Production of monoclonal antibodies against target schistosomal antigen secreted in the urine of Schistosoma mansoni-infected patients.

This study was undertaken to develop an immunodiagnostic test of active human schistosomiasis mansoni using a monoclonal antibody which targets urinary schistosomal antigen. Polyclonal antisera raised in rabbits against the processed urine of Schistosoma mansoni-infected patients showed very high and significant reactivity with ES product of ova compared with other different S. mansoni antigens. The monoclonal antibody (4.23) was reactive with repetitive epitopes of S. mansoni soluble egg antigen and ES product of ova with molecular mass range of 65-23 kDa and 80-23 kDa, respectively. It recognised different stages of the parasite life-cycle, with no cross reaction with Fasciola or hydatid antigen. MAbs were characterised by isotyping, immunoelectrophoresis, SDS-PAGE and the enzyme-linked immunoelectrotransfer blot technique, ELISA, and their recognition of carbohydrate or protein antigenic epitopes by periodate oxidation and trichloroacetic acid treatment of the antigen. It was used for detection of circulating schistosomal antigen in an antigen capture antibody sandwich ELISA on sera and urines of 58 S. mansoni-infected patients, 17 S. haematobium-infected patients, 15 parasite-free negative healthy controls and sera from 13 schistosomiasis-free patients harbouring Fasciola or hydatid infections. The percentage sensitivity of the assay in the serum of S. mansoni-infected patients was 98.4% and in urine 94.8%. A positive correlation was found between the number of faecal S. mansoni eggs and the circulating antigen, both in serum and in urine. Antigen circulating in urine correlated with that in the sera of S. mansoni patients. These data provide a sensitive and non-invasive method almost comparable with the use of sera for immunodiagnosis of schistosomiasis and an indirect way to reflect the intensity of infection.

A monoclonal antibody diagnoses active Fasciola infection in humans.

Monoclonal antibodies (MABs) were produced after fusion of spleen cells of Fasciola antigen immunized BALB/c mice and non secreting murine myloma cells (P3x63 Ag8). Six MAbs, showing the highest reactivity with purified Fasciola antigen, were prepared. All 6 MABs were IgG2 with Kappa light chain. Reactive epitopes recognized by the six MAbs were glycoprotein in nature, and each MAb recognized a single epitope of Fasciola antigen. No cros reactions were observed with Schistosomal AWSA, hydatid Ag and Entamoeba Ag. EITB technique showed a specific diagnostic band at 17.5 kDa for each of the six MAbs. Anti-Fasciola MAb (AD2) was conjugated with peroxidase and was used with anti-rabbit anti-Fasciola polyclonal antibody in sandwich-ELISA to detect circulating Fasciola antigen in serum and urine samples of 57 fascioliasis patients, 51 schistosomiasis patients, 45 patients infected with other parasites and 47 healthy controls. Sensitivity of the assay in detection of circulating Fasciola antigen in sera and urines of Fasciola infected patients was 100%. The specificity of the assay was calculated among patients infected with schistosomiasis and other parasites and was 98% in serum and 97% in urine. A positive correlation was found between levels of circulating Fasciola antigen in serum and urine samples of fascioliasis patients (r = 0.825, p < 0.05).

Efficacy of an antipathology vaccine in murine schistosomiasis administered with and without chemotherapy.

This study was undertaken to study the efficacy of praziquantel (PZQ) in potentially tolerized Schistosoma mansoni infected, egg-injected C57BL/6 mice, receiving multiple administrations of soluble egg antigen (SEA) intravenously (i.v.). Four animal groups were studied. Experimental group I received four injections of SEA (10 micrograms) intravenously on days -7, -5, -3 and -2 before infection and PZQ orally (500 mg/kg over two consecutive days 7 weeks post-infection. Three control groups received the following treatment: group II received the same tolerizing dose of SEA without PZQ, group III received PZQ in the same dose and at the same timing. Group IV received S. mansoni infection and egg injection 8 weeks post-infection and served as an infected, egg-injected control. Egg injection was conducted 8 weeks post-infection using viable S. mansoni eggs via the tail vein. Animals were killed 16 days post-egg injection, i.e. 10 weeks post-infection. After sacrifice, lungs and livers were removed for histopathological study and measurement of granuloma diameters. Spleens and serum were collected for the assay of lymphoproliferative response to SEA and antischistosomal immunoglobulins. The worm and egg burdens were also studied. Compared to infected, egg-injected untreated controls, repeated i.v. administrations of SEA down-regulated egg-injected (pulmonary) and egg-deposited (hepatic) granulomas and the lymphoproliferative response to SEA. Antischistosomal IgG level was increased. Susceptibility to S. mansoni infection was not found to be different from that in the infected, egg-injected controls. PZQ in the dose used caused complete eradication of worms, disappearance of immature egg stages, decrease in the number of mature eggs and an increase in the number of dead eggs. Hepatic granuloma diameter, lymphoproliferative response to SEA and IgG level were reduced. In mice receiving a combined regimen of multiple SEA administrations and PZQ with down-regulated granuloma and reduced lymphoproliferative response to SEA, the efficacy of PZQ was the same as in mice receiving PZQ alone. This was shown by comparable grades of worm and egg reduction. The histopathological examination of liver and lung sections in the different treated groups revealed moderate to small-sized hypocellular granulomas. Although no statistically significant difference was recorded between the mean granuloma diameters of the experimental group receiving both the tolerizing dose of SEA and PZQ compared to the group receiving the tolerizing dose of SEA without chemotherapy, this experimental group showed the least associated histopathological parenchymal changes. It appears from this work that combined SEA and PZQ provided many complementary goals; a reduction of egg-induced pathology, minimal parenchymal changes and the eradication of worms.

Detection of circulating schistosome antigens in serum and urine of schistosomiasis patients and assessment of cure by a monoclonal antibody.

From a panel of monoclonal antibodies (MAb), an IgM monoclonal antibody (7F1/6B) reactive with repetitive epitopes on S. mansoni soluble egg antigen was selected. This MAb was employed both as antigen capture and detection antibody in a sandwich ELISA and had a detection limit < 1 ng S. mansoni SEA/mi. Serum and urine samples were collected from rural students who had S. mansoni (169 subjects) or mixed S. mansoni and S. haematobium (64 subjects) infections. Samples were collected before and at 4, 8 and 12 weeks after praziquantel therapy. Circulating schistosome antigens (CSA) were demonstrated in 90% of sera and 97% of urine samples of S. mansoni group and in 91% of sera and 100% of urine samples of mixed infection group. All sera from 29 uninfected individuals, 30 patients with other parasites and 70% of 55 S. haematobium-infected subjects were negative in this assay. CSA level in serum and urine samples correlated positively with the number of S. mansoni eggs/g stool in both groups. A significant reduction in CSA level was observed in serum and urine samples after praziquantel therapy. By 12 weeks post-treatment, negativity was 98% in sera and 97% in urine of S. mansoni-infected group and 98% in sera and 91% in urine of mixed infection group. The data demonstrate that the use of MAb 7F1/6B for the detection of CSA provides a sensitive method for immunodiagnosis of schistosomiasis and monitoring of cure.

Immunoregulatory potential of exogenous Schistosoma mansoni soluble egg antigen in a model of experimental schistosomiasis--I. Regulation of granuloma formation in vivo.

This study was undertaken to assess the optimum conditions required to reduce the vigorous host granulomatous reaction around Schistosoma mansoni eggs. Soluble schistosomal egg antigen (SEA) at a concentration of 10 or 100 micrograms protein was administered i.p. or i.v. into unprimed C57BL/6 mice. SEA was injected either alone or in combination with cyclophosphamide (CY) 100 or 50 mg/kg via i.p. route. Seven or 14 days later viable eggs of S. mansoni were injected via the tail vein into treated groups and untreated normal controls. Mice were sacrificed 8, 16 and 24 days after the injection of eggs. The lungs were removed for histopathological study, measurement of granuloma diameter and phenotypic analysis of granuloma intralesional T-cell subsets. Compared to untreated controls, the lower concentration of SEA (10 micrograms) administered by the i.v. route 7 days before egg injection, induced a significant reduction in granuloma diameter 16 days after egg injection than that by the i.p. route or at a higher SEA concentration (100 micrograms). Compared to untreated controls, the higher dose of CY (100 mg/kg), given i.p. alone or in combination with 10 micrograms SEA by the i.v. or i.p. route, induced a significant reduction in granuloma diameter, while 50 mg/kg CY did not cause any reduction. The reduction in granuloma diameter by i.v. administration of low SEA concentration alone or in combination with CY IP, was associated with a decrease in the granuloma intralesional L3T4+/Lyt2+ ratio. The decrease in the ratio was due to an increase in Lyt2+ cells. The results suggest that the use of low dose SEA by the i.v. route alone or combined with an immunosuppressive drug ameliorates pathological changes concurrent with S. mansoni infection.

Evaluation of specific Fasciola antigen in the immunodiagnosis of human fascioliasis in Egypt.

Enzyme-linked immunosorbent assay (ELISA) and enzyme linked immunoelectrotransfer blot technique (EITB) were employed for the detection of circulating Fasciola antibodies in infected human sera using a specific Fasciola antigen, prepared by immunoaffinity purification of homogenates of Fasciola hepatica adult worms. Ninety two individuals diagnosed clinically and parasitologically were classified into: Fascioliasis group (21 patients), schistosomiasis group (21 patients) and subjects harbouring other parasitic infections (50 patients). Eighteen healthy individuals served as normal controls. ELISA was 100% sensitive and 93% specific with 96.5% diagnostic efficacy, whereas EITB was 100% sensitive and specific with 100% diagnostic efficacy. Our data revealed that ELISA can be used as a good screening test while EITB can serve as a confirmatory test for immunodiagnosis of fascioliasis.

Use of Kato and nucleopore techniques for qualitative diagnosis of schistosomiasis.

A comparison on qualitative basis, is attempted between merthiolate-iodine-formaldehyde concentration (MIFC) and Kato thick smear techniques for diagnosis of schistosome eggs in stools. As well, the centrifugation-sedimentation method was compared with the Nucleopore filtration technique for schistosome eggs in urine. Using MIFC and Kato techniques, 149 out of 185 subjects were found to have Schistosoma mansoni infection, 41 of them were diagnosed by Kato alone, while no case was solely MIFC positive. The sensitivity of MIFC compared to kato was 72.3% and both techniques were 100% specific. For diagnosis of S. haematobium infection, 78 out of 103 subjects were positive by centrifugation- sedimentation and/or Nucleopore techniques. 42 of them were diagnosed by Nucleopore alone and none was positive by centrifugation- sedimentation only. The sensitivity of the latter technique was 46.2% and both techniques were 100% specific. The study demonstrates that Kato thick smear and Nucleopore filtration are highly sensitive techniques that can be used for routine qualitative diagnosis of schistosomiasis. Under field conditions, they are qualitatively and quantitatively useful. The Kato technique besides its high sensitivity is very cheap. The only limitation for the Nucleopore technique is its relative high expenses.

Purification and characterization of a specific Fasciola antigen.

Specific Fasciola antigen was prepared from homogenates of Fasciola hepatica adult worms. The homogenate was ultracentrifuged and the supernatant containing crude Fasciola antigen was then passed over a cyanogen bromide activated sepharose 4B column coupled with antiserum against Schistosoma mansoni adult worm surface antigen. The specific, Schistosoma-free Fasciola antigen was tested for its specificity by immunodiffusion. Characterization of the specific Fasciola antigen was done by gradient poly-acrylamide gel electrophoresis and immunoblotting technique. The electrophoresis migration pattern of specific Fasciola antigen, stained with Coomassie blue, showed 7 bands in the 12-54 kDa regions. Using the immunoblotting technique, a batch of positive fascioliasis sera recognized two specific bands at the 33 and 54 kDa regions.

Antifilarial IgM versus IgG antibody determination in the diagnosis of Wuchereria bancrofti infection in Egyptians.

Circulating antifilarial IgM and IgG antibodies were assessed by indirect ELISA in 184 serum specimens from 80 patients with clinically and parasitologically diagnosed filarial infections (20 with acute filariasis 40 with chronic filariasis & 20 asymptomatic microfilaraemic subjects), 64 individuals with other parasitic infections, 20 parasitologically-free subjects from filariasis endemic areas and 20 normal healthy controls. A soluble surface membrane extract from Dirofilaria immitis worms was used as the antigen. Using a single serum dilution of 1:128 and optical densities (OD) at 492 nm, the respective cut off values for IgM and IgG were found to be 0.24 and 0.22. All healthy non-endemic controls were seronegative by IgM and IgG ELISAs. The highest antifilarial IgM OD492 values were obtained in 20 patients with acute filariasis (95% sensitivity), while the highest antifilarial IgG OD492 values were observed n 40 patients with chronic filariaisis (97.5% sensitivity). Asymptomatic microfilaraemic subjects gave IgM and IgG OD492 values which were significantly lower than those of other forms of clinical disease and endemic control subjects. The antifilarial IgM and IgG respective sensitivities in asymptomatic subjects were 75% and 70%. Endemic controls had positive antifilarial IgM (65%) and IgG (75%) levels. Of 64 subjects with other parasites only one with Ancylostoma duodenale had positive IgM level (98.4% specificity); while 9 patients with nematodal infections mainly had false positive antifilarial IgG antibody levels (85.9% specificity). These results suggest that measuring circulating antifilarial IgM antibody level may have some diagnostic advantage over measuring IgG antibody level for the detection of active filarial infection and consequently better management of the disease.

Evaluation of different immunodiagnostic techniques for diagnosis of hydatidosis in Egypt.

In this study detection of the circulating hydatid antibodies was performed by three serological tests: Immunoelectrophoresis (IEP), Enzyme linked immunosorbent assay (ELISA) and Enzyme linked immunoelectrotransfer blot (EITB) technique. The antigen which was used in these tests was Echinococcus granulosus antigen free of host (camel) antigen by immunoaffinity chromatography. Most serum samples were pre-selected from the patients on the basis of clinical syndrome or past history of possible diagnosis of echinococcosis. In addition, physical aids to diagnosis, especially sonographic and plain X-ray examination have already been carried out. The hydatidosis group was subgrouped according to the site of the cysts in different organs. The surgical removal of hydatid cysts was taken as a sure diagnosis of hydatid disease. The outcome of comparison between the three serological tests in surgically confirmed cases, showed that ELISA technique is a good screening test (88.2% sensitivity, 88.8% specificity and 88.5% diagnostic efficacy) and EITB is a good confirmatory test (100% sensitivity, 100% specificity and 100% diagnostic efficacy).

Identification and characterization of specific hydatid antigen fraction(s).

A specific hydatid antigen was prepared in this study from Echinococcus granulosus cyst in livers and lungs of camels. Elimination of host "camel" protein from crude hydatid fluid was achieved by two methods: Salting out using ammonium sulfate precipitation method and immunoaffinity purification using coupled anticamel antibody to cyanogenbromide activated sepharose 4B gel. Testing the prepared hydatid antigen against anticamel serum, using immunodiffusion method, indicated that the affinity purified hydatid antigen was almost completely purified from camel protein. Characterization of the affinity purified hydatid antigen, using immunoelectrophoresis, showed positive arc 5 precipitation when tested against known positive antihydatid sera. Further characterization with gradient gel electrophoresis, showed with silver stain that the dominant and most consistently demonstrable proteins occurred as a complex in the 52/62 KDa region. Strong reaction with the 52/62 KDa complex was consistently observed when the affinity purified hydatid antigen was probed with known positive reference antihydatid sera. The identified hydatid antigen fraction(s) with 52/62 KDa complex can provide promising non-invasive parameter for diagnosis of Hydatidosis.

Detection of circulating anodic antigen before and after specific chemotherapy in experimental murine Schistosomiasis mansoni.

In two groups of mice infected with 60 (group I) and 120 (group II) Schistosoma mansoni cercariae, respectively, the effects of intensity and duration of infection, and of praziquantel therapy (curative vs subcurative dose) on the levels of circulating anodic antigen (CAA), were studied. CAA was measured in trichloracetic acid-treated serum samples with an avidin-biotin enzyme-linked immunosorbent assay (AB-ELISA) using the monoclonal anti-CAA antibody. Total worm burdens, oogram patterns and ova counts/g liver and intestine were followed up. The lowest detectable level of CAA was about 1.0 ng/ml, and was positive with a worm load of 3-5/mouse. CAA levels became already detectable as early as 1-2 weeks post-infection (pi) before any parasitological parameter and showed a significant drop from the 11th-12th week pi onwards. A positive correlation was demonstrated between the CAA level and worm load. Following successful praziquantel therapy, CAA disappeared earlier than any of the other parameters studied.

Effect of specific chemotherapy on the immune response and resistance to reinfection in experimental Schistosomiasis mansoni.

Mice infected for 45 days with 120 Schistosoma mansoni cercariae and treated with praziquantel in a dose of 500 mg/kg for two consecutive days had a significant lower resistance to reinfection when challenged two weeks after treatment (45% compared to 88% in infected challenged untreated mice). In praziquantel-treated mice, the reduction in the per cent resistance was accompanied by a diminution in the size of hepatic granulomata and its in vivo correlate the delayed foot pad swelling. Moreover, the granuloma proportionate T-cell subset enumeration revealed a significant reduction in the number of T-helper cells. The humoral immune response as measured by the immediate foot pad swelling was not affected by praziquantel. Results reveal besides the diminution of the state in resistance to reinfection after praziquantel, possible involvement of egg-related pathology as a T-cell mediated reaction and as a mechanical obstacle in maintenance of this resistance.

Levamisole restored the compromised state of immunity after specific chemotherapy in experimental schistosomiasis mansoni.

Mice infected for 45 days with 120 Schistosoma mansoni cercariae and treated with levamisole (25 mg/kg subcutaneously) have more efficient acquired immunity when challenged with 240 Schistosoma mansoni cercariae the same day of treatment (97.7% # 87.7% in infected challenged controls). In praziquantel-treated mice (500 mg/kg for 2 days orally), the reduction in the percent resistance (45.5%) was accompanied by a significant diminution in the size of granuloma, delayed foot pad swelling and granuloma proportionate T-helper cells number. Levamisole when given two weeks post praziquantel treatment and with the challenge infection increased the percent resistance to 79.2%. The increase in percent resistance recorded in mice receiving both praziquantel and levamisole was accompanied by restoration of granuloma size, delayed foot pad swelling and granuloma proportionate T-helper cells number to infected challenged untreated control values. Results reveal-beside efficacy of levamisole as immunoregulant in schistosome immunity--a possible role for the granuloma as a T-cell mediated response in maintenance of immunity.

Association between HLA-A, B, C and DR antigens and clinical manifestations of Schistosoma haematobium in the bladder.

This study is a search for the genetic susceptibility of Egyptians to Schistosoma haematobium infestation with its various bladder complications, including cancer. 80 bilharzial patients, 20 with simple bilharzial bladder cystitis, 30 with bilharzial bladder lesions, and 30 with bilharzial bladder cancer, as well as 35 normal Egyptian controls were studied. All patients were typed for HLA-A, B, C and DR antigens using the microlymphocytotoxicity test. HLA-A9 and its split Aw24 antigens were found to be negatively associated with the disease. As for the antigens with positive associations, HLA-B7 was significantly increased in the simple bilharzial cystitis group. In the bilharzial bladder cancer group, HLA-B16 and Cw2 antigens had positive associations. These findings might support the genetic control of the disease or the presence of an immune response and/or immune suppression genes which are in linkage disequilibrium with these HLA antigens and they control the susceptibility and pathological sequences of the disease.

Effect of praziquantel on certain immune responses of schistosomal Egyptian patients. I. Changes of specific immunoglobulins.

Specific antischistosomal IgG, IgM, and IgE were estimated by ELISA in 117 rural school students before specific treatment with praziquantel monthly for 3-4 months thereafter. IgG and IgM were estimated as percentage of bound antibodies. IgE was estimated by avidin-biotin ELISA (AB-ELISA) as IU/ml using a panel of known IgE standards. Soluble surface Schistosoma mansoni adult worm antigen was used for all estimates. Total IgE was estimated in a smaller group by an ELISA kit. The percentage of specific IgE was calculated. A group of endemic controls (22 students) and non-endemic controls (17 cases) were included. Statistical analysis of results showed the specific immunoglobulins to be significantly reduced 2 months after treatment of the schistosomal cases. These reduced levels, however, were still significantly higher than those of controls. The presence of early hepatosplenomegaly and the co-existence of other parasites had no significant influence on the results. No correlation could be established between the levels of specific antischistosomal IgG, M and E and the intensity of infection. The significance of these results is discussed.

Effect of praziquantel on certain immune responses of schistosomal Egyptian patients. II. Cell-mediated lymphoproliferative response.

The lymphoproliferative blastogenic responses to mitogens, PHA and Con A, and to schistosome-derived antigens. S. mansoni worm and egg, were tested in 35 schistosomal patients and 10 healthy controls. Of the former group, 18 patients had intestinal mansoniasis and 17 had mansoniasis with hepatosplenomegaly. The test was repeated 2 weeks and 1 and 2 months after treatment with praziquantel. The delayed intradermal test for schistosomiasis was performed on 25 of the schistosomal patients and was repeated 1 month after treatment. Statistical analysis of results of lymphoproliferative blastogenic responses showed no significant differences between the control and the two schistosomal groups in response to mitogens. The group with intestinal mansoniasis responded significantly to both schistosomal antigens, compared to the control and hepatosplenic groups. Their proliferative responses showed a significant rise 2 weeks after treatment, then a gradual drop at 1 and 2 month intervals. The hepatosplenic group responded significantly to worm antigen before treatment; their proliferative responses to both schistosomal antigens showed a significant rise 2 weeks after treatment and remained raised thereafter. No relationship was established between either of the two schistosomal groups for age, intensity of infection or positive delayed intradermal reaction.

Prevalence of HBs-Ag in schistosomiasis. (A) general aspects.

Nine hundred and sixteen schistosomal patient together with 97 non-schistosomal controls were examined for the presence of HBs-Ag and anti-HBs in their sera by counter-immunoelectrophoresis (CIEP). The results are reported and statistically analysed. HBs-Ag, anti-HBs and the exposure rate were found significantly higher in schistosomal patients than in controls. The frequency of HBs-Ag was not significantly different in the active and inactive schistosomal groups, while the anti-HBs was significantly higher in the inactive group. S. mansoni-infected cases showed significantly higher frequency of HBs-Ag and anti-HBs than cases with S. haematobium. The prevalence of HBs-Ag was highest between 20-39 years of age in the schistosomal group, while the anti-HBs was maximum at the age of 30-39 years. There were no sex differences in the prevalence of HBs-Ag and anti-HBs in both schistosomal and control groups. However, a significantly higher frequency of anti-HBs was found between male patients and male controls. The same was true for females. Patients with history of parenteral anti-bilharzial treatment and blood transfusions were accompanied with significantly higher percentage of HBs-Ag and anti-HBs. The importance and significance of the results are discussed.

Prevalence of HBs-Ag in schistosomiasis: B-frequency in various stages of schistosomiasis.

The study included 916 schistosomal patients and 97 controls. The prevalence of HBs-Ag and anti-HBs was significantly higher in the bilharzial patients compared to controls. Their frequency was higher in the ascitic than the hepatosplenic group, and the difference between each and the simple group was highly significant. Cases with current jaundice showed highly significant frequency of both HBs-Ag and anti-HBs compared to those with no history or manifest jaundice at the time of study. In addition, cases with raised bilirubin, SGPT and SGOT showed significantly higher frequency of HBs-Ag and anti-HBs compared to cases having normal levels. On the other hand, the frequency was not affected by the level of serum alkaline phosphatases. As regards liver pathology, cases with mixed pathologic picture showed significantly higher frequency of both HBs-Ag an anti-HBs compared with those having pure schistosomal lesions.

Preliminary investigation of bilharzial patients for the presence of Australia antigen using latex agglutination test.

THE AUSTRALIA antigen was studied by the use of a reversed passive latex agglutination test in the sera of 250 cases, mostly bilharzial. The results were analysed in relation to age, clinical presentation, bilharzial infestation, antibilharzial treatment, jaundice and serum bilirubin. It was found that, the test was significantly more positive in hepatosplenic cases, in those with past history of bilharziasis, in those with jaundice and in those with higher serum bilirubin level. While no significant difference was found in relation to age, and to history of antibilharzial treatment.