Performance of Xpert MTB/RIF RUO Assay and IS 6110 Real-Time PCR for Mycobacterium tuberculosis Detection in Clinical Samples

Abstract

The Cepheid Xpert MTB/RIF research-use-only (RUO) assay and a laboratory-developed test (LDT) targeting IS6110 were evaluated and compared to mycobacterial culture as the gold standard. The performance characteristics of both molecular assays were determined by using 112 specimens from 90 patients, including 89 pulmonary specimens and 23 extrapulmonary specimens. Of the specimens tested, 37 (33%) were culture positive for the Mycobacterium tuberculosis complex; 29 were pulmonary, and 8 were extrapulmonary. Of these culture-positive specimens, 83% of the pulmonary specimens and 50% of the extrapulmonary specimens were smear positive. There was complete concordance between the smear-positive culture-positive specimens, independent of the anatomical site (100% sensitivity). The sensitivity of the MTB/RIF RUO assay for smearnegative specimens was 60% for pulmonary and 75% for extrapulmonary specimens, while the IS6110 LDT sensitivities were 40% and 0%, respectively. There was also complete concordance among the culture-negative specimens tested. Both assays showed 95% specificity, with four culture-negative specimens testing as positive. A review of patient records indicated that there was a high likelihood of the presence of M. tuberculosis complex DNA in the false-positive specimens. Biosafety analysis was performed and showed an acceptable reduction in organism viability using the processing methods described above. Both molecular assays are suitable for the detection of M. tuberculosis isolates in smear-positive pulmonary and extrapulmonary specimens, while the sensitivity of the detection of M. tuberculosis isolates in smear-negative specimens was variable.Tuberculosis remains an important worldwide health concern, with over 9 million new cases and approximately 2 million deaths annually. Currently in the United States, there is only one FDA-approved nucleic acid amplification test (NAAT) for the Mycobacterium tuberculosis complex, the Gen-Probe Amplified Mycobacterium Tuberculosis Direct (MTD) test (San Diego, CA). The Roche Amplicor Mycobacterium tuberculosis complex (MTB) test was discontinued in 2010. A recently introduced research-use-only (RUO) NAAT for the M. tuberculosis complex is the GeneXpert MTB/RIF assay (Cepheid, Sunnydale, CA), which simultaneously detects the presence of the M. tuberculosis complex and rifampin resistance directly from respiratory specimens. The Xpert MTB/RIF assay is a semiquantitative, nested, real-time PCR designed to amplify a 192-bp segment of the M. tuberculosis complex rpoB gene using five overlapping molecular beacon probes that span the entire 81-bp rifampin-resistance-determining region. The MTB/RIF assay also contains an internal control in which the detection of lyophilized Bacillus atrophaeus subsp. globigii spores serves as an internal processing and amplification control. Previous work has demonstrated that the Xpert MTB/RIF assay displays high percentages of sensitivity and specificity for the detection of M. tuberculosis complex isolates, particularly in smear-p...

Laboratory evaluation of a specimen transport medium for downstream molecular processing of sputum samples to detect Mycobacterium tuberculosis

“…Performance was in line with generally accepted characteristics of molecular testing for M. tuberculosis: 90-100% sensitivity in microscopy smear-positive sputum specimens and 55-75% among smearnegative cases (Armand et al, 2011, Dinnes et al, 2007, Miller et al, 2011, Steingart et al, 2014. There was no apparent effect of chemical composition of PS-MTM on detection of lower bacillary loads as diagnostic performance among smear-negative cases was adequate.We did not test amplification suppression, but think that the effect, if present, was minimal as we were able to successfully amplify M. tuberculosis at relatively low CFU/ml as well as detect smear negative TB cases in the clinical laboratory evaluation.…”

Gene Xpert MTB/RIF Assay: A New Hope for Extrapulmonary Tuberculosis

“…In one previous study, Armand et al (2011) demonstrated the results from just 32 nonrespiratory samples and found an overall sensitivity of 53% that was substantially lower than that obtained using an in-house IS6110-based real-time PCR assay (78%) [27]. By contrast, the sensitivity of the Xpert assay was 75% while the IS6110 real time assay's sensitivitiy was 0% in smear negative extrapulmonary specimens [28].…”

Clinical Laboratory Diagnostics for Mycobacterium tuberculosis

“…The Xpert assay provides excellent sensitivity and specificity from direct specimens while providing a same day turn-around time for results (Helb et al, 2010;Boehme et al, 2011;Marlowe et al, 2011;Rachow et al, 2011;Scott et al, 2011). In addition, the assay provides immediate information on RIF resistance and has recently been successfully evaluated using non-respiratory specimens (Ioannidis et al, 2011;Miller et al, 2011).…”