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

INTRODUCTION: Tuberculosis 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...

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Autores principales: Takawira, Faustinos Tatenda, Mandishora, Racheal Shamiso Dube, Dhlamini, Zephaniah, Munemo, Ellen, Stray-Pedersen, Babill
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The African Field Epidemiology Network 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5567934/
https://www.ncbi.nlm.nih.gov/pubmed/28904673
http://dx.doi.org/10.11604/pamj.2017.27.145.10883
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author Takawira, Faustinos Tatenda
Mandishora, Racheal Shamiso Dube
Dhlamini, Zephaniah
Munemo, Ellen
Stray-Pedersen, Babill
author_facet Takawira, Faustinos Tatenda
Mandishora, Racheal Shamiso Dube
Dhlamini, Zephaniah
Munemo, Ellen
Stray-Pedersen, Babill
author_sort Takawira, Faustinos Tatenda
collection PubMed
description INTRODUCTION: Tuberculosis 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. METHODS: PCR 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. RESULTS: The 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. CONCLUSION: These 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.
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spelling pubmed-55679342017-09-13 Mutations in rpoB and katG genes of multidrug resistant mycobacterium tuberculosis undetectable using genotyping diagnostic methods Takawira, Faustinos Tatenda Mandishora, Racheal Shamiso Dube Dhlamini, Zephaniah Munemo, Ellen Stray-Pedersen, Babill Pan Afr Med J Research INTRODUCTION: Tuberculosis 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. METHODS: PCR 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. RESULTS: The 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. CONCLUSION: These 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. The African Field Epidemiology Network 2017-06-28 /pmc/articles/PMC5567934/ /pubmed/28904673 http://dx.doi.org/10.11604/pamj.2017.27.145.10883 Text en © Faustinos Tatenda Takawira et al. http://creativecommons.org/licenses/by/2.0/ The Pan African Medical Journal - ISSN 1937-8688. This is an Open Access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research
Takawira, Faustinos Tatenda
Mandishora, Racheal Shamiso Dube
Dhlamini, Zephaniah
Munemo, Ellen
Stray-Pedersen, Babill
Mutations in rpoB and katG genes of multidrug resistant mycobacterium tuberculosis undetectable using genotyping diagnostic methods
title Mutations in rpoB and katG genes of multidrug resistant mycobacterium tuberculosis undetectable using genotyping diagnostic methods
title_full Mutations in rpoB and katG genes of multidrug resistant mycobacterium tuberculosis undetectable using genotyping diagnostic methods
title_fullStr Mutations in rpoB and katG genes of multidrug resistant mycobacterium tuberculosis undetectable using genotyping diagnostic methods
title_full_unstemmed Mutations in rpoB and katG genes of multidrug resistant mycobacterium tuberculosis undetectable using genotyping diagnostic methods
title_short Mutations in rpoB and katG genes of multidrug resistant mycobacterium tuberculosis undetectable using genotyping diagnostic methods
title_sort mutations in rpob and katg genes of multidrug resistant mycobacterium tuberculosis undetectable using genotyping diagnostic methods
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5567934/
https://www.ncbi.nlm.nih.gov/pubmed/28904673
http://dx.doi.org/10.11604/pamj.2017.27.145.10883
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