Direct resazurin microplate assay in drug susceptibility testing of smear-positive sputum samples against mycobacterium tuberculosis

Abstract

Background: A new direct microplate-based colorimetric drug susceptibility test that omits the initial isolation of Mycobacterium tuberculosis from sputum specimens was evaluated. Methods: A total of 51 M. tuberculosis acid fast bacilli (AFB) smear-positive sputum specimens were inoculated directly into drug-free and serial dilutions of drug-containing Middlebrook 7H9 broth media. With this direct resazurin micro plate assay (REMA) method, resazurin dye was used as a growth indicator in microplate wells. The minimum inhibitory concentrations (MIC) of isoniazid (INH) and rifampicin (RIF) were compared with those of the ‘gold standard’ absolute concentration method (ACM). The turnaround time (TAT) of the direct REMA and the ACM were also determined. Results: At the selected cut-off points (INH: 0.0625 µg/mL; RIF: 0.125 µg/mL), good drug susceptibility test results were obtained for INH and RIF with an average sensitivity, specificity and accuracy of 90%, 100% and 97%, respectively, with a TAT of 15 days. The REMA method also correctly classified the resistant isolates with positive predictive values of 95% and negative predictive values of 98% for the two drugs. Conclusions: The direct REMA was reliable in routine diagnostic laboratories for the drug susceptibility testing of M. tuberculosis and the rapid detection of multi-drug-resistant tuberculosis.