A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis
Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a ra...
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Instituto Oswaldo Cruz, Ministério da Saúde
2016-02-01
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doaj-f2ebe14a8d034062a7eeb807dd1bbdf72020-11-24T21:35:19ZengInstituto Oswaldo Cruz, Ministério da SaúdeMemórias do Instituto Oswaldo Cruz.1678-80602016-02-0111129310010.1590/0074-02760150306S0074-02762016000200093A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosisLuz Maira Wintaco MartínezGloria Puerto CastroMartha Inírida GuerreroDeveloping a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762016000200093&lng=en&tlng=entuberculosismultidrug-resistant tuberculosisextensively drug-resistant tuberculosismolecular diagnosticmutationsSNPreverse hybridisation |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Luz Maira Wintaco Martínez Gloria Puerto Castro Martha Inírida Guerrero |
spellingShingle |
Luz Maira Wintaco Martínez Gloria Puerto Castro Martha Inírida Guerrero A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis Memórias do Instituto Oswaldo Cruz. tuberculosis multidrug-resistant tuberculosis extensively drug-resistant tuberculosis molecular diagnostic mutations SNP reverse hybridisation |
author_facet |
Luz Maira Wintaco Martínez Gloria Puerto Castro Martha Inírida Guerrero |
author_sort |
Luz Maira Wintaco Martínez |
title |
A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis |
title_short |
A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis |
title_full |
A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis |
title_fullStr |
A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis |
title_full_unstemmed |
A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis |
title_sort |
molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in colombian isolates of mycobacterium tuberculosis |
publisher |
Instituto Oswaldo Cruz, Ministério da Saúde |
series |
Memórias do Instituto Oswaldo Cruz. |
issn |
1678-8060 |
publishDate |
2016-02-01 |
description |
Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours. |
topic |
tuberculosis multidrug-resistant tuberculosis extensively drug-resistant tuberculosis molecular diagnostic mutations SNP reverse hybridisation |
url |
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762016000200093&lng=en&tlng=en |
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