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Previous Article | Next Article 
Clinical Microbiology Reviews, January 2003, p. 18-36, Vol. 16, No. 1
0893-8512/03/$08.00+0 DOI: 10.1128/CMR.16.1.18-36.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Molecular Parasitology Laboratory, Australian Centre for International and Tropical Health and Nutrition, The Queensland Institute of Medical Research and The University of Queensland, Brisbane, Queensland 4029, Australia,1 Veterinary Research Institute, Xinjiang Academy of Animal Science, Urumqi, Xinjiang 830000, People's Republic of China2
SUMMARY INTRODUCTION IMMUNITY IN THE INTERMEDIATE HOST Innate Resistance and Early Immunity Primary infection. Secondary infection. Established Cysts Inhibition of Cyst Growth Evading the Immune System IMMUNITY IN THE DEFINITIVE HOST VACCINES AND VACCINOLOGY Vaccination of the Intermediate Host Vaccination of the Definitive Host DIAGNOSIS Immunodiagnosis of Cystic Echinococcosis in Humans Antigen B. Antigen 5. Limitations of Current Tests Diagnosis for Monitoring Treatment of Cystic Echinococcosis in Humans Brief Comments on the Diagnosis of Alveolar Echinococcosis in Humans Immunodiagnosis of Cystic Echinococcosis in Animals Diagnosis of Echinococcosis in Definitive Hosts ACKNOWLEDGMENTS REFERENCES
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| INTRODUCTION |
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Hydatid cysts of E. granulosus develop in internal organs (mainly the liver and lungs) of humans and intermediate hosts (herbivores such as sheep, horses, cattle, pigs, goats and camels) as unilocular fluid-filled bladders (Fig. 1 to 3). These consist of two parasite-derived layers, an inner nucleated germinal layer and an outer acellular laminated layer surrounded by a host-produced fibrous capsule. Brood capsules and protoscoleces (PSC) bud off from the germinal membrane. Definitive hosts are carnivores such as dogs, wolves, and foxes (Fig. 1). Sexual maturity of adult E. granulosus occurs in the host small intestine within 4 to 5 weeks after the host ingests offal containing viable PSC. Gravid proglottids or released eggs are shed in the feces, and following their ingestion by a human or ungulate host, an oncosphere larva is released that penetrates the intestinal epithelium and enters the lamina propria. The larva is then transported passively through the blood or lymph to the target organs, where it develops into a hydatid cyst. Since the life cycle relies on carnivores eating infected herbivores, humans are usually a dead end for the parasite. Adult worm infections by E. multilocularis occur mainly in red and arctic foxes, although dogs and cats can also act as definitive hosts. Small mammals (usually microtine and arvicolid rodents) act as intermediate hosts. The metacestode of E. multilocularis (Fig. 2) is a tumor-like multivesicular, infiltrating structure consisting of numerous small vesicles embedded in stroma of connective tissue; the larval mass usually contains a semisolid matrix rather than fluid (245). CE and AE are both serious diseases, the latter especially so, with a high fatality rate and poor prognosis in the absence of careful clinical management.
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| IMMUNITY IN THE INTERMEDIATE HOST |
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After infection, the earliest detectable immunoglobin G (IgG) response to hydatid cyst fluid (HCF) antigens occurs after 2 to 11 weeks in mice and sheep, respectively (247, 266), and after 4 weeks in vervet monkeys (227). Early infections may be associated with a significant cellular inflammatory response (172, 210) that may cause pathologic changes (12, 80) since there is an increased leukocytosis, mainly of eosinophils, lymphocytes, and macrophages (195). With oncospheres, necrosis of surrounding cells is followed by infiltration of neutrophils and macrophages 3 to 5 days after infection in sheep (195). Experiments in vitro have shown also that neutrophils, in association with antibody, can bring about the killing of E. granulosus oncospheres (226), suggesting a possible role for antibody-dependent cell-mediated cytotoxicity reactions. At the early stages of disease, there is a marked activation of cell-mediated immunity to the parasite (81).
Secondary infection. In experimentally induced secondary infections in mice, intraperitoneally injected PSC are surrounded by a considerable cellular infiltration within 3 days, initially involving activated macrophages and subsequently including neutrophils, eosinophils, and lymphocytes (208, 221, 222). Interleukin-10 (IL-10), IL-4, and IL-5 secreted in vitro by splenocytes can be detected as early as week 1 postinfection (55). High levels of tumor necrosis factor alpha (TNF), gamma interferon (IFN), IL-6, and specific IgG1 were detectable in serum, and IgG3 was measurable in the peritoneal cavity using protoscolex somatic antigens (55, 111). These data suggest that polarized Th2 reactions are evoked at the very beginning of the immune response to secondary infection. E. granulosus PSC contain immunogenic T-independent antigens (29). Primary antibody responses to protoscolex somatic antigens and the production of IgM and IgG3 in early infection appear to be stimulated mainly by a T-independent mechanism (29).
Similar to E. granulosus, differences in susceptibility to E. multilocularis have been shown in both primary and secondary infection of different mouse strains (26, 53, 74, 75, 106, 184). Susceptibility and resistance are based on the activation of different CD4+ T-cell immune responses (26, 53, 198). Experiments with mice infected with eggs showed that IFN-gamma-, IL-2-, and IL-4-expressing cells in the parasitic lesions were not detectable at the early phase of the infection but were present at the end (26). Similarly, low levels of cytokines in the sera were measurable at the beginning of the infection and high levels were detectable subsequently (26). IL-10 was the most prominent cytokine measurable throughout the course of the infection. Correspondingly, only small amounts of IgM, IgG1, IgG2a, and IgG3 could be detected early on, and higher levels were detectable later (190). A strong, specific intestinal immune response was found in the early stage (190). Both subsets of CD4+ T cells (Th1 and Th2) are involved in primary murine alveolar echinococcosis (26, 74).
In secondary AE in mice, very low levels of Th2 cytokines and IgG1, IgG2a, IgG3, and IgM are produced at the early infection stage, but these levels subsequently increase significantly (73). The degree of antibody response by the murine host does not correlate with susceptibility to E. multilocularis (198).
Also involved in the establishment phase is cellular infiltration, which includes eosinophils, neutrophils, macrophages, and fibrocytes (8, 9, 17, 18, 208, 219, 222, 240). However, this generally does not result in a severe inflammatory response and aged cysts tend to become surrounded by a fibrous layer that separates the laminated layer from host tissue. Eosinophilia and the production of high levels of IgE are the common consequences of infection by helminths (30, 37). It has been suggested that the eosinophil has evolved especially as a defense against the tissue stages of parasites that are too large to be phagocytosed (112) and that the IgE-dependent mast cell reaction has evolved primarily to localize eosinophils near the parasite and then enhance their antiparasitic functions (30). Eosinophils are less phagocytic than neutrophils, but, like neutrophils, they can kill larval stages of parasites (204) such as Echinococcus (179) by both dependent and independent mechanisms; their activities are also enhanced by cytokines (36).
Like other helminth infections (12, 80, 159, 180, 193), echinococcosis induces two very distinct Th1 and Th2 cytokine secretion patterns. Th1 cells produce IL-2, IFN-
, and lymphotoxin, whereas Th2 cells express IL-4, IL-5, IL-6, IL-10, and an induced gene (p600) of unknown function. In tissue culture, the Th1- and Th2-cell patterns are well defined and stable. They are generally cross-inhibitory. IFN- inhibits Th2-cell proliferation, whereas IL-10 inhibits the synthesis of Th1 cytokines. In hydatid infections, both cell population profiles remain highly expressing, at least in cysts that survive the immune response. Elevated levels of IL-4, IL-5, IL-6, and IFN- are produced in vitro by peripheral blood mononuclear cells (PBMC) isolated from infected human subjects and stimulated by HCF antigens (213-219). Elevated cytokine levels were also measurable in the sera from hydatid disease patients with lung and liver involvement (249). The coexpression of IL-10 and IFN- at high levels in human hydatidosis suggests that the immune response to E. granulosus infection is possibly regulated by both Th1 (or Th0) and Th2 profiles. It is not understood why hydatid infection can induce high levels of both Th1 and Th2 cytokines since they usually down-regulate each other (193). It may be due to the very complex mixture of antigens in HCF (177), which probably contain distinct epitopes for each T-cell subset. However, the situation with human subjects is difficult to explain since the involvement of Th0 cells in a late chronic infection is rare (1).
In human subjects undergoing chemotherapy treatment, a Th1 cytokine profile, rather than a Th2 profile, typically dominates (215). It has been suggested that this could be one of the killing mechanisms that set in during the later stages of infection (225). Significantly, increased production of IL-4 and IL-10 in hydatid disease patients corresponds to high levels of IgE and IgG4 (215). Therefore, both IL-4 and IFN- regulate the IgE and IgG4 responses (153, 154, 180). AE patients experiencing a relapse of the disease have a tendency to increased production of IL-5 but lower IFN- production accompanied by significantly higher levels of IgE and IgG4 compared to patients with a primary infection (94).
In addition to IL-4 and IL-10 production, two other Th2 cytokines, IL-5 and IL-6, are produced in large quantities by hydatid disease patients. IL-5 was shown to be specifically induced by parasite antigens in 90% of patients while control subjects were negative (219). Other studies have shown that IL-5 is associated with the regulation of specific IgE and IgG4 expression (219). In general, IL-5 also regulates the eosinophilic response (45, 125). However, some patients infected with E. granulosus (11%) (218) and most patients infected with E. multilocularis have limited eosinophilia (219, 244).
Sera from patients with active cysts have a range (2 to 500 U/ml) of concentrations of IL-6. The major role of this cytokine is to induce differentiation of B cells into plasma cells, thus contributing to the development of antigen-specific humoral responses (254).
CE patients with relapsing disease have high levels of IgE and IgG4, increased levels of IL-5, IL4, and IL-10, and low levels of IFN- produced in vitro by PBMC compared to patients with a primary infection (215, 219). Patients with a primary infection have higher levels of IL-2, IFN-, and IL-5. The high level of IL-5 is in agreement with the high levels of IgG4 and IgE observed (219). IFN- and IL-6 activities were undetectable in sera from two liver hydatidosis patients who relapsed (250). There is a significant correlation between IgE and IgG4 production in sera from patients with hydatid disease and a trend toward increased IL-4 and IL-10 levels in patients who are high producers of IgE and IgG4 (214).
When specifically stimulated with HCF antigen, PBMC from hydatid disease patients produced higher levels of IL-2 did than those from uninfected donors (125). The apparent bias toward a Th2 response appeared to be related to clinical status and was suggestive of a putative role of Th2-like responses in susceptibility to reinfection by E. granulosus (125). Clearly, these results merit further study.
Primary and secondary infections elicit similar responses, which include elevated levels of TNF-
, IL-1, IFN-, IL-6, and IL-10 (111) and detectable levels of specific IgG1 and IgG3 isotypes (55). The levels of IgM and IgG2a are slightly increased following infection and remain elevated throughout the first 18 weeks of infection. During the 129- to 209-day period following the onset of infection, there is an increase in the level of secreted IL-10 and a slow decrease in the levels of IL-6 and IFN-. IgM, IgG, IgG1, and IgG2a levels plateau during this period, whereas IgG3 and TNF- levels peak on day 190 postinoculation. These data suggest that induction of Th2 antibody-mediated immunity with a parallel expansion of Th1-mediated inflammatory responses is an important mechanism of host defense against the metacestode (111). Local inflammatory reactions to PSC at the site of injection are intense, involving neutrophils, eosinophils, macrophages, and mast cells (221).
Significantly higher levels of IL-10 (257) and IL-5 (222, 244) have been found in AE patients than in controls. In contrast, IL-4 was measurable in only a minority of patients and controls. IL-12 levels were comparable between AE patients and controls and showed a similar distribution pattern to IL-10 with regard to disease progression. These studies suggest that a Th2-dominated immune response occurs in AE in vivo. AE infection results in a strong Mac-1+ cell infiltration of the peritoneal cavity and spleen (53). Peritoneal cells from mice infected with AE at the 1-month stage were rich in macrophages and expressed significantly higher levels of transcripts for the inflammatory cytokine IL-1ß and for TNF- and inducible nitric oxide synthase (257).
and nitric oxide production may play a role (249). Complement through C5-mediated effectors contributes to host defenses by both restricting the establishment of infection and controlling the growth of established cysts. This contribution may be associated with the ability of C5a to promote eosinophil infiltration (65, 266). Lysis in both immune and normal serum is antibody dependent and complement mediated (116). Protoscoleces of E. multilocularis and E. granulosus are lysed by fresh serum of many different species of mammals (121, 152). The presence of Echinococcus cysts appears to deplete host complement (121, 150, 151). The rapid development of E. multilocularis infection is associated with depletion of serum complement; the use of cobra venom factor to deplete complement results in faster growth of E. multilocularis cyst masses.
In hydatid infections, IL-6 seems to be produced nonspecifically (254) whereas IL-5 production appears antigen specific. The effect of IL-5 on human B cells is controversial (45), but a significant correlation between IL-5 production and IgE and IgG4 expression has been found in hydatid disease patients (219). When CE cysts grow, IgG1 and IgG4 levels are elevated, whereas the concentrations of specific IgG1 and IgG4 decline in cases characterized by cyst infiltration or calcification. This indicates that the IgG4 antibody response is also associated with cystic development and growth and with disease progression whereas the IgG1, IgG2 and IgG3 responses occur predominantly when cysts became infiltrated or are destroyed by the host (52).
In experimental infection, fewer than 10% of PSC survive to form cysts (194, 272). The majority of parasite killing occurs within the first 2 weeks postinfection. Activated macrophages are involved in the killing of Echinococcus PSC (8, 9, 24, 142). Studies in vitro indicate that macrophage-dependent killing of PSC can be increased by IFN- (148) and decreased by some cytokines such as IL-10 or IL-4 (142). Therefore, it seems likely that during a secondary infection, an initial Th0 or Th1 response effective in killing parasites becomes polarized to a Th2-type response and that this response seems less effective. This is supported by studies with patients undergoing albendazole chemotherapy who responded better to treatment when they possessed a more dominant Th1-type cytokine profile than when they had a more dominant Th2-type profile (125).
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The initial response of the host abates with time and is minimal 6 months after infection (81). IFN--, IL-2-, and IL-4-expressing cells could not be detected in lesions of the early phase of infection (26), possibly indicating host immunosuppression. Furthermore, about 30% of CE patients have undetectable antibody levels in their sera (40, 48, 216), a feature which also appears to occur in ovine infections (145, 169, 265). The mechanisms behind this are unclear. Circulating parasite antigen could be mopping up specific antibody, since both circulating antigen and immune complexes are detectable in some seronegative individuals (46). The possibility of antigen-induced specific immunological tolerance has also been raised, suggesting that antibody production during the course of the infection may be regulated, perhaps through periodic release of antigen from cysts and/or general down-regulation of B cells through Th-cell activity.
When using susceptible C57BL/6 mice, spleen cells supplemented with peritioneal cells from E. multilocularis-infected mice induced a complete suppression of splenic proliferation at the early and late stages of infection, and this suppression was reversed to a large extent by the addition of NG-monomethyl-L-arginine and partially by anti-IFN- antobodies (53). Spleen cells from late-stage-infected mice express only background levels of IL-10 but greatly increased levels of inducible nitric oxide synthase. The immunosuppression observed in chronic AE is not primarily dependent on IL-10 but rather on nitric oxide production by macrophages from infected animals (53, 74).
Proliferative responses and IL-2 production induced by concanavalin A (ConA) in spleen cells from BALB/c mice are significantly depressed at an early stage after infection (157). With E. multilocularis PSC, addition of plastic-adherent cells from normal syngeneic mice to the nonadherent spleen cells from infected mice did not restore the depressed ConA responsiveness. On the other hand, exogenous IL-2 completely reconstituted the proliferative responses to ConA. Flow cytometry analysis revealed that the number of CD4- CD8+ cells with a low density of CD8 antigen (CD8dull cells) increased in spleens from infected mice 2 weeks after inoculation. Addition of the spleen cell subpopulation containing the CD8dull cells, but not that depleted of the CD8dull cells, to normal spleen cells resulted in marked suppression of the ConA responses. These findings suggest that the CD8dull cells detected in the spleens of mice inoculated with E. multilocularis PSC may play a key role in the suppressive regulation of immune responses (157).
Little is known about antigenic drift or shift in Echinococcus infections compared with some other parasites (50, 104, 105, 108-110). The production of proteases has been reported for a range of helminths and is considered important for the conversion of host tissues into nutrients (128), for host invasion (126), and for migration through host tissues (127). It is unknown whether proteases from Echinococcus function in the cleavage of IgG, as has been recorded in other helminths (19, 20, 158, 202, 203). Nevertheless, secretions from the penetration glands of hatched and activated oncospheres of E. granulosus cause lysis of host tissues; these secretions may protect the parasite against the host immune response while the laminated layer develops (126).
| IMMUNITY IN THE DEFINITIVE HOST |
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An increase in the level of immune mediators, sheep mast cell protease larval migration inhibition components, and peptidyl leukotriene is correlated with the clearing of T. colubriforms infection (144) and a reduction in fecal egg counts (69). The secretion of leukotrienes and larval migration inhibition from MMC is thought to be a major mechanism of parasite removal (70). IgA and IgE are important in mucosal immunity since they bind directly to antigens and also attract effector cells that bind the constant region of the antibody. Eosinophils and MMC bind immunoglobulin constant regions via Fc receptors, becoming activated to degranulate when bound to the opsonized parasite. This method of antibody-dependent cell-mediated cytotoxicity is well established as an important mechanism by which the host can damage a multicellular parasite (211).
As reviewed by Heath (113, 114), the scolex of adult Echinococcus worms is normally in close contact with the canine intestinal submucosa, but mucosal immune responses, leading to the production of neutralizing IgA antibodies to deal with the secretions of the strobila, have no effect on the scolex. The scolex is in intimate contact with the systemic circulation, even in the Peyer's patches, and it appears to maintain its privileged integrity by suppression of cytotoxic and effector cell activity in the region of the scolex.
Experiments with immunosuppressed golden hamsters subsequently infected with E. multilocularis showed that worms developed faster than in normal animals (147). In addition, dogs that were immunosuppressed and then challenged with PSC of E. granulosus were shown to harbor more worms than did nontreated dogs, suggesting that the definitive host may have some innate resistance to infection by adult worms (D. Heath, personal communication).
Cells from Peyer's patches of dogs infected with E. granulosus produce specific immunoglobulin in vitro (61). Infection depresses the ability of unstimulated cells to proliferate in response to HCF protein but enhances the response to ConA (10). Dogs with enhanced reactivity to ConA and another mitogen, phytohemagglutinin, have significantly fewer worms and a lower number of mature worms than do dogs with less reactivity. After infection, the concentrations of IgG and IgA increased in serum and IgA levels increased in feces. Dogs with high-titer anti-HCF antigen serum antibodies were better protected than were dogs with low titers in serum (10).
| VACCINES AND VACCINOLOGY |
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The immunity (mediated by complement-fixing antibodies) generated by two injections of the vaccine given 1 month apart persists for at least 12 months (115). Thereafter, annual vaccination of domestic livestock is recommended (115, 167). The shelf life of the formulated vaccine is at least 12 months (115). One liter of Escherichia coli culture can yield more than 10,000 vaccine doses, which means that the vaccine could be manufactured cheaply if produced on a large scale. Other noteworthy features of the EG95 vaccine are that immunity can be transferred passively to neonates with antibody from vaccinated dams (115) and that the protection induced is associated with conformational epitopes (261-264). Furthermore, the vaccine conferred a high degree of protection against challenge with different geographical isolates of E. granulosus (166), indicating that it could have wide applicability as a new tool for use in hydatid disease control campaigns. The vaccine therefore provides a valuable new tool to aid in the control of transmission of this important human pathogen and also has the potential to prevent hydatid disease directly through vaccination of humans. Recent research indicates that the EG95-encoding gene belongs to a gene family of six or more genes (43). A closely related protein (designated EM95) that can induce significant levels of protection against challenge infection with E. multilocularis eggs in mice has also been identified in E. multilocularis (90).
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The immunodiagnosis of echinococcosis has been comprehensively reviewed in a series of early articles by Schantz and Gottstein (232), Rickard and Lightowlers (209), Lightowlers and Gottstein (165) and Gottstein (100). Here, more recent progress in the development and application of specific diagnosis of Echinococcus infection in humans, animal intermediate hosts and definitive hosts is assessed. Readers should also refer to excellent recent comprehensive reviews of the field (192). Over the past decade, diagnosis of CE and AE has improved due to the use of new or more optimal methods for purification of Echinococcus antigens from somatic materials, by the application of molecular tools for parasite identification and the synthesis of recombinant diagnostic antigens and immunogenic peptides. These approaches have not only improved the sensitivity and specificity of tests for diagnosis of AE and CE but also allowed more reliable characterization of the biological status of parasite materials (reviewed in references 237 and 245).
Hydatid serological testing has a very long history, and almost all serological tests that have been developed have been used in the diagnosis of human cases. There are considerable differences among the various tests in both specificity and sensitivity. As the sensitivity of a test increases, so generally does the demand for improved antigens so that sufficient specificity can be achieved to take advantage of the greater sensitivity. An optimum test should be specific with high sensitivity. Insensitive and nonspecific tests, including the Cassoni intradermal test, the complement fixation test, the indirect haemagglutination test, and the latex agglutination test, have been replaced by the enzyme-linked immunosorbent assay (ELISA), the indirect immunofluorescence antibody test, immunoelectrophoresis (IEP), and immunoblotting (IB) in routine laboratory application (165).
Chordi and Kagan (42) were the first to analyze antibody responses in human hydatid infection by IEP with sera from patients and HCF of sheep origin as antigen. Further extensive studies have also focused on HCF antigens that are still considered an invaluable source of antigenic material for immunodiagnosis (Table 2). This research evaluated the immunoreactivity of these antigens with sera from patients with hydatid infection and resulted in the development of new techniques for the preparation of purified antigens (160, 183, 186, 196, 200, 201, 259). Antigen prepared from human HCF was found to be unsuitable for diagnosis because it contains host proteins such as IgG (27). Sheep HCF obtained from fertile cysts has been used routinely to prepare and standardize antigen. Bovine HCF can be used as an alternative antigen source; indeed, it can improve diagnostic sensitivity (130, 206). HCF of camel origin has also been used as antigen in an ELISA format to measure total E. granulosus-specific IgG antibodies and IgG subclasses (206). The diagnostic value of measuring IgG1 (97.7%), as assessed by a rating index (J) for combined sensitivity and specificity, was superior to the use of total IgG (65.1%) and IgG2 to IgG4 (77.8, 57.9, and 39.6%, respectively) (206). These findings have set the stage for field evaluation of the IgG1 assay in areas where human CE is endemic.
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Antigen B. AgB is a polymeric lipoprotein with a molecular mass of 120 kDa (185). It can be measured in patient blood as circulating antigen (149, 171), and it has been suggested that it plays an important role in the biology of the parasite and its relationship with the host (212, 235). AgB is a highly immunogenic molecule (42, 186), a characteristic that underpins its value in serodiagnosis (Table 3). It appears ladder-like under reduced condition on SDS-PAGE, with three bands with molecular sizes of approximately 8 or 12, 16, and 24 kDa (42, 162, 168, 186, 236), suggesting that it comprises polymers of 8-kDa subunits (95, 168). The smallest subunit has proved the most useful target in diagnostic studies (188, 229).
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In further efforts to standardize CE diagnosis, Barbieri et al. (22) compared the diagnostic value of p65 and GU4, a 34-mer synthetic peptide corresponding to the C-terminal end of the AgB8/2 subunit (Tables 3 and 4) with that of p89-122, a synthetic peptide derived from Ag5 (Table 5). The p65 peptide provided three- to fourfold higher sensitivity but 30% lower specificity than the other two peptides. A further study showed that a highly antigenic region of AgB resides in the N-terminal extension of the AgB8/1 subunit; an ELISA based on the use of a single peptide designated p176, a 38-mer peptide from the N -terminus of the AgB8/1 subunit, exhibited a diagnostic performance that was superior to that obtained by the use of native AgB (97). The results of this and other studies investigating the sensitivity and specificity of recombinant AgB and AgB peptides in CE diagnosis are summarized in Table 4.
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Using pooled hydatid disease-specific sera highly reactive with Ag5, a partial cDNA sequence termed Eg6 was isolated (76). The recombinant protein fragment encoded by the sequence was recognized by a monoclonal antibody specific for Ag5 (38). In addition, antibodies eluted from this recombinant protein recognized the 38-kDa subunit of Ag5. Another clone, designated Eg14, was selected and shown to code for an amino acid sequence partially homologous to Eg6 identified with the same monoclonal antibody. Using Eg6 sequence primers, a novel sequence coding for a 29-kDa antigen (termed P-29) was amplified from PSC of E. granulosus. The sequence has 100% identity to the amino acid sequence encoded by Eg6. Additional work has shown that P-29 and Ag5 are immunologically related but are nevertheless different proteins, raising questions about the current state of knowledge of Ag5 (96). Results of studies investigating the sensitivity and specificity of native and recombinant Ag5 and Ag5 peptides in CE diagnosis are summarized in Table 5.
One recent study highlights the need to standardize techniques and antigenic preparations and to improve the performance of immunodiagnosis by characterizing new antigens and detecting distinct immunoglobulin classes. The diagnostic sensitivity and specificity of IEP, ELISA, and IB in detecting IgG antibodies to native and recombinant AgB and a hydatid fluid fraction in patient sera were compared (188). Sera tested were from patients who had CE grouped according to their type of cysts, from patients with other parasitic diseases, lung or liver carcinomas, or serous cysts, and from healthy controls. Hydatid fluid fraction-IB gave the highest sensitivity (80%) followed by ELISA (72%) and IEP (31%). The diagnostic sensitivity decreased significantly as cysts matured (from type I-II to type VII, classified by ultrasonography). Recombinant and native AgB-IB yielded similar sensitivities (74%), but a large number of clinically or surgically confirmed CE patients (20%) were negative. In these patient sera, the use of IB to assess the usefulness of another recombinant E. granulosus molecule (elongation factor 1ß/
[EF-1ß/]) in detecting IgE antibodies yielded 33% positivity (188).
The results of this and other studies suggest that hydatid serological testing may be improved by combining several defined antigens (including synthetic peptides) and by designing new E. granulosus-specific peptides that react with otherwise false-negative sera.
PBMC isolated from CE patients can be driven in vitro by HCF antigens (125, 216) to produce large amounts of cytokines. IL-4 detection may be useful in the follow up of patients with CE. Furthermore, this can be combined with reverse transcriptase PCR to determine the mRNA expression of cytokines in PBMC to complement the biological assays in the follow-up (213). Detection of circulating antigens is also relevant as a method of post-surgical follow-up of patients and for monitoring the growth dynamics and/or the activity of cysts (78, 165, 207).
EF-1ß/, a parasite protein present both in PSC and hydatid fluid, is a sensitive marker of infection (176). The higher percentage of humoral immune responses to EF-1ß/ observed in CE patients with calcified cysts than in patients with active cysts suggests that the protein is released into the hydatid fluid after the degeneration of PSC and indicates its possible use in immunosurveillance of CE. Furthermore, EF-1ß/ may play a key role in the allergic disorders (urticaria, itching, and anaphylactic shock) that often complicate the course of CE (176, 187).
Em2, a species-specific native antigen isolated from the metacestode of E. multilocularis (101), has been used for immunodiagnosis of human AE with encouraging results (99). The sensitivities of Em2 in ELISA varied depending on the geographical origin of the patient; they ranged between 77 and 92% (103). The Em2plus ELISA, a combination of Em2 with a recombinant protein designated II/3-10, increased the sensitivity to 97%. The Em2plus assay exhibits cross-reaction with CE (in 25.8% of cases), which is higher than for the individual Em2 (5.6%) and 11/3-10 (6.5%), but limited cross-reactivity with other diseases. The Em2plus ELISA has been commercialized for clinical diagnosis of AE (103) and for population screening (32). More recently, an 18-kDa antigen (Em18) from PSC of AE was reported as being a highly species-specific (96.8%) and sensitive (97%) antigen with potential not only for differentiation of AE from either CE or other helminth infections but also for differentiation of active from inactive AE (131-136, 143). Both Em2plus ELISA and Em18 in an IB format have been used for long-term follow-up monitoring of AE patients following pharmacological treatment (173). Furthermore, Em13 and Em10, recombinant proteins expressed from cloned cDNAs from PSC of E. multilocularis, are also valuable in serodiagnosis of AE (Table 6). A purified alkaline phosphatase from E. multilocularis metacestodes has been shown to have exceptional diagnostic characteristics, with 100% specificity without any decrease in sensitivity (100%), and has significant potential for use in routine diagnosis and follow-up of AE patients (231).
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ELISA techniques using a variety of antigens have been applied to the immunodiagnosis of animal CE (138, 141, 266). In experimentally infected sheep, antibodies to hydatid antigens can be detected as early as 4 to 6 weeks postinfection (266) and persist for at least 4 years (138). However, as referred to above, serological cross-reactions between E. granulosus and other cestodes limit the specific diagnosis of hydatid infection by ELISA with crude parasite antigens (138, 266). Affinity purification of crude antigens with antibodies from animals immunized with homologous antigen (141) or affinity depletion of cross-reactive antigens with monoclonal antibody (138) only partly reduces the cross-reactivity. Components of ovine HCF can bind to sheep immunoglobulin nonspecifically and contribute to false-positive reactions, even with sera from cestode-free animals (138). After affinity depletion of crude antigen with both monoclonal antibody and sheep immunoglobulin from animals not infected with hydatid disease, background reactions were greatly reduced. Using this affinity-depleted antigen, it was possible to differentiate serologically between a flock of sheep with hydatid infection and uninfected sheep from the same locality; however, specific diagnosis of infection in individual sheep from another locality was low, and variation in antibody responses to different parasite strains was suggested as a possible cause of these differences (138).
Polysaccharide antigens from either the secretions produced during in vitro cultivation of E. granulosus PSC or from mouse hydatid cyst membranes by phenol extraction have been used to test sera from sheep (140). Although the antibody responses were significantly higher than those of sheep infected with T. hydatigena or T. ovis, very high cross-reacting antibody responses in the sera from T. hydatigena-infected animals were detected with the antigenic secretions from PSC. Neither antigen was sufficiently sensitive or specific for routine serodiagnostic use (140).
DNA techniques are now available that allow the identification of Echinococcus species and of E. granulosus strains by using metacestode material from intermediate hosts (245).
Specific serum antibodies were shown to be detectable in the blood of dogs after experimental infection with taeniid cestodes, including E. granulosus, using metacestode antigen preparations in ELISA (138-141). Subsequently, others confirmed the appearance of specific antibodies, following experimental infection with E. granulosus in dogs, detected using antigens derived from the oncosphere (25, 238, 239). These latter results suggested that some of the eggs released into the small intestine, following apolysis of proglottids, may hatch and penetrate the intestinal wall, resulting in immunological stimulation of the host. Detection of circulating anti-Em2 antibodies by ELISA may be useful for primary screening of fox populations, but antibody prevalence does not correlate with the actual prevalence of the E. multilocularis intestinal infection (58, 102). Overall, the available ELISA-based methods have poor sensitivity, the specificity is unclear, and there is no correlation with worm burden (85-89); therefore, their usefulness, other than in population-based studies of canine hosts, is questionable.
The other major approach to diagnosis of Echinococcus infection in the definitive host is through detection of adult worm products in feces by using the sandwich ELISA. Craig et al. (47, 49) used a monoclonal antibody specific for an antigen on the surface of E. granulosus oncospheres to distinguish E. granulosus eggs in perianal swabs or samples from environmental sites. This indirect immunofluorescence test is relatively cumbersome, and its sensitivity can be affected by the periodic absence of eggs in fecal samples (47). Nevertheless, the approach has been developed further and used successfully by a number of groups to detect coproantigens of Echinococcus spp. in canine host feces by antibody capture ELSA (11, 59); at least two commercial ELISA kits are now available. Although the various tests that have been developed show some cross-reactivity with other cestode infections (72), they exhibit a high probability of correlation with current infection (57, 59, 137). Further, these tests are capable of detecting patent and prepatent infections with a high degree of sensitivity and specificity, making them reliable tools for epidemiologic investigations (11, 57-60, 72, 184). Some details of investigations showing the sensitivity and specificity of the coproantigen ELISA are shown in Table 7.
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| ACKNOWLEDGMENTS |
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We much appreciate the typing and formatting skills of Ian Dillon and his help in producing Figure 1. We also acknowledge Wen Hao, Xinjiang Medical University, Xinjiang, People's Republic of China, for providing the clinical pictures.
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