Pyrazinamide susceptibility testing of Mycobacterium tuberculosis by high resolution melt analysis.
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.
Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA.
Suporn
S
Pholwat
Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA.
Suzanne
S
Stroup
Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA.
Jean
J
Gratz
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Varittha
V
Trangan
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Suporn
S
Foongladda
Kilimanjaro Clinical Research Institute, Moshi, Kilimanjaro, Tanzania.
Happiness
H
Kumburu
Kilimanjaro Clinical Research Institute, Moshi, Kilimanjaro, Tanzania.
Saumu Pazia
SP
Juma
Kilimanjaro Clinical Research Institute, Moshi, Kilimanjaro, Tanzania.
Gibson
G
Kibiki
Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA. Electronic address: erh6k@virginia.edu.
Eric
E
Houpt
D000293:Adolescent; D000328:Adult; D000368:Aged; D000581:Amidohydrolases / Q000187:drug effects / Q000302:isolation & purification*; D000995:Antitubercular Agents / Q000494:pharmacology*; D002648:Child; D002675:Child, Preschool; D024901:Drug Resistance, Multiple, Bacterial; D005260:Female; D005838:Genotype; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D011159:Population Surveillance; D011718:Pyrazinamide / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction; D018088:Tuberculosis, Multidrug-Resistant / Q000188:drug therapy* / Q000235:genetics / Q000382:microbiology
D000995:Antitubercular Agents; D011718:Pyrazinamide; D000581:Amidohydrolases; C487496:PncA protein, Mycobacterium tuberculosis
Genotypic; High resolution melt; MDR-TB; Pyrazinamide