Affiliation:
1. Department of Tuberculosis, Beijing Chest Hospital, Capital Medical University, Beijing, China
2. Centre for Infectious Diseases and Microbiology-Public Health (CIDM-PH), Institute of Clinical Pathology and Medical Research (ICPMR), Westmead, New South Wales, Australia
3. Sydney Institute for Emerging Infectious Diseases and Biosecurity, University of Sydney, Sydney, NSW 2006, Australia
Abstract
ABSTRACT
A high-resolution melting analysis (HRMA) assay was developed to detect isoniazid, rifampin, and ofloxacin resistance in
Mycobacterium tuberculosis
by targeting resistance-associated mutations in the
katG
,
mabA-inhA
promoter,
rpoB
, and
gyrA
genes. A set of 28 (17 drug-resistant and 11 fully susceptible) clinical
M. tuberculosis
isolates was selected for development and evaluation of HRMA. PCR amplicons from the
katG
,
mabA-inhA
promoter,
rpoB
, and
gyrA
genes of all 28 isolates were sequenced. HRMA results matched well with 18 mutations, identified by sequencing, in 17 drug-resistant isolates and the absence of mutations in 11 susceptible isolates. Among 87 additional isolates with known resistance phenotypes, HRMA identified
katG
and/or
mabA-inhA
promoter mutations in 66 of 69 (95.7%) isoniazid-resistant isolates,
rpoB
mutations in 51 of 54 (94.4%) rifampin-resistant isolates, and
gyrA
mutations in all of 41 (100%) ofloxacin-resistant isolates. All mutations within the HRMA primer target regions were detected as variant HRMA profiles. The corresponding specificities were 97.8%, 100%, and 98.6%, respectively. Most false-positive results were due to synonymous mutations, which did not affect susceptibility. HRMA is a rapid, sensitive method for detection of drug resistance in
M. tuberculosis
which could be used routinely for screening isolates in countries with a high prevalence of tuberculosis and drug resistance or in individual isolates when drug resistance is suspected.
Publisher
American Society for Microbiology
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