Rapid detection of Mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology

J Clin Microbiol. 2010 Jan;48(1):229-37. doi: 10.1128/JCM.01463-09. Epub 2009 Oct 28.

Abstract

Current nucleic acid amplification methods to detect Mycobacterium tuberculosis are complex, labor-intensive, and technically challenging. We developed and performed the first analysis of the Cepheid Gene Xpert System's MTB/RIF assay, an integrated hands-free sputum-processing and real-time PCR system with rapid on-demand, near-patient technology, to simultaneously detect M. tuberculosis and rifampin resistance. Analytic tests of M. tuberculosis DNA demonstrated a limit of detection (LOD) of 4.5 genomes per reaction. Studies using sputum spiked with known numbers of M. tuberculosis CFU predicted a clinical LOD of 131 CFU/ml. Killing studies showed that the assay's buffer decreased M. tuberculosis viability by at least 8 logs, substantially reducing biohazards. Tests of 23 different commonly occurring rifampin resistance mutations demonstrated that all 23 (100%) would be identified as rifampin resistant. An analysis of 20 nontuberculosis mycobacteria species confirmed high assay specificity. A small clinical validation study of 107 clinical sputum samples from suspected tuberculosis cases in Vietnam detected 29/29 (100%) smear-positive culture-positive cases and 33/39 (84.6%) or 38/53 (71.7%) smear-negative culture-positive cases, as determined by growth on solid medium or on both solid and liquid media, respectively. M. tuberculosis was not detected in 25/25 (100%) of the culture-negative samples. A study of 64 smear-positive culture-positive sputa from retreatment tuberculosis cases in Uganda detected 63/64 (98.4%) culture-positive cases and 9/9 (100%) cases of rifampin resistance. Rifampin resistance was excluded in 54/55 (98.2%) susceptible cases. Specificity rose to 100% after correcting for a conventional susceptibility test error. In conclusion, this highly sensitive and simple-to-use system can detect M. tuberculosis directly from sputum in less than 2 h.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Antitubercular Agents / pharmacology*
  • Bacteriological Techniques / methods*
  • Drug Resistance, Bacterial*
  • Female
  • Humans
  • Male
  • Middle Aged
  • Mycobacterium tuberculosis / drug effects*
  • Mycobacterium tuberculosis / isolation & purification
  • Point-of-Care Systems*
  • Polymerase Chain Reaction / methods
  • Rifampin / pharmacology*
  • Sensitivity and Specificity
  • Sputum / microbiology
  • Tuberculosis / diagnosis*
  • Tuberculosis / microbiology
  • Uganda
  • Vietnam
  • Young Adult

Substances

  • Antitubercular Agents
  • Rifampin