Capsular typing method for Streptococcus agalactiae using whole genome sequence data

Group B streptococcus (GBS) capsular serotype is a major determinant of virulence, and affects potential vaccine coverage. Here we report a whole genome sequencing-based method for GBS serotype assignment. This shows high agreement (kappa=0.92) with conventional methods, and increased serotype assignment (100%) to all ten capsular types

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

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

Capsular typing method for Streptococcus agalactiae using whole genome sequence data

Group B streptococcus (GBS) capsular serotype is a major determ inant of virulence, and affects potential vaccine coverage. Here we report a whole genome sequencing-based method for GBS serotype assignment. This shows high agree ment (kappa=0.92) with conventional methods, and increased serotype assignment (100%) to all ten capsular types.</p

DNA extraction from primary liquid blood cultures for bloodstream infection diagnosis using whole genome sequencing

Purpose Speed of bloodstream infection diagnosis is vital to reduce morbidity and mortality. Whole genome sequencing (WGS) performed directly from liquid blood culture could provide single-assay species and antibiotic susceptibility prediction; however, high inhibitor and human cell/DNA concentrations limit pathogen recovery. We develop a method for the preparation of bacterial DNA for WGS-based diagnostics direct from liquid blood culture. Methodology We evaluate three commercial DNA extraction kits: BiOstic Bacteraemia, Amplex Hyplex and MolYsis Plus. Differential centrifugation, filtration, selective lysis and solid-phase reversible immobilization bead clean-up are tested to improve human cells/DNA and inhibitor removal. Using WGS (Illumina/MinION), we assess human DNA removal, pathogen recovery, and predict species and antibiotic susceptibility inpositive blood cultures of 44 Gram-negative and 54 Staphylococcus species. Results/Key findings BiOstic kit extractions yield the greatest mean DNA concentration, 94–301 ng µl−1, versus 0–2.5 ng µl−1 using Amplex and MolYsis kits. However, we note higher levels of inhibition (260/280 ratio 0.9–2.1) and human DNA (0.0–4.4×106 copies) in BiOstic extracts. Differential centrifugation (2000  g , 1 min) prior to BiOstic extraction reduces human DNA by 63–89 % with selective lysis minimizing by a further 62 %. Post-extraction bead clean-up lowers inhibition. Overall, 67 % of sequenced samples (Illumina MiSeq) contain &lt;10 % human DNA, with &gt;93 % concordance between WGS-based species and susceptibility predictions and clinical diagnosis. If &gt;60 % of sequencing reads are human (7/98 samples) susceptibility prediction becomes compromised. Novel MinION-based WGS (n=9) currently gives rapid species identification but not susceptibility prediction. Conclusion Our method for DNA preparation allows WGS-based diagnosis direct from blood culture bottles, providing species and antibiotic susceptibility prediction in a single assay

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

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