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Title: Rapid first- and second-line drug susceptibility assay for mycobacterium tuberculosis isolates by use of quantitative PCR
Authors: Suporn Pholwat
Scott Heysell
Suzanne Stroup
Suporn Foongladda
Eric Houpt
University of Virginia
Mahidol University
Keywords: Medicine
Issue Date: 1-Jan-2011
Citation: Journal of Clinical Microbiology. Vol.49, No.1 (2011), 69-75
Abstract: The slow turnaround time for Mycobacterium tuberculosis drug susceptibility results is a barrier to care. We developed a rapid quantitative PCR (qPCR)-based phenotypic antimicrobial susceptibility test that utilizes amplification of the M. tuberculosis 16S rRNA gene after 3 days of incubation with antituberculosis drugs. To decrease background from killed organisms, we used propidium monoazide (PMA), a DNA-binding dye that penetrates damaged bacterial cells and renders DNA unamplifiable. M. tuberculosis was cultured in broth media containing PMA with or without drugs for 3 days prior to DNA extraction and real-time PCR ampli-fication. 16S rRNA qPCR exhibited a significant decrease in threshold cycle (C T ) time values (C T control - C T drug treated) with drug-susceptible strains compared with resistant strains. Susceptibility data were reported as ΔCT or as 2 ΔCT and with appropriate cutoffs yielded an accuracy of 89 to 100% on 38 susceptible, multidrug-resistant, and extensively drug-resistant strains compared with conventional agar proportion susceptibility results for isoniazid, rifampin, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide, para-aminosalicylic acid, linezolid, and cycloserine and compared with Bactec MGIT results for pyrazinamide. This PMA-qPCR assay is useful as a rapid 3-day first- and second-line drug susceptibility test for M. tuberculosis. Copyright © 2011, American Society for Microbiology. All Rights Reserved.
ISSN: 1098660X
Appears in Collections:Scopus 2011-2015

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