[PMID]: | 29458686 |
[Au] Autor: | Anson LW; Chau K; Sanderson N; Hoosdally S; Bradley P; Iqbal Z; Phan H; Foster D; Oakley S; Morgan M; Peto TEA; Modernizing Medical Microbiology Informatics Group Mmmig; Crook DW; Pankhurst LJ |
[Ad] Endereço: | 1​Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK. |
[Ti] Título: | DNA extraction from primary liquid blood cultures for bloodstream infection diagnosis using whole genome sequencing. |
[So] Source: | J Med Microbiol;67(3):347-357, 2018 Mar. |
[Is] ISSN: | 1473-5644 |
[Cp] País de publicação: | England |
[La] Idioma: | eng |
[Ab] Resumo: | 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 , versus 0-2.5 ng µl using Amplex and MolYsis kits. However, we note higher levels of inhibition (260/280 ratio 0.9-2.1) and human DNA (0.0-4.4×10 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. |
[Mh] Termos MeSH primário: |
Bacteriemia/diagnóstico Hemocultura DNA Bacteriano/isolamento & purificação Genoma Bacteriano Sequenciamento Completo do Genoma
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[Mh] Termos MeSH secundário: |
Bacteriemia/microbiologia Infecções Relacionadas a Cateter/diagnóstico Infecções Relacionadas a Cateter/microbiologia DNA Bacteriano/análise DNA Bacteriano/genética Escherichia coli/genética Seres Humanos Testes de Sensibilidade Microbiana Técnicas de Diagnóstico Molecular/métodos Kit de Reagentes para Diagnóstico Análise de Sequência de DNA/métodos Staphylococcus aureus/genética
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[Pt] Tipo de publicação: | EVALUATION STUDIES; JOURNAL ARTICLE |
[Nm] Nome de substância:
| 0 (DNA, Bacterial); 0 (Reagent Kits, Diagnostic) |
[Em] Mês de entrada: | 1803 |
[Cu] Atualização por classe: | 180308 |
[Lr] Data última revisão:
| 180308 |
[Sb] Subgrupo de revista: | IM |
[Da] Data de entrada para processamento: | 180221 |
[St] Status: | MEDLINE |
[do] DOI: | 10.1099/jmm.0.000664 |
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