Evaluation of whole genome sequencing for Mycobacterial species identification and drug susceptibility testing in a clinical setting: a large-scale prospective assessment of performance against line-probe assays and phenotyping

Abstract

Use of whole genome sequencing (WGS) for routine Mycobacterial species identification and drug susceptibility testing (DST) is becoming a reality. We compared performance of WGS and standard laboratory workflows prospectively, by parallel processing at a major Mycobacterial Reference Service over one year, for species identification, first-line Mycobacterium tuberculosis (TB) resistance prediction, and turnaround time. Of 2039 isolates with line-probe results for species identification, 74 (3.6%) failed sequencing or WGS species identification. Excluding these, clinically important species were identified in 1902 isolates, of which 1825 (96.0%) were identified by WGS as the same species. 2157 line-probe test results assaying resistance to the first-line drugs isoniazid and rifampicin were available from 728 TB complex isolates. Excluding 216 (10.0%) cases where there was insufficient sequencing data for WGS to make a prediction, overall concordance was 99.3% (95% CI 98.9-99.6), (sensitivity 97.6% (91.7-99.7), specificity 99.5% (99.0-99.7)). 2982 phenotypic DST results were available from 777 TB complex isolates. Of these, 356 (11.9%) had no WGS comparator due to insufficient sequencing data, and in 154 (5.2%) cases the WGS prediction was indeterminate due to discovery of novel, previously uncharacterized mutations. Excluding these, overall concordance was 99.2% (98.7-99.5), (sensitivity 94.2% (88.4-97.6), specificity 99.4% (99.0-99.7)). Median processing time for the routine laboratory versus WGS was similar overall, at 20 days (IQR 15,31) and 21 days (15,29) respectively (p=0.41). In conclusion, WGS predicts species and drug susceptibility with great accuracy but work is needed to increase the proportion of predictions made