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The influence of human leukocyte antigens (HLA) on the immune response is well established. We investigated the regulatory role of HLA-DRB1 alleles on cytokine response to live M. tuberculosis and its culture filtrate antigen (CFA) in normal healthy subjects (NHS) and pulmonary tuberculosis (PTB) patients. Th1 (IFN-gamma and IL-12p40), Th2 (IL-4 and IL-5), pro-inflammatory (IL-6 and IL-8) and anti-inflammatory (TGF-beta and IL-10) cytokines were measured by ELISA in 72-h-old peripheral blood mononuclear cell culture supernatants from 58 NHS and 48 PTB patients. HLA-DRB1 genotyping was carried out by polymerase chain reaction and dot-blot hybridization with biotinylated sequence-specific oligonucleotide probes and detection by chemiluminescence. In response to live M. tuberculosis and CFA, significantly increased levels of IL-6, IL-8 and TGF-beta and decreased IFN-gamma, IL-12p40 and IL-10 were seen in PTB patients compared to NHS. We observed a significantly increased IFN-gamma response in HLA-DRB1*03-positive NHS (p=0.03) and decreased IFN-gamma response in HLA-DRB1*15-positive patients (p=0.04) than respective allele-negative individuals. An increased level of IL-12p40 in DRB1*10 (p=0.02) and IL-10 in DRB1*12- (p=0.03) positive NHS and an increased level of IL-6 in DRB1*04- (p=0.02) positive patients were observed. The study suggests that HLA-DRB1 alleles differentially modulate the various cytokine responses to M. tuberculosis antigens, which may influence the cellular and humoral immune responses to M. tuberculosis infection in a susceptible host.

Mannose binding lectin (MBL) plays an important role in innate immunity. Plasma MBL levels and MBL2 gene polymorphisms were studied in HIV-1 infected patients without tuberculosis (HIV+TB-) (n=151) and with tuberculosis (HIV+TB+) (n=109), HIV negative tuberculosis patients (HIV-TB+) (n=148) and healthy controls (n=146) by ELISA and genotyping by polymerase chain reaction based methods. MBL levels were significantly increased among HIV-TB+ and HIV+TB+ patients than controls and HIV+TB- patients (P<0.05). A significantly increased frequency of OO genotype of structural polymorphism and YY genotype of -221Y/X was observed among HIV-TB+ patients than controls. In HIV+TB+ patients, a significantly increased frequency of YA/YA diplotype (associated with very high MBL levels) was observed compared to controls (P=0.03). In HIV+TB+ patients, a significantly decreased frequency of medium MBL expression diplotypes (XA/XA and YA/YO) were noticed compared to HIV+TB- and healthy controls. The results suggest that YA/YA diplotype associated with very high MBL levels may predispose HIV-infected patients to tuberculosis while O/O genotype associated with very low MBL levels may be associated with susceptibility to tuberculosis in HIV uninfected individuals. Medium MBL expression diplotypes might protect against development of TB in HIV-infected patients.

Setting: A rural community in Chingleput district in Tamil Nadu state in south India., Objective: To determine the value of dual testing with PPD-S and PPD-B for identifying subjects with a tuberculous infection., Design: About 240,000 subjects in rural south India, all of whom were tested initially with PPD-S and PPD-B, were followed up for 15 years, mainly by total population survey once in every 212 years. The incidence of culture-positive tuberculosis was estimated using life-table technique., Results: Among 17,530 subjects with an intermediate reaction (8-11 mm) to PPD-S at intake, 285 with an induration to PPD-S exceeding the induration to PPD-B by at least 2mm, had a significantly higher incidence of culture-positive tuberculosis than the remaining (154 and 93 per 100,000), and similarly 481 who had an induration of <10mm to PPD-B compared to those with >or=10 mm (131 and 93 per 100,000). These subjects may be regarded as having a tuberculous infection. Infection with non-tuberculous mycobacteria conferred protection of about 30% against the development of tuberculosis over a 15-year period., Conclusion: In subjects with an intermediate reaction (8-11 mm) to PPD-S, dual testing with PPD-B enabled identification of those with a tuberculous infection. Most of the reactions were due to non-tuberculous mycobacteria.

India has a long and distinguished tradition of research in the field of tuberculosis (TB). Pioneering studies from India demonstrated the efficacy and safety of domiciliary treatment, the necessity of direct observation of treatment, the feasibility of case detection through sputum smear microscopy in primary health care institutions, and the effectiveness of intermittent short-course chemotherapy. These findings laid the foundation of directly observed treatment, short course (DOTS), which has been adopted by nearly 150 countries worldwide. Today, India has the second-largest and the fastest-growing DOTS programme in the world. A strong component of programme evaluation and operational research is needed to sustain and expand DOTS in the context of a suboptimal primary health care system, a large and unregulated private health care system, and the dual threats of HIV and multidrug-resistant TB (MDR-TB). Therefore, the focus of TB research in India has shifted to the following operational research areas: evaluating models to involve the private health sector; assessing the role of incentives in increasing treatment compliance; examining gender differentials in the access to TB services; assessing risk factors for delay in diagnosis; evaluating diagnosis, treatment and prevention of TB among HIV-infected persons; monitoring MDR-TB; estimating cost-effectiveness of the DOTS programme; monitoring the quality of smear microscopy services; and measuring the current burden of TB. Research for developing newer diagnostic tools, drugs and vaccines remains a long-term priority. Greater networking is needed among national researchers, programme managers and policy-makers to translate the findings of research into policies and programmes to make TB control in India more effective and efficient.

Setting: A study was undertaken to understand the non-major histocompatibility restricted cytotoxicity in order to delineate the role of natural killer (NK) cells towards the development of host immunity to tuberculosis., Objective: (a) Enumeration of NK cell numbers and activity in normal individuals (35), pulmonary tuberculosis patients (32), HIV-infected TB patients (20) and patient contacts (10), (b) effect of treatment on NK status, (c) enumeration of effector-target conjugates and (d) effect of in vitro cytokine stimulation on NK activity., Design: NK cells were enumerated by flow cytometry. NK activity was assessed by chromium release assay before and after treatment for tuberculosis and after stimulation with IL-2/IL-12. Novel flow cytometric method was standardized to enumerate effector-target conjugates., Results: No changes were seen between different groups as far as number of NK cells and relative proportions of different conjugate types were concerned, but there was a decrease in NK activity in TB patients which increased after treatment. Augmentation of NK activity was observed after cytokine stimulation., Conclusion: Lowered NK activity during tuberculosis infection is probably the 'effect' and not the 'cause' for the disease as demonstrated by the follow-up study. Similar number of conjugates in both groups indicates no defect in the recognition/binding step but probably at subsequent steps of the cytotoxic process. Augmentation of NK activity with cytokines implicates them as potential adjuncts to tuberculosis chemotherapy., (Copyright 2001 Harcourt Publishers Ltd.)

Setting: A study of tumour necrosis factor alpha and beta (TNFalpha and beta) gene polymorphism and haplotype analysis with HLA in pulmonary tuberculosis., Objective: To determine whether TNFalpha (-238 and -308) and TNFbeta (Nco I polymorphism in intron 2) genes either alone or in combination with human leucocyte antigens (HLA) as haplotypes afford susceptibility or resistance to pulmonary tuberculosis as well as bacteriological relapse of the disease., Design: Tumour necrosis factor alpha -238, -308 (TNFalpha -238, -308) and TNFbeta (Nco I) gene polymorphisms were carried out in HLA-A,B and DR typed pulmonary tuberculosis patients (n=210) and 120 normal healthy control subjects., Results: No difference in the genotype frequencies of TNFalpha-238 and -308 and TNFbeta was seen between control subjects and pulmonary tuberculosis patients. Of the HLA-TNF haplotypes analysed, the infrequent allele (A) of TNFalpha238 was in strong linkage disequilibrium with HLA-A1 (P corrected: Pc=0.001), B17 (Pc<0.0001) and DR7 (Pc=0.01) in control subjects and with B17 (Pc<0.0001) in pulmonary tuberculosis. The infrequent allele 2 of TNFalpha-308 and the infrequent allele 2 of TNFbeta were in strong linkage disequilibrium with HLA-B21. An increased haplotype frequency of HLA-B17-TNFalpha-238/A (P=0.05), B17-TNFalpha308/2 (P=0.03) and B17-TNFalpha308/2 (P=0.01) was observed in bacteriological relapse patients than quiescent patients., Conclusion: The present study suggests that TNFalpha (-238 and -308) and TNFbeta gene variants are not associated independently with the susceptibility to pulmonary tuberculosis. However, in combination with the HLA genes/gene products such as HLA-A1, B17, B21 and DR7, the TNFalpha and beta genes as haplotypes are associated with protection against the disease as well as an increased susceptibility to bacteriological relapse., (Copyright 2001 Harcourt Publishers Ltd.)

The objective was to apply the purified 38kDa protein antigen of Mycobacterium tuberculosis in ELISA to estimate the IgG, IgA and IgM antibody levels in sera and circulating immune complexes of tuberculosis patients. Sera from smear and culture positive tuberculosis patients were positive for anti 38kDa IgG, IgA and IgM antibodies, with a sensitivity of 61%, 30% and 10%, respectively, and with a specificity of 100% for IgG. The sensitivity of the test improved to a level of 68% for IgG+IgA and of 71.4% for IgG+IgA+IgM without significantly compromising the specificity (IgG of 100%, IgG+IgA of 96%, IgG+IgA+IgM of 90%). Among the smear, culture-negative but X-ray-positive cases, 60% were serum positive for IgG antibody, while in smear-negative but culture-positive cases, 54% were positive for IgG antibody. Measurement of 38kDa antibodies showed a greater than 95% sensitivity in smear and culture-positive, and smear-negative and culture-positive patients, through a combination of assays for serum IgG and circulating immune complex antibodies, while the specificity was 100%., (Copyright 2001 Harcourt Publishers Ltd.)

Setting: Mannose binding protein gene polymorphism in pulmonary tuberculosis in India., Objective: To find out whether non-HLA genes such as mannose binding protein (MBP) genes are associated in the susceptibility to pulmonary tuberculosis., Design: Genotyping of MBP 52, 54 and 57 wild and mutant alleles was carried out in HLA-DR typed pulmonary tuberculosis patients (n = 202) and control subjects (n = 109). Since HLA-DR2 is associated with pulmonary-TB, the interaction of MBP genes on -DR2 and non-DR2 genes on the susceptibility was also studied., Results: A significantly increased genotype frequency of MBP functional mutant homozygotes (including 52, 54 and 57) was seen in pulmonary tuberculosis (PTB) patients (10.9%) than in control subjects (1.8%; P = 0.008; odds ratio: 6.5). Analysis of interaction of MBP genes and HLA-DR2 on the susceptibility to PTB revealed that these genes are associated with PTB independent of each other., Conclusion: The present study shows that functional mutants of MBP are associated with PTB. Apart from HLA-DR2 association, association of non-HLA genes in the susceptibility to PTB is evident. This suggests that multigenetic factors (candidate genes) may be involved in the susceptibility/resistance to PTB.

Setting: Influence of HLA-DR antigens and lymphocyte responses in pulmonary TB patients., Objective: To elucidate the role of HLA-DR genes/gene products on lymphocyte responses to Mycobacterium tuberculosis antigens and mitogens, the present study was carried out in pulmonary tuberculosis during active and cured stage of the disease., Design: Serological determination of HLA-DR antigens was carried out in 50 active TB patients, 44 cured TB patients and 58 normal healthy control subjects. The influence of HLA-DR antigens on peripheral blood lymphocyte responses to M. tuberculosis culture filtrate antigens and mitogens such as phytohaemagglutinin (PHA) and concanavalin-A (Con-A) was studied in the patients as well as normal healthy control subjects., Results: Of all the DR antigens studied, patients (active TB and cured TB) with DR2 antigen showed an increased lymphocyte response (stimulation index) to a higher dose of antigenic (10 micrograms/ml) stimulation. A significantly lower lymphocyte response to antigen and mitogens was seen in HLA-DR3 positive normal healthy subjects than non-DR3 (DR3 negative) subjects., Conclusion: The present study suggests that HLA-DR genes/gene products may be playing an immunoregulatory role in eliciting an immune response against M. tuberculosis antigens and mitogens induced lymphocyte response in pulmonary TB patients and normal healthy subjects.

Setting: HLA and tuberculin status in pulmonary tuberculosis patients. Tuberculosis Research Centre, Indian Council of Medical Research, Madras, India., Objective: To elucidate the role of HLA-class-II genes/gene products on tuberculin reactivity in pulmonary tuberculosis patients., Design: Serological determination of HLA-DR and -DQ antigens was carried out in 62 healthy control subjects and 146 pulmonary tuberculosis patients. The tuberculin reaction pattern of pulmonary tuberculosis patients to PPD was studied and the role of HLA-DR and -DQ antigens (class-II gene products) on tuberculin reaction was analysed., Results: HLA-DR and -DQ antigens did not influence high, medium and low tuberculin reaction dramatically in active pulmonary tuberculosis patients. However, a heterozygous combination of various HLA-DR antigens influenced the tuberculin reaction., Conclusion: The HLA-genetic make up (heterozygous combination) of the individual may influence the tuberculin reaction pattern in pulmonary tuberculosis.

Setting: Madras, India., Objective: To explore the utility of a standardized IS6110/PvuII deoxyribonucleic acid (DNA) fingerprinting restriction fragment length polymorphism (RFLP) typing method for distinguishing between isolates of Mycobacterium tuberculosis, and to assess the potential for distinguishing between relapse versus reinfection rates., Design: To assess RFLP heterogeneity in the population, initial isolates, obtained from the sputum of tuberculous 98 patients in diagnosis and follow-up during short-course chemotherapy, were stored and compared. To assess the frequency of disparity between the RFLP type of the initial isolate and one obtained after successful completion of chemotherapy, either during relapse or as an isolated positive culture, 124 isolates comprising 62 such pairs were coded and compared both blind and after decoding., Results: Although a wide variety of DNA band patterns (fingerprints) was present, the isolates from 39 (40%) of the patients showed a single copy of IS6110. Only 15 pairs of coded initial and follow-up isolates could be identified as having the same band pattern when isolates with zero or single bands were excluded. Nevertheless, after decoding, in a retrospective analysis that included all isolates, those isolates that bacteriologically defined a patient's relapse more often showed RFLP type identity with the initial isolate (19 of 30 comparisons) than did isolates that were obtained as isolated positive cultures (3 of 32 comparisons) (X2 P < 0.001). Tests of sensitivity to chemotherapeutic drugs, catalase activity and resistance to thiophene-2-carboxylic acid hydrazide were of minimal value in discriminating between isolates., Conclusions: Despite the high frequency of single- and zero-band isolates in this population, the discriminatory power of RFLP typing with IS6110 is sufficiently high to be useful for clinical and epidemiological studies.

Studies of the HLA class II genes were performed in patients and multiple families with pulmonary tuberculosis from South India to seek any association of disease susceptibility with an individual allele or haplotype. TaqI RFLP analysis of HLA-DRB, -DQA and -DQB genes was done in 38 patients and 36 healthy control subjects. No significant association with any particular allele or haplotype was obtained. Linkage analysis performed in 12 families and haplotype sharing analysis in 9 families showed that the genetic susceptibility to pulmonary tuberculosis was not linked to the HLA region. The results suggest that the RFLP patterns of HLA class II genes DRB, DQA and DQB are not associated with susceptibility to pulmonary tuberculosis and that the genes controlling susceptibility or resistance may be located outside the HLA region.