Multicenter proficiency testing of nucleic acid amplification methods for the detection of enteroviruses.

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.

Multicenter evaluation of the Amplicor Enterovirus PCR test with cerebrospinal fluid from patients with aseptic meningitis. The European Union Concerted Action on Viral Meningitis and Encephalitis.

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.

The role of HSV-induced Fc- and C3b (i)-receptors in bacterial adherence.

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.

A flow cytometric rosetting assay for the analysis of Fc receptors and C3 receptors on HSV-infected cells.

A sensitive and reproducible flow cytometric assay was developed for the analysis of Fc gamma and C3b(i) receptors on HSV-infected cells. The method is based on a rosette technique using fluorochrome-labeled erythrocytes sensitized with IgG or C3b(i). A comparison of flow cytometric and microscopic quantitation demonstrated that the binding of EIgG, EC3b(i) to HSV-infected cells were correlated. Flow cytometric analysis provides the opportunity to study simultaneously the distribution of E per HSV-infected cell and the total binding of E to the whole population of HSV-infected cells. Receptor activity and HSV glycoprotein cell surface expression were shown to be correlated in a linear fashion. The assay could be applied to other Fc gamma R- and C3b(i)R-bearing cells.

Direct evidence for antibody bipolar bridging on herpes simplex virus-infected cells.

Cells infected with herpes simplex virus type 1 (HSV-1) express a cell-surface receptor able to bind the Fc portion of immunoglobulin G (IgG). In this study we provide direct evidence that bipolar bridging of antibodies, bound to the surface antigens on HSV-infected cells and to the Fc-receptor through the Fc part, offers the virus a survival advantage. Evidence was obtained by comparing the binding of FITC-labelled protein A, which has a similar binding site on IgG as the HSV-FcR, to cell-bound antibodies on HSV-infected cells and non-infected cells. The effectiveness of antibody bipolar bridging was dependent on the concentration of cell-bound IgG. At low concentrations of serum (0.1%) an 80% reduction in protein A-FITC binding to HSV-infected cells compared to non-infected cells was found. Even at higher concentrations of serum, antibody bipolar bridging resulted in a 40% reduction in the number of 'free' available Fc parts on HSV-infected cells compared to non-infected cells. Furthermore, these findings could be confirmed in a functional assay. The Fc-mediated attachment of granulocytes was significantly lower in HSV-infected cells compared to non-infected cells. From this study we conclude that HSV-FcR, by binding immune IgG in a bipolar fashion, provides the virus with an effective defence mechanism.

Suppression of the cellular adjuvanticity of lipophilic amines by a polyanion.

Modulation of delayed-type hypersensitivity reaction (DTH) in mice by synthetic adjuvants and the mode of their action were investigated. Intracutaneous injection of azobenzenearsonate coupled to phosphatidylethanolamine (A-PE) without adjuvant did not induce DTH. Administration of A-PE with the quaternary amines dimethyldioctadecylammonium bromide (DDA) or N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)propane diamine (CP-20,961) induced a strong response. Other surfactants, dextran sulfate (DXS) and dextran were not effective. In combination with 200 nmol DDA the optimal dose of antigen was 5 nmol A-PE, while at higher antigen doses DTH was diminished. Responses on combination of two adjuvants and A-PE revealed that DXS counteracted the stimulatory effects of both DDA and CP-20,961. In vitro, DDA formed insoluble complexes with 14C-A-PE and at optimal antigen concentration more than 90% of the antigen was bound to the adjuvant. The percentage of 14C-A-PE bound to 200 nmol DDA decreased with increasing doses of 14C-A-PE. Addition of DXS to the mixture of 14C-A-PE and DDA reduced the percentage of 14C-A-PE bound to DDA. Dose-response curves demonstrated a close relationship between the inhibitory effects of DXS on the DTH and the A-PE/DDA complex formation. Nonsulfated dextran affected neither the DTH nor the formation of complexes in vitro. In conclusion, cellular adjuvanticity of DDA for the lipophilic antigen A-PE is probably the result of formation of insoluble complexes with the antigen. Free A-PE suppresses the cellular response to A-PE/DDA complexes. The adjuvant DXS inhibits DTH by reducing the amount of immunogenic A-PE/DDA complexes and thus increasing the amount of free, immunosuppressive A-PE.