Use of multiplex allele-specific polymerase chain reaction (MAS-PCR) to detect multidrug-resistant tuberculosis in Panama.
The frequency of individual genetic mutations conferring drug resistance (DR) to Mycobacterium tuberculosis has not been studied previously in Central America, the place of origin of many immigrants to the United States. The current gold standard for detecting multidrug-resistant tuberculosis (MDR-TB) is phenotypic drug susceptibility testing (DST), which is resource-intensive and slow, leading to increased MDR-TB transmission in the community. We evaluated multiplex allele-specific polymerase chain reaction (MAS-PCR) as a rapid molecular tool to detect MDR-TB in Panama. Based on DST, 67 MDR-TB and 31 drug-sensitive clinical isolates were identified and cultured from an archived collection. Primers were designed to target five mutation hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and MAS-PCR was performed. Whole-genome sequencing confirmed DR mutations identified by MAS-PCR, and provided frequencies of genetic mutations. DNA sequencing revealed 70.1% of MDR strains to have point mutations at codon 315 of the katG gene, 19.4% within mabA-inhA promoter, and 98.5% at three hotspots within rpoB. MAS-PCR detected each of these mutations, yielding 82.8% sensitivity and 100% specificity for isoniazid resistance, and 98.4% sensitivity and 100% specificity for rifampin resistance relative to DST. The frequency of individual DR mutations among MDR strains in Panama parallels that of other TB-endemic countries. The performance of MAS-PCR suggests that it may be a relatively inexpensive and technically feasible method for rapid detection of MDR-TB in developing countries.
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
Bing-Shao
BS
Chia
Fedora
F
Lanzas
Dalin
D
Rifat
Aubrey
A
Herrera
Elizabeth Y
EY
Kim
Christine
C
Sailer
Edith
E
Torres-Chavolla
Purvaja
P
Narayanaswamy
Viktor
V
Einarsson
Jaime
J
Bravo
Juan M
JM
Pascale
Thomas R
TR
Ioerger
James C
JC
Sacchettini
Petros C
PC
Karakousis
D000483:Alleles; D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D002374:Catalase / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D025202:Molecular Diagnostic Techniques; D060885:Multiplex Polymerase Chain Reaction; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D009876:Operon; D010088:Oxidoreductases / Q000235:genetics; D010176:Panama; D017354:Point Mutation; D012293:Rifampin / Q000494:pharmacology; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis* / Q000382:microbiology; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis* / Q000382:microbiology
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D010088:Oxidoreductases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; C085257:InhA protein, Mycobacterium; D007538:Isoniazid; D012293:Rifampin