Abstract
Increasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid characterization of a DR-TB patient's complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing of Mycobacterium tuberculosis DNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of ≤1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool.
Highlights
Tuberculosis (TB) infects an estimated 9.6 million people and is the cause of 1.5 million deaths per year [1]
Next-generation sequencing (NGS) is much simpler to validate for the detection of multiple resistance mutations in M. tuberculosis, as its performance does not depend the number of mutations present
Fifty-four samples (22.0%) failed across all three replicates of rpoB-specific quantitative PCR (qPCR) and were not included in the Gen-RDST analysis, due to DNA volume constraints dictating that multiple attempts for Gen-RDST were not possible
Summary
Tuberculosis (TB) infects an estimated 9.6 million people and is the cause of 1.5 million deaths per year [1]. Current diagnostic tools for broad drug susceptibility testing (DST) of Mycobacterium tuberculosis isolates are culture based, slow (take weeks to months), not standardized, and require complex laboratory infrastructure [3,4,5] These complexities lead to systematic errors in the resistance profiles obtained. We demonstrated the advancement of amplicon sequencing using overlapping reads to examine subpopulations at extremely low levels of antituberculosis drug-resistant bacteria [20] This pilot study evaluates the performance and clinical utility of rapid amplicon sequencing of DNA from patient sputum samples using a tabletop next-generation sequencing technology and automated data analysis scripts to provide an accessible rapid DST ( Gen-RDST) solution. Utilizing a well-characterized set of DNA from a previous study, we compared these newly generated Gen-RDST results against previous phenotypic DST and pyrosequencing results from the same clinical sputum DNA samples
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