Serum pretreatment with Schistosoma mansoni antigens for serological diagnosis of fascioliasis.

Cross-reactivity between Fasciola and Schistosoma often causes false positive results in serological assays for diagnosis of fascioliasis. The authors tried to reduce cross-reactivity in ELISA for diagnosis of fascioliasis by preincubation of serum samples with a mixture of Schistosoma mansoni adult worm and egg antigens. This method was evaluated using serum samples from 4 groups: 25 patients infected with Fasciola, 40 healthy controls, 113 patients infected with S. mansoni and a group of 100 patients with suspected Fasciola infection. In group with confirmed Fasciola infection, the sensitivity of ELISA was 96% without any change after serum pretreatment while, in control group, the specificity was elevated from 90% to 97.5% after serum pretreatment with S. mansoni antigens. In S. mansoni infection and suspected Fasciola infection groups, there was a highly significant reduction in number of ELISA positive cases after serum pretreatment with S. mansoni antigens (McNemar P < 0.001 for each). In logistic regression model, seroconversion showed significant dependence on presence of S. mansoni infection (P = 0.012). The probability of seroconversion was more than three times higher in S. mansoni infected individuals than in non-infected ones (Odds ratio = 3.5).

Oral immunization of mice against Schistosoma mansoni using drinking water from trays containing Biomphalaria alexandrina infected with Schistosoma mansoni.

Water collected from trays containing Biomphalaria alexandrina infected with Schistosoma mansoni at the time of cercariae shedding (SmISW) and trays containing clean, non-infected, B. alexandrina (NISW) and underground water (UW), were filtered used as a drinking water for 3 groups of albino mice males. After two months, blood samples were collected from the 3 groups and serum was tested for anti-cercarial IgG, then mice were infected with 150 S. mansoni cercariae. Eight weeks after infection, mice were perfused and adult S. mansoni worms were counted. Anti-cercarial IgG was positive in 23 (82.1%) out of the 28 samples collected from mice drinking SmISW and only in 2 (9.5%) out of the 21 samples collected from mice drinking NISW, while all samples collected from mice drinking UW were negative for anti-cercarial IgG (X2=45.897; P<0.001). Worm load was significantly lower in the group of mice drinking SmISW than mice drinking NISW (P=0.032) and mice drinking UW (P=0.02). In mice drinking SmISW, adult worm count showed significant negative correlation with anti-cercarial IgG concentration (Kendall's taub =-0.325 and P=0.018). The results indicate that antigens present in drinking water stimulate a level of immunity against schistosomiasis, (inhabitants of endemic areas) resulting in a lower intensity and severity of infection. Also, it may reduce the specificity of serological tests used for diagnosis of Schistosoma infection, based on antibody determination.

Quantitative inhibition ELISA for diagnosis of schistosomiasis using human IgG labeled with fluorescein and anti-fluorescein/peroxidase.

Usually mouse monoclonal antibodies are used in inhibition assays for antibody determination. Interference may occur in these inhibition assays due to presence of naturally occurring anti-mouse antibodies in some human serum samples. To avoid such interference, human IgG isolated from a pool of serum samples of S. mansoni patients and highly positive for IgG against S. mansoni soluble egg antigen (SEA) was used in inhibition ELISA for diagnosis of S. mansoni infection. The assay was based on inhibition of binding of human IgG labeled with fluorescein to S. mansoni SEA coating microtitration plates by tested serum samples. Plates were washed and labeled human IgG reacted with SEA was linked to peroxidase enzyme by incubation with anti-fluorescein/peroxidase conjugate. The assay showed 90% sensitivity and 96.3% specificity. The level of inhibition in ELISA showed highly significant positive correlation with stool egg output (Kandall's tau b = 0.512, P < 0.001). To make the assay quantitative, serial dilutions of the highly positive human serum pool, used for preparation of human IgG, were applied in each plate and concentration of anti-SEA antibodies in serum samples tested was calculated from a 4-parameters logistic curve equation. The highly positive serum pool used as a standard was considered to contain one million arbitrary units of immunoglobulins against S. mansoni SEA. Human IgG is expected to be more practical in inhibition assays than mouse monoclonal antibodies due to elimination of interference caused by naturally occurring human anti-mouse antibodies. Also, large amount of human IgG could be purfied from remnants of serum samples highly positive for the proposed antibodies. A higher specificity and sensitivity could be obtained if IgG is isolated by affinity purification instead of ammonium sulphate precipitation. In conclusion, human IgG isolated from highly positive serum samples could be used in sensitive and specific diagnostic antibody determination inhibition assays for diagnosis of infectious and autoimmune diseases.

Opportunistic intestinal protozoal infections in immunocompromised children.

Hundred immunocompromised children and 100 house contact controls were chosen. Patients included: 52 nephrotic syndrome children receiving corticosteroids for more than one month (age 5.28 +/- 2.32 years), 14 protein-calorie malnutrition (PCM) patients (8 cases of marasmus aged 6 +/- 2.27 months and 6 cases of marasmic kwashiorkor aged 1.39 +/- 0.88 years) and 34 lymphomas patients (22 cases of Hodgkin's disease and 12 cases of non-Hodgkin's lymphoma; age 4.5 +/- 3.54 years). Examination of concentrated stool was done using iodine stain of fresh mounts and modified Ziehl-Neelsen (cold acid-fast) to fixed smears. T-cell subsets were counted after staining with mouse monoclonal antibodies against CD4 and CD8 labeled with fluorescein. Both nephrotic syndrome and lymphomas groups showed affection of cellular immunity in the form of significant decrease in T-helper and H/S ratio and significant increase in suppressor T-cell subsets. Giardia lamblia, Entamoeba histolytica, Cryptosporidium parvum and Blastocystis hominis were the most frequent in patients group and were significantly more prevalent among patients than controls. No significant difference in the prevalence of Entamoeba coli and Chylomastix mesnili between the two groups. C. parvum infection were strictly confined to groups with T-cell subsets abnormalities i.e. nephrotic syndrome and lymphomas groups.

Anti-schistosomal IgE and its relation to gastrointestinal allergy in breast-fed infants of Schistosoma mansoni infected mothers.

To study the relationship between presence of gastrointestinal allergic manifestations in breast-fed infants and presence of IgE against Schistosoma mansoni antigens, sixty breast-fed infants of S. mansoni infected mothers were selected. Of them, thirty infants were suffering from manifestations of gastrointestinal allergy (patients) and the other thirty were not suffering from such manifestations (controls). Levels of IgE against S. mansoni adult worm antigen (AWA), soluble egg antigen (SEA) and cercarial antigen (CA) were determined, by ELISA, in sera of these infants. There was significant association between presence of allergic manifestations and presence of IgE against AWA (P = 0.018), SEA (P < 0.001) and CA (P = 0.002). Also, concentration of IgE against AWA was significantly higher in patients group than the control group (P = 0.024). IgE against AWA showed significant negative correlation with haemoglobin concentration (P = 0.009) and serum albumin level (P = 021) and significant positive correlation with absolute eosinophilic count (P = 0.005). Also, IgE against CA showed significant negative correlation with haemoglobin concentration (p = 0.047) and serum albumin level (0 = 0.036). It was concluded that gastrointestinal allergy in breast-fed infants of S. mansoni infected mothers may be due to hypersensitivity of Schistosoma mansoni antigens present in mothers' milk. Schistosoma mansoni should be investigated and treated in mothers from endemic localities when their breast-fed infants are suffering from manifestations suggestive of gastrointestinal allergy.