title: Direct RNA-based detection of CTX-M β-lactamases in human blood samples.
abstract: Bloodstream infections with ESBL-producers are associated with increased mortality, which is due to delayed appropriate treatment resulting in clinical failure. Current routine diagnostics for detection of bloodstream infections consists of blood culture followed by species identification and susceptibility testing. In attempts to improve and accelerate diagnostic procedures, PCR-based methods have been developed. These methods focus on species identification covering only a limited number of ESBL coding genes. Therefore, they fail to cover the steadily further evolving genetic diversity of clinically relevant β-lactamases. We have recently designed a fast and novel RNA targeting method to detect and specify CTX-M alleles from bacterial cultures, based on an amplification-pyrosequencing approach. We further developed this assay towards a diagnostic tool for clinical use and evaluated its sensitivity and specificity when applied directly to human blood samples. An optimized protocol for mRNA isolation allows detection of specific CTX-M groups from as little as 100 CFU/mL blood via reverse transcription, amplification, and pyrosequencing directly from human EDTA blood samples as well as from pre-incubated human blood cultures with a turnaround time for test results of <7 h.
authors.affiliation: Center for Infectious Diseases and Infection's Control, Jena University Hospital, Erlanger Allee 101, D-07747 Jena, Germany. Electronic address: claudia.stein@med.uni-jena.de.
authors.forename: Claudia
authors.initials: C
authors.lastname: Stein
authors.affiliation: Center for Infectious Diseases and Infection's Control, Jena University Hospital, Erlanger Allee 101, D-07747 Jena, Germany.
authors.forename: Oliwia
authors.initials: O
authors.lastname: Makarewicz
authors.affiliation: Nosocomial Pathogens and Antibiotic Resistance, Robert Koch Institute, Burgstraße 37, D-38855 Wernigerode, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Pfeifer
authors.affiliation: Center for Infectious Diseases and Infection's Control, Jena University Hospital, Erlanger Allee 101, D-07747 Jena, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Brandt
authors.affiliation: Center for Infectious Diseases and Infection's Control, Jena University Hospital, Erlanger Allee 101, D-07747 Jena, Germany.
authors.forename: Mathias W
authors.initials: MW
authors.lastname: Pletz
mesh_terms: D016470:Bacteremia / Q000175:diagnosis* / Q000382:microbiology; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001769:Blood / Q000382:microbiology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012680:Sensitivity and Specificity; D013997:Time Factors; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D012329:RNA, Bacterial; D012333:RNA, Messenger; D001618:beta-Lactamases
keywords: Blood culture; Degenerate primers; EDTA blood; Extended-spectrum beta-lactamases (ESBL); Pyrosequencing; qRT-PCR