV by rabies preexposure and postexposure vaccination (7), immune system components other than neutralizing antibodies may be involved in protection. Therefore, in the absence of an alternative vaccine, rabies vaccination and postexposure treatment should still be advised because of potential cross-reactivity. This report demonstrated the value of cytochrome b DNA sequencing for accurately identifying the host in a rabies case. Diagnostic laboratories do not routinely receive the complete carcass of suspected rabid animals, and identification is dependent on reports of persons who captured the animal or removed its brain before submission to the diagnostic facility. Host identity is rarely a problem in domestic animals, but wildlife species show potential uncertainty, such as demonstrated in the case reported here. One important aspect of disease epidemiology is accurate information about the host species involved, which enables informed decisions to be made with regard to the epidemiologic patterns and potential threats to public and veterinary health. Identification of the first case of LBV in a mongoose underscores the need for surveillance of rabies-related viruses and the need for accurate identification of lyssavirus genotypes even if the host involved is normally only associated with RABV. With respect to LBV, we have recently reported the likely persistence of this virus in pteropid bats in South Africa, which implicates continuous opportunity for spillover into terrestrial species (9). In determining the extent of risk to human and veterinary public health, it is important to establish the prevalence of LBV not only in bats but also in potential terrestrial animal vectors, to which mongoose species should be added, based on the finding in this report. The origin of mongoose rabies in South Africa is not clear (14). Epidemiologic cycles among yellow mongooses and other Herpestidiae are well established and shown to be impossible to extinguish or control by the attempted eradication or control of vector and host density (26). With respect to more modern or scientific approaches, no vaccination strategy has been considered feasible in tackling this complicated and entrenched wildlife rabies epidemic. 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