Performance of TaqMan array card to detect TB drug resistance on direct specimens

Abstract

Culture based phenotypic drug susceptibility testing (DST) for Mycobacterium tuberculosis (TB) is time consuming therefore rapid genotypic methods are increasingly being utilized. We previously developed and evaluated on TB isolates a rapid genotypic TaqMan array card (TAC) that detects mutations in several resistance-associated genes using dozens of primer pairs, probes, and high resolution melt analysis, with >96% accuracy versus Sanger sequencing. In this study we examined the performance of TAC on sputum, comparing results between 71 paired sputum and TB isolates of which 62 were MDR-TB. We also adapted the TAC to include wild-type probes and broadened coverage for rpoB and gyrA mutations. TAC was 89% successful at detecting wild-type or mutations within inhA, katG, rpoB, eis, gyrA, rplC, and pncA on smear positive sputa and 33% successful on smear negative sputa. The overall accuracy of these detections as compared to the TAC results of the paired isolate was 95% ± 7 (average sensitivity 98% ± 3; specificity 92% ± 14). Accuracy of sputum TAC results versus phenotypic DST for isoniazid, rifampin, ofloxacin/moxifloxacin, and pyrazinamide was 85% ± 12. This was similar to that of the isolate TAC results (accuracy 88% ± 13), thus inaccuracies primarily reflected intrinsic genotypic-phenotypic discordance. The TAC is a rapid, modular, comprehensive, and accurate TB DST for the major first and second line TB drugs and could be used for supplemental testing of GeneXpert resistant smear positive sputum.

Fig 1. TB drug resistance TAC version 2.

TAC tests 8 samples and compartmentalizes 48 assays per sample. Assays are grouped according to drug isoniazid (INH), rifampin (RIF), amikacin (AMK), kanamycin (KAN), ofloxacin (OFX), moxifloxacin (MXF), linezolid (LZD), and pyrazinamide (PZA). Each assay is shown on the basis of gene nucleotide or codon. Wt = wild-type. A/B indicates duplex assays.

Fig 2. Amplification Ct cut-off.

For each of 71 sputum samples the average SYTO9 Ct value for each of the 11 amplicons is shown. Average Ct are organized according to whether the sputum probe result was wild-type (black -), mutant (black x), or negative. Sputum probe negative results are further stratified into whether the cultured isolate was found to be wild-type (red +), mutant (red -), or to possess an “other” mutation (green X). ROC analysis was performed to examine the optimal Ct whereby SYTO9 to interpret the probe results.