Genotyping of mutations detected with GeneXpert

Abstract

Objective/background: Tuberculosis remains an important cause of mortality worldwide. Previous tuberculosis treatment is a strong determinant of multi-drug resistant tuberculosis. The study objective was to describe the mutations detected of Mycobacterium tuberculosis (MTB) complex clinical strains screened with GeneXpert isolated from previously treated patients in Côte d'Ivoire. Methods: Sputum collected and decontaminated by the n-acetyl-l-cysteine method was used to perform Ziehl–Neelsen staining, GeneXpert MTB/rifampicin, and culture on Lowenstein–Jensen medium. Drug susceptibility testing (DST) for first-line drugs was performed in a Bactec 960 Automated System. After strain identification by antigen MPT64 detection, DNA extraction, and genotyping with MTBDRplus assay was performed and interpreted. The strains muted in rpoB without a specific protein identified and were sequenced. Results: Mutant sequences were detected in 60 sputum samples with GeneXpert MTB/rifampicin of which 55 were confirmed multi-drug resistant MTB strains after DST. The most frequent mutations responsible for rifampin resistance were detected with MTBDRplus assay for 49 (81.7%) clinical strains, while sequencing was required for 11 (18.3%). H526Q mutation, L533P, and D516V associated respectively with L533P, A532A, and S522L, and were observed for three relapse cases. For these cases, GeneXpert and sequencing results were concordant. Discrepancies between GeneXpert and mycobacteria growth indicator tube-DST for rifampin were observed for three strains, on which D516Y, H526C, and L533P were identified. Conclusion: In the setting of a high prevalence of drug resistance, characterization of the genetic basis of MTB strains resistant to rifampin could be screened first with MTBDRplus.

Keywords

• Molecular assays
• Resistance
• Rifampin
• Tuberculosis