Immuno-PCR for detection of antigen to Angiostrongylus cantonensis circulating fifth-stage worms.

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 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.

Kinetics of change in the eotaxin concentration in serum and cerebrospinal fluid of mice infected with Angiostrongylus cantonensis.

The kinetics of changes in the eotaxin concentration in the serum and cerebrospinal fluid (CSF) of BALB/c mice after infection with Angiostrongylus cantonensis and the correlation between the concentration of eotaxin and worm recovery were investigated. The mean concentration of eotaxin in serum of infected mice gradually increased from 46.3+/-6.5 pg/ml at week 0 to 104.9+/-44.8 pg/ml at week 3 after infection, while the mean eotaxin level in the CSF of infected mice rapidly increased from 18.7+/-2.1 pg/ml to 193.2+/-23.6 pg/ml 1 week after infection and then increased further to 507.8+/-167.9 pg/ml at week 3. The concentrations of eotaxin in the CSF of infected mice each week after infection were all significantly higher than those in serum ( P<0.0001). In parallel with the increase in eotaxin in the CSF, infected mice showed gradual increases in CSF eosinophilia and a reduction in intracranial worm recovery. The concentration of eotaxin in CSF was higher in infected mice with more worms in the brain, except when the number of worms in the brain was >30. In addition, when the worm counts in the brains of infected mice were <30, eotaxin concentrations in the CSF were positively correlated with worm counts in the brain ( P<0.001). Thus, the release of eotaxin in the CSF of mice infected with A. cantonensis observed in this study was time dependent and worm-load dependent, and in parallel with the increase in eotaxin in the CSF, and gradual decreases in worm counts in the brains of infected mice.