期刊
JOURNAL OF MEDICAL MICROBIOLOGY
卷 67, 期 3, 页码 347-357出版社
MICROBIOLOGY SOC
DOI: 10.1099/jmm.0.000664
关键词
bloodstream infection; sepsis; bacteraemia; whole genome sequencing
类别
资金
- Wellcome Trust [WT098615/Z/12/Z]
- Department of Health [T5-358]
- NIHR Health Protection Research Unit (NIHR HPRU) in Healthcare Associated Infections and Antimicrobial Resistance at University of Oxford with Public Health England (PHE) [HPRU-2012-10 041]
- National Institute for Health Research (NIHR) Oxford Biomedical Research Centre based at Oxford University Hospitals NHS Trust
- University of Oxford
- Wellcome Trust/Royal Society Sir Henry Dale Fellowship [102541/Z/13/Z]
- Wellcome Trust PhD studentship
- Wellcome Trust [102541/Z/13/Z] Funding Source: Wellcome Trust
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 mu l(-1), versus 0-2.5 ng mu l(-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 x 10(6) 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 < 10% human DNA, with > 93% concordance between WGS-based species and susceptibility predictions and clinical diagnosis. If > 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.
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