title: Evaluation of loop-mediated isothermal amplification for the rapid identification of bacteria and resistance determinants in positive blood cultures.
abstract: The use of molecular assays to rapidly identify pathogens and resistance genes directly from positive blood cultures (BCs) contribute to shortening the time required for the diagnosis of bloodstream infections. In this work, loop-mediated isothermal amplification (LAMP) assays have been examined for their potential use in BC diagnosis. Three different assays were applied. The commercially available eazyplex® MRSA test detects Staphylococcus aureus, S. epidermidis, mecA, and mecC. Two in-house assays [Gram-positive (GP) and Gram-negative (GN)] have been developed for the detection of streptococci, enterococci, vanA, vanB, Pseudomonas spp., Enterobacteriaceae, and the blaCTX-Mfamily. A total of 370 positive BCs were analyzed. LAMP test results were obtained within 30 min, including sample preparation. Amplification was measured by real-time fluorescence detection. The threshold time for fluorescence intensity values ranged from 6.25 to 13.75 min. The specificity and sensitivity of the assays varied depending on the target. Overall, from 87.7% of BCs, true-positive results were obtained, compared to routine standard diagnosis. Twenty-one tests were true-negative because of the lack of an appropriate target (5.7%). The concordance of positive test results for resistance genes with subsequent antibiotic susceptibility testing was 100%. From 15 BC bottles with mixed cultures, eazyplex® assays produced correct results in 73% of the cases. This study shows that LAMP assays are fast and cost-saving tools for rapid BC testing in order to expedite the diagnostic report and improve the antibiotic stewardship for sepsis patients.
authors.affiliation: Institute of Medical Microbiology, Jena University Hospital, Jena, Germany. juergen.roedel@med.uni-jena.de.
authors.forename: J
authors.initials: J
authors.lastname: Rödel
authors.affiliation: Institute of Medical Microbiology, Jena University Hospital, Jena, Germany.|Friedrich Loeffler Institute of Medical Microbiology, University Medicine Greifswald, Greifswald, Germany.
authors.forename: J A
authors.initials: JA
authors.lastname: Bohnert
authors.affiliation: Institute of Medical Microbiology, Jena University Hospital, Jena, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Stoll
authors.affiliation: AmplexDiagnostics GmbH, Gars Bahnhof, Germany.
authors.forename: L
authors.initials: L
authors.lastname: Wassill
authors.affiliation: Institute of Medical Microbiology, Jena University Hospital, Jena, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Edel
authors.affiliation: Institute of Medical Microbiology, Jena University Hospital, Jena, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Karrasch
authors.affiliation: Institute of Medical Microbiology, Jena University Hospital, Jena, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Löffler
authors.affiliation: Institute of Medical Microbiology, Jena University Hospital, Jena, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Pfister
mesh_terms: D016470:Bacteremia / Q000175:diagnosis*; D001419:Bacteria / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification*; D000071997:Blood Culture; D024881:Drug Resistance, Bacterial; D060005:Genotyping Techniques / Q000379:methods; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors