title: A qPCR and multiplex pyrosequencing assay combined with automated data processing for rapid and unambiguous detection of ESBL-producers Enterobacteriaceae.
abstract: Rapid and specific detection of extended-spectrum β-lactamase-producing (ESBL) bacteria is crucial both for timely antibiotic therapy when treating infected patients as well as for appropriate infection control measures aimed at curbing the spread of ESBL-producing isolates. Whereas a variety of phenotypic methods are currently available for ESBL detection, they remain time consuming and sometimes difficult to interpret while being also affected by a lack of sensitivity and specificity. Considering the longer turnaround time (TAT) of susceptibility testing and culture results, DNA-based ESBL identification would be a valuable surrogate for phenotypic-based methods. Putative ESBL-positive Enterobacteriaceae isolates (n = 330) from clinical specimen were prospectively collected in Bulgaria, Romania and Democratic Republic of Congo and tested in this study. All isolates were assessed for ESBL-production by the E-test method and those giving undetermined ESBL status were re-tested using the combination disk test. A genotypic assay successively combining qPCR detection of blaCTX-M, blaTEM and blaSHV genes with a multiplex pyrosequencing of blaTEM and blaSHV genes was developed in order to detect the most common ESBL-associated TEM and SHV single nucleotides polymorphisms, irrespective of their plasmid and/or chromosomal location. This assay was applied on all Enterobacteriaceae isolates (n = 330). Phenotypic and genotypic results matched in 324/330 (98.2%). Accordingly, real-time PCR combined with multiplex pyrosequencing appears to be a reliable and easy-to-perform assay with high-throughput identification and fast TAT (~5 h).
authors.affiliation: Defence Laboratories Department, Belgian Armed Forces, Brussels, Belgium.
authors.forename: Yann
authors.initials: Y
authors.lastname: Deccache
authors.forename: Leonid M
authors.initials: LM
authors.lastname: Irenge
authors.forename: Jérôme
authors.initials: J
authors.lastname: Ambroise
authors.forename: Encho
authors.initials: E
authors.lastname: Savov
authors.forename: Dan
authors.initials: D
authors.lastname: Marinescu
authors.forename: Raphael B
authors.initials: RB
authors.lastname: Chirimwami
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Gala