title: Rapid first- and second-line drug susceptibility assay for Mycobacterium tuberculosis isolates by use of quantitative PCR.
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 amplification. 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(Δ)(CT) 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.
authors.affiliation: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Suporn
authors.initials: S
authors.lastname: Pholwat
authors.forename: Scott
authors.initials: S
authors.lastname: Heysell
authors.forename: Suzanne
authors.initials: S
authors.lastname: Stroup
authors.forename: Suporn
authors.initials: S
authors.lastname: Foongladda
authors.forename: Eric
authors.initials: E
authors.lastname: Houpt
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001386:Azides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D050296:Microbial Viability / Q000187:drug effects; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011419:Propidium / Q000031:analogs & derivatives / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013997:Time Factors; D014376:Tuberculosis / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D001386:Azides; D004791:Enzyme Inhibitors; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; C533957:propidium monoazide; D011419:Propidium