Diagnostic value of in situ polymerase chain reaction in leprosy
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《美国医学杂志》
1 Department of Pediatrics, S.N. Medical College,Agra, India
2 Central JALMA Institute for Leprosy and other Mycobacterial Diseases (ICMR), Agra, India
Abstract
Objective : This prospective study was carried out to assess the diagnostic value of in situ Polymerase Chain Reaction in leprosy, particularly in enhancing the histopathological diagnosis. Method : Clinical examination of 20 patients (<16 yr) was done and skin smear for AFB was prepared. Biopsy of lesion site was taken for histopathological examination and in situ PCR testing. Results : The histopathological examination confirmed the clinical diagnosis in 45% cases only; non-specific histopathology was reported in the remaining 55% cases. In situ PCR showed a positivity of 57.1% in early/localized form of leprosy (IIBT) and 61.5% in (BB/BL) group. When compared to histopathology examination, a significant enhancement of 15% in diagnosis was seen. With in situ PCR, the diagnosis could be confirmed in 4/11 (36.3%) cases with non-specific histopathological features, (which is common in early disease) in addition to confirmation of 8/9 (88.8%) histopathologically-confirmed tissue sections. Histopathology and in situ PCR, combined together, confirmed the diagnosis in 13/20 cases (65% of total cases). Conclusion : Thus, in situ PCR is an important diagnostic tool especially in early and doubtful cases of leprosy.
Keywords: Leprosy; Histopathology; In situ Polymerase Chain Reaction
Leprosy, a chronic granulomatous disease which primarily affects the peripheral nervous system and skin, has a worldwide distribution; the total number of cases is estimated to be around 53400. India accounts for 64% of leprosy prevalence and 76% of the new cases detected worldwide.[1] In India, in 2003-2004, the prevalence rate was 2.44 per 10,000 population and new case detection rate was 3.37 per 10,000 population.[2] While there has been a dramatic fall in prevalence rate of leprosy in India, the new case detection rate has not reduced concomitantly.[3]
The diagnosis of leprosy is primarily clinical. Anesthetic skin lesions with or without thickened peripheral nerves are virtually pathognomonic of leprosy. A full thickness skin biopsy from an anesthetic lesion showing granuloma and lymphocytic infiltration of nerves essentially confirms the diagnosis. Acid fast bacilli (AFBs) are rarely found in patients with indeterminate or tuberculoid disease. In the established form of the disease, the diagnosis is relatively simple. However, the established forms are decreasing in number due to widespread use of multidry therapy (MDT), and early disease is being reported more frequently. Routine histopathological examination by Hematoxylin and Eosin and Fite-Faraco staining of sections can confirm the diagnosis in only about 35% of such early cases[4],[5], because of paucity of acid fast bacilli and absence of infiltration inside the dermal nerves which are considered essential criteria for diagnosis of leprosy. Instead, they show non-specific histopathology in the form of chronic inflammatory cell infiltrate at various locations, which is common to many dermatological conditions. This group, therefore, requires additional methods to confirm the diagnosis, like demonstrating antigen at the site of lesion by immunostaining, or demonstrating nucleic acid sequences specific to the pathogen by using in situ hybridization and amplification by in situ polymerase chain reaction. In situ detection of PCR amplified (DNA/RNA) products has been described and was initially used to detect viral DNA and single copy genes.[6],[7],[8] This technique allows cellular localization of low-level of nucleic acid within the tissue and cells[9] and can aid in a significant way to augment the sensitivity of histopathological diagnosis. When compared to in vitro PCR, the in situ PCR procedure offers the distinct advantage of providing structural correlates, thereby permitting the concomitant study of tissue pathology; also, the possibility of contamination by foreign DNA/RNA does not exist. However, hardly any studies have been done to study the diagnostic value of in situ PCR in childhood leprosy. With this background, we conducted this study to evaluate the diagnostic value of in situ PCR in cases of leprosy.
Materials and Methods
This study was conducted at S.N. Medical College, and Central JALMA Institute of Leprosy and Other Mycobacterial Diseases, Agra. Cases were chosen from the patients attending the outpatient departments of these hospitals. A total of 20 cases, less than 16 yr of age, who had not taken any treatment were selected, and were categorized as I (Indeterminate), BT (Borderline Tuberculoid), BB (Borderline Borderline) and BL (Borderline Lepromatous) types according to Ridley Jopling classification.[10] Due to paucity of cases of early leprosy and difficulties in taking biopsy in young children with early lesions, well-established cases were also included in the study and the overall sensitivity of the diagnostic procedure of in situ PCR was determined. Skin smears were taken from lesion site and stained with Ziehl-Neelsen's staining method. Skin biopsy from the lesion site was taken after taking informed consent. The tissue specimens were processed for routine histopathological examination (i.e. staining with Hematoxylin-Eosin and Fite-Faraco stains) and in situ PCR. A definitive histological diagnosis of leprosy requires: a) presence of infiltration within dermal nerves and b) the presence of Acid Fast Bacilli (AFB).
IN-SITU PCR
In situ PCR is a powerful technique in which gene fragments are amplified within tissue sections. The procedure of in situ PCR was performed in 3 steps on routinely processed (Formalin fixed paraffin embedded) tissue specimens, as follows: (1) pretreatment of tissue sections was done with proteolytic enzymes; (2) amplification; in this step a solution containing Reaction buffer, Digoxogenin labeled nucleotide (d UTP, dNTP, dCTP) and primers 19 mer and 18 mer was added to the tissue sections. These primers were specific to M.leprae and amplified the 530 bp fragment of 36 kDa protein encoding gene sequence in the M. leprae genome.[11] After this amplification, reaction was done in a programmable thermal cycler with attachment for slide (PTC100, Model 60 MJ Research); (3) post- amplification labeling and detection. In this step, an enzyme, substrate-chromogen combination (containing Anti-Digoxogenin Antibody, Alkaline phosphatase, NBT/BCIP), was added. This was followed by visualization under microscope. A blue black color was visualized at the site where M. leprae was present in the tissue section Figure1.
Results
In the present study, 20 untreated patients, under 16 yr, were included. Maximum number of patients were males (85%) and fell in the 12-16 yr age group. They belonged to the BB (Borderline Borderline) variety table1.
Maximum number of the cases (85%) presented with hypopigmented, ill-defined and macular skin lesions present both over covered and uncovered parts of the body. Most of the patients had more than 4 lesions and no deformities. 20% cases had skin lesions only, while 80% had both skin lesions and nerve thickening. Only 20% cases had history of contact with leprosy patients. All the patients with I, BT, and BB lesions were smear negative for AFB; 2/4 (50%) of the cases with BL lesions were smear positive table2.
All cases in the present study were examined histopathologically. It was observed that confirmation of clinical diagnosis by histopathology could be made in 9/20 (45%). Among these histopathological confirmed cases, AFB positivity in 7/9 (77.7%) was noted in sections. Non-specific pathology was seen in 55% of cases. When compared with skin smear examination, the results of histopathology were statistically significant (p<0.05).
In the final phase of the study, in situ PCR was performed in all tissue specimens. It was positive in 60% of the total no. of cases. (i.e. 57.1% among early/localized form of leprosy (I/BT) and 61.5% in BB/BL leprosy cases).
The results of in situ PCR were definitely better than the results of histopathology, and this improved the diagnosis. However, overall positivity was not statistically significant. This could be due to small sample size.
In the present study, non-specific histopathology was reported in 11/20 (55%) cases (I= 2/3, BT=1/4, BB= 6/9, BL=2/4), which needed further confirmation of diagnosis. In situ PCR was performed on all 20 (100%) cases. With in situ PCR, the authors had an overall positivity of 60% and could diagnose 4/11 (36.3%) cases with non specific histopathology. In addition to these 4/11 cases, the diagnosis of leprosy could also be confirmed in 8/9 histopathological confirmed tissue sections. Thus, the total number of cases diagnosed with both histopathology and in situ PCR was 13/20 (65%) table3.
Discussion
The present study comprised of 20 cases. Maximum number of children had hypopigmented, macular lesions, with ill-defined margins (85%) present over both covered and uncovered parts of the body (75%). The results are comparable to the findings reported earlier by the present authors and others. [12],[13],[14],[15] In the present study 75% children had 4-10 skin lesions; 10% had less than 3 skin lesions. Patients' disability/deformity were graded according to WHO protocol. Only 20% patients had grade-I disability; no patient had grade-II disability. This reflects a good trend of reporting early for diagnosis and treatment. 20% of that cases had history of contact with leprosy patients in the family; these results are comparable to studies by other authors.[14],[15] All cases of Indeterminate, Borderline Tuberculoid, and Borderline Borderline leprosy were smear negative. Skin smear was positive in 10% of total cases which were of BL type.
Diagnosis of leprosy is easy when cardinal signs are present, and specific tissue histopathology, namely detection of AFB and infiltration inside the dermal nerves, is seen. However, in early and doubtful cases of leprosy, sensitivity and specificity of cardinal signs and histopathology become low. A non-specific histopathology is often encountered in these cases. Such cases require sophisticated and specific methods to prove the diagnosis. Such modalities will become even more important in the post-elimination era. The extent to which histology can confirm the clinical diagnosis of early leprosy cannot be stated with uniformity as it varies from situation to situation. It ranges from a high 86.6% to a low 30%, with AFB positivity ranging from 10% to 63%.[16] In the present series, histological confirmation could be done in 9/20 (45%) cases with AFB positivity of 77.7% in tissue specimens. During the last few years, PCR based tests for detection of M. leprae in clinical specimens have been described[17],[18],[19] and in situ PCR has emerged as a promising technique for diagnosis of various infectious diseases.[6],[7],[8] The detection limit ranges from 1 to 100 bacilli and signal positivity ranges from 45-75%.[19], [20] Despite the advancement of the technique of PCR, its major limitation has been that it is not possible to correlate the results with the pathological features of the disease. In the present study with in situ PCR, the authors observed 57.1% positivity in early and localized leprosy (I/BT) and 61.5% positivity in BB/BL group, with an overall positivity of 60% which is comparable to studies by other investigators who reported an overall positivity of 58.7% in early leprosy group using in vitro PCR.[20] The present results are better than those reported by Singh et al[19]. In situ PCR when compared to histopathology improved the diagnosis by 15%.
Hardly any literature is available on the use of in situ PCR in the diagnosis of leprosy in children. The present pioneer work showed an overall positivity of 60% and a positivity of 100% in Ziehl-Neelsen's Staining AFB positive specimens. Further, the authors could diagnose 4/11 (36.3%) cases with non-specific histopathology with in situ PCR. Total cases diagnosed with both histopathology and in situ PCR were 13/20 (65%), which is a substantial improvement over 45% with histopathology alone. In situ PCR was 15% more sensitive than histopathology alone. The reasons for negative in situ PCR results in 7 children with non-specific histopathology could be:
(a) low bacterial load
(b) faulty technique of sampling of sections.
Keeping in mind that in situ PCR offers excellent structural correlation permits the concomitant study of tissue pathology, and contamination by foreign DNA/RNA does not exist; the authors recommend that large studies should be carried out to further explore the diagnostic potential of this technique.
Contributors : The concept and design of the study was provided by RD, PPM, VMK, KK and MN, which was carried out by SPS.SPS analyzed the data, drafted the manuscript which was finally revised and approved by RD and VMK. RD will act as the guarantor.
Competing interests : None
Funding : Central JALMA Institute for Leprosy and other
Mycobacterial Diseases (ICMR), Agra.
Word count of the text : 1480
References
1. WHO. WHO Leprosy elimination project: Status Report, 2003.
2. Dhillon GPS. An overview of the challenges plans and achievements in India's leprosy particularly during the past two decades. Bull Lepr Eliminat Alliance 2004; 1, 2 : 5-9.
3. Subramaniam M, Showkath Ali MK, Throat DM, Muthukumar M, Satish Kumar E, Ramadoss C, Ali Khan M. Leprosy situation in endemic states of India and prospects of elimination of disease. Indian J Lepr 2003; 75 : 325-345.
4. Fine PEM, Job CK, MC Dougall ACM et al. Comparability among histopathologists in the diagnosis and classification of lesions suspected of leprosy in Malawri. Int J Lepr 1986; 61 : 614-625.
5. Ramu G, Karthikeyan S, Balakrishanan S et al. Histological and Immunological correlation of suspected leprosy patients. Indian J Lepr 1996; 68 : 153.
6. Arnoldi J, Schindler C, Ducknow M, Hubner L, Ernst M, Teske A, Flad HD, Gerdes J, Bottger EC. Species specific assessment of Mycobacterium leprae in skin biopsies by in situ hybridizationand Polymerase chain reaction. Lab Invest 1992; 66 : 618-623.
7. Komminoth P, Long AA. In situ Polymerase chain reaction. An overview of methods, applications and limitations of a new molecular technique. Virchows Arch B cell Pathol Incl Mol Pathol 1993; 64 : 67-73.
8. Nuovo GJ, Gallery F, Mac Connel P, Becker J and Bloch W. An improved technique for the in-situ detection of DNA after polymerase chain reaction. Am J Pathol 1991; 139 : 1239-1244.
9. Komminoth P, Long AA. In situ polymerase chain reaction-methodlogy, applications and non specific pathways. In : Boerhinger Mannheim. PCR Application Manual Mannheim , Germany 1995; 97-106.
10. Ridley DS. Histological classification and the immunological specturm of leprosy. Bull WHO 1974; 51 : 451-465.
11. Hartskeel RA, Dewit MYL and Klatser PR. Polymerase chain reaction for detection of M leprae. J Gen Microbiol 1989; 135 : 2357-2364.
12. Dave MS, Agarwal SK. Prevalence of leprosy in children of Leprosy patients. Lepr India 1984; 56 : 615-621.
13. Dayal R, Hashmi NA, Mathur PP, Prasad R. leprosy in children. Indian Pediatr 1990; 27 : 170-180.(Dayal R, Singh SP, Mathur)
2 Central JALMA Institute for Leprosy and other Mycobacterial Diseases (ICMR), Agra, India
Abstract
Objective : This prospective study was carried out to assess the diagnostic value of in situ Polymerase Chain Reaction in leprosy, particularly in enhancing the histopathological diagnosis. Method : Clinical examination of 20 patients (<16 yr) was done and skin smear for AFB was prepared. Biopsy of lesion site was taken for histopathological examination and in situ PCR testing. Results : The histopathological examination confirmed the clinical diagnosis in 45% cases only; non-specific histopathology was reported in the remaining 55% cases. In situ PCR showed a positivity of 57.1% in early/localized form of leprosy (IIBT) and 61.5% in (BB/BL) group. When compared to histopathology examination, a significant enhancement of 15% in diagnosis was seen. With in situ PCR, the diagnosis could be confirmed in 4/11 (36.3%) cases with non-specific histopathological features, (which is common in early disease) in addition to confirmation of 8/9 (88.8%) histopathologically-confirmed tissue sections. Histopathology and in situ PCR, combined together, confirmed the diagnosis in 13/20 cases (65% of total cases). Conclusion : Thus, in situ PCR is an important diagnostic tool especially in early and doubtful cases of leprosy.
Keywords: Leprosy; Histopathology; In situ Polymerase Chain Reaction
Leprosy, a chronic granulomatous disease which primarily affects the peripheral nervous system and skin, has a worldwide distribution; the total number of cases is estimated to be around 53400. India accounts for 64% of leprosy prevalence and 76% of the new cases detected worldwide.[1] In India, in 2003-2004, the prevalence rate was 2.44 per 10,000 population and new case detection rate was 3.37 per 10,000 population.[2] While there has been a dramatic fall in prevalence rate of leprosy in India, the new case detection rate has not reduced concomitantly.[3]
The diagnosis of leprosy is primarily clinical. Anesthetic skin lesions with or without thickened peripheral nerves are virtually pathognomonic of leprosy. A full thickness skin biopsy from an anesthetic lesion showing granuloma and lymphocytic infiltration of nerves essentially confirms the diagnosis. Acid fast bacilli (AFBs) are rarely found in patients with indeterminate or tuberculoid disease. In the established form of the disease, the diagnosis is relatively simple. However, the established forms are decreasing in number due to widespread use of multidry therapy (MDT), and early disease is being reported more frequently. Routine histopathological examination by Hematoxylin and Eosin and Fite-Faraco staining of sections can confirm the diagnosis in only about 35% of such early cases[4],[5], because of paucity of acid fast bacilli and absence of infiltration inside the dermal nerves which are considered essential criteria for diagnosis of leprosy. Instead, they show non-specific histopathology in the form of chronic inflammatory cell infiltrate at various locations, which is common to many dermatological conditions. This group, therefore, requires additional methods to confirm the diagnosis, like demonstrating antigen at the site of lesion by immunostaining, or demonstrating nucleic acid sequences specific to the pathogen by using in situ hybridization and amplification by in situ polymerase chain reaction. In situ detection of PCR amplified (DNA/RNA) products has been described and was initially used to detect viral DNA and single copy genes.[6],[7],[8] This technique allows cellular localization of low-level of nucleic acid within the tissue and cells[9] and can aid in a significant way to augment the sensitivity of histopathological diagnosis. When compared to in vitro PCR, the in situ PCR procedure offers the distinct advantage of providing structural correlates, thereby permitting the concomitant study of tissue pathology; also, the possibility of contamination by foreign DNA/RNA does not exist. However, hardly any studies have been done to study the diagnostic value of in situ PCR in childhood leprosy. With this background, we conducted this study to evaluate the diagnostic value of in situ PCR in cases of leprosy.
Materials and Methods
This study was conducted at S.N. Medical College, and Central JALMA Institute of Leprosy and Other Mycobacterial Diseases, Agra. Cases were chosen from the patients attending the outpatient departments of these hospitals. A total of 20 cases, less than 16 yr of age, who had not taken any treatment were selected, and were categorized as I (Indeterminate), BT (Borderline Tuberculoid), BB (Borderline Borderline) and BL (Borderline Lepromatous) types according to Ridley Jopling classification.[10] Due to paucity of cases of early leprosy and difficulties in taking biopsy in young children with early lesions, well-established cases were also included in the study and the overall sensitivity of the diagnostic procedure of in situ PCR was determined. Skin smears were taken from lesion site and stained with Ziehl-Neelsen's staining method. Skin biopsy from the lesion site was taken after taking informed consent. The tissue specimens were processed for routine histopathological examination (i.e. staining with Hematoxylin-Eosin and Fite-Faraco stains) and in situ PCR. A definitive histological diagnosis of leprosy requires: a) presence of infiltration within dermal nerves and b) the presence of Acid Fast Bacilli (AFB).
IN-SITU PCR
In situ PCR is a powerful technique in which gene fragments are amplified within tissue sections. The procedure of in situ PCR was performed in 3 steps on routinely processed (Formalin fixed paraffin embedded) tissue specimens, as follows: (1) pretreatment of tissue sections was done with proteolytic enzymes; (2) amplification; in this step a solution containing Reaction buffer, Digoxogenin labeled nucleotide (d UTP, dNTP, dCTP) and primers 19 mer and 18 mer was added to the tissue sections. These primers were specific to M.leprae and amplified the 530 bp fragment of 36 kDa protein encoding gene sequence in the M. leprae genome.[11] After this amplification, reaction was done in a programmable thermal cycler with attachment for slide (PTC100, Model 60 MJ Research); (3) post- amplification labeling and detection. In this step, an enzyme, substrate-chromogen combination (containing Anti-Digoxogenin Antibody, Alkaline phosphatase, NBT/BCIP), was added. This was followed by visualization under microscope. A blue black color was visualized at the site where M. leprae was present in the tissue section Figure1.
Results
In the present study, 20 untreated patients, under 16 yr, were included. Maximum number of patients were males (85%) and fell in the 12-16 yr age group. They belonged to the BB (Borderline Borderline) variety table1.
Maximum number of the cases (85%) presented with hypopigmented, ill-defined and macular skin lesions present both over covered and uncovered parts of the body. Most of the patients had more than 4 lesions and no deformities. 20% cases had skin lesions only, while 80% had both skin lesions and nerve thickening. Only 20% cases had history of contact with leprosy patients. All the patients with I, BT, and BB lesions were smear negative for AFB; 2/4 (50%) of the cases with BL lesions were smear positive table2.
All cases in the present study were examined histopathologically. It was observed that confirmation of clinical diagnosis by histopathology could be made in 9/20 (45%). Among these histopathological confirmed cases, AFB positivity in 7/9 (77.7%) was noted in sections. Non-specific pathology was seen in 55% of cases. When compared with skin smear examination, the results of histopathology were statistically significant (p<0.05).
In the final phase of the study, in situ PCR was performed in all tissue specimens. It was positive in 60% of the total no. of cases. (i.e. 57.1% among early/localized form of leprosy (I/BT) and 61.5% in BB/BL leprosy cases).
The results of in situ PCR were definitely better than the results of histopathology, and this improved the diagnosis. However, overall positivity was not statistically significant. This could be due to small sample size.
In the present study, non-specific histopathology was reported in 11/20 (55%) cases (I= 2/3, BT=1/4, BB= 6/9, BL=2/4), which needed further confirmation of diagnosis. In situ PCR was performed on all 20 (100%) cases. With in situ PCR, the authors had an overall positivity of 60% and could diagnose 4/11 (36.3%) cases with non specific histopathology. In addition to these 4/11 cases, the diagnosis of leprosy could also be confirmed in 8/9 histopathological confirmed tissue sections. Thus, the total number of cases diagnosed with both histopathology and in situ PCR was 13/20 (65%) table3.
Discussion
The present study comprised of 20 cases. Maximum number of children had hypopigmented, macular lesions, with ill-defined margins (85%) present over both covered and uncovered parts of the body (75%). The results are comparable to the findings reported earlier by the present authors and others. [12],[13],[14],[15] In the present study 75% children had 4-10 skin lesions; 10% had less than 3 skin lesions. Patients' disability/deformity were graded according to WHO protocol. Only 20% patients had grade-I disability; no patient had grade-II disability. This reflects a good trend of reporting early for diagnosis and treatment. 20% of that cases had history of contact with leprosy patients in the family; these results are comparable to studies by other authors.[14],[15] All cases of Indeterminate, Borderline Tuberculoid, and Borderline Borderline leprosy were smear negative. Skin smear was positive in 10% of total cases which were of BL type.
Diagnosis of leprosy is easy when cardinal signs are present, and specific tissue histopathology, namely detection of AFB and infiltration inside the dermal nerves, is seen. However, in early and doubtful cases of leprosy, sensitivity and specificity of cardinal signs and histopathology become low. A non-specific histopathology is often encountered in these cases. Such cases require sophisticated and specific methods to prove the diagnosis. Such modalities will become even more important in the post-elimination era. The extent to which histology can confirm the clinical diagnosis of early leprosy cannot be stated with uniformity as it varies from situation to situation. It ranges from a high 86.6% to a low 30%, with AFB positivity ranging from 10% to 63%.[16] In the present series, histological confirmation could be done in 9/20 (45%) cases with AFB positivity of 77.7% in tissue specimens. During the last few years, PCR based tests for detection of M. leprae in clinical specimens have been described[17],[18],[19] and in situ PCR has emerged as a promising technique for diagnosis of various infectious diseases.[6],[7],[8] The detection limit ranges from 1 to 100 bacilli and signal positivity ranges from 45-75%.[19], [20] Despite the advancement of the technique of PCR, its major limitation has been that it is not possible to correlate the results with the pathological features of the disease. In the present study with in situ PCR, the authors observed 57.1% positivity in early and localized leprosy (I/BT) and 61.5% positivity in BB/BL group, with an overall positivity of 60% which is comparable to studies by other investigators who reported an overall positivity of 58.7% in early leprosy group using in vitro PCR.[20] The present results are better than those reported by Singh et al[19]. In situ PCR when compared to histopathology improved the diagnosis by 15%.
Hardly any literature is available on the use of in situ PCR in the diagnosis of leprosy in children. The present pioneer work showed an overall positivity of 60% and a positivity of 100% in Ziehl-Neelsen's Staining AFB positive specimens. Further, the authors could diagnose 4/11 (36.3%) cases with non-specific histopathology with in situ PCR. Total cases diagnosed with both histopathology and in situ PCR were 13/20 (65%), which is a substantial improvement over 45% with histopathology alone. In situ PCR was 15% more sensitive than histopathology alone. The reasons for negative in situ PCR results in 7 children with non-specific histopathology could be:
(a) low bacterial load
(b) faulty technique of sampling of sections.
Keeping in mind that in situ PCR offers excellent structural correlation permits the concomitant study of tissue pathology, and contamination by foreign DNA/RNA does not exist; the authors recommend that large studies should be carried out to further explore the diagnostic potential of this technique.
Contributors : The concept and design of the study was provided by RD, PPM, VMK, KK and MN, which was carried out by SPS.SPS analyzed the data, drafted the manuscript which was finally revised and approved by RD and VMK. RD will act as the guarantor.
Competing interests : None
Funding : Central JALMA Institute for Leprosy and other
Mycobacterial Diseases (ICMR), Agra.
Word count of the text : 1480
References
1. WHO. WHO Leprosy elimination project: Status Report, 2003.
2. Dhillon GPS. An overview of the challenges plans and achievements in India's leprosy particularly during the past two decades. Bull Lepr Eliminat Alliance 2004; 1, 2 : 5-9.
3. Subramaniam M, Showkath Ali MK, Throat DM, Muthukumar M, Satish Kumar E, Ramadoss C, Ali Khan M. Leprosy situation in endemic states of India and prospects of elimination of disease. Indian J Lepr 2003; 75 : 325-345.
4. Fine PEM, Job CK, MC Dougall ACM et al. Comparability among histopathologists in the diagnosis and classification of lesions suspected of leprosy in Malawri. Int J Lepr 1986; 61 : 614-625.
5. Ramu G, Karthikeyan S, Balakrishanan S et al. Histological and Immunological correlation of suspected leprosy patients. Indian J Lepr 1996; 68 : 153.
6. Arnoldi J, Schindler C, Ducknow M, Hubner L, Ernst M, Teske A, Flad HD, Gerdes J, Bottger EC. Species specific assessment of Mycobacterium leprae in skin biopsies by in situ hybridizationand Polymerase chain reaction. Lab Invest 1992; 66 : 618-623.
7. Komminoth P, Long AA. In situ Polymerase chain reaction. An overview of methods, applications and limitations of a new molecular technique. Virchows Arch B cell Pathol Incl Mol Pathol 1993; 64 : 67-73.
8. Nuovo GJ, Gallery F, Mac Connel P, Becker J and Bloch W. An improved technique for the in-situ detection of DNA after polymerase chain reaction. Am J Pathol 1991; 139 : 1239-1244.
9. Komminoth P, Long AA. In situ polymerase chain reaction-methodlogy, applications and non specific pathways. In : Boerhinger Mannheim. PCR Application Manual Mannheim , Germany 1995; 97-106.
10. Ridley DS. Histological classification and the immunological specturm of leprosy. Bull WHO 1974; 51 : 451-465.
11. Hartskeel RA, Dewit MYL and Klatser PR. Polymerase chain reaction for detection of M leprae. J Gen Microbiol 1989; 135 : 2357-2364.
12. Dave MS, Agarwal SK. Prevalence of leprosy in children of Leprosy patients. Lepr India 1984; 56 : 615-621.
13. Dayal R, Hashmi NA, Mathur PP, Prasad R. leprosy in children. Indian Pediatr 1990; 27 : 170-180.(Dayal R, Singh SP, Mathur)