The World Health Organization has identified India as a major hot-spot region for Mycobacterium tuberculosis infection. We have characterized the sequences of the loci associated with multidrug resistance in 126 clinical isolates of M. tuberculosis from India to identify the respective mutations. The loci selected were rpoB (rifampin), katG and the ribosomal binding site of inhA (isoniazid), gyrA and gyrB (ofloxacin), and rpsL and rrs (streptomycin). We found known as well as novel mutations at these loci. Few of the mutations at the rpoB locus could be correlated with the drug resistance levels exhibited by the M. tuberculosis isolates and occurred with frequencies different from those reported earlier. Missense mutations at codons 526 to 531 seemed to be crucial in conferring a high degree of resistance to rifampin. We identified a common Arg463Leu substitution in the katG locus and certain novel insertions and deletions. Mutations were also mapped in the ribosomal binding site of the inhA gene. A Ser95Thr substitution in the gyrA locus was the most common mutation observed in ofloxacin-resistant isolates. A few isolates showed other mutations in this locus. Seven streptomycin-resistant isolates had a silent mutation at the lysine residue at position 121. While certain mutations are widely present, pointing to the magnitude of the polymorphisms at these loci, others are not common, suggesting diversity in the multidrug-resistant M. tuberculosis strains prevalent in this region. Our results additionally have implications for the development of methods for multidrug resistance detection and are also relevant in the shaping of future clinical treatment regimens and drug design strategies.

Proteins released from Mycobacterium tuberculosis (Mtb) during late logarithmic growth phase are often considered candidate components of immunogenic or autolysis markers. One such protein is isocitrate dehydrogenase (ICD), a key regulatory enzyme in the citric acid cycle. We have evaluated the immunogenic properties of two isoforms of Mtb ICD and compared them with the control antigens heat-shock protein 60 and purified protein derivative (PPD). PPD lacks the sensitivity to distinguish between bacillus Calmette-Gué rin (BCG)-vaccinated and tuberculosis (TB)-infected populations, and, therefore, epidemiological relevance of PPD in BCG-vaccinated regions is debatable. We show that Mtb ICDs elicit a strong B cell response in TB-infected populations and can differentiate between healthy BCG-vaccinated populations and those with TB. The study population (n ‫؍‬ 215) was categorized into different groups, namely, patients with fresh infection (n ‫؍‬ 42), relapsed TB cases (n ‫؍‬ 32), patients with extrapulmonary TB (n ‫؍‬ 35), clinically healthy donors (n ‫؍‬ 44), nontuberculous mycobacteria patients (n ‫؍‬ 30), and non-TB patients (culture negative for acid-fast bacteria but carrying other infections, n ‫؍‬ 32). The Mtb ICDs showed statistically significant antigenic distinction between healthy BCG-vaccinated controls and TB patients (P < 0.0001) and those with other infections. Although extrapulmonary infections could not be discriminated from healthy controls by heat-shock protein 60 (P ‫؍‬ 0.2177), interestingly, the Mtb ICDs could significantly (P < 0.0001) do so. Our results highlight the immunodominant, immunosensitive, and immunospecific nature of Mtb ICDs and point to an unusual property of this tricarboxylic acid energy cycle enzyme. T uberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major threat to the human population, being responsible for Ϸ2-3 millions deaths every year worldwide (1-3). The secret of the pathogen's success is its ability to escape the host immune system and remain undetected in lungs for decades. In only 10% of infected people, the number being higher in immunocompromised patients, does TB erupt as a full-blown disease (4). Delay in diagnosis and treatment impedes the downstream management and control of the disease. With the increasing emergence of multidrug resistant strains and coinfection with HIV, the problem is further compounded (5-7). Early diagnosis, therefore, is a matter of utmost concern not just for TB disease management but also for epidemiological investigations (8). Current diagnostic tools for TB often lack sensitivity and can be time consuming. TB diagnosis in developing countries largely banks on tuberculin skin tests and staining and culture methods. The epidemiological relevance of the tuberculin test with purified protein derivative (PPD) is questionable in areas where bacillus Calmette-Guérin vaccination is compulsory because PPD is not sensitive enough to distinguish between vaccinated and infected individuals (9). Microscopic determination ...

The current vaccine against tuberculosis (TB), Mycobacterium bovis BCG, fails to protect against the most prevalent disease form, pulmonary TB in adults. It is generally assumed that active TB occurs because of a weakening of the immune system, which keeps Mycobacterium tuberculosis in check as long as it is fully competent. M. tuberculosis does not induce the optimum protection because the pathogen is not eradicated, and it has now been shown that exogenous reinfection does occur, suggesting that natural immunity is insufficient (26) and fails to control the pathogen in the long run. Hence, other mycobacterial strains which share cross-reactive antigens (Ags) with M. tuberculosis have also been considered as alternatives to M. bovis for vaccine use. One strain, "Mycobacterium w," had been evaluated for its immunomodulatory properties in leprosy. M. w is a nonpathogenic, cultivable mycobacterium (18) which has been found to improve immunity to leprosy (30). A vaccine against leprosy based on M. w is approved for human use, where it has resulted in clinical improvement, accelerated bacterial clearance, and increased immune responses to Mycobacterium leprae Ags (13,21,25). M. w shares Ags not only with M. leprae but also with M. tuberculosis (29), and initial studies have shown that vaccination with killed M. w induces protection against TB in animal models (22, 23) and also resulted in early sputum conversion in TB patients (17). Recently it has been suggested that M. w be referred to as Mycobacterium indicus pranii to avoid confusion with M. tuberculosis-W (Beijing strain) (24). It is generally known that live bacteria impart greater protection than killed bacteria. It may be that persistence of live bacteria in the host for some time results in a robust memory response (12). Another important factor is that secretory proteins which are absent in the killed bacterial vaccines have been shown to play an important role in protection. In this study, we analyzed the M. tuberculosis-specific immune response induced in mice immunized with live or killed M. w and compared it with the BCG-induced immune response and also compared the protective efficacies of the two mycobacteria.As the lung is the primary target organ of this disease, immunization potential by the aerogenic route was also studied. Inhalation of aerosols provides a noninvasive delivery system that physically targets the lung as the desired site of the pharmacological effect. This route of immunization has emerged a very attractive route of vaccine delivery, inducing both local and systemic immunity (7, 10).

BackgroundThe 9-month-long chemotherapy of tuberculosis often results in poor compliance and emergence of drug-resistant strains. So, improved therapeutic strategy is urgently needed. Immunotherapy could be beneficial for the effective management of the disease. Previously we showed the protective efficacy of Mycobacterium indicus pranii (MIP) when given as prophylactic vaccine in animal models of tuberculosis.MethodsWe sought to investigate whether MIP can be used as an adjunct to the chemotherapy in guinea pig models of tuberculosis. Efficacy of MIP was evaluated when given subcutaneously or by aerosol.ResultsMIP-therapy as an adjunct to the chemotherapy was found to be effective in accelerating bacterial killing and improving organ pathology. MIP-immunotherapy resulted in higher numbers of activated antigen-presenting cells and lymphocytes in the infected lungs and also modulated the granulomatous response. Early increase in protective Th1 immune response was observed in the immunotherapy group. Following subsequent doses of MIP, decrease in the inflammatory response and increase in the immunosuppressive response was observed, which resulted in the improvement of lung pathology.ConclusionMIP immunotherapy is a valuable adjunct to chemotherapy for tuberculosis. Aerosol route of immunotherapy can play a crucial role for inducing immediate local immune response in the lung.

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A substantial percentage of the outpatients seen in the clinics of the district hospital in Uttar Pradesh harbor HIV and viral hepatitis infections, which otherwise would remain undiagnosed without serological screening.

The gene for histone-like protein (hupB [Rv2986c]) of Mycobacterium tuberculosis has been identified as a singular target which allows differentiation of two closely related mycobacterial species, namely, M. tuberculosis and M. bovis of the MTB complex, by a PCR assay. The N and S primer-generated PCR amplicons differed in M. tuberculosis and M. bovis; these amplicons were determined to be 645 and 618 bp, respectively. This difference was localized to the C-terminal part of the gene by using primers M and S. The C-terminal PCR amplicons of M. tuberculosis and M. bovis were determined to be 318 and 291 bp, respectively. The differences in the C-terminal portion of the gene were confirmed by restriction fragment length polymorphism analysis and sequencing. Sequence analysis indicated that in M. bovis there was a deletion of 27 bp (9 amino acids) in frame after codon 128 in the C-terminal part of the hupB gene. In the present study 104 mycobacterial strains and 11 nonmycobacterial species were analyzed for hupB gene sequences. Of the 104 mycobacterial strains included, 62 belonged to the MTB complex and 42 were non-MTB complex strains and species. Neither the hupB gene-specific primers (N and S) nor the C-terminal primers (M and S) amplify DNA from any other mycobacteria, making the assay suitable for distinguishing members of the MTB complex from other mycobacterial species, as well as for differentiating between members of the MTB complex, namely, M. tuberculosis and M. bovis.Early and reliable detection of pathogenic mycobacteria in clinical samples is a major limitation in the control of human tuberculosis. At present, a battery of tedious tests (microbiological, biochemical, etc.) requiring more than several days or weeks are routinely used to identify clinical mycobacterial isolates. The criteria used for the differentiation of Mycobacterium tuberculosis and M. bovis have been colony morphology, nitrate reduction, niacin test, and sensitivity or resistance to pyrazinamide. Deviations from standard patterns in all of the above tests have been reported, making it virtually impossible to differentiate between M. bovis, M. tuberculosis, and M. africanum (12, 34, 38, 48). The high degree of variability in the phenotypic characteristics has made it important to develop reliable techniques to distinguish between members of the Mycobacterium tuberculosis and M. bovis (MTB) complex (22,41). Techniques based on the amplification of mycobacterial DNA sequences by PCR have been introduced in many laboratories as a promising alternative rapid, sensitive, and specific detection of M. tuberculosis in clinical specimens (2,7,14).Novel targets have been exploited for diagnostic purposes by using PCR, namely, the devR response regulator gene (42, 43), rRNA (5), selected chromosomal fragments (3,18,25,29), genes coding for the 65-kDa heat shock protein (36), the 38-kDa protein antigen (44), the dnaJ gene (47), and insertion sequences such as IS6110, IS990, and IS1081 (1,15,16,23); these are all examples of diverse targets that ...

For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast-and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria.Several species of environmental mycobacteria have been known to be important human pathogens (12). Further exposure to them is believed to alter immunity to vaccines like Mycobacterium bovis BCG (11). Isolation of mycobacteria from environmental samples is difficult because other microbes are also present in the environment. All mycobacterial species are not equally resistant to the different decontamination procedures. For the isolation of mycobacteria from environmental samples, such as soil and water, different methods have been described by various workers (1-10). These are not universally applicable because of differences between floras. No studies of this issue had previously been carried out in the northern parts of India. For this reason, the present study was undertaken to select or develop an improved, appropriate decontamination method(s) for the isolation of mycobacteria from the predominantly hot, dry environment of Agra, India (annual temperature, maximum of 16 to 47°C and minimum of 3 to 30°C; humidity, maximum of 49 to 100% and minimum of 28 to 70%).After we tried different permutations and combinations in controlled experiments, the following procedure was standardized ( Fig. 1). Wet soil samples of approximately 5 g were collected from a depth of 3 cm, and 50-ml water samples were collected from ditches, ponds, lakes, and rivers in the Agra region throughout the year. Soil was suspended in 20 ml of double-distilled autoclaved water (D/W) in polycarbonate centrifuge tubes. After being shaken manually for 60 s, the suspension was centrifuged at 600 ϫ g for 5 min at 4°C to pellet the soil particles. The turbid supernatant (10 ml) was transferred into other sterile centrifuge tubes and centrifuged at 8,000 ϫ g for 15 min at 4°C. Water samples were centrifuged at 8,000 ϫ g for 15 min at 4°C. Pellets from the soil and water samples were resuspended in 20 ml of treatment solution (3% sodium dodecyl sulfate [SDS] plus 4% NaOH) and then divided into two parts: A and B. Part A was incubated at room temperature (RT) for 15 min to obtain the growth of rapid growers, and part B was incubated at RT for 30 min to obtain the growth of slow growers. After incubation, both the suspensions were centrifuged at 8,000 ϫ g for 15 min at 4°C, and then the supernatants were decanted. Sediments were processed for cetrimide treatment. In the initial pilot experiments, various incubation periods with 2% cetrimide treatment were tried for slow and rapid growers. The pellets were resuspended in 20 ml of 2% cetrimide. Part A was incubated at RT for 5 min to obtain the g...