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. 2014 Jan;94(1):20-5.
doi: 10.1016/j.tube.2013.10.006. Epub 2013 Nov 1.

Pyrazinamide susceptibility testing of Mycobacterium tuberculosis by high resolution melt analysis

Suporn Pholwat  1 , Suzanne Stroup  1 , Jean Gratz  1 , Varittha Trangan  2 , Suporn Foongladda  2 , Happiness Kumburu  3 , Saumu Pazia Juma  3 , Gibson Kibiki  3 , Eric Houpt  4
Affiliations

Pyrazinamide susceptibility testing of Mycobacterium tuberculosis by high resolution melt analysis

Suporn Pholwat et al. Tuberculosis (Edinb). 2014 Jan.

Abstract

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.

Keywords: Genotypic; High resolution melt; MDR-TB; Pyrazinamide.

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Conflict of interest statement

COMPETING INTERESTS: None.

Figures

FIG. 1
FIG. 1
High resolution melt analysis of the entire pncA gene and upstream regions. Normalized (left panel) and difference graphs (right panel) of 7 amplicons are shown: pncA1 (A), pncA2 (B), pncA3(C), pncA4 (D), pncA5 (E), pncA6 (F), and pncA7 (G). Each line indicates the melt curve profile for an individual sample. In the difference plot, the melt curve profile of M. tuberculosis H37Rv was compared with the curve profiles of all other samples. The baseline represents M. tuberculosis H37Rv and other wild-type isolates. Isolates with “Variation” or mutant profiles, as determined by HRM software as <70% similar to wild-type, are shown.
FIG. 2
FIG. 2
Discrepancies by sequencing and HRM. HRM showed variation for 4 samples whose sequencing revealed mixed population of sequences (arrows).
See this image and copyright information in PMC

Comment in

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