title: A rapid, highly sensitive and culture-free detection of pathogens from blood by positive enrichment.
abstract: Molecular diagnostics is a promising alternative to culture based methods for the detection of bloodstream infections, notably due to its overall lower turnaround time when starting directly from patient samples. Whole blood is usually the starting diagnostic sample in suspected bloodstream infections. The detection of low concentrations of pathogens in blood using a molecular assay necessitates a fairly high starting volume of blood sample in the range of 5-10mL. This large volume of blood sample has a substantial accompanying human genomic content that interferes with pathogen detection. In this study, we have established a workflow using magnetic beads coated with Apolipoprotein H that makes it possible to concentrate pathogens from a 5.0mL whole blood sample, thereby enriching pathogens from whole blood background and also reducing the sample volume to ~200μL or less. We have also demonstrated that this method of enrichment allows detection of 1CFU/mL of Escherichia coli, Enterococcus gallinarum and Candida tropicalis from 5mL blood using quantitative PCR; a detection limit that is not possible in unenriched samples. The enrichment method demonstrated here took 30min to complete and can be easily integrated with various downstream molecular and microbiological techniques.
authors.affiliation: Technology Center, Siemens Healthcare Private Limited, Bengaluru 560100, India.
authors.forename: Manjula Ramya
authors.initials: MR
authors.lastname: Vutukuru
authors.affiliation: Technology Center, Siemens Healthcare Private Limited, Bengaluru 560100, India.
authors.forename: Divya Khandige
authors.initials: DK
authors.lastname: Sharma
authors.affiliation: Technology Center, Siemens Healthcare Private Limited, Bengaluru 560100, India.
authors.forename: M S
authors.initials: MS
authors.lastname: Ragavendar
authors.affiliation: Strategy, Siemens Healthcare GmbH, Erlangen 91052, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Schmolke
authors.affiliation: Technology Center, Siemens Healthcare GmbH, Erlangen 91058, Germany.
authors.forename: Yiwei
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Technology Center, Siemens Healthcare GmbH, Erlangen 91058, Germany.
authors.forename: Walter
authors.initials: W
authors.lastname: Gumbrecht
authors.affiliation: Technology Center, Siemens Healthcare Private Limited, Bengaluru 560100, India. Electronic address: nivedita.mitra@siemens.com.
authors.forename: Nivedita
authors.initials: N
authors.lastname: Mitra
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D001769:Blood / Q000382:microbiology*; D017848:Blood-Borne Pathogens / Q000302:isolation & purification*; D041022:Candida tropicalis / Q000235:genetics / Q000302:isolation & purification; D002478:Cells, Cultured; D015169:Colony Count, Microbial / Q000379:methods; D004269:DNA, Bacterial / Q000097:blood; D004271:DNA, Fungal / Q000097:blood; D016983:Enterococcus / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D005658:Fungi / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D015894:Genome, Human; D006801:Humans; D057230:Limit of Detection; D008828:Microbiological Techniques / Q000379:methods*; D057089:Pathology, Molecular / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D018805:Sepsis / Q000097:blood / Q000175:diagnosis* / Q000382:microbiology*; D013997:Time Factors; D053482:beta 2-Glycoprotein I / Q000008:administration & dosage
chemical_list: D004269:DNA, Bacterial; D004271:DNA, Fungal; D053482:beta 2-Glycoprotein I
keywords: Apolipoprotein H; Blood stream infections; Culture-free detection; Pathogen enrichment; Quantitative PCR; Sepsis