Mutations in rpoB and katG genes of multidrug resistant mycobacterium tuberculosis undetectable using genotyping diagnostic methods

Abstract

IntroductionTuberculosis remains the leading causes of death worldwide with frequencies of mutations in rifampicin and isoniazid resistant Mycobacterium tuberculosis isolates varying according to geographical location. There is limited information in Zimbabwe on specific antibiotic resistance gene mutation patterns in MTB and hence, increased rate of discordant results and mortality due to inappropriate antibiotic prescriptions. The rpoB and katG genes molecular markers are used for detecting rifampicin and isoniazid resistance respectively. Some mutations within these gene sequences are associated with drug resistance as they directly alter gene function. The objectives of this research was to determine the drug resistance profiles in M. tuberculosis isolates that are phenotypically resistant but not detected by the GeneXpert and MTBDRplus kit and also to detect mutations in the rpoB and katG genes which are not detected by the Hain Genotype MTBDRplus kit and GeneXpert diagnosis.MethodsPCR was used for the amplification of the rpoB and katG genes from MTB isolates collected from human clinical samples between 2008 and 2015. The genes were sequenced and compared to the wild type MTB H37Rv rpoB (accession number L27989) and kat G genes (KP46920), respectively. Sequence analysis results were compared to genotyping results obtained from molecular assays and culture results of all isolates.ResultsThe most frequent mutation responsible for rifampicin resistance was (25/92) S531L that was detected by using all molecular assays. Some inconsistencies were observed between phenotypic and genotypic assay results for both katG and rpoB genes in 30 strains. For these, eight codons; G507S, T508A, L511V, del513-526, P520P, L524L, R528H, R529Q and S531F were novel mutations. In addition, the I572P/F, E562Q, P564S, and Q490Y mutations were identified as novel mutations outside the rifampicin resistance determining region. In katG gene, amino acid changes to threonine, asparagine and isoleucine exhibited high degrees of polymorphism such as V473N, D311N, and L427I. The R463L (20/92) amino acid substitution was most common but was not associated with isoniazid resistance.ConclusionThese finding indicate that molecular assay kit diagnosis that is based on the rpoB and katG genes should be improved to cater for the genetic variations associated with the geographic specificity of the target genes and be able to detect most prevalent mutations in different areas.

Rifampicin resistance patterns and dynamics of tuberculosis and drug-resistant tuberculosis in Enugu, South Eastern Nigeria

“…suggested that variations may exist in different ethnic groups and geographic locations [24], most reports have shown mutations in codon 531 to be the most frequent, leading to the conclusion that codon 531 is the most prevalent region associated with rifampicin resistance [24][25][26][27][28][29][30]. Given the observation in this study, however, there may be need to carry out further investigation into the geographical variations.…”

Occurrence of mutations associated with rifampicin and isoniazid resistant in Mycobacterium tuberculosis isolates from patients in Burkina Faso

“…39,40 To cater the geographic specificity of the target genes and be able to detect more possible mutations in different geographical areas, it is necessary to improve molecular assay kit diagnosis. 41,42…”

Classification of Mycobacterium tuberculosis DR, MDR, XDR Isolates and Identification of Signature Mutation Pattern of Drug Resistance

“…This evidence of association between codon variation and the resistant strains can be used further in targeted mutation screening for identification of drug resistance and non-drug resistant regions can be new targets for drug discovery process. The demonstrations of codon variation in hotspot region and also outside resistant determining region will have implications in diagnostics of TB and drug development process 23.…”