A multicenter study of molecular detection of enteroviruses was conducted using a proficiency panel. Of 70 data sets, 46 (66%) reported correct results for samples containing at least 1 50% infective dose per ml and for negative samples. Variation in performance between laboratories demonstrates the need for ongoing quality control.Nucleic acid amplification methods are widely used for enterovirus (EV) detection because they are sensitive, specific, and rapid (1,3,6,10,12,17). However, reliable molecular diagnosis requires assay standardization and continuous monitoring of sensitivity and specificity. A quality assessment program for EV detection was therefore established within the framework of the European Union Concerted Action on Quality Control of Nucleic Acid Amplification in Diagnostic Virology (QCCA) to assess the proficiency of laboratories using molecular EV detection methods. Here we describe results of 70 data sets reported by 59 laboratories upon testing an EV proficiency panel. To our knowledge, this represents the largest such study reported to date.The proficiency panel consisted of 12 coded samples, including a coxsackievirus A9 (CVA9) dilution series, other enterovirus serotypes representing different genetic clusters of human enteroviruses (5, 11), human parechovirus type 1 (HPEV1; formerly classified as echovirus 22 but now known to be genetically distinct from enteroviruses), and negative controls (Table 1). The sources, production, and characterization of the viruses used are described elsewhere (7,15). Titration of viral infectivity was performed on the original virus stocks (50% tissue culture infective doses [TCID 50 ] per milliliter). Virus stocks were inactivated at 56°C for 30 min, freeze-dried in 1-ml volumes, and stored at 4°C. Freeze drying resulted in an approximately 10-fold reduction in levels of PCR-detectable viral RNA. Full details of the production and evaluation of the proficiency panel are available on the QCCA website (www .qcca.org.uk). Prior to release, external quality control testing was performed by two reference laboratories to assess sample quality and homogeneity. Four vials of each sample were analyzed in duplicate using the Amplicor EV-PCR assay (Roche Molecular Systems, Branchburg, N.J.) and an in-house PCR. A stochastic distribution of positive results was found in the highly diluted samples close to the detection limit of the assay in use (data not shown). To assess the effects of storage and of transportation on sample stability, samples were tested before and after 1, 2, 4, and 7 days of storage at room temperature. Identical results were obtained, with the exception of one borderline sample, which produced a 2.5-fold decrease in optical density values in the Amplicor EV-PCR assay over a period of 7 days (data not shown). Storage of the samples for 1 year at 4°C demonstrated no detectable loss of activity.The proficiency panel was distributed at ambient temperatures to 63 European laboratories, which were asked to reconstitute each sample with 1 ml of wate...

The Amplicor Enterovirus PCR test was compared with viral culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. In a multicenter study in which nine laboratories participated, a total of 476 CSF specimens were collected from patients with suspected aseptic meningitis. Sixty-eight samples were positive by PCR (14.4%), whereas 49 samples were positive by culture (10.4%), demonstrating that the Amplicor Enterovirus PCR test was significantly more sensitive than culture (P < 0.001). After discrepancy analysis the sensitivity and specificity of the Amplicor Enterovirus PCR test obtained by using viral culture as the “gold standard” were 85.7 and 93.9%, respectively. Our results with the CSF specimens collected in different countries demonstrate that the Amplicor test is capable of detecting a large variety of enterovirus serotypes and epidemiologically unrelated isolates in CSF specimens from patients with aseptic meningitis. The Amplicor Enterovirus PCR test is a rapid assay which can be routinely performed with CSF samples and is an important improvement for the rapid diagnosis of enteroviral meningitis.

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Summary.Herpes simplex virus type-1 (HSV-1) induces Fc-and C3b(i)-receptors on infected cells. The role of these receptors in bacterial superinfection was studied by comparing the adherence of non-opsonised and opsonised bacteria to HSV-infected and non-infected HEp-2 cells. A flow cytometric adherence assay, based on the fluorescent quantitation of FITC-labelled bacteria, was developed. Opsonisation of Staphylococcus epidermidis with human serum, resulted in a marked increase in adherence to HSV-infected cells and revealed a role for C3b(i)R-and FcR-mediated adhesion. However, the enhanced adherence never exceeded the level of attachment to non-infected cells. Increased adherence of other pathogenic bacteria, including Escherichia coli, Streptococcus pneumoniae, Haemophilus influenzae and Pseudomonas aeruginosa was not observed, indicating that the HSV-receptors play a minor role in secondary infections. Bacterial adhesion factors such as the fimbriae of E. coli played a more dominant role in the adherence of bacteria to HSV-infected cells.