Hydatid Antigen Research Articles

Detection of hydatid antigen by co-agglutination in fluid samples from hydatid cysts

The co-agglutination (Co-A) procedure was standardized and evaluated to detect hydatid antigen in fluid samples aspirated from hydatid cysts. Samples were collected from 14 hydatid cysts in different organs (liver, 7; lung, 5; multiple abdominal cysts, 1; and kidney, 1) by surgery. Echinococcus granulosus scolices and booklets were seen in only 6 (42.85%) samples by routine microscopy. In contrast, hydatid antigen was demonstrated in all the samples by Co-A (100% sensitivity). Co-A could be employed as a simple and rapid diagnostic procedure, as an alternative to microscopy, to confirm the hydatid aetiology of a suspected cyst.

Dot immunobinding assay as a new diagnostic test for human hydatid disease

Bovine and human hydatid antigens collected from hepatic cysts and characterized by SDS-PAGE immunoblotting show similar patterns. The bovine hydatid antigen has been used to develop a simple and fast in vitro diagnostic assay for human hydatidosis. This method, named HA-DIA (hydatid antigen dot immunobinding assay), consists of incubation of a serum sample with a textile colloidal dye (pink) and a nitrocellulose stick to which the hydatid antigen has been bound. The presence of parasite-specific antibodies leads to dyeing of the stick reactive area, and a coloured spot appears. HA-DIA sensitivity and specificity have been studied in comparison with RAST-IgE and ELISA-IgG by testing 17 sera of patients with hydatid disease and 36 control sera from patients affected with other parasitic and non-parasitic diseases. HA-DIA showed positive results in all the patients' sera and in none of the control sera. Correlation with ELISA-IgG and RAST-IgE was significant. HA-DIA has been demonstrated to be of good predictive value, allowing a speedy diagnosis of hydatid disease. In view of its simplicity, not requiring any laboratory instruments, it is particularly suitable for large-scale field screening.

IgG Subclasses in Human Hydatid Disease: Prominence of the lgG4 Response

To assess the participation of the four subclasses of IgG in the humoral response to Echinococcus granulosus infection, we determined total and parasite-specific IgG1, IgG2, IgG3 and IgG4 in sera from 46 patients with hydatid disease using an enzyme-linked immunosorbent assay (ELISA). Parasite-specific IgG subclass antibodies were quantitatively measured by means of standard curves obtained by affinity chromatography. Sera from 35 healthy individuals served as controls. The total component of IgG1, IgG2, and IgG3 showed a slight increase in patients with hydatidosis in comparison to normal control subjects with no significant differences. For the IgG4 subclass, however, a marked elevation was found in the patients group (p = 0.001 by analysis of variance). IgG1 and IgG4 subclasses showed a high anti-echinococcus antibody response, whereas there was a low parasite-specific IgG2 and IgG3 response. Indeed IgG-specific antibodies were found to belong mainly to IgG1 (63%) and to IgG4 (30%) and to a lesser extent to IgG2 (5%) and IgG3 (2%). The percentage of the total serum IgG4 antibodies that were specific for hydatid antigen reached a mean level of 18%, significantly higher than that of any of the other three IgG subclasses (p < 0.001 by Student's t test). Thus, the continuous antigenic stimulation of hydatidosis may result in an enhanced IgG4 subclass response.

The significance of free and immune-complexed hydatid-specific antigen(s) as an immunodiagnostic tool for human hydatidosis.

Micro-enzyme-linked immunosorbent assay (Micro-ELISA) systems were developed and evaluated for the detection of circulating (free or immune-complexed) hydatid antigens in the sera of patients with hydatidosis, by employing monospecific antibodies to hydatid-specific antigens of 8-kDa and 116-kDa. Fifteen (75%) of 20 sera from patients with hydatidosis had both 8-kDa and 116-kDa antigens freely circulating in their sera while three and two samples, respectively, had only 8-kDa or 116-kDa antigen. All the surgically confirmed cases of hydatidosis had detectable levels of both 8-kDa and 116-kDa circulating immune complexes in glycine HCl-treated sera. However, none of the sera from control subjects (patients with cysticercosis, ascariasis, ancylostomiasis, hymenolepiasis, amoebic liver abscess or viral hepatitis) had any detectable level of either type of circulating specific antigen. These results suggest that the demonstration of either 8- or 116-kDa antigen(s) in free or immune-complex form could confirm the diagnosis of hydatidosis.

Diagnostic value of a synthetic peptide derived from Echinococcus granulosus recombinant protein.

A specific monoclonal antibody (MAb; EG 02 154/12) directed against a protein epitope of Echinococcus granulosus antigen 5 was used to screen a cDNA library constructed from E. granulosus protoscoleces RNA. One clone designated Eg14 was selected and shown to code for an amino acid sequence partially homologous to that of the clone Eg6 previously identified with the same MAb. Hydrophobic cluster analysis showed that both recombinant antigens may adopt a similar alpha-helical organization and share a common conformational epitope. A synthetic peptide (89-122) mimicking the conformational site of Eg6 and Eg14 was constructed and demonstrated to be able to inhibit binding of the MAb and human hydatid sera to the Eg6 fusion protein (FP6) or to native hydatid antigens. To assess the diagnostic value of the peptide 89-122, we tested sera from patients infected with different parasites for their antibody reactivity with this peptide in ELISA. A high binding sensitivity and specificity of IgG-A-M antibodies were obtained with E. granulosus-infected patient sera. Moreover, the peptide 89-122 was found to be specifically recognized by IgE antibodies from patients with hydatid disease. These results indicate the particular interest of this synthetic peptide as a standardized antigen in diagnosis and treatment surveillance of hydatidosis.

Specific antibodies in serum of patients with hydatidosis recognised by immunoblotting

Hydatid fluids from sheep, goat, pig and man, after resolution by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions, revealed at least 15 discrete polypeptide bands of 8-116 Kda. By ELISA, sera from all 20 cases of hydatidosis showed anti-hydatid antibody, but so did 11 (73%) of 15 sera samples from cysticercosis patients, eight (67%) of 12 sera from patients with other parasitic infections (amoebic liver abscess or hymenolepiasis) and one (4%) of 25 sera from healthy controls. Antibody to cysticercus antigen was found in 14 (93%) of 15 sera from cysticercosis patients, 17 (85%) of 20 sera from hydatid patients, six (50%) of 12 sera from patients with other parasitic infections and one (4%) of 25 sera from healthy controls. Sera from 17 (85%) of 20 hydatid patients, 11 (73%) of 15 cysticercosis patients and five (42%) of 12 patients with other parasitic infections had antibodies to both hydatid and cysticercus antigens. Sera from 20 surgically confirmed cases of hydatidosis reacted with 12 polypeptides of 8-116 Kda in Western immunoblot with hydatid antigens. Polypeptides of 16, 24, 38, 45 and 58 Kda were recognised by all hydatidosis sera but also by many sera from patients with other infections. However, polypeptides of 8 and 116 Kda were recognised by all hydatidosis sera but not by any sera from patients with cysticercosis, other parasitic infections or viral hepatitis, or from healthy controls. Thus, recognition of 8- and 116-Kda hydatid antigens by a patient's serum appears to be a specific test confirming a clinical diagnosis in an individual case of hydatidosis.

Counter-current immunoelectrophoresis test for serodiagnosis of hydatid disease by detection of circulating hydatid antigen

Counter-current immunoelectrophoresis (CIEP) was standardized and evaluated to demonstrate circulating hydatid antigen in sera for the diagnosis of hydatid disease. The test is moderately sensitive and it could detect circulating antigen in 55.55% of surgically proved and 100% ultrasound proved hydatid disease cases. It is highly specific and no false positive reactions were observed with the sera from diseased or healthy controls. This test is simple, inexpensive and rapid with the results being obtainable within ≈ 45–60 min after the receipt of the sera. This is a test with a potential for wider application in the serodiagnosis of hydatid disease in poorly equipped laboratories of developing countries.

Diagnostic value of anti-arc 5 IgG antibody and analysis of the IgG subclasses in sera of patients with cystic hydatid disease

Serodiagnosis of cystic hydatid disease (CHD) due to the metacestode of Echinococcus granulosus depends on detecting antibodies specific to hydatid antigen, but cross-reactivity with other parasites is one of the major draw-backs. We used a commercially-available antigen that elicits the arc 5 in immunoelectrophoresis and analysed the immunoglobulin class and the IgG subclass response by an ELISA. We tested sera from patients with confirmed CHD, cystic mass/lesions (CML) of non-hydatid origin, cysticercosis and healthy controls. High levels of antibodies to the hydatid antigen in all three classes (IgG, IgM and IgA) were observed only in the CHD patients. Significantly, only IgG antibody levels were discriminative and of diagnostic value. Also discussed is the validity of reading the significant cut-off point in IgG-ELISA in relation to a clinically important group of patients such as those with a CML of non-hydatid origin rather than healthy controls. Analysis of the anti-arc 5 IgG subclass responses demonstrated high antibody responses in all subclasses among the hydatid patients, with IgG3 the most discriminatory. The significance of the elevation of all four subclasses and more specifically of IgG4 in CHD is discussed in relation to certain biological activities of these immunoglobulin molecules.

Hydatid disease: analysis of parasite antigens in circulating immune complexes and in preformed hydatid antigen-antibody complexes.

Fifty-three sera from 29 patients with hydatid disease, all but one positive for specific anti-parasite antibodies and all negative for specific circulating antigens, were studied for the presence of circulating immune complexes (CIC) by conglutinin binding-assay (KgBA). Fourteen serum samples (26%) from eight patients (27%) were positive. These positive sera were pooled for each patient and the eight samples were PEG-precipitated and analysed for the presence of specific Echinococcus granulosus antigens in the CIC using a human anti-human-hydatid cyst fluid antiserum capable of recognizing the major antigenic systems of the parasite namely, antigens 4 and 5. The assays utilized for detecting antigen in CIC were: (a) blotting on nitrocellulose paper after sodium dodecil sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and specific immunological detection; (b) ultracentrifugation in acid buffer and subsequent detection of antigens by a sandwich-radioimmuno assay (RIA); (c) protein separation by isoelectric focusing (IEF) and specific immunological recognition. In addition, all positive sera were analysed for the presence of antigen in the CIC by a modified KgBA and by polyethylenglicol (PEG)-precipitation in acid buffer followed by immunological recognition of antigen. All tests gave negative results with the patients' samples, but were positive with preformed in vitro complexes between parasite antigens and corresponding antibodies. Failure to detect antigen in the CIC could be due to: 1) insufficient sensitivity of the assays used to detect hydatid antigens in CIC; 2) rapid clearance of antigen or CIC from the circulation; 3) presence of parasite antigen not recognized by the antiserum employed; 4) production of CIC as a result of polyclonal B-cell activation. This last hypothesis is supported by the demonstration of IgM-rheumatoid factor (RF) and anti-F(ab')2 antibodies respectively in 11 (44%) and 13 (52%) out of 25 patients.

New experimental criteria for optimization of solid-phase antigen concentration and stability in ELISA

A peroxidase saturation technique for the determination of coating antigen concentration necessary to saturate polystyrene plates with a wide range of different antigens is described. The same technique has also been used to compare the stability of antigen-polystyrene bonds for native and denaturated antigens. Furthermore, the inappropriate selection of solid-phase antigen concentration and its influence on ELISA results is analyzed and an experimental criterion to select the optimum antigen concentration is proposed. Two different antigens, BSA-Ar and hydatid antigen, were used for ELISA determination of specific antibodies as a model system. Optimum solid-phase antigen concentration wasdetermined by two different methods: (1) peroxidase saturation, in which binding of peroxidase to non-antigen-occupied polystyrene surface sites was used to evaluate the degree of coating by antigen; (2) chequer-board titration, using several immune sera of different affinity. Optimum antigen concentration selected by chequer-board titration using low affinity sera was similar to that selected with peroxidase saturation. On the other hand, lower antigen concentration would be selected by chequer-board titration using high affinity sera. For this reason, the concentrations of low affinity antibodies would be underestimated using the chequer-board titration. These results indicate that peroxidase saturation should be used to avoid avidity-dependent artifacts in ELISA.

Ovine hydatidosis: some immunological characteristics of the seronegative host.

The immune status of a group of sheep naturally infected with hydatid cysts was investigated. Low specific anti-hydatid antibody (Sab) titres and negative results in intradermal and leucocyte migration tests occurred in hosts with high hydatid antigen-stimulated lymphocyte transformation (LT). Total (non-specific) IgG was elevated in infected sheep and was positively correlated with LT. Circulating hydatid antigen (cAg) was detectable in some infected sheep, but not in all of those with low Sab. Relationships between cAg, Sab and immune complex levels suggest that most cAg is present in immune complexes.

Application of antigens from Taenia hydatigena cyst fluid for the immunodiagnosis of human hydatidosis.

Fluid was collected from cysts of Taenia hydatigena in 60 adult sheep and fluid from each animal pooled separately. By double diffusion antigen 5 was demonstrated in all pools but one. The criteria are described for selection and standardization of these preparations for use as antigens for the immunodiagnosis of human hydatid disease. Sera from 50 persons harbouring hydatid cysts and from 50 patients with other disease conditions were examined by the arc-5 double-diffusion test, using two antigens prepared from Echinococcus granulosus and T. hydatigena cyst fluids, respectively. The results showed that a higher diagnostic sensitivity was obtained with the hydatid antigen. The significance of the findings is discussed in terms of their application to human immunodiagnosis in areas where hydatidosis, but not cysticercosis, is rare in livestock.

Activation of human complement by crude and purified antigens from Echinococcus granulosus.

Hydatid cyst fluid of Echinococcus granulosus was found to interact with human complement. This interaction resulted in the depletion of hemolytic complement in normal serum and in the electrophoretic conversion of C3 and factor B. The results provided evidence of an activation of complement via the alternative pathway when hydatid cyst fluid was used in the mixtures. Also purified hydatid antigen preparations were found to interact with complement in vitro; this interaction resulted in the consumption of complement not only via the alternative pathway, but also via the classical pathway.

Membranous glomerulonephritis secondary to hydatid disease

To study the association of hydatid disease of the liver with a nephrotic syndrome we processed renal biopsy tissue for light microscopy, immunofluorescence and electron microscopy studies. These procedures disclosed the typical features of a membranous nephropathy whereas indirect immunofluorescence revealed glomerular deposits of hydatid antigen. Rabbits, previously immunized with bovine hydatid fluid, provided the specific antiserum. Clearly, the pathogenicity of this parasitosis derives, in part, from its immunologic repercussions. In this regard, our results support an etiopathogenic role for hydatid antigen in an immune-mediated glomerulonephritis.

Echinococcus multilocularis: Cell-mediated immune response in early and chronic alveolar murine hydatidosis

The in vivo cell-mediated immune response of C57L/J mice infected with Echinococcus multilocularis cysts was investigated using a homologous antigen, a T cell-dependent heterologous antigen (oxazolone) and an alloantigen at 6 and 12 weeks postinfection. Mice infected for 12 and 14 weeks harbored a 9 to 18 times larger larval cyst mass than those infected for 6 weeks and showed splenomegaly and elevated antihydatid antibody titers. In mice infected for 6 weeks and challenged with hydatid antigen the 48-hr footpad swelling and histological picture of footpad were consistent with cell-mediated immunity. These parameters were significantly reduced in the mice infected for 12 weks. Their response to skin allograft and oxazolone, however, remained comparable to the mice 6 weeks postinfection and uninfected controls. The lymphoid tissues of the mice 6 weeks postinfection were histologically intact and showed moderate to intense lymphoproliferation. In the mice 14 weeks postinfection disorganization of lymphoid tissue, plasma-, and histiocytosis in the thymus-dependent areas coincided with a large larval cyst mass.

Ultrastructural immunocytochemical localization of two hydatid fluid antigens (antigen 5 and antigen B) in the brood capsules and protoscoleces of ovine and equine Echinococcus granulosus and E. multilocularis

The unlabelled antibody method was used in the ultrastructural localization of two hydatid fluid antigens, antigen 5 and antigen B, in brood capsules and protoscoleces of Echinococcus granulosus and E. multilocularis. Antigen 5 was found in the parenchyma cells of the protoscolex and brood capsule wall and to a lesser extent in the walls of the flame cells and collecting ducts of the excretory system and in the surrounding interstitial material. It is suggested that, while some excretion of this antigen may occur from the protoscolex, it could also be liberated into the cystic cavity by degeneration of protoscoleces and parenchymal cells of the brood capsule wall. Antigen B was found mainly in the distal cytoplasm and perinuclear cytoplasm of the tegument anterior to the suckers. It is apparently secreted to the outside and was present in the brood capsule contents; it adheres to the anterior surface and the posterior periodic acid-Schiff (PAS)-positive glycocalyx of the protoscolex and to the inner surface of the brood capsule wall. The protoscolex tegument posterior to the suckers was negative. The parenchyma cells of the protoscolex and brood capsule wall were also positive although the intensity of the reaction product was variable.

The host response in secondary hydatidosis of mice. II. Cell mediated immunity.

The presence of cell mediated immunity to Echinococcus granulosus antigen was detected in syngeneic mice with secondary hydatidosis. Significant increases of thymidine uptake were observed in spleen cells from infected animals exposed to protoscolex or hydatid fluid antigens. A definite decrease in the number and size of cysts occurred in mice following the simultaneous intraperitoneal injection of viable protoscolices and a suspension of 1 x 10(7) sensitized spleen cells.

Evaluation of Whole and Purified Hydatid Fluid Antigens in the Diagnosis of Human Hydatidosis by the Immunoelectrophoresis Test *

Whole sheep hydatid cyst fluid (WHF) and purified hydatid fluid antigens (PHF) were evaluated in the immunoelectrophoresis (IEP) test for the diagnosis of human hydatid disease. A higher sensitivity was obtained using the PHF than with the WHF antigen but false positive results were observed with the former in 22.4% of the 102 non-hydatid sera examined. Using the presence of arc 5 as the criterion of positivity however, no false positives were observed in non-hydatid sera with the WHF antigen. It is postulated that the number of bands other than arc 5 may be of value in the diagnosis of hydatid disease by the IEP test, in cases where this diagnostic arc is absent.

HYDATID DISEASE OF THE LUMBAR VERTEBRAE

1. A thirty-two-year-old man has been under treatment for twelve years for primary hydatid disease of the body of the third lumbar vertebra. 2. The second lumbar vertebra became involved, as were the soft tissues over a wide area, but the spinal canal was not affected. 3. A hydatid cyst was found in the left lung. 4. Management included stabilisation of the spine by bone graft, desensitisation to hydatid antigen, removal of the diseased vertebral bodies, pedicles and processes, the use of saturated solution of sodium chloride as a scolicide and the dilation of secondary ureteral strictures. 5. The patient, although not cured, has been enabled to "live with his disease" to earn his living and enjoy tennis and swimming.

Antibodies Against Sheep Erythrocytes Produced by the Injection of Hydatid Liquid in Patients with Hydatid Cyst

Summary The injection of hydatid fluid in patients with hydatid cyst provokes the increase in their serum of agglutinins and hemolysins for the red cells of sheep and agglutinins for the red cells of rabbits. The increase in the titer of these heterologous antibodies is the more marked, the more intense is the response of the patient to intracutaneous injection of hydatid fluid (Casoni's reaction). In subjects without hydatid cysts and in noninfected rabbits the injection of hydatid fluid does not cause the appearance of such antibodies. Taking into account the results of these investigations, we present the hypothesis that for the appearance of this type of heterophilic antibodies the previous sensitization of tissues to the hydatid antigen is necessary.