NITRATE REDUCTASE ASSAY FOR SUSCEPTIBILITY TESTING TO SECOND-LINE ANTI-TUBERCULOUS DRUGS WITH DETECTION OF GYRAD94G AND RRS A1401G MUTATIONS

Abstract

Introduction: The rising number of multidrug-resistant Mycobacterium tuberculosis (MDR M. tuberculosis )strains and the emergence of extensively drug-resistant (XDR) strains substantiate the urgent demand for rapid and reliable techniques for susceptibility testing in M. tuberculosis. Phenotypic determination has been a common practice for years, whereas more recently the genetic detection of mutations that confer antituberculous drug resistance has been established. Aim of the Work: This study aimed to evaluate direct and indirect Nitrate Reductase Assay (NRA) for susceptibility testing of MDR M. tuberculosis to second-line anti-tuberculous drugs compared to the gold standard indirect proportional method (PM) and to assess the performance of multiplex allele-specific polymerase chain reaction (MAS-PCR) assays targeting GyrA D94G and rrs A1401G mutations for the identification of resistance to ofloxacin and kanamycin. Patients and Methods: Susceptibility of MDR M. tuberculosis was evaluated against 5 second-line antituberculous drugs: Ethionamide (ETH), kanamycin monosulfate (KAN), cycloserine (CYL), ofloxacin (OFX), and para-aminosalicylic acid (PAS) by NRA. The test was performed directly on 25 sputum samples from MDR tuberculosis (TB) patients and indirectly on 40 MDR M. tuberculosis isolates generously supplied by Reference TB laboratory - Central laboratories - Egyptian Ministry of Health. GyrA D94G and rrs A1401G MAS-PCR assays were carried out on a total of 36 isolates. The results were compared to those obtained by indirect PM. Results: For PAS, ETH OFX, KAN and CYL, the agreement of direct NRA, with indirect PM, was found to be 94.7%, 89.4%, 94.7%, 100% and 94.7% and for indirect NRA the agreement was found to be 100%, 90.9%, 97.7%, 93.1% and 95.4%, respectively. MAS-PCR assays indicated clinical sensitivity of 77.7% and 90.9% for the GyrA D94G and rrs A1401G assays, respectively and specificity of 100% for both assays for detection of OFX and KAN resistance. Conclusion: Indirect NRA showed higher agreement with standard indirect PM than direct NRA for susceptibility testing of MDR M. tuberculosis to second-line anti-tuberculous drugs with preserving the advantage of shorter turnaround time. The simplicity and cost-effectiveness, of the NRA, make it suitable for large scale surveillance studies in resource-limited settings. MAS-PCR molecular assays may not replace phenotypic drug susceptibility testing, however they may provide a reliable method for predicting aminoglycoside and fluoroquinolone resistance, with inclusion of a wider range of targeted mutations.