title: Evaluation of the analytical performance of the Xpert MTB/RIF assay.
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.
authors.affiliation: Division of Infectious Disease, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07103, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Blakemore
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Story
authors.forename: Danica
authors.initials: D
authors.lastname: Helb
authors.forename: JoAnn
authors.initials: J
authors.lastname: Kop
authors.forename: Padmapriya
authors.initials: P
authors.lastname: Banada
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Owens
authors.forename: Soumitesh
authors.initials: S
authors.lastname: Chakravorty
authors.forename: Martin
authors.initials: M
authors.lastname: Jones
authors.forename: David
authors.initials: D
authors.lastname: Alland
mesh_terms: D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis; D012321:DNA-Directed RNA Polymerases; D016014:Linear Models; D008826:Microbial Sensitivity Tests / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results; D012293:Rifampin / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology
chemical_list: D000904:Antibiotics, Antitubercular; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin