Peptide Nucleic Acid Fluorescence In Situ Hybridization-Based Identifi
http://www.100md.com
《微生物临床杂志》
University of Maryland School of Medicine, Division of Infectious Diseases, Baltimore, Maryland 21201
University of Maryland School of Dentistry, Baltimore, Maryland 21201
University of Maryland Medical Center, Department of Pharmacy, Baltimore, Maryland 21201
University of Maryland School of Medicine, Department of Pathology, Baltimore, Maryland 21201
ABSTRACT
The impact of rapid identification of Candida albicans blood isolates by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) on the selection and expenditure of antifungal therapy was evaluated. PNA FISH was 100% sensitive and specific in the rapid identification of 31 out of 72 candidemias as C. albicans and resulted in a significant reduction of caspofungin usage, with an overall cost savings of $1,729 per patient.
TEXT
The peptide nucleic acid fluorescence in situ hybridization (PNA FISH) assay is a rapid test that utilizes fluorescent-labeled peptide nucleic acid probes targeting the specific rRNA sequences of Candida albicans (5, 9, 12). The aims of this study were to demonstrate the accuracy of implementing PNA FISH testing for C. albicans and the actual financial impact on antifungal therapy costs at the University of Maryland Medical Center (UMMC) over a 1-year period.
(This work was presented in part as an abstract at the 43rd Infectious Diseases Society of America Meeting, San Francisco, Calif., 6 to 10 October 2005.)
Following Institutional Review Board approval, clinical and laboratory data were obtained from all patients that had PNA FISH testing performed. Positive blood cultures confirmed as yeast by Gram stain morphology from the beginning of 2003 to the end of 2004 were evaluated. Duplicate yeast-positive blood cultures from the same individual or cultures with Cryptococcus neoformans were excluded from the study. The blood culture system utilizes Bactec 9000 (Becton Dickinson, Maryland) bottles, which are then placed in a continuous automated detection incubator (BacT/Alert) upon arrival at the laboratory.
Yeast isolates were identified using a Quali Test Albicans (Qual Tech, California), a Vitek II system (bioMerieux, North Carolina), germ tube formation, and determination of morphology on cornmeal agar according to standard laboratory protocols (2, 4, 10).
In 2004 only, blood cultures positive for yeast by Gram stain had the PNA FISH test (AdvanDx, Inc., Woburn, MA) performed on smears directly from the blood cultures according to the manufacturer's instructions. A positive C. albicans test, where yeast cells emit green fluorescence, was reported to clinicians within 3 h (5, 12). During the study period, the PNA FISH test was batched once a day, with results made available by 1 p.m. During 2004, fluconazole susceptibility testing was performed on the isolates and was interpreted according to the guidelines outlined in CLSI document M27-A on agar disk diffusion and broth microdilution methods (4). A disk diffusion assay was performed using a 25-μg fluconazole diffusion disk (Pfizer, New York, NY) on Mueller-Hinton agar supplemented with glucose and methylene blue. The MIC was recorded as the minimal concentration resulting in 80% growth inhibition (2, 4, 6).
Yeast-positive blood cultures were called in to the primary clinical service per normal laboratory protocol. One member of the antimicrobial management team, the Infectious Diseases attending, the fellow on call, or the clinical pharmacist, was also notified of all PNA FISH results, as their approval was required to release antifungal therapy to treating physicians. A test positive for C. albicans ensured the preferred use of fluconazole; however, the antifungal choice for a negative test would depend on the patient's clinical condition (hemodynamic instability or endocarditis), host factors (AIDS or neutropenia), and prior history of Candida colonization and antifungal therapy or prophylaxis. The antifungals used and antifungal defined daily doses (DDD) were obtained from the pharmacy database and were calculated using standard World Health Organization guidelines (11). The wholesale acquisition costs used to standardize and calculate the costs for the antifungals between 2003 and 2004 were the following: fluconazole, 400 mg oral, $2; fluconazole, 400 mg intravenous (i.v.), $94; caspofungin, 70 mg i.v., $429; caspofungin, 50 mg i.v., $330; and amphotericin B lipid complex (ABLC), 50 mg i.v., $157. Statistical analysis was performed using the Mann-Whitney test, chi square analysis, and Fisher's exact test where appropriate, with SPSS software (version 13 for Windows).
Results demonstrated that the PNA FISH test accurately identified all 31 C. albicans isolates (of 72 total candedimias [43%]) in 2004. In addition, six Candida dubliniensis isolates initially identified as C. albicans by culture failed to hybridize with the C. albicans-specific probe and were later confirmed by genotypic testing to be C. dubliniensis, indicating a 100% sensitivity and specificity for PNA FISH (3). The numbers and species identification of the recovered Candida isolates are shown in Table 1. There were no episodes of mixed-species candidemia reported during the study period. PNA FISH significantly reduced the median time required for the identification of C. albicans to 9.5 h (range, 3 to 17 h), compared to the standard culture median time of 44 h (range, 36 to 92 h) (P < 0.001), while the median time for the final identification of Candida species other than C. albicans by culture was even longer (61 h; range, 36 to 124 h).
In comparing the effects of the PNA FISH test on antifungal usage between 2003 and 2004, the most pronounced effect of the PNA FISH test was on caspofungin usage in patients with candidemia due to C. albicans. In this group (Table 2), there was a significant reduction in the DDD/patient usage of caspofungin (P < 0.05), with a corresponding decrease in antifungal costs of $1,978 per patient. Although a drop in the usage and cost of caspofungin following PNA FISH implementation was also noted for the group of Candida species other than C. albicans, the reduction was not significant. Overall, there was a total cost savings of $130,231, or $1,808 per patient, despite a 20% increase in fluconazole use. Our costs for running the PNA FISH test included a start-up outlay of $1,000 for purchasing a water bath, lens filter, and UV light source microscope, technician time costs (45 min, or $12 in labor), and the reagent kit's list price of $68 ($30 is reimbursed by insurance), with a total cost of $5,760 for the year. Therefore, our overall cost savings per patient was $1,729.
Two Candida glabrata isolates out of 16 and 2 C. albicans isolates out of 31 demonstrated fluconazole MICs of >64 μg/ml. Three of the four patients with isolates with high MICs had a history of fluconazole prophylaxis, while the fourth had endocarditis. All patients received appropriate initial antifungal therapy based on their histories. Table 3 summarizes the initial and subsequent antifungal therapies used. The most frequent reasons treating physicians preferred the use of caspofungin or ABLC over fluconazole were the identification of C. glabrata prior to susceptibility results and recent bacterial endocarditis or endocarditis in patients with prosthetic material.
By use of a decision analytic tool with the PNA FISH test, a cost minimization model was recently developed by Alexander et al. (1). In their study, they predicted that in an institution with a rate of 40% for C. albicans candidemias, the test would result in a cost savings of about $1,800 per patient from reduced caspofungin usage (1). Our paper uses clinical data to show the effect of PNA FISH testing for C. albicans and validates the decision model Alexander et al. describe in their paper (1). In our experience, the financial savings in reducing caspofungin usage surpassed the cost of the PNA FISH test and has led to the development of straightforward hospital-specific treatment algorithms. We have since increased the frequency of PNA FISH to twice daily to further decrease the reporting time. An interesting finding from this study was the overall low level of fluconazole resistance (5%) in our institution, especially with C. glabrata (7, 8). Important clinical limitations of the PNA FISH test are that it has not been validated with specimens other than blood and that only a C. albicans-specific probe is currently available. Although we did not have any polyfungemias in our study, an additional benefit to the PNA FISH test is that it allows for the discrimination between varied species morphologies in specimens of mixed C. albicans species and Candida species other than C. albicans under a fluorescent microscope. The availability of specific probes for Candida species other than C. albicans, especially C. glabrata, would be of great benefit both clinically and financially.
In conclusion, the PNA FISH test is an accurate and affordable test for the rapid identification of C. albicans from positive blood cultures leading to a significant reduction in using caspofungin for treating C. albicans. Further development of probes for other Candida species should offer additional benefits.
ACKNOWLEDGMENTS
G.N.F. and R.A.V. received speakers' honoraria from AdvanDx. J.K.J. and D.P.L. received travel support from AdvanDx.
FOOTNOTES
Corresponding author. Mailing address: University of Maryland School of Medicine, Division of Infectious Diseases, 20 Penn St., Rm. S403B, Baltimore, MD 21201. Phone: (410) 706-5680. Fax: (410) 706-8700. E-mail: gforrest@medicine.umaryland.edu.
REFERENCES
Alexander, B. D., E. D. Ashley, L. B. Reller, and S. D. Reed. 2006. Cost savings with implementation of PNA FISH testing for identification of Candida albicans in blood cultures. Diagn. Microbiol. Infect. Dis. 54:277-282.
Cuenca-Estrella, M., W. Lee-Yang, M. A. Ciblak, B. A. Arthington-Skaggs, E. Mellado, D. W. Warnock, and J. L. Rodriguez-Tudela. Comparative evaluation of NCCLS M27-A and EUCAST broth microdilution procedures for antifungal susceptibility testing of Candida species. Antimicrob. Agents Chemother. 46:3644-3647.
Jabra-Rizk, M. A., J. K. Johnson, G. Forrest, K. Mankes, T. F. Meiller, and R. A. Venezia. 2005. Prevalence of Candida dubliniensis fungemia at a large teaching hospital. Clin. Infect. Dis. 41:1064-1067.
NCCLS. 2002. Reference method for broth dilution antifungal susceptibility testing of yeasts: approved standard M27-A. NCCLS, Wayne, Pa.
Oliveira, K., G. Haase, C. Kurtzman, J. J. Hyldig-Nielsen, and H. Stender. 2001. Differentiation of Candida albicans and Candida dubliniensis by fluorescent in situ hybridization with peptide nucleic acid probes. J. Clin. Microbiol. 39:4138-4141.
Pfaller, M. A. 2005. Antifungal susceptibility testing methods. Curr. Drug Targets 6:929-943.
Pfaller, M. A., D. J. Diekema, M. G. Rinaldi, R. Barnes, B. Hu, A. V. Veselov, N. Tiraboschi, E. Nagy, D. L. Gibbs, and the Global Antifungal Surveillance Group. 2005. Results from the ARTEMIS DISK Global Antifungal Surveillance Study: a 6.5-year analysis of susceptibilities of Candida and other yeast species to fluconazole and voriconazole by standardized disk diffusion testing. J. Clin. Microbiol. 43:5848-5859.
Pfaller, M. A., S. A. Messer, L. Boyken, S. Tendolkar, R. J. Hollis, and D. J. Diekema. 2003. Variation in susceptibility of bloodstream isolates of Candida glabrata to fluconazole according to patient age and geographic location. J. Clin. Microbiol. 41:2176-2179.
Rigby, S., G. W. Procop, G. Haase, D. Wilson, G. Hall, C. Kurtzman, K. Oliveira, S. Von Oy, J. J. Hyldig-Nielsen, J. Coull, and H. Stender. 2002. Fluorescence in situ hybridization with peptide nucleic acid probes for rapid identification of Candida albicans directly from blood culture bottles. J. Clin. Microbiol. 40:2182-2186.
Sutton, D. A. 2003. Specimen collection, transport, and processing: mycology, p. 1659-1667. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of clinical microbiology. ASM Press, Washington, D.C.
WHO Collaborating Centre for Drug Statistics Methodology. 1996. Guidelines for ATC classification and DDD assignment. WHO Collaborating Centre for Drug Statistics Methodology, Oslo, Norway.
Wilson, D. A., M. J. Joyce, L. S. Hall, L. B. Reller, G. D. Roberts, G. S. Hall, B. D. Alexander, and G. W. Procop. 2005. Multicenter evaluation of a Candida albicans peptide nucleic acid fluorescent in situ hybridization probe for characterization of yeast isolates from blood cultures. J. Clin. Microbiol. 43:2909-2912.(G. N. Forrest, K. Mankes, M. A. Jabra-Ri)
University of Maryland School of Dentistry, Baltimore, Maryland 21201
University of Maryland Medical Center, Department of Pharmacy, Baltimore, Maryland 21201
University of Maryland School of Medicine, Department of Pathology, Baltimore, Maryland 21201
ABSTRACT
The impact of rapid identification of Candida albicans blood isolates by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) on the selection and expenditure of antifungal therapy was evaluated. PNA FISH was 100% sensitive and specific in the rapid identification of 31 out of 72 candidemias as C. albicans and resulted in a significant reduction of caspofungin usage, with an overall cost savings of $1,729 per patient.
TEXT
The peptide nucleic acid fluorescence in situ hybridization (PNA FISH) assay is a rapid test that utilizes fluorescent-labeled peptide nucleic acid probes targeting the specific rRNA sequences of Candida albicans (5, 9, 12). The aims of this study were to demonstrate the accuracy of implementing PNA FISH testing for C. albicans and the actual financial impact on antifungal therapy costs at the University of Maryland Medical Center (UMMC) over a 1-year period.
(This work was presented in part as an abstract at the 43rd Infectious Diseases Society of America Meeting, San Francisco, Calif., 6 to 10 October 2005.)
Following Institutional Review Board approval, clinical and laboratory data were obtained from all patients that had PNA FISH testing performed. Positive blood cultures confirmed as yeast by Gram stain morphology from the beginning of 2003 to the end of 2004 were evaluated. Duplicate yeast-positive blood cultures from the same individual or cultures with Cryptococcus neoformans were excluded from the study. The blood culture system utilizes Bactec 9000 (Becton Dickinson, Maryland) bottles, which are then placed in a continuous automated detection incubator (BacT/Alert) upon arrival at the laboratory.
Yeast isolates were identified using a Quali Test Albicans (Qual Tech, California), a Vitek II system (bioMerieux, North Carolina), germ tube formation, and determination of morphology on cornmeal agar according to standard laboratory protocols (2, 4, 10).
In 2004 only, blood cultures positive for yeast by Gram stain had the PNA FISH test (AdvanDx, Inc., Woburn, MA) performed on smears directly from the blood cultures according to the manufacturer's instructions. A positive C. albicans test, where yeast cells emit green fluorescence, was reported to clinicians within 3 h (5, 12). During the study period, the PNA FISH test was batched once a day, with results made available by 1 p.m. During 2004, fluconazole susceptibility testing was performed on the isolates and was interpreted according to the guidelines outlined in CLSI document M27-A on agar disk diffusion and broth microdilution methods (4). A disk diffusion assay was performed using a 25-μg fluconazole diffusion disk (Pfizer, New York, NY) on Mueller-Hinton agar supplemented with glucose and methylene blue. The MIC was recorded as the minimal concentration resulting in 80% growth inhibition (2, 4, 6).
Yeast-positive blood cultures were called in to the primary clinical service per normal laboratory protocol. One member of the antimicrobial management team, the Infectious Diseases attending, the fellow on call, or the clinical pharmacist, was also notified of all PNA FISH results, as their approval was required to release antifungal therapy to treating physicians. A test positive for C. albicans ensured the preferred use of fluconazole; however, the antifungal choice for a negative test would depend on the patient's clinical condition (hemodynamic instability or endocarditis), host factors (AIDS or neutropenia), and prior history of Candida colonization and antifungal therapy or prophylaxis. The antifungals used and antifungal defined daily doses (DDD) were obtained from the pharmacy database and were calculated using standard World Health Organization guidelines (11). The wholesale acquisition costs used to standardize and calculate the costs for the antifungals between 2003 and 2004 were the following: fluconazole, 400 mg oral, $2; fluconazole, 400 mg intravenous (i.v.), $94; caspofungin, 70 mg i.v., $429; caspofungin, 50 mg i.v., $330; and amphotericin B lipid complex (ABLC), 50 mg i.v., $157. Statistical analysis was performed using the Mann-Whitney test, chi square analysis, and Fisher's exact test where appropriate, with SPSS software (version 13 for Windows).
Results demonstrated that the PNA FISH test accurately identified all 31 C. albicans isolates (of 72 total candedimias [43%]) in 2004. In addition, six Candida dubliniensis isolates initially identified as C. albicans by culture failed to hybridize with the C. albicans-specific probe and were later confirmed by genotypic testing to be C. dubliniensis, indicating a 100% sensitivity and specificity for PNA FISH (3). The numbers and species identification of the recovered Candida isolates are shown in Table 1. There were no episodes of mixed-species candidemia reported during the study period. PNA FISH significantly reduced the median time required for the identification of C. albicans to 9.5 h (range, 3 to 17 h), compared to the standard culture median time of 44 h (range, 36 to 92 h) (P < 0.001), while the median time for the final identification of Candida species other than C. albicans by culture was even longer (61 h; range, 36 to 124 h).
In comparing the effects of the PNA FISH test on antifungal usage between 2003 and 2004, the most pronounced effect of the PNA FISH test was on caspofungin usage in patients with candidemia due to C. albicans. In this group (Table 2), there was a significant reduction in the DDD/patient usage of caspofungin (P < 0.05), with a corresponding decrease in antifungal costs of $1,978 per patient. Although a drop in the usage and cost of caspofungin following PNA FISH implementation was also noted for the group of Candida species other than C. albicans, the reduction was not significant. Overall, there was a total cost savings of $130,231, or $1,808 per patient, despite a 20% increase in fluconazole use. Our costs for running the PNA FISH test included a start-up outlay of $1,000 for purchasing a water bath, lens filter, and UV light source microscope, technician time costs (45 min, or $12 in labor), and the reagent kit's list price of $68 ($30 is reimbursed by insurance), with a total cost of $5,760 for the year. Therefore, our overall cost savings per patient was $1,729.
Two Candida glabrata isolates out of 16 and 2 C. albicans isolates out of 31 demonstrated fluconazole MICs of >64 μg/ml. Three of the four patients with isolates with high MICs had a history of fluconazole prophylaxis, while the fourth had endocarditis. All patients received appropriate initial antifungal therapy based on their histories. Table 3 summarizes the initial and subsequent antifungal therapies used. The most frequent reasons treating physicians preferred the use of caspofungin or ABLC over fluconazole were the identification of C. glabrata prior to susceptibility results and recent bacterial endocarditis or endocarditis in patients with prosthetic material.
By use of a decision analytic tool with the PNA FISH test, a cost minimization model was recently developed by Alexander et al. (1). In their study, they predicted that in an institution with a rate of 40% for C. albicans candidemias, the test would result in a cost savings of about $1,800 per patient from reduced caspofungin usage (1). Our paper uses clinical data to show the effect of PNA FISH testing for C. albicans and validates the decision model Alexander et al. describe in their paper (1). In our experience, the financial savings in reducing caspofungin usage surpassed the cost of the PNA FISH test and has led to the development of straightforward hospital-specific treatment algorithms. We have since increased the frequency of PNA FISH to twice daily to further decrease the reporting time. An interesting finding from this study was the overall low level of fluconazole resistance (5%) in our institution, especially with C. glabrata (7, 8). Important clinical limitations of the PNA FISH test are that it has not been validated with specimens other than blood and that only a C. albicans-specific probe is currently available. Although we did not have any polyfungemias in our study, an additional benefit to the PNA FISH test is that it allows for the discrimination between varied species morphologies in specimens of mixed C. albicans species and Candida species other than C. albicans under a fluorescent microscope. The availability of specific probes for Candida species other than C. albicans, especially C. glabrata, would be of great benefit both clinically and financially.
In conclusion, the PNA FISH test is an accurate and affordable test for the rapid identification of C. albicans from positive blood cultures leading to a significant reduction in using caspofungin for treating C. albicans. Further development of probes for other Candida species should offer additional benefits.
ACKNOWLEDGMENTS
G.N.F. and R.A.V. received speakers' honoraria from AdvanDx. J.K.J. and D.P.L. received travel support from AdvanDx.
FOOTNOTES
Corresponding author. Mailing address: University of Maryland School of Medicine, Division of Infectious Diseases, 20 Penn St., Rm. S403B, Baltimore, MD 21201. Phone: (410) 706-5680. Fax: (410) 706-8700. E-mail: gforrest@medicine.umaryland.edu.
REFERENCES
Alexander, B. D., E. D. Ashley, L. B. Reller, and S. D. Reed. 2006. Cost savings with implementation of PNA FISH testing for identification of Candida albicans in blood cultures. Diagn. Microbiol. Infect. Dis. 54:277-282.
Cuenca-Estrella, M., W. Lee-Yang, M. A. Ciblak, B. A. Arthington-Skaggs, E. Mellado, D. W. Warnock, and J. L. Rodriguez-Tudela. Comparative evaluation of NCCLS M27-A and EUCAST broth microdilution procedures for antifungal susceptibility testing of Candida species. Antimicrob. Agents Chemother. 46:3644-3647.
Jabra-Rizk, M. A., J. K. Johnson, G. Forrest, K. Mankes, T. F. Meiller, and R. A. Venezia. 2005. Prevalence of Candida dubliniensis fungemia at a large teaching hospital. Clin. Infect. Dis. 41:1064-1067.
NCCLS. 2002. Reference method for broth dilution antifungal susceptibility testing of yeasts: approved standard M27-A. NCCLS, Wayne, Pa.
Oliveira, K., G. Haase, C. Kurtzman, J. J. Hyldig-Nielsen, and H. Stender. 2001. Differentiation of Candida albicans and Candida dubliniensis by fluorescent in situ hybridization with peptide nucleic acid probes. J. Clin. Microbiol. 39:4138-4141.
Pfaller, M. A. 2005. Antifungal susceptibility testing methods. Curr. Drug Targets 6:929-943.
Pfaller, M. A., D. J. Diekema, M. G. Rinaldi, R. Barnes, B. Hu, A. V. Veselov, N. Tiraboschi, E. Nagy, D. L. Gibbs, and the Global Antifungal Surveillance Group. 2005. Results from the ARTEMIS DISK Global Antifungal Surveillance Study: a 6.5-year analysis of susceptibilities of Candida and other yeast species to fluconazole and voriconazole by standardized disk diffusion testing. J. Clin. Microbiol. 43:5848-5859.
Pfaller, M. A., S. A. Messer, L. Boyken, S. Tendolkar, R. J. Hollis, and D. J. Diekema. 2003. Variation in susceptibility of bloodstream isolates of Candida glabrata to fluconazole according to patient age and geographic location. J. Clin. Microbiol. 41:2176-2179.
Rigby, S., G. W. Procop, G. Haase, D. Wilson, G. Hall, C. Kurtzman, K. Oliveira, S. Von Oy, J. J. Hyldig-Nielsen, J. Coull, and H. Stender. 2002. Fluorescence in situ hybridization with peptide nucleic acid probes for rapid identification of Candida albicans directly from blood culture bottles. J. Clin. Microbiol. 40:2182-2186.
Sutton, D. A. 2003. Specimen collection, transport, and processing: mycology, p. 1659-1667. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of clinical microbiology. ASM Press, Washington, D.C.
WHO Collaborating Centre for Drug Statistics Methodology. 1996. Guidelines for ATC classification and DDD assignment. WHO Collaborating Centre for Drug Statistics Methodology, Oslo, Norway.
Wilson, D. A., M. J. Joyce, L. S. Hall, L. B. Reller, G. D. Roberts, G. S. Hall, B. D. Alexander, and G. W. Procop. 2005. Multicenter evaluation of a Candida albicans peptide nucleic acid fluorescent in situ hybridization probe for characterization of yeast isolates from blood cultures. J. Clin. Microbiol. 43:2909-2912.(G. N. Forrest, K. Mankes, M. A. Jabra-Ri)