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 the performances of WGS and standard laboratory workflows prospectively, by parallel processing at a major mycobacterial reference service over the course of 1 year, for species identification, first-line Mycobacterium tuberculosis resistance prediction, and turnaround time. Among 2,039 isolates with line probe assay results for species identification, 74 (3.6%) failed sequencing or WGS species identification. Excluding these isolates, clinically important species were identified for 1,902 isolates, of which 1,825 (96.0%) were identified as the same species by WGS and the line probe assay. A total of 2,157 line probe test results for detection of resistance to the first-line drugs isoniazid and rifampin were available for 728 M. tuberculosis complex isolates. Excluding 216 (10.0%) cases where there were insufficient sequencing data for WGS to make a prediction, overall concordance was 99.3% (95% confidence interval [CI], 98.9 to 99.6%), sensitivity was 97.6% (91.7 to 99.7%), and specificity was 99.5% (99.0 to 99.7%). A total of 2,982 phenotypic DST results were available for 777 M. tuberculosis 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 data, overall concordance was 99.2% (98.7 to 99.5%), sensitivity was 94.2% (88.4 to 97.6%), and specificity was 99.4% (99.0 to 99.7%). Median processing times for the routine laboratory tests versus WGS were similar overall, i.e., 20 days (interquartile range [IQR], 15 to 31 days) and 21 days (15 to 29 days), 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.

Keywords: WGS; line probe assay; mycobacteria; phenotype; whole-genome sequencing.

FIG 1

Isolates included in the study, identified by MGIT culture between 20 April 2015 and 31 March 2016.

FIG 2

WGS species prediction versus line probe assay results. The bars represent the species identified by the line probe assay. (A) Concordance of WGS prediction at the species level. In cases where the line probe assays used were known to be unable to distinguish between certain species (i.e., M. chelonae/M. immunogenum, M. intracellulare/M. chimaera, M. fortuitum/M. mageritense, M. malmoense/M. palustre, and M. peregrinum/M. septicum), the results were considered concordant if WGS identified either species in the pair. (B) Isolates for which WGS identified a species different from that identified by the LPA.

FIG 3

WGS susceptibility predictions versus those of MTBDRplus and phenotypic testing for the drugs isoniazid and rifampin. R, resistant; S, susceptible; U, uncharacterized mutations found. MTBDRplus results were treated as resistant if the result was mutation or loss of wild type. The MTBDRplus test does not claim to predict susceptibility (only the absence of known resistance mutations), but we labeled MTBDRplus “wild type” calls “susceptible” in order to aid comparisons.

FIG 4

Differences in processing times for complete first-line susceptibility reporting for MTBC isolates. The horizontal line shows the median processing time for the routine laboratory, starting from the date that the MGIT tube was flagged as positive. The vertical lines show the different stages in the WGS process, starting from the removal of an aliquot from the positive MGIT tube. The WGS infrastructure was substantially upgraded partway through the study, with original report dates unavailable, so the typical processing time (1 day) of the new pipeline was used instead. Each line represents one isolate.