DeepMelt Assay Detects 0.01% Isoniazid-Resistant TB Mutants Through Selective Wild-Type Clamping
The DeepMelt melting curve analysis method uses selective clamping of wild-type sequences to detect isoniazid-resistant M. tuberculosis mutants at frequencies as low as 0.01% in mixed populations. Tested on 602 clinical isolates and 109 sputum specimens, it improved sensitivity from 83-94% to 92-96% compared to standard MeltPro assays while maintaining specificity, helping bridge the gap between genotypic and phenotypic drug susceptibility testing.
The original study
Highly Sensitive Detection of Isoniazid Heteroresistance in Mycobacterium tuberculosis by DeepMelt Assay.
- Authors
- Liang B, Tan Y, Li Z, Tian X, Du C, Li H, et al.
- Journal
- Journal of clinical microbiology
- Type
- Journal Article, Multicenter Study, Research Support, Non-U.S. Gov't
- PMID
- 29118176
Original abstract
Detection of heteroresistance of Mycobacterium tuberculosis remains challenging using current genotypic drug susceptibility testing methods. Here, we described a melting curve analysis-based approach, termed DeepMelt, that can detect less-abundant mutants through selective clamping of the wild type in mixed populations. The singleplex DeepMelt assay detected 0.01% katG S315T in 105M. tuberculosis genomes/μl. The multiplex DeepMelt TB/INH detected 1% of mutant species in the four loci associated with isoniazid resistance in 104M. tuberculosis genomes/μl. The DeepMelt TB/INH assay was tested on a panel of DNA extracted from 602 precharacterized clinical isolates. Using the 1% proportion method as the gold standard, the sensitivity was found to be increased from 93.6% (176/188, 95% confidence interval [CI] = 89.2 to 96.3%) to 95.7% (180/188, 95% CI = 91.8 to 97.8%) compared to the MeltPro TB/INH assay. Further evaluation of 109 smear-positive sputum specimens increased the sensitivity from 83.3% (20/24, 95% CI = 64.2 to 93.3%) to 91.7% (22/24, 95% CI = 74.2 to 97.7%). In both cases, the specificity remained nearly unchanged. All heteroresistant samples newly identified by the DeepMelt TB/INH assay were confirmed by DNA sequencing and even partially by digital PCR. The DeepMelt assay may fill the gap between current genotypic and phenotypic drug susceptibility testing for detecting drug-resistant tuberculosis patients.