Simplified Microarray System for Simultaneously Detecting Rifampin,Isoniazid, Ethambutol,and Streptomycin Resistance Markers in Mycobacterium tuberculosis

We developed a simplified microarray test for detecting and identifying mutations in rpoB, katG, inhA, embB, and rpsL and compared the analytical performance of the test to that of phenotypic drug susceptibility testing (DST). The analytical sensitivity was estimated to be at least 110 genome copies per amplification reaction. The microarray test correctly detected 95.2% of mutations for which there was a sequence-specific probe on the microarray and 100% of 96 wild-type sequences. In a blinded analysis of 153 clinical isolates, microarray sensitivity for first-line drugs relative to phenotypic DST (true resistance) was 100% for rifampin (RIF) (14/14), 90.0% for isoniazid (INH) (36/40), 70% for ethambutol (EMB) (7/10), and 89.1% (57/64) combined. Microarray specificity (true susceptibility) for first-line agents was 95.0% for RIF (132/139), 98.2% for INH (111/113), and 98.6% for EMB (141/143). Overall microarray specificity for RIF, INH, and EMB combined was 97.2% (384/395). The overall positive and negative predictive values for RIF, INH, and EMB combined were 84.9% and 98.3%, respectively. For the second-line drug streptomycin (STR), overall concordance between the agar proportion method and microarray analysis was 89.5% (137/153). Sensitivity was 34.8% (8/23) because of limited microarray coverage for STR-conferring mutations, and specificity was 99.2% (129/130). All false-susceptible discrepant results were a consequence of DNA mutations that are not represented by a specific microarray probe. There were zero invalid results from 220 total tests. The simplified microarray system is suitable for detecting resistance-conferring mutations in clinical M. tuberculosis isolates and can now be used for prospective trials or integrated into an all-in-one, closed-amplicon consumable.