Improving the detection limit of quantitative diagnosis of anti-S. haematobium antibodies using Falcon Assay Screening Test (FAST) ELISA by developing a new standard curve.

Immunodiagnosis of schistosomiasis are currently based on parasitological examinations of stool and urine for egg detection, which is laborious and lacks sensitivity. There are many assays that detect the anti-schistosomal antibodies in patient sera. One of these assays is the Falcon assay screening test (FAST) ELISA that uses adult worm microsomal antigen for Schistosoma haematobium and Schistosoma mansoni, HAMA, MAMA antigen, respectively. This assay depends on quantitative detection of anti-schistosome antibodies, using a standard curve, but the detection limit of FAST-HAMA assay is low due to the small range of the standard curve. In our study, a new wide range (0-80 µl/µl) of standard curve for FAST-HAMA assay was constructed, and the cut-off value of the assay, using the new curve, was determined to be 1.2 units/µl. Screening of 41 S. haematobium-infected sera with FAST-HAMA, using the new constructed curve, showed a sensitivity of 95%. The purity of HAMA antigen and highly specific Abs for S. haematobium lends the FAST-HAMA with the new constructed wide range standard curve as a diagnostic assay with high detection limit for S. haematobium infection.

Identification, cloning and expression of core protein gene of hepatitis C genotype 4a.

Although Egypt has very high rates of HCV, not much is known about genotype 4a which is the most predominant genotype in Egypt. In the present study, core (C_ED43) gene of the Egyptian strain ED43 of HCV genotype 4a was first analyzed using PC/GENE program. Computer analysis of Core region of the isolate ED43 revealed that the Egyptian genotype 4a is different from those isolated from Europe and Central Africa and that it is closely related to genotype 1b. The DNA region coding for the Core was amplified from HCV_ED43/PUC19 plasmid. The PCR product was then cloned and expressed in E. coli M15 using pQE-30 vector. The expression and antigenicity of the core (Core_4a) protein in E. coli was confirmed by SDS-PAGE and western blotting, which will make it useful for developing assay systems for detecting anti-HCV antibodies and HCV antigen, respectively and which might help in the design of a vaccine against the Egyptian genotype 4a.

Characterization, cloning and expression of NS3 protein gene of hepatitis C genotype 4a.

Clone and express NS3 gene of the Egyptian strain ED43 of HCV genotype 4a in E. coli was studied. Gene and protein sequences of NS3 gene of the ED43 strain were first analyzed using PC/GENE program. DNA homology was 89% the homologies and that of the protein was 78.8% indicating that NS3 gene of the genotype 4a is different from those isolated from other strains. DNA of NS3 region of genotype 4a was amplified from HCV_ED43/PUC19 plasmid. The PCR product was cloned and expressed in E. coli M15 using pQE-30 vector. Fusion protein containing the peptides coded by HCV NS3 (NS3_4a) was expressed by Escherichia coli. The specific HCV antigenicity of the NS3_4a fusion protein was identified by western blotting.

Cytokine secretion profile associated with periportal fibrosis in S. mansoni-infected Egyptian patients.

Periportal fibrosis (PPF) is a major pathological consequence of S. mansoni infection. Ultrasonography is a well-established tool for diagnosis and grading of schistosomiasis-related pathology. This work is performed to study the effect of schistosomiasis mansoni infection on the cytokine secretion profile in S. mansoni-infected patients at various grades of fibrosis, as determined by ultrasonography using Cairo Working Group classification. The levels of IL-2, IL-4, IL5, IL-10, IFN-gamma, and TNF-alpha were measured in the absence of in vitro antigen stimulation and after stimulation with worm and egg antigens. Simple intestinal (INT) patients are characterized by strong proliferation to worm antigen and high levels of IL-10 and TNF-alpha compared to patients at various grades of infection. GradeII (GdII)-infected patients are characterized by higher IL-5 production than are patients with other clinical forms of the disease. Sharp reduction of almost all cytokines in response to both worm and egg antigens was detected in GdIII-infected patients. These results stressed the role of both IL-10 and TNF-alpha in the early stages of hepatic fibrosis, while IL-5 could be employed as a potential predictive marker for advanced stages. In conclusion, PPF is associated with cytokine production profiles that vary with the magnitude of the fibrosis.

Patients with papular urticaria have IgG antibodies to bedbug (Cimex lectularius) antigens.

IgG and IgE against salivary gland proteins of bedbug (Cimex lectularius) were assessed in comparison with mosquito (Culex pipiens) and flea (Pulex irritans) antigens in the sera of papular urticaria patients (group I), siblings without papular urticaria (group IIa), patients' parents (group IIb), and healthy controls (group III) (Immunoblotting). Anti-C. lectularius IgG was significantly recognized at 66 and 10 kDa in 40% of group I, besides others ranging from 45 to 107 kDa. Group IIa significantly reacted with 70 kDa (57.1%). Group IIb reacted with 21 and 8.5 kDa (26.7%). Sixty percent of group IIb and 100% of group III significantly identified a band of 12.5 kDa. IgG against C. pipiens was significantly recognized at a range of 18-105 kDa in group I, IIb (115, 7 kDa), and III (58, 50 kDa). Anti-P. irritans IgG was significantly recognized by group I (100, 70 kDa) and group IIa (60, 35 kDa). IgE response was confined to C. pipiens at 115 and 54 kDa in groups I and III, respectively, besides 68 and 58 kDa in group IIa. It is concluded that IgG is present against C. lectularius, C. pipiens, and P. irritans in papular urticaria and may contribute to its pathogenesis.

T cells are depleted in HCV-Induced hepatocellular carcinoma patients: possible role of apoptosis and p53.

Egypt has possibly the highest Hepatitis C Virus (HCV) prevalence worldwide. A high proportion of HCV infections become chronic and lead to liver cirrhosis and hepatocellular carcinoma (HCC). The cellular and molecular mechanisms behind HCV infection complication are not completely understood although apoptosis has been implicated in this process. Using flowcytometry, we examined whether T lymphocyte; isolated from patients with HCV and HCV-associated HCC (HCV-HCC); are predestined in vivo to undergo spontaneous apoptosis. Also, the role of p53; a key protein in apoptotic process; in the development of HCC was examined. Our data showed that T cells were severely depleted in HCV-HCC patients and its spontaneous apoptosis was higher in patient groups as compared to normal controls. In addition, p53 expression in liver tissue (determined by ELISA) was higher in the HCC patient groups as compared to normal controls and correlated well with the HCC grade. In conclusion, HCV infection induces peripheral T cell apoptosis, depletion and subsequently immune-suppression and this may lead to persistence of infection. Also, p53 is implicated in the poor prognosis of HCV-HCC and could be used as a predictive marker to assess the prognosis of HCC patients.

Exposure to hepatitis C virus induces cellular immune responses without detectable viremia or seroconversion.

Sporadic cases of cell-mediated immunity (CMI) in persons exposed to hepatitis C (HCV) but evidently uninfected have been reported. To further define this, we measured CMI in individuals without evidence of HCV infection, that is, negative for HCV-antibodies (anti-HCV) and RNA, residing in a rural Egyptian community where prevalence of anti-HCV was 24%. Cell-mediated immunity (CMI) measured by interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay, confirmed by intracellular staining using flow cytometry, against HCV peptides was measured in seronegative individuals with high-risk (HR) and low-risk (LR) exposures to HCV. Thirteen of 71 (18.3%) HR subjects but only 1 of 35 (2.9%) LR subjects had detectable CMI (P = 0.032). These data are compatible with the hypothesis that exposures to HCV may lead to development of HCV-specific CMI without anti-HCV and ongoing viral replication. We speculate induced CMI clears HCV sometimes when anti-HCV is not detectable, and HCV-specific CMI is a useful surrogate marker for exposure to HCV.

Circulating adhesion molecules in patients with different clinical forms of S. mansoni infection.

Elucidating the mechanisms that regulate the severity of schistosomiasis has been a major research objective over the past several years. In this study, morbidity of S. mansoni infection was assessed using an ultrasonographic staging system of periportal fibrosis of the liver. Sera of S. mansoni infected patients with different clinical forms of the disease were assayed for the presence of intercellular adhesion molecule-1 (ICAM-1), soluble endothelial cell adhesion molecule-1 (sE-selectin), leukocyte adhesion molecule-1 (sL-selectin) and granule membrane protein-140 (P-selectin). Association between serum levels of adhesion molecules and ultrasonographic data was evaluated. Fifty-one subjects with pure hepatic schistosomiasis having ultrasonographic assessment of periportal fibrosis (PPF) were grouped according to the thickness of their portal tracts: simple intestinal =<3mm, grade I=3-5mm, grade II=>5-7mm and grade III=>7mm. Greater diameter of portal vein and larger spleen size were associated with increasing the thickness of portal tract. All groups had elevated levels of slCAM-1 compared with normal controls. Patients in grade III had significantly higher levels of serum slCAM-1 than those in other grades of infection. The sE- and sL-selectin were comparable in the sera of all patient groups. sP-selectin was significantly elevated in the sera of grade II patients compared with other patients of various clinical groups. Positive correlation was recorded between slCAM-1 level and degree of PPF, but not with other adhesion molecules. These data suggested that, the main criteria of diagnosis of S. mansoni infection using ultrasonography include periportal fibrosis, hypertrophy of left hepatic lobe, widening of the portal veins and splenomegaly. slCAM-1 may participate in the pathology associated with schistosomiasis infection. It could be employed as a potential morbidity marker in schistosomiasis mansoni infection.

Immunological identification of Fasciola hepatica antigens containing major human T-cell and B-cell epitopes.

Fasciola hepatica whole worm homogenate (Fhwwh) separated fractions were used in enzyme linked immunoelectrotransfer blot (EITB) to identify the antigen(s) which induces antibody formation in human fascioliasis. The immuno-reactive antigens recognized by the infected patients were 25-29 kDa and 12 kDa. Antigens were biochemically purified by model 491-prep cell fraction (BIO-RAD). The capability of the purified target antigens to induce humoral and cellular responses with cells and sera of infected patients was investigated using enzyme linked immunosorbent assay (ELISA) and lymphoproliferative responses techniques. The 25-29 kDa cluster of antigen(s) were found to be more efficient in inducing lymphoproliferative response than 12 KDa, thus, it was considered as the target antigens used in the generation of human monospecific polyclonal immunopurified antibody probes (IPAb). The specificity and immunoreactivity of the IPAb with Fhwwh, F. hepatica excretory secretory products (FhESP) and S. mansoni adult worm antigen (SAWA) were evaluated by using EITB. Results showed that IPAb was immunoreactive with 25-29 and 12 kDa antigens of both Fhwwh and FhESP. It was concluded that the most immunogenic F. hepatica target antigen(s) were 25-29 and12 KDa antigens and there is a cross-reactivity between 25-29 and 12 KDa antigens in both Fhwwh and FhESP. The involvement of IPAb in antibody dependent cell mediated cytotoxicity in-vitro technique was studied. Results indicated that human neturophils were more effective in adhering to the IPAb-coated flukes at early stages of development.

Cloning and sequence analysis of genes encoding Fasciola hepatica immunodominant antigens.

Fasciola hepatica lambdagt11 cDNA expression library was immuno-screened with IPAb, two clones were isolated and identified as Fhlambda400 Fhlambda800. Both clones were sequenced, FhA400 contained 305 translated bases encoding 11.509 kDa and designated as SFh12, while Fhlambda800 contained 311 translated bases encoding protein of 11.058 kDa designated as SFh11. The DNA sequence homology search of Fhlambda400 revealed a relatively high degree of identity with F. hepatica amoebapore-like protein mRNA (GenBank accession No. AF286903). However, Fhlambda800 revealed the highest similarity with F. hepatica tegumental antigen (T1) mRNA (GenBank accession No. AF153056). The protein homology search of SFh12 gave 100% identity with amoebapore-like protein (APLP), while SFh-\11 showed 75% identity with F. hepatica tegumenttal antigen (TA). The biochemical analysis of the deduced proteins was identified; in addition the predicted T- & B-cell epitopes have been also evaluated. However, histological localization of identified antigens was achieved using the IPAb in an indirect immunoflorescent antibody assay (IFA). Results revealed that the IPAb labeled the outer glycocalyx in a characteristic, pattern, which proved that the identified antigens were tegumental in origin and that infected Fasciola subjects induced antibodies directed mainly against tegumental components.

Application and assessment of a dipstick assay in the diagnosis of hydatidosis and trichinosis.

The aim of the present work was to apply and evaluate a dipstick assay for the serodiagnosis of human hydatidosis as well as human and experimental trichinosis using camel hydatid cyst fluid (HCF) and Trichinella spiralis muscle larval (TSML) antigens, respectively, and compare this to enzyme-linked immunoelectrotransfer blot (EITB) and Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA). Sera samples were collected from patients with confirmed hydatidosis and trichinosis and with other parasitic diseases as well as from normal healthy individuals. Also, sera samples were collected from mice experimentally infected with T. spiralis which were sacrificed at different time points post-infection (PI). HCF and TSML antigens were used in EITB after separation and characterization of their antigenic components using 5-22.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis under non-reducing condition. For the diagnosis of hydatidosis, the sensitivity, specificity and diagnostic accuracy of the dipstick assay and EITB were 100, 91.4 and 95.1% while those of FAST-ELISA were 96.2, 100 and 98.4%, respectively. For the diagnosis of human trichinosis, the sensitivity, specificity and diagnostic accuracy of the dipstick assay and EITB were 100% while those of FAST-ELISA were 85.7%. FAST-ELISA proved to be more sensitive in the early diagnosis of experimental T. spiralis infection (100% sensitivity from the second week PI) than the dipstick and EITB (100% sensitivity from the third week PI). All tests retained their sensitivity till the 12th week PI. Since the dipstick assay is extremely easy to perform with a visually interpreted result within 15 min, in addition to being both sensitive and specific, the test could be an acceptable alternative for use in clinical laboratories lacking specialized equipment and the technological expertise needed for EITB and FAST-ELISA.

In vitro cellular and humoral responses to Schistosoma mansoni vaccine candidate antigens.

Few studies comparing schistosomiasis vaccine candidate antigens between laboratories have been carried out and published. Generally, only the investigators who discovered the molecules have evaluated them in either experimental animal models or in human correlate studies. In an attempt to identify responses against specific antigens and investigate their association with resistance versus susceptibility to re-infection, we studied the serological reactions and the cytokine responses stimulated by a panel of 10 candidate vaccine molecules in 225 long-term residents of an area endemic for Schistosoma mansoni in Egypt. The panel consisted of four recombinant antigens (Sm62-Irv5, Sm37-G3PDH, Sm28-GST and Sm14-FABP), one full-length native protein (Sm97-paramyosin), two synthetic peptides (MAP3 and MAP4) and three unpublished antigens (PR52-filamin, PL45-phosphoglycerate kinase, PN18-cyclophilin). Two different study designs, one based on retrospective and the other on prospective parasitological data were applied in the evaluation of the immune responses. Using historical data collected over the previous 5 years, correlations between frequency of re-infection and antigen-specific immune responses were investigated. In the prospective arm of the study, the subjects were followed over time after treatment with praziquantel with periodic immunological tests and stool examinations. Thus, highly specific humoral and cellular immune reactions in response to the 10 antigens described above could be correlated, both prospectively and retrospectively, with detailed epidemiological data covering a 66-month period. The immune response profiles produced were unique to each antigen but no clear "winner" or "winners" were identified. However, markers for both resistance and susceptibility to re-infection were identified for each molecule indicating which types of responses to aim for in vaccination and which ones to avoid. The insights gained from this approach should be useful for antigen selection and ultimately for vaccine formulation prior to Phase I/II trials in humans.

Cellular and humoral immune responses to recombinant Smp17.7 Schistosoma mansoni antigen.

To determine the immunological responses to S. mansoni antigen rSmp17.7, a total of 184 subjects, 174 patients from a schistosomiasis endemic area, and 10 controls were used. Proliferation, cytokine profile in culture supernatants from antigen-stimulated peripheral blood mononuclear cells and specific IgG1, IgG3, IgG4, IgA, IgM & IgE levels were assessed. The highest stimulation index to rSmp17.7 was detected in S. mansoni patients. The evaluation of the cytokine profile [IL-2, IL-4 & IFN-gamma] in response to this antigen showed a significant increase as demonstrated by ELISA. Specifically, IFN-gamma and IL-2 were significantly detected by flow cytometry. IgG1 and IgM were the only Igs which showed a significant increase. These results highlight the importance of rSmp17.7 as a candidate vaccine for schistosomiasis. The results pave the way to understand the mechanism of schistosome-vaccine efficacy.

A novel and sensitive method to monitor helminth infections by faecal sampling.

The Kato-Katz technique is the method routinely used for diagnosing human schistosomiasis mansoni by estimating faecal egg burdens. To improve the sensitivity of faecal diagnosis, we established and validated a novel separation technique based upon the greater density of viable schistosome eggs relative to faecal material. Subsequently, it was used for faecal examination of 27 schistosomiasis patients in El-Sharkia, Egypt, with Kato-Katz smears as criterion standard. Low intensity infections (<100 eggs/g) were only detected by our technique. Moreover, triple Kato-Katz analysis on consecutive samples still missed 7.4% of all human patients, whereas the new method diagnosed 100% of samples correctly on second analysis. We conclude that in endemic areas many patients are being systematically missed by routine diagnosis. Moreover, the sensitivity of our method allows its use in proposed pre-clinical and clinical vaccine trials in non-human primates and humans, where reliable estimates of faecal egg counts are essential.

Blueprint for schistosomiasis vaccine development.

A number of different schistosome antigens are capable of partially protecting experimental animals from challenge infection. More than 100 such antigens have been identified, about 15% of which are strongly protective and deemed promising though they do not reach the level close to sterile immunity seen after vaccination with irradiated cercariae. Studies of human correlate reactions, i.e. serological reactions and cytokine responses to schistosomiasis antigens, in individuals living in areas endemic for schistosomiasis have shown associations between certain antigen-specific immune responses and lack of re-infection over time. This approach was applied in Brazil and Egypt where it was possible to epidemiologically follow cohorts of individuals in endemic areas for extended periods of time correlating infection status with immune responses against a panel of well-researched, highly purified vaccine candidates. The immune correlates found were unique to each antigen and could be either positive or negative, i.e. associated with resistance or with susceptibility to re-infection. However, few antigens were clear-cut in this respect, i.e. the majority of them induced ambiguous responses. For example, a single antigen might have a significant positive correlation when antigen-driven interferon (INF)-gamma production is measured but also show a significant negative correlation with respect to the IgG1 titre induced. These observations suggest that there are desirable, antigen-specific immune responses that would be valuable in a vaccine but they also indicate that there are responses that must be avoided. The insights gained should be useful not only for antigen selection but also for vaccine formulation prior to Phase I/II trials in humans. It would be of great value if similar independent, long-term human correlate studies could also be undertaken in areas endemic for Schistosoma japonicum.