Lower touch sensibility in the extremities of healthy Indians: further deterioration with age. (In press with Journal of Peripheral Nervous System): Touch sensibility testing is a cost effective, psychophysical measure of peripheral nerve function and impairment. Using Semmes – Weinstein monofilaments (SWM), we studied 568 healthy Indian volunteers without any clinical evidence of peripheral nerve disease. Touch sensibility was evaluated bilaterally in palms, feet and heels. The lowest target force detected ranged from 0.4gm to 2 gm in the palms and 1.4 gm to 15 gm in the feet. These values showed further increase with age. Women as compared to men had higher sensibility in the palms in most age groups. Touch sensibility thresholds recorded in a large group of Indians were higher than that reported in other populations. This is the largest study reported to date and influences diagnosis and draws attention to geographic variations in touch sensation.

Clinical profile of leprosy in urban and semi-rural field areas: A retrospective analysis was undertaken to study the trends and the risk factors for Erythema Nodosum Leprosum (ENL) in smear positive multibacillary (MB) cases for the period of 1991 to 2000 from LEPRA field projects of Andhra Pradesh and Orissa (HYLEP, KORALEP, JUNLEP AND BOLEP). A total number of 14,338 MB cases were analyzed. Of these 3923 were smear positive. Percentage of smear positivity was similar in all the field areas except in one urban area. Maximum numbers of MB+ patients (44.8%) were in the age group of 20-49 years. In general, males predominated over females across all the age groups in 4 project areas. A decreased trend was observed from LL (51.9% to 21.7%) to BT (5.1% to 33.3%). Pattern of skin lesion shows a decreasing trend from skin infiltration (52.8% to 30.6%) to multiple patches (43.9% to 61.8%).Delay in attending leprosy clinic had effect on classification of disease and bacteriological Index. Age has influence on classification of leprosy. Percentage of LL increases with age. Percentage of ENL varies from project to project. It ranges from 6% from BOLEP to 17% to JUNLEP and HYLEP. Gender, age, LL, delay in presentation, bacteriological Index, infiltration and number of truncal nerves were observed to be significant risk factors for ENL

A retrospective study of Neuritis cases from 2000 to 2006: Peripheral nerve damage is a major challenge in leprosy. Neuritis may occur with or without Type I and II reaction. A retrospective study was undertaken to gain insight into factors contributing towards neuritis. 41.9% (292/699) patients had reaction of which 49% had neuritis. All the neuritis cases presented with weakness of which 65% of patients presented with anesthesia. Clinical and histological classification correlated in 66 % nerve biopsy and 9.5% of skin biopsy. Proportion of neuritis was more in BL, LL and PNL patients. 11.5% patients had neuritis even after completion of MDT. 27% of neuritis patients were associated with Type I or Type II reactions.69.2% of acute onset neuritis became chronic as the treatment duration with steroids was more than six months.Number of lesions (>10), Body areas (>2), BI (>2) and involvement of truncal nerves (>3) were found to be risk factors associated with neuritis. Improvement in motor function was more evident than sensory improvement.

Pattern of relapse in leprosy in an out patient clinic in Hyderabad: Duration of multi drug therapy (MDT) regimen reduced from 24 months to 12 months may result in more relapses in leprosy patient. This prospective study was undertaken to document the pattern of relapse. 31 relapse cases were enrolled with relapse from 2003 to 2007. These cases were confirmed histologically. Median age of relapse in patient’s receiving monotherapy - 20 years; PBMDT - 4 years and MBMDT - 14 years. Localized relapse was seen in patients treated in monotherapy era. Clinical and histological classification correlated in 74.2 % of relapsed cases. Minimum and maximum duration of relapse after release from treatment (RFT) in MB cases was 8 to 38 years respectively, while in PB cases it was 1 to 3 ½ years. 86% of the relapse cases presented with the same type of leprosy as seen previously. PB cases relapsed earlier than MB cases. Molecular tools will help us to confirm relapse due to reinfection / reactivation or drug resistance.

Thalidomide is re-introduced in the treatment ENL and about 52 patients are enrolled for thalidomide treatment.  In 2007 around 28 leprosy cases are put on thalidomide.  Several patients who were steroid dependent one completely weaned from steroids and has been economically independent after returning to their avocation.

Ultrasonography of peripheral Nerves in Leprosy Patients: The aim of this study is to determine the role of imaging by ultrasound in asserting the nerve involvement in leprosy. The specific objectives of the study are as follows: a) To know the clinical usefulness of imaging modalities in indicating the long term prognosis or the likely progression of nerve damage in leprosy patients; b) To diagnose the early reactions in leprosy  and c) To monitor the response of steroid treatment in reactions by ultra sonography.

Study of viability of M.leprae in clinical samples and possibility of its presence in the environment using nucleic acid amplification techniques (funded by Indian Council of Medical Research (ICMR)): To investigate whether the bacteria causing leprosy survive outside the human host, we studied soil samples from 50 households of leprosy patients using 16s RNA to identify viable leprosy bacilli  M.leprae 16s RNA was not detectable in any of the soil samples as indicated below where a representative gel with soil from 17 households is shown.  In contrast bacilli from fresh clinical sample showed evidence of 16s RNA (as indicated by the arrow in lane  P but absent in other lanes (1-17) which represent soil samples.

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Study of T regulatory cells (TREG cells) in human leprosy: Treg cells  are being investigated for the lack of specific immunity in leprosy patients. 45 leprosy patients from across the clinical spectrum have been enrolled.  We have been successful in demonstrating FOXP3 cells in stored paraffin blocks which is more difficult to establish than in frozen sections. Gene expression for FOXP3 was also demonstrated by reverse transcription PCR in and fresh tissues. 

Figures below shows a) skin with brown nuclear stained cells and b) cDNA representing mRNA expression of FOXP3 in stimulated cells and skin biopsy of lepromatous leprosy patients in ENL reaction as in lanes 2, 3, 4, 5, 6. lanes 7, 8 indicate the house keeping gene control.

The role of genetic factors relevant to cytokines and immune responses in leprosy: There are limited reports on the influence of the immune regulatory genes on the clinical manifestations of leprosy. This study was designed to evaluate the influence of genotype of interferon-gamma (IFN-γ +874 A/T) and interleukin-10 (IL-10 -1082 G/A) on susceptibility to leprosy. Leprosy patients (356) and healthy controls (112) were screened for single point mutations in the genes (SNP –single nucleotide polymorphism) encoding for these cytokines by using PCR methodology. Significant association of AA genotype in the IFN-γ gene and GA genotype in the IL-10 gene, was observed, suggesting that an individual with these may be susceptible to leprosy. 

IFN γ (+874 A/T)                                                IL-10 (-1082 G/A)

Molecular epidemiology and drug resistance screening in leprosy (funded by NIH, USA): Leprosy research this year was primarily concerned with developing rapid molecular tools for studying transmission and diagnosing drug resistance. VNTR (Variable Nucleotide Tandem Repeats) or short stretches of the alphabet of the DNA code were analysed in bacilli from 15 patients.  Tracking of infection as well diagnosing whether relapse or reinfection with bacillus from another patient has occurred would be possible by such analysis.

The table gives the details of the VNTR pattern for each strain.

Importantly we were able to detect strain differences from ear lobe slit skin smears with BI as low as 1. In addition mutations in genes responsible for drug resistance were undertaken on similar DNA samples with assistance from Colorado University and Dr Niyaz Ahmed of CDFD. Such scans reveal the mutations in one sample where GCC replaced ACC in folp gene indicating resistance to dapsone. Resistance to other drugs were not observed to date.

Mutation in folp gene – Dapsone resistance (MEL/BPRC002 folp gene sequence)

2. Tuberculosis:

Mycobacterial species causing lymphadenitis (accepted for publication in Southeast Asian J Trop Med Public Health): A prospective study evaluated the non-tuberculous mycobacterial (NTM) cases of lymphadenitis. A total of 76 isolates of mycobacteria were obtained from 200 lymph node aspirates suspected of tuberculosis, 74 of which were Mycobacterium tuberculosis, one was Mycobacterium fortuitum and one Mycobacterium kansasii. These results indicate the re-emergence of NTM as potential lymph node pathogens from lymph node sites in this part of the country in the context of prevailing HIV epidemic. Further studies on a larger scale are needed to delineate the association between NTM infections in HIV positive and negative subjects.

Mycobacterial culture of fine needle aspirate- A useful tool in diagnosing tuberculous lymphadenitis (accepted for publication by Indian Journal of Medical Microbiology): A prospective study was undertaken on 157 patients suspected with lymph node TB (LNTB), to know the utility of mycobacterial culture of fine needle aspirate in the diagnosis of lymph node tuberculosis. 127/ 157 (80%) aspirates were positive for cytological examination.  Acid fast bacilli by ZN smear were demonstrated in 28 (18%) aspirates. Mycobacterium tuberculosis was isolated from culture of 70 (45%) aspirates. Eight aspirates (5%), which were negative for tuberculosis by cytology, yielded positive cultures for Mycobacterium tuberculosis out of which four were from HIV positive patients. Our observations suggest that FNA cytology may be the most simple and sensitive test for diagnosing LNTB. However, supplementing it with mycobacterial culture would increase the sensitivity in addition to giving a highly specific diagnosis.

Fig: Diagnosis of TB lymphadenitis- Fine needle aspiration

Fig: Diagnosis of TB lymphadenitis - % positivity by smear and culture

Invitro anti mycobacterial drug resistance and its effect on treatment outcome in tuberculosis patients from an urban and semi urban community in South India (Accepted by Transactions of Royal Society of Tropical Medicine and Hygeine): A record based study was undertaken on new and retreated pulmonary TB patients registered under the DOTS programme of LEPRA Society, a non governmental organization in Hyderabad, India. TB Patients (n=605) registered during 2004-2005 and whose pre treatment sputum yielded M tuberculosis, were studied for the association of anti TB drug resistance and treatment outcome. Out of 605 patients studied, 554 (92%) have shown favourable outcome, 51(8%) had shown unfavorable outcome. Logistic regression analysis of the data found that male sex (Wald p< 0.026) and MDR (Wald p<0.001) were associated with unfavourable outcome. Proportion of treatment failure (6%) was high in previously treated cases as compared to the new cases (2%). Neither patient age nor the treatment category was associated with treatment outcome. Drug resistance including MDR was high among the retreated cases (p<0.001). Considering the frequency of the treatment failures and a high prevalence of MDR, treatment guided by in vitro drug susceptibility tests may therefore be more effective in this group. It may also be feasible to spend on DST, than the expenditure in terms of the costs and the time on the retreatment of patients who fails the standard treatment.

Fig: % Favourable vs. unfavourable treatment outcome

Bacteriological aspects of the infected ulcers in Leprosy patients (presented in biennial conference of IAL): Total 597 leprosy patients with infected plantar ulcers were studied. Pus from the ulcers was inoculated on nutrient agar, blood agar and Mcconkey agar and incubated at 370c for over night for isolating organisms infecting the ulcer. Antibiotic susceptibility testing (AST) was carried out by Kirby-Bauer disc diffusion method as per standard protocols.  

A simple cost analysis has been carried out from patient records, on the usefulness of AST in treating plantar ulcers. The analysis has shown that the cost incurred on the treatment before the culture and AST including the cost of empirical antibiotic treatment seems to be higher than that incurred on culture and AST followed by treatment with specific antibiotics.

Fig: Infected plantar ulcers in leprosy patients- Cost of empirical vs specific treatment

Age related waning of the effect of BCG vaccination in children: India has the greatest burden of TB with the highest number of estimated cases (WHO Report 2005). At present BCG is the only vaccine available against tuberculosis. The effect of the vaccine wanes with age. Our laboratory is exploring the possibility of a booster vaccine using r32 kDa protein of BCG.  Figure shows T-cell assays with BCG r32kDa:  Stimulation Indices (SI) & Interferon- γ levels in different age-groups of BCG-vaccinated children (n=45).

Interferon γ low producer genotype +874 over represented in BCG non responding children: There are limited reports on the influence of the immune regulatory genes on the efficacy of BCG vaccination. A study was designed to evaluate the influence of the cytokine genotype interferon-gamma (IFN-γ) +874 A/T on T cell in vitro assays in BCG non-responders (negative to either in vivo or in vitro test with PPD or both). Majority of the vaccinated children with TT genotype showed high levels of IFN-γ. The results suggest that BCG vaccine may not benefit children with an ‘AA’ (low producer) IFN-γ (+874) genotype. The Pediatric Infectious Disease Journal (In press).