Evaluation of a Rapid Molecular Drug-Susceptibility Test for Tuberculosis

Abstract

Background: Fluoroquinolones and second-line injectable drugs are the backbone of treatment regimens for multidrug-resistant tuberculosis, and resistance to these drugs defines extensively drug-resistant tuberculosis. We assessed the accuracy of an automated, cartridge-based molecular assay for the detection, directly from sputum specimens, of Mycobacterium tuberculosis with resistance to fluoroquinolones, aminoglycosides, and isoniazid.

Methods: We conducted a prospective diagnostic accuracy study to compare the investigational assay against phenotypic drug-susceptibility testing and DNA sequencing among adults in China and South Korea who had symptoms of tuberculosis. The Xpert MTB/RIF assay and sputum culture were performed. M. tuberculosis isolates underwent phenotypic drug-susceptibility testing and DNA sequencing of the genes katG, gyrA, gyrB, and rrs and of the eis and inhA promoter regions.

Results: Among the 308 participants who were culture-positive for M. tuberculosis, when phenotypic drug-susceptibility testing was used as the reference standard, the sensitivities of the investigational assay for detecting resistance were 83.3% for isoniazid (95% confidence interval [CI], 77.1 to 88.5), 88.4% for ofloxacin (95% CI, 80.2 to 94.1), 87.6% for moxifloxacin at a critical concentration of 0.5 μg per milliliter (95% CI, 79.0 to 93.7), 96.2% for moxifloxacin at a critical concentration of 2.0 μg per milliliter (95% CI, 87.0 to 99.5), 71.4% for kanamycin (95% CI, 56.7 to 83.4), and 70.7% for amikacin (95% CI, 54.5 to 83.9). The specificity of the assay for the detection of phenotypic resistance was 94.3% or greater for all drugs except moxifloxacin at a critical concentration of 2.0 μg per milliliter (specificity, 84.0% [95% CI, 78.9 to 88.3]). When DNA sequencing was used as the reference standard, the sensitivities of the investigational assay for detecting mutations associated with resistance were 98.1% for isoniazid (95% CI, 94.4 to 99.6), 95.8% for fluoroquinolones (95% CI, 89.6 to 98.8), 92.7% for kanamycin (95% CI, 80.1 to 98.5), and 96.8% for amikacin (95% CI, 83.3 to 99.9), and the specificity for all drugs was 99.6% (95% CI, 97.9 to 100) or greater.

Conclusions: This investigational assay accurately detected M. tuberculosis mutations associated with resistance to isoniazid, fluoroquinolones, and aminoglycosides and holds promise as a rapid point-of-care test to guide therapeutic decisions for patients with tuberculosis. (Funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and the Ministry of Science and Technology of China; ClinicalTrials.gov number, NCT02251327 .).

Figure 1. Participant Enrollment and Testing in the Main Analysis Population

Among the 12 patients who were excluded from the analysis of drug-susceptibility testing (DST) because of a lack of a reference-test result, 10 had DNA that was of insufficient quality or quantity for sequencing, and 2 had uninterpretable MGIT phenotypic DST results because of contamination. Complete DST reference-standard results were not achievable for 4.3% of participants (14 of 322) whose culture was positive for Mycobacterium tuberculosis. Among the 308 participants in the main analysis population for DST, 152 were excluded from the reflex-test analysis population (146 with an Xpert MTB/RIF result indicating that rifampin resistance was not detected, 4 with an Xpert MTB/RIF result indicating that M. tuberculosis was not detected, and 2 with an indeterminate Xpert MTB/RIF result with regard to rifampin resistance); the reflex-test analysis population therefore included 156 participants. AMK denotes amikacin, FQ fluoroquinolone, INH isoniazid, KAN kanamycin, MXF moxifloxacin, and OFL ofloxacin.

Figure 2. Sequencing Analysis of Isolates with Discrepant Investigational Assay and Phenotypic DST Results

Panel A shows the results of sequencing analysis of isolates that were found by the investigational assay to be susceptible (i.e., to have a wild-type melting temperature) and were found by phenotypic DST to be resistant. Panel B shows the results of sequencing analysis of isolates that were found by the investigational assay to be resistant (i.e., to have a mutant melting temperature detected) and were found by phenotypic DST to be susceptible. A full list of the genotypes found by DNA sequencing is provided in the Supplementary Results section in the Supplementary Appendix.