Abstract
We performed the first studies of analytic sensitivity, analytic specificity, and dynamic range for the new Xpert MTB/RIF assay, a nucleic acid amplification-based diagnostic system that detects
Mycobacterium tuberculosis
and rifampin (RIF) resistance in under 2 h. The sensitivity of the assay was tested with 79 phylogenetically and geographically diverse
M. tuberculosis
isolates, including 42 drug-susceptible isolates and 37 RIF-resistant isolates containing 13 different
rpoB
mutations or mutation combinations. The specificity of the assay was tested with 89 nontuberculosis bacteria, fungi, and viruses. The Xpert MTB/RIF assay correctly identified all 79
M. tuberculosis
isolates and correctly excluded all 89 nontuberculosis isolates. RIF resistance was correctly identified in all 37 resistant isolates and in none of the 42 susceptible isolates. Dynamic range was assessed by adding 10
2
to 10
7
CFU of
M. tuberculosis
into
M. tuberculosis
-negative sputum samples. The assay showed a log-linear relationship between cycle threshold and input CFU over the entire concentration range. Resistance detection in the presence of different mixtures of RIF-resistant and RIF-susceptible DNA was assessed. Resistance detection was dependent on the particular mutation and required between 65% and 100% mutant DNA to be present in the sample for 95% certainty of resistance detection. Finally, we studied whether assay specificity could be affected by cross-contaminating amplicons generated by the GenoType MTBDRplus assay.
M. tuberculosis
was not detected until at least 10
8
copies of an MTBDRplus amplicon were spiked into 1 ml of sputum, suggesting that false-positive results would be unlikely to occur.