Enzyme-linked immunosorbent assay for diagnosis of Amphimerus spp. liver fluke infection in Humans

BACKGROUND Amphimerus spp. is a liver fluke that infects humans and domestic animals. It is highly prevalent in some Ecuadorian communities. Currently, diagnosis is based on the microscopic observation of eggs in faeces, but this has variable sensitivity. More sensitive methods are needed for diagnostic testing. OBJECTIVE The main objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) using crude antigens from Amphimerus spp. adult worms to detect anti-Amphimerus IgG in human sera. METHODS Crude somatic antigens were obtained from adult Amphimerus spp. worms. Human sera from 119 patients were tested: 48 from individuals with a confirmed Amphimerus spp. infection, 78 from non-infected Ecuadorians living in the endemic region, 60 from persons living in non-endemic areas (20 Ecuadorians, 20 Europeans, and 20 Africans), and 33 who had other parasitic and non-parasitic infections. PRINCIPAL FINDINGS Results were analysed using the receiver-operator characteristic (ROC) curve analysis with an area under curve (AUC) value of 0.967. The accuracy of the ELISA was high. The sensitivity was 85.0% [95% confidence interval (CI): 80.3-89.7%] and the specificity was 71.0% (95% CI: 65.2-76.8%). Some cross reactivity was detected against Paragonimus mexicanus, Fasciola hepatica, Schistosomiasis, Taenia solium, Strongyloides stercoralis, Mansonella spp., and Vampirolepis nana. MAIN CONCLUSIONS We have developed the first ELISA technique that detects anti-Amphimerus IgG in human sera with good sensitivity, repeatability and reproducibility. However, more specific antigens are needed to further enhance performance of this assay. Regardless, this ELISA test could be useful for early diagnosis and prompt treatment of human Amphimerus spp. infections.

Enzyme-linked immunosorbent assay for diagnosis of Amphimerus spp. liver fluke infection in Humans.

BACKGROUND: Amphimerus spp. is a liver fluke that infects humans and domestic animals. It is highly prevalent in some Ecuadorian communities. Currently, diagnosis is based on the microscopic observation of eggs in faeces, but this has variable sensitivity. More sensitive methods are needed for diagnostic testing. OBJECTIVE: The main objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) using crude antigens from Amphimerus spp. adult worms to detect anti-Amphimerus IgG in human sera. METHODS: Crude somatic antigens were obtained from adult Amphimerus spp. worms. Human sera from 119 patients were tested: 48 from individuals with a confirmed Amphimerus spp. infection, 78 from non-infected Ecuadorians living in the endemic region, 60 from persons living in non-endemic areas (20 Ecuadorians, 20 Europeans, and 20 Africans), and 33 who had other parasitic and non-parasitic infections. PRINCIPAL FINDINGS: Results were analysed using the receiver-operator characteristic (ROC) curve analysis with an area under curve (AUC) value of 0.967. The accuracy of the ELISA was high. The sensitivity was 85.0% [95% confidence interval (CI): 80.3-89.7%] and the specificity was 71.0% (95% CI: 65.2-76.8%). Some cross reactivity was detected against Paragonimus mexicanus, Fasciola hepatica, Schistosomiasis, Taenia solium, Strongyloides stercoralis, Mansonella spp., and Vampirolepis nana. MAIN CONCLUSIONS: We have developed the first ELISA technique that detects anti-Amphimerus IgG in human sera with good sensitivity, repeatability and reproducibility. However, more specific antigens are needed to further enhance performance of this assay. Regardless, this ELISA test could be useful for early diagnosis and prompt treatment of human Amphimerus spp. infections.

A sero-epidemiological survey on the occurrence of opisthorchiid liver flukes in red foxes ( Vulpes vulpes) in Berlin, Germany.

Serum samples collected from red foxes in the city of Berlin between 1996 and 1999 were analysed for the presence of antibodies against Opisthorchis felineus and Metorchis bilis using an indirect ELISA. Out of 1,000 specimens, 30.6% and 46.5% reacted positively with specific O. felineus and M. bilis antigens, respectively. Seroprevalence in adult foxes was always higher than in juveniles. While no significant differences were observed in adult foxes throughout the period, in juvenile specimens seroprevalence declined from 1996 to 1997, then stayed at a comparable level in 1998 and increased in 1999. A varying availability of fresh cyprinid fish in different years seems to be the reason for changes in seroprevalence. By grouping the samples from juvenile foxes by season, antibodies against both Metorchis and Opisthorchis antigens started to appear between April and June, increased between July and September()and reached a level comparable to adult foxes in the October to December quarter. The lowest seroprevalence was found in Pankow, which is the district with the lowest share of the surface water.

Detection of antibodies in sera from patients with Opisthorchiasis.

The indirect immunofluorescent antibody technique (IFA) was used for detection of antibodies in sera of patients with Opisthorchiasis. Antibodies to fluke worm and egg antigens were detected in 166 of 205 (81%) patients. The test showed that only the IgG class of antibodies reacting exclusively with integumental wall of the worm (AW) were positive in 46.8% (96/205), reacting only with the wall of intact eggs in 11 out of 205 (5.4%) and antibodies to both fluke and their egg antigens were present in 28.8% (59/205). In addition, 5.4% (11/205) of patients' sera were positive for autoantibodies producing a speckled antinuclear antibodies (ANA) pattern. The sera positive for only AW contained detectable autoantibodies to other cell antigens including: anti-smooth muscle antibodies of 9.4% (9/96), antimitochondrial antibodies of 3.1% (3/96), anti-liver/kidney microsomes of 1% (1/96) and anti-parietal cell antibodies of 1% (1/96). Autoantibodies were undetectable in sera from normal subjects. Among the ANA positive sera, 55% (6/11) exhibited antibodies against an extractable nuclear antigen (ENA) by a tanned red cell hemagglutination assay. This finding may suggest that the autoantibody response was due to the cross reaction between worm antigen and self antigen or it may be the result of polyclonal activation of B lymphocytes in these patients.