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.
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.
When C57BL/6 mice were infected with Angiostrongylus cantonensis, the percentage of T helper (CD4+) cells and T supressor (CD8+) cells in peripheral blood increased weekly until the third and seventh week respectively, and then gradually decreased. C57BL/6 mice were depleted of CD4+ and CD8+ T cells by in vivo injection of anti-CD4 and anti-CD8 monoclonal antibodies, respectively, and then infected with A. cantonensis. There were significantly more and less worms recovered in the mice depleted of CD4+ and CD8+ T cells respectively than in undepleted mice. Discrete subpopulations of T cells from mice exposed to A. cantonensis for 3 weeks or 7 weeks were adoptively transferred to syngeneic recipients which were then given a challenge infection. Protection was mediated by a CD4+ T cell population present in mice after 3 weeks of infection but was not demonstrable with cells taken 7 weeks after infection. When CD4+ T cells obtained from 3-week infected mice were mixed with 5% CD8+ T cells obtained from mice infected for 7 weeks, no significant transfer of resistance was observed. Thus, immune responses to A. cantonensis in mice were regulated by discrete subpopulations of T lymphocytes.
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.
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