Molecular epidemiology of tuberculosis

Molecular epidemiology (ME), a blend of molecular biology and epidemiology, is very useful to study the spread of tubercle bacilli in mini epidemics, outbreaks, to analyse the transmission dynamics of tuberculosis (TB) and to determine the risk factors for TB transmission in a community. ME has a great role in distinguishing between exogenous reinfection and endogenous reactivation. In the laboratory, molecular epidemiology can be used to identify cross contamination. Many new DNA typing methods have been introduced after the initial introduction of restriction fragment length polymorphism (RFLP) in 1993. An internationally accepted, standardized protocol for RFLP typing of the Mycobacterium tuberculosis complex using IS6110 was published in 1993 and is still used today. Most of the newer DNA typing methods are PCR based and microarray based methods are also available. This will enable individual strains of M. tuberculosis or clonal groups to be identified by specific phenotypic traits. ME will continue to be a useful tool in future to measure the impact of any public health intervention strategy for control of tuberculosis in the community

Differential expression of a unique protein by intracellular Mycobacterium tuberculosis complex

We have investigated the changes in the protein synthesis pattern in guinea pig peritoneal macrophages following infection with virulent Mycobacterium tuberculosis H37Rv in vitro. By 35S methionine labelling of the newly synthesized proteins followed by ultracentrifugation, SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and autoradiography, the protein synthesis pattern of the control uninfected macrophages and the infected macrophages in vitro were compared. By adding cycloheximide to the macrophage cultures, the protein synthesis of macrophages was inhibited and the protein synthesis pattern of M. tuberculosis has been analysed. We have identified a mycobacterial protein of molecular weight 17 kDa which was expressed exclusively in the cytosolic fraction of M. tuberculosis- infected guinea pig macrophages in vitro

Involvement of Serine Threonine Protein Kinase, PknL, from Mycobacterium Tuberculosis H37Rv in Starvation Response of Mycobacteria

The adaptation to nutrient depletion in bacteria involves a highly organized series of intracellular events that enable them to adapt to starvation conditions. The regulatory effect of serine threonine protein kinase, PknL, from Mycobacterium tuberculosis strain H37Rv was investigated under nutrient deprived conditions that simulate circumstances leading to latency. Recombinant PknL was expressed in Mycobacterium smegmatis strain mc2155 in its wild type and mutant forms. In vitro growth kinetics experiments revealed that clone expressing active PknL had a significant growth advantage under nutrient limiting conditions. Experiments were conducted to ascertain the in silico predictions of the involvement of PknL in regulating glutamine metabolism in mycobacteria. Furthermore, a role for PknL in cell wall biogenesis/cell division was shown by scanning electron microscopy

Molecular analysis of isoniazid-resistant clinical isolates of Mycobacterium tuberculosis from India

The presence of mutations in specific regions of katG, inhA, oxyR–ahpC and kasA associated with isoniazid (INH)-resistant clinical isolates of Mycobacterium tuberculosis from India were analysed by DNA sequencing. Point mutations in the katG gene at codon 315 and a mutation at codon 138 were detected in 64.3% (45/70) and 4% (1/25) of isolates, respectively. Polymorphisms at codon 463 of the katG gene were found both in resistant and sensitive isolates. Mutation at the inhA and oxyR–ahpC promoter regions occurred in 11.4% (8/70) and 35.0% (14/40) of the isolates, respectively. No mutation was found to occur in kasA and inhA structural gene regions. Of the 70 resistant isolates studied, 55 (78.6%) showed mutation in the regions sequenced. This is the first comprehensive molecular analysis of INH resistance in India, which suggests that point mutation rather than deletion and insertion is the major cause of INH resistance

Effect of oral exposure of mycobacterium avium intracellulare on the protective imunity induced by BCG

The relative protective efficacy of oral administration of mycobacteria as compared to the conventional intradermal route of vaccination has been assessed in guinea pigs. Skin test reactivity to partially purified protein derivative and protective immunity to challenge with virulent Mycobacterium tuberculosis were used as parameters of protective immunity. Oral immunisation of guinea pigs either with BCG or with Mycobacterium avium intracellulare induces skin test reactivity and protective immunity comparable to that induced by intradermal route of vaccination. Oral exposure of Mycobacterium avium intracellulare prior to oral or intradermal dose of BCG did not interfere with the protective immunity induced by BCG in guinea pigs challenged with Mycobacterium tuberculosis H37Rv

Diagnosing genital tuberculosis in female infertility by clinical, histopathological, culture and polymerase chain reaction techniques: an evaluative study

Background: In developing countries, the genital tract tuberculosis is one of the common causes of tubal damage leading to infertility. Objective of present study was to evaluate the efficacy of Histopathological examination (HPE), culture and Polymerase Chain Reaction Technique in diagnosing genital tuberculosis.Methods: It was a prospective evaluative study. 173 women were subjected to investigations for tuberculosis. AFB smear, culture and HPE examination and PCR testing were carried out on 173 endometrial samples, 81 POD fluid and 52 urine samples. Based on the clinical profile and laparoscopic findings a diagnostic criterion was derived to suspect GTB and the specific diagnostic tests were evaluated against this diagnostic criterion.Results: Based on the diagnostic criteria, tuberculosis was suspected in 61 of the 153 cases.   AFB smear was positive in 4.6%, culture was positive in 3.5%, HPE positive in 4.0% and PCR was positive in 28.1% of cases. On evaluating against the diagnostic criteria, the sensitivity of PCR, HPE, culture and AFB smear were; 44.3%, 8.2%, 6.6% and   6.7% respectively.  PCR was positive in 18 of the 92 cases in whom GTB was not suspected. The PCR results were negative in 34 of the 61 clinically suspected cases.Conclusions: This study has shown that HPE, AFB smear and culture have low pick up rates. PCR is found to be useful in confirming diagnosis in clinically suspected cases. False negative PCR was an important limitation in this study

CMI response of tuberculosis patients and volunteers to mitogens and mycobacterial antigens by LTT

Various mechanisms have been proposed in the past to explain the inability of the body’s cell mediated immunity (CMI) to cope with the infecting organism in myco bacterial disease such as leprosy. They include short lived suppressor cells, n-2 (IL2) defect and Prostaglandin mediated suppression1,2,3. In leprosy hanisms have been studied using the lymphocyte transformation test (LTT) to elucidate CMI in vitro. The present study was designed to study the regulation of CMI in tuberculosis patients and normal individuals with regard to induction, expression, inhibition and modulation due to prior exposure to environmental mycobacteria

Development of DNA probes for M. tuberculosis

Attempts were made to develop DNA probes for M. tuberculosis. Random library of M. tuberculosis was constructed in plasmid pGEM -4. Selection of recombinant clones was made by hybridisation with 32P labelled M. tuberculosis probe. Ten recombinant clones were selected on the basis of strong signals from the random library. These 10 clones named pTRC1-10 were subjected to tests for specificity and sensitivity. On this basis, pTRC4 was chosen and this is also, useful in restriction fragment length polymorphism (RFLP) studies

Protein kinase E of Mycobacterium tuberculosis has a role in the nitric oxide stress response and apoptosis in a human macrophage model of infection

Mycobacterium tuberculosis, an intracellular pathogen, inhibits macrophage apoptosis to support survival and replication inside the host cell. We provide evidence that the functional serine/threonine kinase, PknE, is important for survival of M. tuberculosis that enhances macrophage viability by inhibiting apoptosis.Apromoter of PknE identified in this study was shown to respond to nitric oxide stress. Deletion of pknE in virulent M. tuberculosis, H37Rv, resulted in a strain that has increased resistance to nitric oxide donors and increased sensitivity to reducing agents. The deletion mutant created by specialized transduction induced enhanced apoptosis while inhibiting necrosis. The pknE mutant also modifies the innate immune response as shown by the marked decline in the pro-inflammatory cytokines in a macrophage model of infection. These findings suggest a novel mechanism,bywhichPknEsensesnitricoxidestress and prevents apoptosis by interfering with host signalling pathways