Circulating antibodies and antigens correlate with egg counts in human fascioliasis
correspondence A.Y. Shehab, Department of Parasitology, Medical Research Institute, 165 El Horreya Avenue, El-Hadara, Alexandria, Egypt. Fax: + 203 35 80 384
Summary We explored the relationships between specific IgG antibody levels and circulating E/S antigen to intensity of Fasciola infection in the human host. Twenty patients with patent infection and six healthy individuals were enrolled in the study. Intensity of infection was determined by repeated egg counts in stools, while IgG antibodies against adult Fasciola gigantica somatic FI, FII and against E/S antigens were measured as ELISA O.D. readings. The level of circulating E/S antigens was determined by IELISA. Positivity as well as levels of antibodies and antigen correlated with infection intensity. These findings may disclose in the future a relation between morbidity in the acute phase and worm load.
In the acute stage of infection, some cases of human fascioliasis present with severe manifestations of pain in the right hypochondrium, high fever of 39 °–40 °C and severe anaemia as low as 4–5 g/dl ( Ragab & Farag 1978; Bendezu et al. 1982 ; Farag et al. 1988 ; Ariona et al. 1995 ). One of the factors responsible for these complications may be a high infection intensity. However, so far there is no way to measure this during the acute stage.
In chronic infection, egg counts are considered the measure of worm load despite various factors that can influence the number of eggs in stools, such as irregular oviposition by the worms and variation in bile flow of the host ( Levine et al. 1980 ; Maklad et al. 1988 ). We tried to explore whether any relationship exists between egg counts and the level of specific antibodies and/or the level of circulating E/S antigens. This may offer in the future a possible explanation for the severe morbidity in some cases of fascioliasis during the acute phase.
Patients, material and methods
Twenty patients with patent fascioliasis were enrolled in the study. They were free from any other parasite as diagnosed by repeated stool examinations. Six healthy parasite-free individuals served as controls. The following investigations were performed:
Stool examination and Fasciola egg counts
Three specimens were obtained from every patient on different days. They were examined by the Kato Katz technique, two slides (41.7 g each) for every specimen were examined microscopically, and the mean of the six egg counts was calculated.
Determination of Fasciola antibody level
Blood was collected from each patient and from the controls. Serum was separated and stored at – 20°C until used. ELISA was used to determine the level of IgG specific antibodies for somatic and E/S antigens. Fasciola gigantica somatic antigen was prepared and fractionated by Sephadex G-75 according to Osman et al. (1992) and peaks I and II were obtained. E/S antigen was obtained from adult worms following the method of Rivera et al. (1988) . The protein content of the three antigens was determined by the method of Lowry et al. (1951) . IgG ELISA was then performed using the three antigens as described by Voller et al. (1975) . 5 μg/ml from all antigens were fixed to the plates and sera were used in a dilution of 1:100. Cut-off levels were determined as the mean O.D. readings of control sera 2 SD.
Detection and determination of circulating E/S antigen
Circulating E/S antigen was detected in the sera using inhibition ELISA as described by Abdel-Hafez et al. (1983) . The percent inhibition was an indicator of the level of antigen in the serum. Cut-off levels were determined according to the findings of De Mol et al. (1985) .
Intensity of infection was classified as light, moderate and heavy using the quartile split. Patients with egg counts less than the 25th percentile (1st quartile) were classified as light (n = 5), those with egg counts ranging between the 25th and 75th percentiles (2nd and 3rd quartiles) as moderate (n = 10), and those above the 75th percentile (4th quartile) as severe (n = 5). The correlation between egg counts and the levels of specific antibodies and circulating antigen was calculated using the Spearman correlation coefficient.
Intensity of infection
In the 20 patients, mean Fasciola egg counts ranged between 48 and 624 eggs/g of stools (EPG) Based on the quartile percentile, intensity was considered light at ≤72 EPG, moderate at 96-≤196 EPG and heavy at ≥216 EPG.
ELISA O.D. readings for Fasciola antibodies
IgG antibodies against somatic fraction I ranged between O.D 0.448 and 0.964. The cut-off level was 0.431, and accordingly all cases were positive. ELISA readings for somatic fraction II varied between 0.247 and 0.618. The cut off-level was 0.411; only 45% of cases had positive readings. For E/S antibodies, ELISA values ranged from 0.513 to 1.160. The cut-off value was 0.464 and all cases were positive. Results are presented in Table 1.
IgG antibodies and circulating E/S antigen in 20 chronic fascioliasis cases
Detection of circulating E/S antigen by IELISA
The cut-off level was set at 30% inhibition. Inhibition values for Fasciola cases ranged between 21.8 and 45.4%; 30% of cases were found negative for antigen, most being light infections ( Table 1).
Results of correlation analysis are presented in Table 2. IgG antibodies against all antigens tested as well as circulating antigen correlated positively with infection intensity.
Relation between intensity of Fasciola infection and antibody and antigen levels
In established fascioliasis, the detection of ova in stools is a sure method of diagnosis. Repeated stool examinations are usually necessary to obtain reliable results of the intensity of infection and to overcome the effect of the factors that may influence the number of excreted ova ( Levine et al. 1980 ; Maklad et al. 1988 ). Serological diagnosis of chronic fascioliasis is known to give false negative results in some cases; similarly failure to detect circulating antigens in chronic patients is reported ( Knobloch 1985). We investigated the intensity of Fasciola infection by repeated egg counts in different stool specimens and explored the relationship, if any, between worm load and IgG antibodies against somatic antigens FI, FII, E/S antigen and circulating E/S antigen.
All cases revealed positive IgG levels against somatic antigen FI and E/S antigens. Antibodies against FII were detected in only 5%; with this antigenic fraction, negativity was observed in all cases with low intensity, and positivity increased gradually in moderate and heavy infections. With all antigens the level of IgG antibodies correlated with infection intensity.
Salem et al. (1987) did not find a relationship between total IgG and intensity of fascioliasis. In a study on schistosomiasis Mott & Dixon (1982) reported that assays measuring egg antibodies revealed a correlation between antibody level and worm burden. Similarly Barakat et al. (1983) reported a positive relationship between IgG antibody levels as measured by ELISA using SEA and egg counts.
Circulating E/S antigens were detected in 70% of sera of chronic cases, while they were absent in cases of light infection. The level of antigen in positive cases correlated with infection intensity. Deelder et al. (1989) reported a strong positive correlation between circulating antigens and worm burden in schistosomiasis.
In conclusion, our findings point to a relation between intensity of Fasciola infection and specific IgG antibody level and circulating E/S antigens. The possibility that such a relationship exists in the acute stage of infection, during which all antigens pass to the circulation, needs further investigation.