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Title: Pyrazinamide susceptibility testing of Mycobacterium tuberculosis by high resolution melt analysis
Authors: Suporn Pholwat
Suzanne Stroup
Jean Gratz
Varittha Trangan
Suporn Foongladda
Happiness Kumburu
Saumu Pazia Juma
Gibson Kibiki
Eric Houpt
University of Virginia
Mahidol University
Kilimanjaro Clinical Research Institute
Keywords: Immunology and Microbiology;Medicine
Issue Date: 1-Jan-2014
Citation: Tuberculosis. Vol.94, No.1 (2014), 20-25
Abstract: Summary Pyrazinamide (PZA) plays the important role in shortening the tuberculosis treatment period and in treating MDR-TB. Phenotypic PZA susceptibility methods are limited because they require specialized acidified media, which increases costs and complexity. In this study we developed a genotypic high resolution melt (HRM) analysis technique to detect pncA mutations associated with PZA resistant Mycobacterium tuberculosis. Seven overlapping primer pairs were designed to cover the entire pncA gene and upstream regions. Each gene segment was individually amplified by real-time PCR followed by HRM analysis. The assay was evaluated on 98 clinical M. tuberculosis isolates (41 PZA susceptible by MGIT method, 55 PZA resistant, 2 undetermined). HRM was 94% concordant to full-length sequencing results, with most discrepancies attributable to mixed populations per HRM or transversions. Sequencing and HRM yielded 82% and 84% concordance, respectively, to phenotypic PZA susceptibilities by MGIT, with most discrepancies attributable to isolates with wild-type pncA but phenotypic PZA resistance. This HRM technique is a simple and high-throughput method for screening clinical M. tuberculosis samples for PZA resistance. © 2013 Elsevier Ltd. All rights reserved.
ISSN: 1873281X
Appears in Collections:Scopus 2011-2015

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