Comprehensive Whole-Genome Sequencing and Reporting of Drug Resistance Profiles on Clinical Cases of Mycobacterium tuberculosis in New York State

Abstract

Whole-genome sequencing (WGS) is a newer alternative for tuberculosis (TB) diagnostics and is capable of providing rapid drug resistance profiles while performing species identification and capturing the data necessary for genotyping. Our laboratory developed and validated a comprehensive and sensitive WGS assay to characterize Mycobacterium tuberculosis and other M. tuberculosis complex (MTBC) strains, composed of a novel DNA extraction, optimized library preparation, pairedend WGS, and an in-house-developed bioinformatics pipeline. This new assay was assessed using 608 MTBC isolates, with 146 isolates during the validation portion of this study and 462 samples received prospectively. In February 2016, this assay was implemented to test all clinical cases of MTBC in New York State, including isolates and early positive Bactec mycobacterial growth indicator tube (MGIT) 960 cultures from primary specimens. Since the inception of the assay, we have assessed the accuracy of identification of MTBC strains to the species level, concordance with culture-based drug susceptibility testing (DST), and turnaround time. Species identification by WGS was determined to be 99% accurate. Concordance between drug resistance profiles generated by WGS and culture-based DST methods was 96% for eight drugs, with an average resistance-predictive value of 93% and susceptiblepredictive value of 96%. This single comprehensive WGS assay has replaced seven molecular assays and has resulted in resistance profiles being reported to physicians an average of 9 days sooner than with culture-based DST for first-line drugs and 32 days sooner for second-line drugs.

Drug resistance mechanisms and drug susceptibility testing for tuberculosis

“…WGS performances have been shown to be accurate and reliable compared to conventional phenotypic diagnostics (species identification: 93%, 95% CI: 90–96%; drug susceptibility accuracy: 93%, 95% CI: 91–95%), with turn‐around time and costs competitive with conventional diagnostics (median of 9 days, interquartile range (IQR): 6–10 for WGS vs a median of 31 days, IQR: 21–44 for conventional diagnostics, with a saving of 7% annually) . The introduction of WGS in routine diagnostics was found accurate (species identification accuracy: 99%; drug susceptibility accuracy: 96% for eight drugs) and rapid (72 h for WGS vs 28 days for DST only), allowing to replace seven different molecular assays and accelerating reporting time . WGS was also successfully used for drug resistance surveillance purposes …”

“…160 The introduction of WGS in routine diagnostics was found accurate (species identification accuracy: 99%; drug susceptibility accuracy: 96% for eight drugs) and rapid (72 h for WGS vs 28 days for DST only), allowing to replace seven different molecular assays and accelerating reporting time. 161,162 WGS was also successfully used for drug resistance surveillance purposes. 163,164 The advances in performances of NGS workflow (from DNA extraction to data analysis) allowed to improve WGS for 'real-time' routine diagnosis in direct clinical samples, with MTB complex detection accuracy of >97%.…”

Whole‐genome sequencing for combatting antibiotic resistance

“…A prospective British study on mycobacterial diagnosis concluded that WGS accurately identified species and antibiotic resistance with 93% accuracy; moreover, WGS diagnosed a case of multidrug‐resistant tuberculosis before the routine diagnosis was accomplished (Pankhurst et al, ). A similar WGS tuberculosis study from the United States reported 99% accuracy, and a concordance of 96% between WGS and culture‐based diagnostics (Shea et al, ). Moreover, the latter study concluded that applying WGS of tuberculosis cases allowed the reporting of resistance profiles to physicians on average 9 days sooner than with culture‐based diagnostics for first‐line antibiotics, and 32 days sooner for second‐line antibiotics.…”

Whole genome sequencing for drug resistance profile prediction inMycobacterium tuberculosis

“…WGS of clinical isolates allows for accurate identification of established-resistance-conferring chromosomal mutations [10, 12, 13] and may ensure adequate treatment in days instead of months. We compared whole genome-based drug resistance profiles with two culture-based quantitative DST methods for a total of 11 drugs, including all first-line drugs (rifampicin, isoniazid, ethambutol, pyrazinamide, streptomycin) and an array of second-line drugs (rifabutin, amikacin, kanamycin A, capreomycin, moxifloxacin, ethionamide).…”