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Background: Definitive diagnosis of infestation withAngiostrongylus 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 immuno-PCR 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.

Two monoclonal antibodies, which recognize the epitope on an antigen with a molecular weight of 204 kD from the fifth-stage worm of Angiostrongylus cantonensis, were previously prepared and used to detect circulating antigens in patients with eosinophilic meningitis or meningoencephalitis and in mice experimentally infected with this parasite by a double-antibody, sandwich enzyme-linked immunosorbent assay (ELISA). The levels of this circulating antigen in experimentally infected mice were significantly higher three weeks after infection. The ELISA values in the detection of circulating antigens in cerebrospinal fluid (CSF) from patients were markedly higher than those in serum. Immunodiagnosis of patients with angiostrongyliasis by this technique proved to be highly specific for circulating antigens in serum and CSF specimens; however, the sensitivity in CSF was significantly higher than in serum.

Suspensions of fertilized eggs of Toxocara canis were mixed with 2% neutral formalin and preserved at 4 degrees C. When, after storage for 0, 12, 18, 21 and 24 months, samples of the eggs were incubated at 30 degrees C for 12 days, 96.8%, 92.6%, 74.1%, 51.0% and 19.3% of the eggs in the samples were found to embryonate. The embryonated eggs produced from the fertilized eggs preserved (in 2% neutral formalin at 4 degrees C) for 0, 12, 18 and 21 months were then tested for their infectivity to BALB/c mice, each mouse being given 800 embryonated eggs. The numbers of larvae recovered from the mice and the sites from which they were recovered, 2 or 14 days post-infection, appeared unaffected by the length of storage of the eggs. The infected mice all had similar eosinophil counts in their peripheral blood and similar serum titres of Toxocara-specific IgM and IgG antibodies, and cultures of their spleen cells produced similar amounts of interleukin-4, interleukin-5 and interferon-gamma when stimulated with concanavalin A. The results of SDS-PAGE indicated that egg preservation for at least 21 months had no effect on the excretory-secretory antigens in samples of medium from cultures of infective larvae released from the eggs. In summary, at least 50% of the fertilized eggs preserved in 2% neutral formalin at 4 degrees C for 21 months could fully embryonate and then had the same infectivity and antigenicity as embryonated fresh eggs.