title: Detection of Mycobacterium tuberculosis directly from sputum specimens & phenotypic drug resistance pattern of M. tuberculosis isolates from suspected tuberculosis patients in Chennai.
abstract: OBJECTIVE
  mRNA is more rapidly destroyed in cells than rRNA or genomic DNA, an assay targeting bacterial mRNA would provide a better guide to mycobacterial viability than amplification tests directed at DNA or rRNA targets. This study was carried out to standardize reverse transcriptase PCR (RT-PCR) targeting 85B gene for the rapid detection of viable Mycobacterium tuberculosis from sputum specimens of suspected TB patients at Chennai, South India and to detect MDR-TB circulating in this population.
  
  
  METHODS
  Sputum samples from clinically suspected tuberculosis patients (n=301) and 78 controls were included in the study. The sputum samples were collected in sterile diethyl pyrocarbonate (DEPC) treated containers and transported in ice to the laboratory within 2 h to prevent degradation of RNA. RT-PCR targeting 85B gene, mycobacterial culture and phenotypic drug susceptibility testing for the first line drugs streptomycin (S), isoniazid (H), rifampicin (R), ethambutol (E) and pyrazinamide (Z) were performed by BACTEC microMGIT culture system for all the sputum specimens.
  
  
  RESULTS
  All the 78 controls were negative for culture and RT-PCR. Among the 301 sputum specimens from patients, 231 (76.8%) were RT-PCR positive and 70 (23.2%) were negative. There were 166 M. tuberculosis isolates, of which 11 (2.9%) were MDR-TB, 33 (8.7%) were polyresistant, 31 (8.2%) were monoresistant and 91 (30.2%) were sensitive to all five first line anti-tuberculous drugs by phenotypic drug susceptibility testing. Monoresistance was higher with Z [20 (20.8%)], followed by S [6 (3%)].
  
  
  CONCLUSIONS
  RT-PCR targeting 85B gene of M. tuberculosis was a specific, rapid, reliable technique to detect the M. tuberculosis directly from sputum specimens. Our results showed that 2.9 per cent of M. tuberculosis isolates in the study population of Chennai were MDR.
authors: L&T Microbiology Research Centre, Vision Research Foundation, Chennai, India. drklt@snmail.org
authors: K Lily
authors: KL
authors: Therese
authors: R
authors: R
authors: Gayathri
authors: L
authors: L
authors: Dhanurekha
authors: R
authors: R
authors: Sridhar
authors: N
authors: N
authors: Meenakshi
authors: H N
authors: HN
authors: Madhavan
authors: S Edwin
authors: SE
authors: Manoj
authors: A Kamala
authors: AK
authors: Vinayagam
mesh_terms: D000217:Acyltransferases / Q000302:isolation & purification; D000942:Antigens, Bacterial / Q000302:isolation & purification; D000995:Antitubercular Agents / Q000627:therapeutic use; D001426:Bacterial Proteins / Q000302:isolation & purification; D006801:Humans; D007194:India; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000302:isolation & purification* / Q000472:pathogenicity; D012333:RNA, Messenger / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013183:Sputum / Q000382:microbiology*; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis / Q000382:microbiology; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis / Q000382:microbiology
chemical_list: D000942:Antigens, Bacterial; D000995:Antitubercular Agents; D001426:Bacterial Proteins; D012333:RNA, Messenger; D000217:Acyltransferases; C121433:antigen 85B, Mycobacterium tuberculosis