Tuberculosis

Projects

Mechanisms of introduction, selection, maintenance and modulation of drug resistance in Mycobacterium tuberculosis through a functional genomics approach

Principal Investigator Dr. Nerges Mistry

Collaborator Prof. K. Muniyappa, Indian Institute of Science

Project team Ms. Desiree D'souza,  Ms. Monica Tolani, Dr. PurvaBhatter, Dr. Anirvan Chatterjee

Funder Department of Biotechnology, Govt. of India

Duration May 2007 - April 2012

Budget Rs.187.21 Lakhs

Background

The Wellcome Trust supported project, revealed a high proportion of MDRTB strains in both previously untreated (24%) and treatment-failure cases (41%). Amongst new cases, resistance to 3 or 4 drug combinations (amplified drug resistance) including isoniazid (H) and rifampicin (R)was greater (20%) than resistance to H and R alone (4%). The initial percentages reflect a high rate of conversion of sensitive and single drug resistant strains to amplified drug resistance even during consistent therapy.  Such results necessitate the in-depth study for deciphering the basis of the process of accumulation and maintenance of resistance in Mycobacterium tuberculosis (M.tb)

Objectives

  • Insights into homologous genetic recombination in Mycobacteria
  • Growth kinetics of Mycobacterium tuberculosis under drug pressure and their relation to compensatory mutations and in vitro virulence parameter

Key Findings

(Above: Molecular typing of M. tuberculosis strains by MIRU-VNTR)

Using pre- and post Directly Observed Treatment Schedule (DOTS) clinical Mycobacterium tuberculosis (M.tb) isolates from the same patient, the study deciphered key mechanisms indicative of rapid amplification of drug resistance. The study reported 60/182 (33%) patients who were infected with drug susceptible (DS) M.tb, but remained sputum positive at the end of a compliant DOTS, and produce MDR M.tb in the post treatment sample. Genotyping of 49/60 isolates identified 16 genotypically identical and 33 genotypically different pre and post M.tbisolates (Tolani et al., 2012). The non-identical DNA fingerprints of longitudinal isolates indicated mixed infection, or exogenous re-infection with a different strain during therapy, possibly, at health posts, where patients spend time for treatment or follow up investigations. Patients with genotypically identical longitudinal isolates, represented evolution of MDR during a single episode of therapy, and may be indicative of selection of MDR mutations under host and drug pressures. This was evinced by global transcriptional profiling of 3 of these strains, which showed increase in efflux, and reduced cellular metabolism and DNA repair in the post DOTS MDR isolate as compare to pre-DOTS DS isolate (Chatterjee et al., 2013).

The study was the first to report drug susceptibility testing and genotyping of pre- and post-DOTS M.tb isolates in India. The genetic identity of these isolates was further confirmed by Next Generation (NGS) ion-torrent whole genome sequencing (WGS) (funded by FMR), which yielded 99.99% (23 - 103 variants in 4.4 Mb genome) genetic similarity. In addition to being the first study to demonstrate transcriptional changes in longitudinal isolates, it also is the only study to perform NGS WGS of longitudinal M.tb isolates.

These genetically identical strains were tested for their fitness, measured as their ability to grow in competition with each other. It was found that the MDR strains had no loss of fitness and grew as well or even better than the progenitor DS strain (Bhatter et al., 2013). A preliminary investigation of the ability of M.tb strains to induce repair of damaged DNA in terms of the levels of recA and recX revealed that the DS strains had a much stable genome inducing lower levels of recA at a longer time post exposure to the damaging agent, whilst MDR strains induced much faster and higher levels of recA post exposure. In both strains regulation via recX induction was found to be unaffected.

Infection Control in Mumbai

Consultant Dr. Rod Escombe, Imperial College, London

Funder Corpus grants to FMR from Jamsetji Tata Trust

The findings of strain change in a large proportion of longitudinally collected samples with amplified drug resistance during therapy raised the issue of cross-infection during AKT. A likely place where this was most likely to occur was the health posts.

Based on indications of nosocomial transmission of M.tb, FMR initiated collaborative effort with Mumbai Municipal Corporation (MCGM) to install and upgrade infection control (IC) practices of health care facilities in Mumbai. Towards that FMR conducted pre-assessment of IC in primary and tertiary TB care facilities, followed by detailed suggestion for improvement of IC by Dr. Rod Escombe (Wellcome Trust Fellow) and Prof. Ed Nardell (Harvard School of Public Health). Dr. Dyson Mysquita was appointed as the IC expert by MCGM.

The presence of rapid amplification of drug resistance through non-traditional mechanisms, which can potentially undermine present and future anti-TB drug therapy, was actively communicated to MCGM, Health Secretary (Govt. of India) and local TB stakeholders (Mumbai alliance against TB). FMR convened various meetings for discussing the urgent need of rapid and repeated drug testing and for deducing appropriate drug combinations to treat MDR, XDR and XXDR.

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