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

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.

FIG. 1.

Limit of detection for the in-cartridge heminested PCR. (A) DNA detection. M. tuberculosis DNA at final concentrations of 0, 1, 1.5, 2.5, 3, or 7.5 genomes per PCR was loaded into cartridges and processed according to the Xpert MTB/RIF protocol. For each genomic concentration tested (n = 5 to 7), the percentage of M. tuberculosis-positive cartridges was plotted. As determined by logistic regression, there is a 95% probability of detecting M. tuberculosis in samples containing at least 4.5 genomes per PCR (95% CI, 3.3 to 9.7). (B) Detection of M. tuberculosis cells in clinical sputum samples. M. tuberculosis cells were added to 1 ml of M. tuberculosis-negative sputum to final concentrations of 10, 50, 100, 150, or 300 CFU/ml (n = 20) and then processed according to the Xpert MTB/RIF protocol. The percentage of assays where M. tuberculosis was detected was then plotted for each concentration of cells. As determined by logistic regression, there was a 95% probability of detecting M. tuberculosis in samples containing at least 131 CFU/ml (95% CI, 106.2 to 176.4).

FIG. 2.

Detection of RRDR mutations. Genomic M. tuberculosis DNA or artificial targets containing clinically relevant mutations were added to the wash buffer of cartridges that were then run with M. tuberculosis-negative sputum. Typical results from six rifampin-susceptible (wild-type RRDR) isolates and 23 RRDR mutants are shown. The results produced by each sample are indicated by a single vertical line on which the C_T of each of the five rpoB-specific molecular beacons (probes A to E) is plotted. ΔC_T values ≥3.5 cycles indicate rifampin resistance. All 23 RRDR mutants were correctly identified as rifampin resistant.

FIG. 3.

Effect of sample reagent. (A) Biohazard reduction. Sputum samples spiked with high concentrations of M. tuberculosis cells were treated with 2 volumes of SR and incubated for 15 min to 5 days (n = 3). M. tuberculosis cells remaining viable after SR treatment were measured by quantitative culture. The log decrease in M. tuberculosis viability, compared to an untreated control, was plotted for each incubation time tested. (B) Assay performance. Sputum spiked with 150 CFU/ml of M. tuberculosis cells was treated with 2 volumes of SR and incubated for 15 min to 5 days and then processed in the Xpert MTB/RIF assay. Average C_T values were plotted for each incubation time (n = 3 to 4).

FIG. 4.

Assay performance with nontuberculosis mycobacteria. (A) Lack of cross-reaction by NTM. A total of 10⁶ CFU/ml of relevant NTM species was added into M. tuberculosis-negative sputum and processed according to the Xpert MTB/RIF protocol. The results produced by each species are indicated by a single vertical line on which the C_T of each of the five rpoB-specific molecular beacons (probes A to E) is plotted. For tuberculosis detection, the GeneXpert software requires a sample to have at least two positive molecular beacon probes with a ΔC_T of <2 cycles. In the example shown, the assay correctly identified members of the M. tuberculosis complex (M. tuberculosis, M. bovis BCG, and Mycobacterium africanum) as M. tuberculosis positive. However, none of the NTM samples produced ΔC_Ts that fulfilled the criteria for M. tuberculosis detection. (B) Lack of interference by NTM. Possible interactions between low concentrations of M. tuberculosis and high concentrations of NTM species were investigated. Sputum containing both 200 CFU/ml of M. tuberculosis and 10⁶ CFU/ml of an NTM species was processed according to the Xpert MTB/RIF protocol, and cycle thresholds for all five rpoB probes were plotted. M. tuberculosis was accurately identified in all of these samples. However, the combination of high levels of M. malmoense with low concentrations of M. tuberculosis resulted in a false rifampin resistance result. Rifampin resistance was not seen when 200 CFU/ml of H37Rv was tested in combination with 10⁵ CFU/ml of M. malmoense or when sputum contained 300 CFU/ml of H37Rv and 10⁶ CFU/ml of M. malmoense (data not shown).