Sub-national TB prevalence surveys in India, 2006-2012: Results of uniformly conducted data analysis.

SETTING: Community based tuberculosis (TB) prevalence surveys in ten sites across India during 2006-2012. OBJECTIVE: To re-analyze data of recent sub-national surveys using uniform statistical methods and obtain a pooled national level estimate of prevalence of TB. METHODS: Individuals ≥15 years old were screened by interview for symptoms suggestive of Pulmonary TB (PTB) and history of anti-TB treatment; additional screening by chest radiography was undertaken in five sites. Two sputum specimens were examined by smear and culture among Screen-positives. Prevalence in each site was estimated after imputing missing values to correct for bias introduced by incompleteness of data. In five sites, prevalence was corrected for non-screening by radiography. Pooled prevalence of bacteriologically positive PTB was estimated using Random Effects Model after excluding data from one site. Overall prevalence of TB (all ages, all types) was estimated by adjusting for extra-pulmonary TB and Pediatric TB. RESULTS: Of 769290 individuals registered, 715989 were screened by interview and 294532 also by radiography. Sputum specimen were examined from 50 852 individuals. Estimated prevalence of smear positive, culture positive and bacteriologically positive PTB varied between 108.4-428.1, 147.9-429.8 and 170.8-528.4 per 100000 populations in different sites. Pooled estimate of prevalence of bacteriologically positive PTB was 350.0 (260.7, 439.0). Overall prevalence of TB was estimated at 300.7 (223.7-377.5) in 2009, the mid-year of surveys. Prevalence was significantly higher in rural compared to urban areas. CONCLUSION: TB burden continues to be high in India suggesting further strengthening of TB control activities.

Rapid detection of ethambutol-resistant Mycobacterium tuberculosis in clinical specimens by real-time polymerase chain reaction hybridisation probe method.

Background: Early diagnosis of drug resistance (DR) to ethambutol (EMB) in tuberculosis (TB) remains a challenge. Simple and reliable method (s) are needed for rapid detection of DR Mycobacterium tuberculosis (MTB) in clinical specimens. Objectives: The aim of this study was to design fluorescence resonance energy transfer hybridisation probe-based real-time polymerase chain reaction (PCR) method for the early detection of EMB-resistant MTB direct from clinical sputa. Materials and Methods: Primers and probes were designed against 306 codon of embB gene which is commonly associated with EMB resistance. A comparative study was done between Lowenstein-Jenson (L-J) proportion and hybridisation probe-based real-time PCR method for susceptibility testing. DNA sequencing was used in nine representative isolates to validate the efficiency of real-time PCR method to detect emb306 mutation of MTB. Results: A total of 52 clinical sputum samples and corresponding culture isolates (from category II pulmonary TB cases) were included in this study. Out of 52 MTB isolates, 32 and 20 were resistant and susceptible to EMB, respectively, as determined by L-J proportion method. Real-time PCR showed 95% specificity, 75% sensitivity and 82.69% accuracy when compared with L-J proportion method. A 100% of concordance was observed by validating the real-time PCR results with DNA sequencing. Conclusions: Our real-time PCR hybridisation probe method promises for rapid detection of EMB-resistant MTB directly from clinical specimens. However, future studies and modifications of method by incorporating other potential loci along with targeted mutation (emb306) are still required to increase the sensitivity of method.

Human immunodeficiency virus and hepatitis B virus co-infections among tuberculosis patients attending a Model Rural Health Research Unit in Ghatampur, North India.

INTRODUCTION: This prospective cross-sectional hospital-based study was carried out in order to assess the prevalence of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) infections among patients with active tuberculosis (TB) disease attending an Outpatient Department (OPD) at the Model Rural Health Research Unit in Ghatampur, a rural village in Kanpur district. MATERIALS AND METHODS: The socio-demographic features and clinical profile of the TB patients were analysed in the context of symptoms at the time of testing. The HIV and HBV status were determined and correlated with clinical features at the time of testing. RESULTS: In our study, the prevalence of HIV infection among TB patients is 1.48% (18/1215) and that of HBsAg reactivity was found to be 2.96% (36/1215). During 2007-2010, the HIV-positivity varied between 1.5% and 1.45% whereas HBV reactivity ranged between 2.4% and 3.63%.A substantial percentage of the TB patients attending the OPD in Ghatampur harbour HIV and HBV infections, which otherwise would remain undiagnosed without serological screening. CONCLUSION: Co infection with HBV among TB patients potentiate the risk of anti-tuberculous therapy-induced hepatotoxicity, therefore, exercising caution and carefully monitoring the patients for drugs associated hepatotoxicity is essential. There is an urgent need to perform population-based surveys of HIV and hepatitis infections among TB patients to assess the true extent of the problem. Efforts should be made to make physicians aware of the peculiarities and manage these patients effectively.

Evaluation of the diagnostic value of immunocytochemistry and in situ hybridization in the pediatric leprosy.

Leprosy is characterized by a long and variable incubation period and a chronic clinical course. Diagnosis of leprosy is essentially based on clinical features. Although the majority of cases can be diagnosed clinically yet alternative methods for diagnosis are required especially for early cases. Immunocytochemistry and in situ hybridization can be a valuable tool for diagnosis for early cases. The present study is aimed to assess the diagnostic value of immunocytochemistry and in situ hybridization in cytological specimens and to compare these techniques with Z.N. staining. This prospective study was carried out in 26 patients below 18 years of age of leprosy. Clinical examination of each patient was done and categorized according to IAL. After taking consent, three skin smears was taken, one for Z.N. staining and remaining two for immunocytochemistry and in situ hybridization respectively. Routine skin smear examination by Z.N. staining method confirmed the diagnosis in 4/26 (15.83%) and these belonged to BB, BL category. Immunocytochemistry showed positivity in 10/15 (66.6%) in BT and 72.7% in BB/BL leprosy. Immunocytochemistry improved the diagnosis by 53.85%, and the results were statistically significant (p < 0.01). In situ hybridization showed the positive results in 80% cases of BT leprosy and 90.9% cases of BB/BL leprosy. In situ hybridization improved the diagnosis by 70% in comparison to ZN staining and the results were statistically significant (p < 0.01). This study supports that immunocytochemistry and in situ hybridization enhance the diagnosis of leprosy when compared to routine skin smears stained by Z.N staining. They are important diagnostictoolsfor definitive diagnosis in early as well as established cases of leprosy.

Clinical and histopathological evaluation of the effect of addition of immunotherapy with Mw vaccine to standard chemotherapy in borderline leprosy.

This study reports detailed analysis of clinical parameters and clearance of granuloma in borderline leprosy patients treated with immunotherapy and chemotherapy. It aims to assess the additive effect of immunotherapy (Mwvaccine) with standard MDT on clinical status of untreated borderline leprosy cases and on granuloma fraction of untreated borderline leprosy cases. Patients attending the OPD were serially recruited in two groups. A total of 150 cases in one treatment (trial) group (Mw vaccine plus MDT) and 120 cases in another treatment (control) group (MDT only) of border line leprosy have been included. After the formal written consent, detailed clinical examination, charting, smear examination of all untreated borderline patients of both groups was done, biopsies were taken from the active lesions of all patients of both groups at start of therapy and every six month thereafter till the completion of therapy. The same procedure was repeated every six months during the follow-up period. Standard MDT was given to all the patients of both groups according to type of disease. Mw vaccine 0.1 ml (0.5 x 10(9) bacilli) was injected intra-dermally at the start of therapy and every six months in addition to chemotherapy to the treatment group. The BT cases were followed up after 6 doses of MDT and 2 doses of Mw vaccine, and, the BB, BL cases were followed up after 24 doses of MDT plus 5 doses of Mw vaccine. Clinically, greater and faster improvement was observed in all the clinical parameters, faster attainment of smear negativity and two episodes of lepra reaction occurred in cases treated with combined chemotherapy and immunotherapy, as compared to controls (chemotherapy alone) wherein clinical improvement was slower in all parameters, slower attainment of smear negativity in bacillary index and seven showed the occurrence of reactions, histipathologically in addition to more rapid clearance of granuloma in immunotherapy treated group, a significant finding was an increase in the epithelioid cells population in this group. This suggests a possible immunoactivation of the macrophages especially in BB/BL immunotherapy group. Overall comparison of regression induced by chemotherapy alone with that induced by combined chemotherapy and immunotherapy shows a greater reduction in clinical parameters as well as granuloma fraction in BT cases as well as in BB/BL cases. This trial shows the potential usefulness of this approach of addition of immunotherapy to standard chemotherapy in borderline leprosy cases which leads to in faster recovery from disease reduced chances of reactions and faster granuloma clearance. Such information is expected to be useful in improving the immunotherapeutic approaches for treatinggranulomatous conditions in general and in leprosy in particular.

Histological diagnosis of early and suspicious leprosy by in situ PCR.

Leprosy is a chronic mycobacterial disease whose diagnosis is primarily based on clinico-pathological examination and supported by slit skin smears for the presence of acid fast bacilli (AFB). However, definitive diagnosis of early leprosy and those suspected to have the disease but not histologically confirmed pose major public health problems. The present study reports the utility of the in situ Polymerase Chain Reaction amplification (PCR) directed at a 530bp fragment of DNA encoding the 36kd antigen of the causative Mycobacterium leprae for the diagnosis of such patients using skin biopsies of lesions. Twenty five adult patients (aged 15-50yrs) each from the clinical categories of Early and clinically Suspect leprosy were selected for the study after obtaining permission. They had solitary lesions, which were negative for AFB on slit skin smear examination. Routine histopathology confirmed the diagnosis of leprosy in 8/25 (32%) cases in the category of Early leprosy with AFB being seen in 2 biopsies, and in 5/25(20%) cases of Suspect leprosy with AFB being seen in a solitary case. The Direct in situ PCR procedure which was performed in the histologically unconfirmed cases improved the diagnosis with positive results observed in 12/17 (70.6%) cases of Early (p=0.001) and in 12/20 (60%) cases of Suspect Leprosy (p=0.005 indicating the usefulness of the Direct in situ PCR to establish the diagnosis of leprosy in histologically doubtful cases.

Evaluation of diagnostic role of in situ PCR on slit-skin smears in pediatric leprosy.

A large proportion of early cases of leprosy in children remain AFB negative in skin smears. Such cases required additional techniques to confirm the diagnosis. In situ PCR on slit- skin smears is minimally invasive and less cumbersome as compared to skin biopsies. This study was initiated in our institute with the objective to evaluate the diagnostic value of in situ PCR on slit- skin smears in pediatric leprosy. A total of 25 cases of leprosy below 16 years of age were included in the study. After detailed history and thorough clinical examination, informed consent was obtained from the parents of children for slit- skin smears from lesion sites for AFB staining and for in situ PCR technique. Cases were clinically categorized according to IAL classification into indeterminate (I), tuberculoid tuberculoid (TT), borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL) and lepromatous (LL). Most of the patients (76%) were between 9-16 years of age and 64% of the cases had history of contact with leprosy patients within the family. Skin smears were positive for AFB in only 20% of the cases. On applying in situ PCR, it was observed that 62.5% cases of I/TT/BT/BB category and 88.8% of BL/LL category gave positive signals. Overall in situ PCR confirmed the diagnosis in 72% cases while by slit smears diagnosis was confirmed in only 20% of cases. Further, out of 20 skin smear negative cases, 13 were positive by in situ PCR. Specificity of the signals of in situ PCR was established by demonstrating the absence of signals in nonleprosy dermatological conditions of vitiligo and P.alba. This study supports the potential usefulness of in situ PCR on slit- skin smears of early pediatric leprosy cases. This strategy will be especially useful in cases where skin smears are negative and in those cases where skin biopsy can not be done either because of unusual locations of lesions or because of sensitive age of the patients.

In vitro effect of fluoroquinolones against Mycobacterium tuberculosis isolates from Agra & Kanpur region of north India.

BACKGROUND & OBJECTIVE: Fluoroquinolones (FQs) are important drugs used for treatment of drug resistant tuberculosis and are also now being considered as first line drugs to shorten the duration of treatment of tuberculosis (TB). In order to find out useful FQs for treatment of tuberculosis, the comparative efficacy of five FQs, namely, ofloxacin (OFL), ciprofloxacin (CIP), sparfloxacin (SPX), gatifloxacin (GAT) and levofloxacin (LEVX) was studied against Mycobacterium tuberculosis (MTB) isolates obtained from both treated and untreated patients from Agra and Kanpur regions of north India. METHODS: A total of 162 MTB isolates [including 110 MTB isolates obtained from untreated patients (Cat-I) and 52 isolates from treated patients (Cat-II)] were tested for their susceptibilities to FQs using standard minimum inhibitory concentration (MIC) method on Löwenstein-Jensen medium. RESULTS: Keeping in view the therapeutically achievable drug levels, it was found that in Cat-I 97.2 per cent (107/110) isolates were sensitive to GAT, 89 per cent (98/110) to LEVX at 1 microg/ml whereas 92.7 per cent (102/110) isolates were inhibited by OFL at 2 microg/ml and 73.6 per cent (81/110) to SPX at 0.5 microg/ml. Only 63.6 per cent (70/110) isolates were found to be sensitive to CIP at 2 microg/ml which increased to 89 per cent (98/110) at 4 microg/ml (higher than achievable peak serum level). On the other hand, among 52 isolates for Cat-II, 37 (71.2%) were found to be sensitive to GAT and 33 (63.5%) to LEVX at 1 microg/ml concentration, 28 (53.8%) to SPX at 0.5 microg/ml whereas 33 (63.5%) and 24 (46.2%) isolates were found to be sensitive to OFL and CIP at 2 microg/ml, respectively. INTERPRETATION & CONCLUSION: It appears that GAT has higher activity against MTB isolates followed by OFL, LEVX and SPX whereas CIP showed the lowest activity. GAT was also found to be the most effective FQ against multi-drug resistant (MDR) isolates both from Cat-I and Cat-II patients. Thus, except CIP, other FQs showed potential to be included in the treatment regimens of tuberculosis including MDR-TB.

Study of rifampicin resistance and comparison of dapsone resistance of M. leprae in pre- and post-MDT era.

The aim of this study to study the drug resistance patterns of dapsone (pre- and post-MDT) and rifampicin (post-MDT era). All the 84 patients from pre-MDT period (1985-1990) and 77 patients for post-MDT period (1990-2002) reporting to a tertiary care hospital-NJIL & OMD, Agra and referred for drug susceptibility testing were included in the study. Drug resistance was studied by mouse foot pad method. Dapsone resistance was high during pre-MDT era i.e. 8.3% (medium) and 19.1% (high) with an overall dapsone resistance of 27.4%. During the post-MDT era, the dapsone resistance was low i.e. 1.3% (medium) and 3.9% (high) respectively (overall dapsone resistance-5.2%). While no comparison with pre-MDT era is available, the rifampicin resistance in these selected self-reporting cases during the post-MDT era was comparatively rather high (9.1%). MDT appears to have been useful in reducing the prevalence of dapsone resistance in leprosy patients reporting to a tertiary care hospital.

Potential of a metabolic gene (accA3) of M. leprae as a marker for leprosy reactions.

Understanding the mechanism(s) of reactions in leprosy remains a challenging task for both clinicians and basic scientists. While there is some understanding of host processes associated with different type of lepra reactions, there is very little information about bacterial factors triggering these inflammatory processes. This study is continuation of our earlier research programme on leprosy genomics in which significant transcription of 11 genes was observed during active disease and these included accA3 gene. In present study, we have investigated the potential of this gene or its gene product as molecular and or immunological marker for studying the reactions. Using quantitative Real-Time RT-PCR significant higher expression (mean log2 ratio=3.39) of accA3 was observed in specimens from leprosy reaction cases compared with cases without reactions. in silico homology model of this protein was analyzed for hydrophilic and B-cell epitope regions. Peptides with maximum antigenecity were selected, cloned, expressed and used to study sero-reactivity across the disease spectrum by indirect ELISA. While sero-reactivity was observed in leprosy cases the antibody levels did not vary significantly between the patient/s of same clinical type with and without reaction thereby indicating the limitation of this approach for this purpose. Measurement of transcription of this gene has, thus, potential as a molecular marker for monitoring the reactions.

International open trial of uniform multi-drug therapy regimen for 6 months for all types of leprosy patients: rationale, design and preliminary results.

OBJECTIVE: To describe the rationale, design and preliminary results of an open trial of 6 months uniform multi-drug therapy (U-MDT) for all types of leprosy patients assuming a cumulative relapse rate not exceeding 5% over 5 years of follow-up. METHODS: We intended to recruit 2500 patients each in multi-bacillary (MB) and pauci-bacillary (PB) groups from India (five centres) and China (two centres). Standardized clinical criteria were used to assess skin lesions in the field. RESULTS: A total of 2912 patients enrolled from November 2003 to May 2007 (India, 2746; China, 166). MB patients constituted 39% and 3% had grade 2 disability. During follow-up, 27 patients (0.9%) developed new lesions. Of these, 78% were on account of reactions. Six patients had clinically confirmed relapse. Clofazimine-related skin pigmentation was short-lived and was acceptable to patients. We analysed data for clinical status of skin lesions. About 2.9% of patients were lost to follow-up; 85.9% completed treatment, of whom 19% had inactive skin lesions. PB patients responded better than MB patients (27%vs. 6%; P < 0.001). At the end of the first (n = 2013) and second year (n = 807) of follow-up post-U-MDT, in 49% and 46% patients, lesions were inactive, respectively (59% and 57% in PB, 37% and 28% in MB; P < 0.001). CONCLUSION: U-MDT appears to be promising with respect to clinical status of skin lesions.

Potential of Mw as a prophylactic vaccine against pulmonary tuberculosis.

Mycobacterium w (Mw), is a cultivable, non-pathogenic mycobacterium and has been tried extensively as an immunomodulator in leprosy. This has been found to be safe and has shown beneficial immunoprophylactic effect in population based, double blind placebo controlled trials in North India. These effects were also observed in the vaccine trials in South India. Keeping in view these beneficial effects and its earlier reported protective effect against tuberculosis in animals, its protective efficacy was evaluated in a rural population of about 28,948 people belonging to 272 villages in Ghatampur, Kanpur (India). The population was vaccinated with two doses (1st dose of 1x10(9) heat killed organisms followed 6 months later with a 2nd dose of 5x10(8) organisms) of Mw 10-13 years ago originally to investigate its effect against leprosy. The vaccine/placebo was given to healthy contacts of leprosy patients who had no evidence of suffering from tuberculosis. Incidence and prevalence of pulmonary tuberculosis in the present study was assessed in a blind manner by an active field survey and also retrospectively by history of anti tuberculosis treatment received by the patient in the intervening period (since vaccination), which was also corroborated by scrutinizing the medical records. Diagnosis was confirmed by standard clinical and bacteriological criteria. A total of 69 patients were diagnosed to be suffering from pulmonary tuberculosis during the survey which included 17 new sputum smear positive cases and 52 previously partially treated but still active pulmonary tuberculosis cases. The difference in the new sputum positive cases between the vaccinated (5/17) and placebo groups (12/17) was significant at 5% level of significance for 1 tailed test (Z>1.64). As 75% (52/69) of the cases had been diagnosed as suffering from pulmonary tuberculosis but had not taken adequate therapy all the cases diagnosed during the intervening period were recorded and re-analysis done. The differences are more significant at 1% level of significance for 1 tail test (Z>2.59) when all cases were analysed as a group. A small proportion 12.85% (total number=3036) of the contacts in the study population had BCG scars. On analysis of results on protection against tuberculosis in this group, BCG did provide protection against tuberculosis (p<0.01). In the placebo group the prevalence of tuberculosis was 1.11% which reduced to 0.70% for those who received Mw vaccine (p<0.01) which further decreased to 0.53% in those who had BCG scars and received Mw. These results thus provide evidence suggesting protective efficacy of Mw against pulmonary tuberculosis and that Mw merits investigation in future prospective immunoprophylactic trials along with other candidates for protection against pulmonary tuberculosis.

Development and evaluation of real-time RT-PCR assay for quantitative estimation of viable Mycobacterium leprae in clinical samples.

Detection of live organisms by molecular methods has special significance in leprosy where causative organism can not be cultivated in vitro. Such techniques would be especially important for monitoring the progress of the disease. While real-time RT- PCR technology will be appropriate for this purpose, there is very little experience of use of such tools in leprosy. This study describes the development of a quantitative RT-PCR targeting 16S rRNA based on primers used in a semi quantitative RT-PCR and its application on clinical samples including slit scraping and biopsies. RNA was extracted from biopsies from 3 lepromatous leprosy (LL) cases and standard curve was generated by plotting crossing over point against the dilutions of input RNA quantity (number of bacilli used for RNA extraction). Real-time RT-PCR was performed for quantitative detection of live M. leprae in 28 slit (13/28 smear positive) scrappings and 32 biopsies (22/32 smear positive). Number of viable bacteria as estimated by solid stained bacilli and real-time PCR correlated (no difference p>0.05). The test achieved a theoretical analytical sensitivity limit of up to single live bacillus even considering 11.3% efficiency of RNA preparation which was calculated by spiking of known number of leprosy bacilli in non leprosy skin biopsies (PCR negative). All smear positive cases were positive by this assay. This assay appears to be a promising tool for detection and quantification of viable bacilli in selected clinical situations and should be of use even in smear negative cases also.

Predominance of Mycobacterium fortuitum-chelonae complex in Ghatampur field area, endemic for leprosy.

Non-tuberculous mycobacteria (NTM) are commonly found in the environment. As exposure to environmental mycobacteria has been reported to immunomodulatory in this study, the presence of environmental mycobacteria was investigated in soil, drinking water and drainage sample in Ghatampur, India, which is known for high endemicity for leprosy. Soil, drinking water from the hand pumps/wells and also drainage water collected in pools was collected in clean containers and cultured for environmental mycobacteria. Samples were processed according to the protocol established earlier. 69 soil, 62 drinking water and 31 drainage water samples were analysed from soil and water collected from 48 villages of this field area. After decontamination, cultures were set upon Lowenstein Jensen (LJ) medium. Mycobacteria were identified using biochemical tests and molecular techniques such as PCR-RFLP targeting hsp65 kD and rpoB region as well as 16S ribosomal sequencing in case of isolates showing variable biochemical features. NTM (non-tubercular mycobacteria) were isolated from 47.82% of soil samples, 20.69% of drinking water samples and 19.35% of the drainage water samples, overall mycobacteria could be isolated 52/162 of samples (32.09%). Among these mycobacteria, M. fortuitum-chelonae complex was predominant in this area; other species isolated were M. phlei, M. vaccae, M. terrae and M. flavescens. Relevance of exposure to these mycobacteria on endemicity needs to be studied by immunological and epidemiological parameters.

Long term follow-up results of 1 year MDT in MB leprosy patients treated with standard MDT + once a month Minocycline and Ofloxacin.

BACKGROUND: This study was initiated in consultation with the National Leprosy Eradication Programme (NLEP) in mid nineties to try new treatment regimens for leprosy which were more robust in terms of control of reactions, long term relapses, operationally easier to undertake and feasible in field conditions. It was also envisaged to see if the addition of newer bactericidal drugs would be beneficial. OBJECTIVES: (i) To test the feasibility, safety and response of the patients to the new regimen. (ii) To observe the incidence of reactions during and after stoppage of therapy, for a period of 8-10 years after release from treatment. MATERIALS AND METHODS: A total of one hundred skin smear positive MB patients (15 LL, 35 BL and 50 BB) patients were included in this study. All the patients received the standard MDT + once a month supervised 100 mg of Minocycline and 400 mg of Ofloxacin for 12 months during the treatment phase. Thereafter, the treatment was stopped in all the patients which were followed-up on placebo (B complex tablets). Of these, 70 patients completed the treatment schedule of one year therapy and the post treatment follow-up of 9 to 10 years. RESULTS: All the patients tolerated the drugs well. The clinical response of the patients to the treatment was very good of which 32.85% of cases had history of reactions before starting treatment. During treatment, the incidence of reactions increased marginally to 38.5%, but these were easily controlled with concurrent administration of steroids. After completion of treatment the incidence was much less i.e. 10% and 3% after 1 and 2 years of post treatment follow-up respectively. The overall relapse rate is 5.7% (4/70) with an incidence density of 0.05/100 patient years. Relapses were confirmed by clinical, bacteriological, molecular biological (rRNA probes and 36 kD targeting PCR) as well as ATP bioluminescence. The relapsed patients presented with the appearance of new lesions, slit-skin smears were again found to become positive after becoming negative. Three of the four cases who relapsed had the initial mean BI of 2 to 2.9+ whereas one had the initial mean BI of 1.5+. Also, 2 of the 4 relapsed patients had positive PCR signals at the time of stoppage of treatment. CONCLUSION: The addition of Minocycline and Ofloxacin to the standard FDT has been observed to be a well tolerated. Overall as of now, the incidence of reactions observed with the newer treatment regimen is found to be significantly lower than that of 2 years fixed duration MB-MDT. The efficacy of this regimen regarding bacteriological clearance and relapse rates could not be compared due to non-availability of the results of experience with standard 1 year MDT regimen. However, this regimen appears to be operationally feasible and safe for the users.

Changing profile of disease in leprosy patients diagnosed in a tertiary care centre during years 1995-2000.

A hospital based retrospective study was carried out to determine change in the profile of disease in leprosy patients taking 1995 as baseline and compared with the profile seen in year 2000. A total of 2149 and 1703 cases were studied respectively of year 1995 and 2000. Male to female ratio slightly increased from 2.95:1 in year 1995 to 3.4:1 in year 2000. Majority of patients were of borderline type in both years. Proportion of cases with MB leprosy was nearly same in females (60.8%) and males (63.1%) in year 1995 and in year 2000 (64.8% females and 67.6% males). Proportion of highly bacillary cases has decreased over the years in females (from 20.95% in 1995 to 11.7% in year 2000, p=0.03) as well as in males (from 25% in 1995 to 15.5% in year 2000, p=0.001). Incidence of total reactions increased from 27.6% to 35.4% over the years which is significant (p<0.01). Proportion of type 1 reactions were more in reproductive age group in females in both years (p<0.05) and of type 2 reactions were significantly (p > or = 0.05) more in males in both years. Incidence of disability (both grade 1 and grade 2) was significantly more in males than in females in both years (p > or = 0.04). Grade 1 disability has significantly increased over years in females from 10.11% to 14.8%(p<0.03) as well as in males from 13.27% to 21.3%(p<0.001). Onset of reactions was associated with pregnancy/lactation in 62% of cases and with menopause in 21% of cases in 2000, which suggests strong correlation with hormonal imbalance. To conclude while leprosy incidence has declined after MDT, recognition and management of reactions in women around changes in their hormonal levels should be properly monitored for early and effective management.

Recent advances in molecular biology of leprosy.

The last three decades have witnessed rapid progress in understanding the molecular biology of Mycobacterium leprae. Following the availability of complete genome sequence of leprosy bacillus in 2001, things have drastically changed. With the information about genetic structure, several techniques have been developed for diagnosis, molecular epidemiology and also detection of drug resistance. With the decline in the prevalence of leprosy globally, there has been some reduction in interest in the molecular methods for diagnosis, yet molecular techniques for studying the transmission dynamics and detection of drug resistance continue to be relevant. Knowledge about complete genome sequence has made it possible to undertake studies that can improve our understanding of the structure and function of this enigmatic organism. Newer information emerging about biology of M. leprae would provide insight into mechanisms of its survival and persistence in host and is likely to lead to better diagnostics and also therapeutics for mycobacterial infections in general.

PCR and in-situ hybridization for diagnosis of leprosy.

OBJECTIVE: To assess the diagnostic value of Polymerase Chain Reaction (PCR) and in situ hybridization. METHODS: This prospective study was carried out in 22 patients RESULTS: The histopathological examination confirmed the clinical diagnosis in 27.2% cases only. In situ hybridization showed a positivity of 42.8% in early (I/BT) and 46.7% in BB/BL group. In situ hybridization thus enhanced the diagnosis by 18.1%. PCR targeting 36 kDa gene of M. leprae was performed on 15 cases. In these 15 cases, histopathology confirmed the diagnosis in 4 cases (26.6%) and PCR confirmed the diagnosis in 10 cases (66.6%), thus enhancing the diagnosis by 40%. CONCLUSION: 36 kDa PCR and in situ hybridization enhance the diagnosis of leprosy when compared to routine histopathology. They are important diagnostic tools for definitive diagnosis in early and doubtful cases of leprosy.

Immunogenetics of mycobacterial infections in the North Indian population.

Several lines of evidence highlight the genetic basis of risk to develop mycobacterial diseases. Human leukocyte antigen (HLA)-DR2 alleles (DRB1*1501 and DRB1*1502) have been found to be strongly associated with mycobacterial disease, especially the more severe forms such as lepromatous leprosy and multidrug-resistant pulmonary tuberculosis. In this study, DNA-based high-resolution typing techniques of polymerase chain reaction-sequence-specific oligonucleotide probe were used to determine the distribution of HLA-DR/DQ alleles in patients with leprosy and pulmonary tuberculosis. Analysis of different DR2 subtypes based on valine/glycine dimorphism at codon beta86 in pocket 1 of HLA-DR showed an inverse relationship of DR2 alleles with V/G as the severity of disease increased both in leprosy and in pulmonary tuberculosis.