Helb D, Jones M, Story E, Boehme C, Wallace E, Ho K, Kop J, Owens MR, Rodgers R, Banada P, Safi H, Blakemore R, Lan NT, Jones-López EC, Levi M, Burday M, Ayakaka I, Mugerwa RD, McMillan B, Winn-Deen E, Christel L, Dailey P, Perkins MD, Persing DH, Alland D
Rapid detection of Mycobacterium tuberculosis and rifampin-resistance using on-demand, near patient technology. [JOURNAL ARTICLE]
J Clin Microbiol 2009 Oct 28.
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 Xpert 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 colony forming units (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 non-tuberculosis 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 22/39 (84.6%) or 38/53 (71.7%) smear-negative culture-positive cases, as determined by growth on solid media or 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 under two hours.
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