Quantitative PCR-enhanced immunoassay for measurement of enteroviral immunoglobulin M antibody and diagnosis of aseptic meningitis

Abstract

A PCR-enhanced immunoassay (PIA) to detect enterovirus (EV) immunoglobulin M (IgM) for diagnosis of recent EV infection was recently developed. This test was compared with another EV IgM capture technique, the solid-phase reverse immunosorbent test (SPRIST). Fourteen of 43 serum samples from aseptic meningitis patients were positive by PIA, whereas 10 were positive by SPRIST. One of 39 control serum samples was weakly positive by PIA. A single-serum-dilution real-time PCR-based PIA for EV IgM (quantitative PIA [QPIA]) was also developed and evaluated against PIA, SPRIST, an EV IgM radioimmunoassay (RIA), and clinical data. A mixture of 12 EVs was used as the antigen. Results from investigating four groups of serum samples were as follows. (i) The nine PIA-positive serum samples in group 1 were all positive by QPIA. (ii) Group 2 consisted of 59 serum samples from aseptic meningitis patients. Nineteen of 30 serum samples (63%) taken at hospital admission were positive by QPIA. Of these, 17 were positive in EV PCR. (iii) None of the 30 control serum samples in group 3 were positive by QPIA. (iv) For the 24 serum samples in group 4, of which 11 were positive and 13 were negative by RIA, the QPIA results were completely concordant. The sensitivity and specificity of QPIA for diagnosis of EV infection were 70 and 80%, respectively. QPIA provides a rational strategy for the detection of EV IgM, allows the use of viral antigens with minimal purification, and needs no virus-specific reagents apart from those in the PCR. QPIA is a generally applicable method for the detection of viral IgM in IgM capture assays.

FIG. 1.

The highest IgM binding was achieved with a serum dilution of 1/100. Nine known EV IgM-positive serum samples were diluted up to 10 million times. Dotted line, theoretical curve for a signal proportional to dilution. The chosen standard dilution of 1/200 is indicated.

FIG. 2.

Distribution of QPIA binding values in four categories of serum samples. Serum samples from 20 patients previously found to be EV IgM positive (groups [gr] 1 and 4), 59 meningitis patients (group 2), and 30 blood donors (group 3) were tested. The 59 group-2 samples were divided into samples from 32 meningitides with known EV infection (QPCR positive in CSF), 11 meningitides for which the cause was unknown (QPCR negative in CSF), and 16 meningitides with known or possible bacterial, herpes simplex, or TBE infection. Results of 13 RIA-negative samples (group 4) are also shown. Values between 0 and 0.5 are plotted only to show the number of observations.

FIG. 3.

Contributions of individual EVs to the amount of EV RNA bound from the viral antigen mixture. The nine serum samples were run with nine EVs, but not with PV1, -2, or -3. CB2, CB1, and E30 dominate the binding. The serum samples were obtained from AM patients during the autumns of 1995 and 1996, when E9, CB5, and E30 had been isolated from AM patients in Uppsala.