title: Rapid detection of multidrug-resistant Mycobacterium tuberculosis by use of real-time PCR and high-resolution melt analysis.
abstract: The current study describes the development of a unique real-time PCR assay for the detection of mutations conferring drug resistance in Mycobacterium tuberculosis. The rifampicin resistance determinant region (RRDR) of rpoB and specific regions of katG and the inhA promoter were targeted for the detection of rifampin (RIF) and isoniazid (INH) resistance, respectively. Additionally, this assay was multiplexed to discriminate Mycobacterium tuberculosis complex (MTC) strains from nontuberculous Mycobacteria (NTM) strains by targeting the IS6110 insertion element. High-resolution melting (HRM) analysis following real-time PCR was used to identify M. tuberculosis strains containing mutations at the targeted loci, and locked nucleic acid (LNA) probes were used to enhance the detection of strains containing specific single-nucleotide polymorphism (SNP) transversion mutations. This method was used to screen 252 M. tuberculosis clinical isolates, including 154 RIF-resistant strains and 174 INH-resistant strains based on the agar proportion method of drug susceptibility testing (DST). Of the 154 RIF-resistant strains, 148 were also resistant to INH and therefore classified as multidrug resistant (MDR). The assay demonstrated sensitivity and specificity of 91% and 98%, respectively, for the detection of RIF resistance and 87% and 100% for the detection of INH resistance. Overall, this assay showed a sensitivity of 85% and a specificity of 98% for the detection of MDR strains. This method provides a rapid, robust, and inexpensive way to detect the dominant mutations known to confer MDR in M. tuberculosis strains and offers several advantages over current molecular and culture-based techniques.
authors.affiliation: Division of Tuberculosis Elimination, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.
authors.forename: Melissa V
authors.initials: MV
authors.lastname: Ramirez
authors.forename: Kelley C
authors.initials: KC
authors.lastname: Cowart
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: Campbell
authors.forename: Glenn P
authors.initials: GP
authors.lastname: Morlock
authors.forename: David
authors.initials: D
authors.lastname: Sikes
authors.forename: Jonas M
authors.initials: JM
authors.lastname: Winchell
authors.forename: James E
authors.initials: JE
authors.lastname: Posey
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D002374:Catalase / Q000235:genetics; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial; D005798:Genes, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D010088:Oxidoreductases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis*
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D010088:Oxidoreductases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; C085257:InhA protein, Mycobacterium; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit
references.citation: J Clin Microbiol. 2008 Apr;46(4):1369-73
references.pmid: 18305131
references.citation: J Clin Microbiol. 2008 Nov;46(11):3660-7
references.pmid: 18784319
references.citation: J Antimicrob Chemother. 2009 Jun;63(6):1121-7
references.pmid: 19369271
references.citation: Tuber Lung Dis. 1998;79(1):3-29
references.pmid: 10645439
references.citation: Genet Sel Evol. 2002 May-Jun;34(3):275-305
references.pmid: 12081799
references.citation: J Clin Microbiol. 1990 Sep;28(9):1913-7
references.pmid: 2229372
references.citation: Clin Microbiol Rev. 1995 Oct;8(4):496-514
references.pmid: 8665467
references.citation: J Clin Microbiol. 1994 Apr;32(4):1095-8
references.pmid: 8027320
references.citation: J Clin Microbiol. 2010 Apr;48(4):1047-54
references.pmid: 20164280
references.citation: J Clin Microbiol. 2009 Dec;47(12):3985-90
references.pmid: 19846642
references.citation: Toxicol Appl Pharmacol. 2009 Mar 1;235(2):163-70
references.pmid: 19063910
references.citation: Annu Rev Microbiol. 2007;61:35-50
references.pmid: 18035606
references.citation: Tuberculosis (Edinb). 2008 Aug;88 Suppl 1:S93-100
references.pmid: 18762157
references.citation: Clin Chem. 2004 Jul;50(7):1156-64
references.pmid: 15229148
references.citation: Int J Tuberc Lung Dis. 2000 Feb;4(2 Suppl 1):S4-10
references.pmid: 10688142
references.citation: MMWR Recomm Rep. 2003 Jun 20;52(RR-11):1-77
references.pmid: 12836625