Opisthorchis viverrini, a food-borne trematode parasite endemic in the lower Mekong countries, is conventionally diagnosed by stool examination. However, parasitological stool-based diagnosis can be unreliable in light infections. The goal of this study was to develop the immunodiagnosis of opisthorchiasis using cathepsin F cysteine protease of O. viverrini in both indirect and sandwich ELISA assays. A recombinant O. viverrini cathepsin F (rOv-CF) of 40 kDa was expressed in E. coli strain BL21 (DE3), affinity purified, and deployed in ELISA assays. Human sera from 272 cases were investigated by indirect rOv-CF based-ELISA. Positive antibody response to rOv-CF was found in 137 out of 272 cases (50.37%) using a cut off OD (0.400) determined by ROC analysis. In comparison to parasitological stool examined for fluke eggs, the gold standard, the rOv-CF indirect ELISA showed a sensitivity and specificity of 62.1% and 84.05%, respectively. Serum antibody levels correlated well with egg counts per gram feces (EPG) (P < 0.001). In addition chicken IgY antibody was raised against rOv-CF was tested in a sandwich ELISA for detection of coproantigen in the feces of experimentally infected hamsters. The sandwich ELISA using this chicken IgY in combination with rabbit antibody to O. viverrini somatic antigens showed sensitivity and specificity of 93.3% and 78.57%, respectively. Together these findings indicated the potential of rOv-CF for diagnosis of opisthorchiasis, including for uses with chicken IgY for detection of coproantigens of O. viverrini.
Diagnosis of Opisthorchis viverrini infection by conventional stool examination is increasingly difficult due to the low intensity of the infection after several rounds of control programmes in endemic regions as well as coinfections with intestinal flukes. Therefore sensitive and specific diagnostic test is needed. In this study, a coproantigen sandwich ELISA using recombinant O. viverrini cathepsin F (rOv-CF) was developed. This sandwich ELISA employing chicken IgY raised against rOv-CF in combination with rabbit IgG antibody to the somatic O. viverrini antigens showed a lower detection limit (LLD) of 70 ng native O. viverrini somatic antigens by spiking the parasite antigens into control feces. When applied to the diagnosis, the IgY-based sandwich ELISA exhibited sensitivity and specificity of 93.3% and 76.7%, respectively, in an investigation of 90 human cases positive or negative for opisthorchiasis. The positive predictive value (PPV) and negative predictive value (NPV) for this coproantigen detection were 66.7% and 95.2%, respectively. This IgY-based sandwich ELISA using parasite cathepsin F detection shows a promising immunodiagnostic alternative for human opisthorchiasis in endemic regions.
Opisthorchiasis caused by Opisthorchis viverrini is a major food-borne zoonosis in Greater Mekong sub-region. Even though campaigns discouraging the consumption of raw fish have been launched to public, the disease still remains highly endemic. The unsuccessful eradication of the disease is probably because of the persistence of the parasite in animal reservoir hosts, particularly felids. Praziquantel (PZQ) is the drug of choice for morbidity control of opisthorchiasis in humans and animals. However, there is no specific study on its dosage regimen for feline opisthorchiasis. Thus, the effective treatment dose of PZQ, as well as its adverse effects, was evaluated in O. viverrini infected cats. Twenty-eight infected male and female cats from the endemic area of Khon Kaen and Mahasarakham Provinces, Thailand were enrolled in this study. Physical, hematological, blood chemical and urine examinations were analyzed, as indicators of health status, on the day before and 30 days after treatment. Intensity of the infections was determined by the formalin-ethyl acetate sedimentation technique. Cats were equally allotted into the low infection group of 14 cats with egg count per gram of feces (EPG) less than 300 and the high infection group of 14 cats with EPG higher than 300. Cats in each group were equally divided into two subgroups of 7 cats; thus, there were two low infection subgroups (L1 and L2 subgroups) and two high infection subgroups (H1 and H2 subgroups). A single dose of 25 mg/kg PZQ was orally administered to each cat in the L1 and H1 subgroups and a single oral dose of 40 mg/kg PZQ was administered to the L2 and H2 subgroups. Complete clearance of O. viverrini eggs was found in all cats in the L1, L2 and H2 subgroups; thus, the cure rate (CR) and egg reduction rate (ERR) were 100%. However, partial clearance was observed in two cats with high EPG (1,502 and 1,518) in the H1 subgroup, which received 25 mg/kg PZQ. Regards, CR and ERR for these two animals was 71.4 and 99.5%. No significant difference among the 4 subgroups was seen. Almost all hematological, blood chemical and urinalysis data were within normal ranges, except for the eosinophilia and an increase of alanine aminotransferase (ALT). Hookworm infection seen in all cats would cause eosinophilia. As for drug safety, there was no side effect observed in any cats. In conclusion, this study suggested that 40 mg/kg PZQ is a highly effective and safe dosage for the treatment of feline reservoir hosts of human opisthorchiasis.
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