Prospective identification of HEV-B enteroviruses during the 2005 outbreak.

Enteroviruses (EVs) represent the main etiological agents of epidemics of viral meningitis and especially the serotypes related to the human enterovirus B species. Genetic typing by sequencing a PCR-amplified portion of the genome has proved to be useful for identifying EVs and is more rapid than standard seroneutralization tests. However, prospective genotyping has not been reported in routine practice within a clinical diagnostic laboratory. A genetic typing assay using two sets of primers was developed for the amplification and sequencing of the VP1 coding sequence of the HEV-B serotypes. Identification was carried out by sequence comparisons with EV sequences in GenBank using the BLAST search tool and confirmed by phylogenetic analysis. This method was used to identify prospectively the 48 enteroviruses isolated in patients with either enterovirus-proved meningitis (n = 41) or other clinical manifestations (n = 7) admitted to the University Hospital of Clermont-Ferrand (France) in 2005. The assay was also used to type retrospectively EVs isolated in cerebrospinal fluid specimens of 25 patients admitted to the Trousseau Paediatric Hospital in Paris (France) between February and August 2005. In both prospective and retrospective investigations of meningitis, echovirus 30 (E30) was the most frequent serotype, followed in decreasing order by E18, E13, coxsackievirus B5, B3, E6, E4, E7, E11, E33, and coxsackievirus A9. In patients with other manifestations, coxsackievirus B3, B5, and E3 were each identified twice, and E2 once. In E30 infected patients, nine different lineages were demonstrated by phylogenetic analysis. Genetic typing allowed the prospective, effective and rapid identification of all EV isolates involved in the 2005 outbreak. Molecular typing in combination with phylogenetic analysis will be a reliable means to confirm the emergence of new EV variants, and is of interest of both individual patients and public health.