Journal of Clinical Virology 47 (2010) 54–59 Contents lists available at ScienceDirect Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv Multicenter evaluation of the ENTEROVIRUS R-gene TM real-time RT-PCR assay for the detection of enteroviruses in clinical specimens Sylvie Pillet a , Geneviève Billaud b , Shabir Omar a , Bruno Lina b , Bruno Pozzetto a , Isabelle Schuffenecker b,∗ a Laboratoire de Bactériologie-Virologie, GIMAP EA 3064 and University Hospital of Saint-Etienne, 42023 Saint-Etienne Cedex 2, France b Centre National de Référence des Enterovirus, Laboratoire de Virologie Est des Hospices Civils de Lyon, Centre Hospitalier Universitaire de Lyon, 59 Boulevard Pinel, 69677 Bron Cedex, France article info Article history: Received 22 September 2009 Accepted 25 September 2009 Keywords: Real-time RT-PCR Human enteroviruses Molecular diagnosis Meningitis Clinical evaluation abstract Background: The rapid molecular diagnosis of enteroviral meningitis has been shown important for an adequate management of the patients. Objectives: A new CE-marked real-time RT-PCR assay (ENTEROVIRUS R-gene TM , Argene) was evaluated in two university hospital virology laboratories. Study design: Reactivity, analytical sensitivity and specificity were evaluated using 54 prototype and 173 clinical human enterovirus (HEV) strains, a 12-sample HEV proficiency panel, and 30 non-HEV microor- ganisms. The clinical performance of the ENTEROVIRUS R-gene TM assay was evaluated by testing 197 cerebrospinal fluid (CSF) and 103 respiratory specimens, comparatively to the routinely used diagnostic techniques. Results: Sixty-four out of the 65 HEV serotypes tested were detected. The analytical sensitivity ranged between 10 -2.64 and 10 2.39 TCID 50 /50 l. Cross-reactivity was observed with four human rhinoviruses. On 59 CSF specimens analyzed prospectively, the results of the ENTEROVIRUS R-gene TM assay showed a 94.8% concordance with those of the Smart enterovirus (EV) assay (Cepheid). On 138 CSF specimens tested retrospectively, the results of the ENTEROVIRUS R-gene TM assay showed a 97.1% concordance with those of either the GeneXpert EV assay (Cepheid) or the in-house RT-PCR HEV assays used at the time of specimen collection. On 103 respiratory specimens, the concordance between the results of the ENTEROVIRUS R-gene TM assay and those of the routine RT-PCRs or viral culture was 90.2% and 96.1% before and after retest, respectively. Conclusions: The new test was found able to detect a large panel of enterovirus serotypes; it was sensitive when used on clinical specimens; and, easy and rapid to perform on a routine basis. © 2009 Elsevier B.V. All rights reserved. 1. Background Human enteroviruses (HEV) are small single-stranded positive RNA viruses belonging to the Enterovirus genus in the Picornaviri- dae family. They are classified into four species (HEV-A to -D) (www.picornaviridae.com) and more than 100 serotypes, includ- ing coxsackieviruses (CV), echoviruses (E), enteroviruses (EV) and polioviruses (PV), have been described to date. 1 HEV are the most common cause of aseptic meningitis in both children and adults (75,000 cases each year in the USA), but are also responsible for a wide variety of clinical manifestations ranging from asymp- tomatic or mild febrile illness to more severe and potentially ∗ Corresponding author. Tel.: +33 4 72 12 96 47; fax: +33 4 72 12 95 00. E-mail address: isabelle.schuffenecker@chu-lyon.fr (I. Schuffenecker). fatal syndromes, including bronchiolitis, paralysis, encephalitis, myocarditis, and neonatal systemic infection. 1–4 Molecular techniques have now been recognized as the “gold standard” for the diagnosis of HEV infections and the rapidity of the molecular diagnosis of HEV meningitis has been shown to be a determining factor in the management of patients. 5–7 Numer- ous RT-PCR assays have been developed using various extraction methods, primer and probes design, amplification and amplicon detection conditions. Whereas the amplification methods are still in-house RT-PCR assays in many laboratories, a few commercial techniques using either end-point 8,9 or real-time detection 9–11 have proved their efficiency in the diagnosis of HEV infections and are progressively superseding the in-house assays. Ideally, they should detect a broad range of serotypes, including the newly described ones, and combine high sensitivity and specificity on clinical specimens. 1386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2009.09.033