Highly Sensitive Detection of Isoniazid Heteroresistance in Mycobacterium tuberculosis by DeepMelt Assay.

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.

• Authors: Bin Liang, Yaoju Tan, Zi Li, Xueshan Tian, Chen Du, Hui Li, Guoli Li, Xiangyang Yao, Zhongan Wang, Ye Xu, Qingge Li
• Journal: Journal of clinical microbiology (J Clin Microbiol) Vol. 56 Issue 2 (02 2018) ISSN: 1098-660X [Electronic] United States
• PMID: 29118176 (Publication Type: Journal Article, Multicenter Study, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2018 American Society for Microbiology.

Chemical References

• Antitubercular Agents
• Bacterial Proteins
• DNA, Bacterial
• Oxidoreductases
• InhA protein, Mycobacterium
• Isoniazid

Topics

• Antitubercular Agents (pharmacology)
• Bacterial Proteins (genetics)
• DNA, Bacterial (genetics)
• Drug Resistance, Bacterial (drug effects, genetics)
• Humans
• Isoniazid (pharmacology)
• Microbial Sensitivity Tests
• Molecular Diagnostic Techniques
• Mycobacterium tuberculosis (drug effects, genetics, isolation & purification)
• Oxidoreductases (genetics)
• Polymerase Chain Reaction
• Sensitivity and Specificity
• Sequence Analysis, DNA
• Sputum (microbiology)
• Tuberculosis, Multidrug-Resistant (diagnosis, microbiology)