Immuno-PCR for Detection of Antigen to Angiostrongylus cantonensis Circulating Fifth-Stage Worms

Abstract

Background: Definitive diagnosis of infestation with Angiostrongylus cantonensis is difficult because the parasitic nematode is undetectable in the cerebrospinal fluid (CSF) of one-half of afflicted patients and the diagnostic sensitivity of ELISA for circulating worm antigens in patient sera is low. We studied immunoPCR as a diagnostic tool. Methods: We studied 30 controls and 60 afflicted patients (30 confirmed by parasitologic analysis of CSF). We used a monoclonal antibody to capture circulating A. cantonensis antigens in serum samples. A DNA label generated by PCR amplification with biotinylated primer was bound by use of streptavidin to a biotinylated third antibody. Circulating antigens sandwiched by monoclonal antibody were detected by PCR amplification of the DNA label. Results: The detection limit of the ELISA was 100-1000 times higher than that of the immuno-PCR. The concentrations of circulating antigens in patients were markedly higher than those in controls (Wilcoxon rank-sum test, P <0.001). At a cutoff of 0.1 ng/L, sensitivity and specificity for immunodiagnosis of patients with angiostrongyliasis by immuno-PCR were 98% (95% confidence interval, 91-99%) and 100% (93-100%), respectively. The test was positive in all parasitologically confirmed cases. Conclusions: Immuno-PCR is a promising technique for diagnosis of A. cantonensis infestation. © 2004 American Association for Clinical Chemistry.