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

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.

Molecular analysis of Mycobacterium tuberculosis causing multidrug-resistant tuberculosis meningitis.

SETTING: Tertiary referral hospitals in southern Vietnam. OBJECTIVE: Molecular characterisation of multidrug-resistant (MDR) tuberculous meningitis (TBM). DESIGN: Mycobacterium tuberculosis isolates from the cerebrospinal fluid (CSF) of 198 Vietnamese adults were compared with 237 isolates from patients with pulmonary tuberculosis (PTB) matched for age, sex and residential district. Isolates resistant to isoniazid or rifampicin (RMP) were sequenced in the rpoB and katG genes, inhA promoter and oxyR-ahpC intergenic regions. RESULTS: While drug resistance rates were lower in the CSF (2.5% MDR) than pulmonary isolates (5.9% MDR), the difference was not significant. The most commonly mutated codons were 531, 526 and 516 in rpoB and 315 in katG. Four novel triple mutants in rpoB were identified. CONCLUSION: RMP resistance is a good surrogate marker for MDR-TBM in this setting. However, probes directed against these three codons would have a maximum sensitivity of only 65%. A rapid phenotypic detection test may be more applicable for the diagnosis of MDR-TBM.