Circulating antibodies and antigens correlate ... - Wiley Online Library

TMIH466 Tropical Medicine and International Health volume 4 no 10 pp 691–694 october 1999

Circulating antibodies and antigens correlate with egg counts in human fascioliasis A.Y. Shehab1, L. M.Abou Basha1, H. N. El-Morshedy2, M.Abdel-Fattah3, M. M. Osman1 and H. F. Farag1 1 Department of Parasitology, Medical Research Institute, University of Alexandria, Egypt 2 Department of Tropical Health, High Institute of Public Health, University of Alexandria, Egypt 3 Department of Medical Statistics, Medical Research Institute, University of Alexandria, Egypt

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.

keywords human fascioliasis, egg counts, circulating antigens, antibodies correspondence A.Y. Shehab, Department of Parasitology, Medical Research Institute, 165 El Horreya Avenue, El-Hadara, Alexandria, Egypt. Fax: 1 203 35 80 384

Introduction 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 8–40 8C 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

© 1999 Blackwell Science Ltd

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 – 208C 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 mg/ml from all 691

Tropical Medicine and International Health A. Y. Shehab et al.

volume 4 no 10 pp 691–694 october 1999

Antibodies and antigens in human fascioliasis

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).

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. 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).

Statistical analysis 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 5), those with egg counts ranging between the 25th and 75th percentiles (2nd and 3rd quartiles) as moderate (n 5 10), and those above the 75th percentile (4th quartile) as severe (n 5 5). The correlation between egg counts and the levels of specific antibodies and circulating antigen was calculated using the Spearman correlation coefficient.

Results 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

IgG ELISA reading Ab to somatic FI Ab to somatic FII Ab to E/S IELISA Circulating E/S antigen

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Correlation studies 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.

Discussion 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

Range for controls

Cut-off level

Range for Fasciola cases

Positives ————— No. %

00.219–0.426 00.184–0.421 00.177–0.434

00.431 00.411 00.464

00.448–0.964 00.247–0.685 00.513–1.160

20 09 20

100 045 100

16.7–22.5

30% 21.8–45.4 inhibition

14

070

Table 1 IgG antibodies and circulating E/S antigen in 20 chronic fascioliasis cases


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A. Y. Shehab et al. Antibodies and antigens in human fascioliasis

Table 2 Relation between intensity of Fasciola infection and antibody and antigen levels Intensity of infection Ab to F I: Range Mean 6 2SE Positives No. % Ab to F II: Range Mean 6 2SE Positives No. % Ab to E/S: Range Mean 6 2SE Positives No. % E/S antigen: Range of inhibition Mean 6 2SE Positives No. %

Egg counts/g stools —————————————————————— #72 96–#196 $216 light (5)** moderate (10)** heavy (5)**

000.448–0.609 000.52 6 0.06

000.574–0.762 000.67 6 0.04

000.565–0.964 000.76 6 0.14

005 100

010 100

005 100

000.247–0.324 000.28 6 0.02

000.250–0.608 000.43 6 0.08

000.313–0.685 000.54 6 0.12

0 0

005 050

004 080

000.513–0.886 000.68 6 0.14

000.649–1.160 000.75 6 0.14

000.694–1.135 000.92 6 0.18

5 100

010 100

005 100

021.8–31.1 026.2 6 3.02

023.6–45.4 034.62 6 4.30

032.4–41.5 036.64 6 4.02

1 20

008 080

005 100

r

P

0.696

0.001

0.640

0.002

0.478

0.003

0.579

0.007

*Spearman correlation coefficient; **Number of patients.

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.


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