Molecular and Immunological Diagnosis ofEchinococcosis · they are widely prevalent and maycause severe disease in humans:Echinococcusgranulosus is the causative agent of cystic echinococcosis,

CLINICAL MICROBIOLOGY REVIEWS, July 1992, p. 248-261 Vol. 5, No. 30893-8512/92/030248-14$02.00/0Copyright X 1992, American Society for Microbiology

Molecular and Immunological Diagnosis of EchinococcosisBRUNO GOTTSTEIN

Institute of Parasitology, University of Zurich, CH-5057 Zurich, Switzerland

INTRODUCTION .......................................................................... 248Biology of E. granulosus and E. multilocularis ..........................................................................248Geographic Distribution ........................................................................... 248Clinical Manifestations .......................................................................... 249E. granulosus ........................................................................... 249E. mulilocularis.......................................................................... 249

IMMUNOLOGY.......................................................................... 250Definitive Hosts.......................................................................... 250Intermediate Hosts and Humans.......................................................................... 251

IMMUNODIAGNOSIS ........................................................................... 252Antibody Detection in Humans with Echinococcosis for Clinical Diagnosis, Seroepidemiology,

and Posttreatment Follow-Up.......................................................................... 253Antigen Detection in Patients with Echinococcosis .....................................................................255Lymphoproliferative Responses in Patients with Echinococcosis ...................................................255Immunodiagnosis in Definitive Hosts .......................................................................... 256

MOLECULAR DIAGNOSIS .......................................................................... 256Recombinant Echinococcus Antigens .......................................................................... 256DNA Hybridization Techniques and PCR.......................................................................... 257

REFERENCES .......................................................................... 257

INTRODUCTION

Echinococcosis is an infectious disease caused by thelarval (metacestode) stages of various cestode (tapeworm)species of the genus Echinococcus. Two of these parasitespecies are of medical and public health importance in thatthey are widely prevalent and may cause severe disease inhumans: Echinococcus granulosus is the causative agent ofcystic echinococcosis, or cystic hydatid disease; and E.multilocularis in humans causes alveolar echinococcosis, or

alveolar hydatid disease. Two other species of the genusEchinococcus, namely, E. vogeli and E. oligarthrus, are

mainly restricted to sylvatic animals and occur in some areasof Central and South America. As cases of the so-calledpolycystic echinococcosis (E. vogeli) are very rare in hu-mans and cases of infections with E. oligarthrus have notbeen reported yet, these two parasite species will not beconsidered in the following text.A brief characterization of organisms causing echinococ-

cosis is given in Table 1.

Biology of E. granulosus and E. multilocularis

E. granulosus is a small tapeworm (rarely exceeding 7 mmin length) that lives firmly attached to the mucosa of thesmall intestine in definitive hosts, usually dogs but occasion-ally other carnivores. Ungulates are intermediate hosts forE. granulosus. E. multilocularis occurs mainly in red andarctic foxes, but dogs and cats can incidentally be involvedin the life cycle as definitive hosts (124). Small mammals(microtine and arvicolid rodents, occasionally muskrats, andothers) are intermediate hosts for E. multilocularis.For both Echinococcus species, sexual maturity of the

adult-stage tapeworms is reached within 4 to 5 weeks. This isfollowed by the shedding of gravid proglottids (each contain-ing several hundred eggs) or released eggs in the feces ofdefinitive hosts. Following ingestion of Echinococcus eggs

by susceptible intermediate hosts and humans, a larva, theoncosphere, is released from the egg envelope. The onco-sphere penetrates through the intestinal epithelium into thelamina propria and is passively transported through blood orlymph vessels to primary target organs such as liver andlungs or, less frequently, to other organs. At these locations,the metacestode stage of the parasite develops. Proto-scolices form, and they grow to the adult stage once ingestedby a definitive host.The life cycles of E. granulosus and E. multilocularis are

shown in Fig. 1 and 2.

Geographic Distribution

Infections with E. granulosus occur worldwide. A so-called European form (100), primarily involving synan-thropic hosts in its cycle, has a nearly cosmopolitan distri-bution. This form is responsible for major public health oreconomic problems in many rural areas of the world. ANorthern form (100) is prevalent in northern parts of NorthAmerica and Eurasia. Areas of endemicity are mainly re-lated to tundra and taiga and are delineated by the southernlimits of the boreal forest.

E. multilocularis seems to occur only in the northernhemisphere. In North America, the cestode is present in thesubarctic regions of Alaska and Canada, including St.Lawrence Island (101) and some other islands (100). Theparasite has been discovered in Manitoba, Canada, andNorth Dakota (78) and, more recently, in Alberta andSaskatchewan, Canada, and Illinois, Nebraska (12), Iowa,South Dakota, Montana, Wyoming, and even South Caro-lina (73), thus indicating an apparent expansion of the focuswithin the north central American continent. In Europe,areas with relatively frequent reports of alveolar echinococ-cosis in humans encompass central and eastern France,Switzerland, Austria, and Germany. These main Europeanareas of endemicity have previously been regarded as iso-

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DIAGNOSIS OF ECHINOCOCCOSIS 249

TABLE 1. Brief characterization of organisms causing echinococcosis and the various forms of disease

Parameter E. granulosus E. multilocularis E. vogeli

Major definitive hosts Dog, wild canids Fox, dog, cat Bush dog (Speothos)

Major intermediate hosts Sheep, cattle, pig, horse, Rodents Pacacamel

Adult tapewormLength (mm) 2-7 1.2-3.7 3.9-5.6No. of proglottids (range) 3 (3-6) 5 (2-6) 3

Larval form in humansShape Fluid-filled single cysts Vesiculated tissue, no fluid, Multiple small cysts, fluid

central necrotic zones filled

Growth Expansive Infiltrative Expansive

Main organ localization Liver (60%), lungs (20%), and Liver (98%) Liver and other organsothers

Echinococcosis form in Cystic Alveolar Polycystichumans

Distribution Worldwide Northern hemisphere Central and South America

lated foci, but recent data suggest that they might beconnected to each other and to other Eurasian or Asian areaswhere E. multilocularis has been reported. In Asia, E.multilocularis occurs in the whole zone of tundra from theWhite Sea eastwards to the Bering Strait, thus appearing inlarge parts of the Soviet Union and smaller parts of othercountries (109, 140).

Clinical Manifestations

E. granulosus. In cystic echinococcosis (hydatidosis) ofhumans, well-delineated spherical primary cysts are formedmost frequently in the liver (approximately 65% of the cases)(Fig. 3), but also in the lungs (25%) and other organs such askidney, spleen, brain, heart, and bone (110). Cysts causepathological damage or dysfunction mainly by the gradualprocess of space-occupying repression or displacement ofvital host tissue, vessels, or organs. Consequently, clinicalmanifestations are primarily determined by the site andnumber of cysts and are quite variable. Accidental rupture ofcysts can be followed by a massive release of cyst fluid anddissemination of protoscolices, resulting occasionally inanaphylactic reactions and/or multiple secondary cysticechinococcosis, since protoscolices have the potential ofdeveloping into cysts within the intermediate host. Success-ful surgical removal of hydatid cysts is frequent, so casefatality rates are low (varying between 1 and 4% for caseswith first surgical intervention [110]), provided modern med-ical facilities are available. The public health importance ismainly reflected by the number of infected persons and theirdiminished capacities, the direct and indirect costs of hospi-talization and recovery from surgery, and any residualdisability or clinical sequelae (116). Globally, few data on theoverall prevalence of human cystic echinococcosis exist.Regions with good documentation of prevalence include thewhole Mediterranean area, the Turkana district of Kenya,large foci in South America, and many other zones in allcontinents. North American experiences have been docu-mented by various groups (24, 29, 76, 112). An updatedsummary of cases in countries of the European Community

and the European Free Trade Association has recently beenpresented (123).E. multilocularis. E. multilocularis metacestodes (larvae)

in humans are found almost exclusively in the liver, butsecondary lesions can form in the lungs, brain, and otherorgans (110). The hepatic lesion usually consists of a dis-persed, spongy, pale tissue consisting of scattered smallcysts and vesicles (Fig. 4). The diffuse borders are com-monly not well delineated from the adjacent liver tissue. Acentral necrotic cavity is often found in the advanced stageof hepatic alveolar echinococcosis. The lesions may be alliedwith focal zones of calcification. Microscopically, there isevidence of a vigorous proliferation of fibrous and germina-tive tissue in the periphery of the metacestode but also thereare regressive changes centrally. In contrast to infection inrodent hosts, lesions from infected humans rarely exhibitprotoscolices, brood capsules, and calcareous corpuscleswithin vesicles and cysts. At diagnosis of human alveolarechinococcosis, the nonspecific clinical symptoms usuallyinclude mild upper-quadrant and epigastric pain, possiblehepatomegaly, and obstructive jaundice.

Occasionally, the initial manifestations are caused bymetastases localized in the lungs or other organs (7, 109,110). As in cystic echinococcosis, few data on the overallprevalence of human alveolar echinococcosis exist. Cases inthe native Eskimo population at risk in western Alaska,including St. Lawrence Island, have been diagnosed at anaverage annual rate of 28/100,000 inhabitants (138). In Swit-zerland, an annual average morbidity rate of 0.18 case per100,000 inhabitants was reported (37). However, in variouscantons of Switzerland, annual morbidity rates are higher(between 0.2 and 0.7 case per 100,000 [122]). Data for someareas of France, Germany, and Austria were similar (139).However, the importance of the disease is not representedby the number of reported cases but rather by the severity ofthe disease in the individual patient and by a frequentlylethal outcome: for cases without radical surgery, mortalitywas found to be 92% within 10 years after primary diagnosis(114). In recent times, the mortality rate has significantly

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250 GOTTSTEIN

FIG. 1. Life cycle of E. granulosus. 1, Adult tapeworms live in the small intestine of dogs. 2, Proglottid, containing tapeworm eggs. 3, Egg.4, Ungulates are the main intermediate hosts, with mostly the lungs being affected. Humans (4a) can become infected accidentally asintermediate hosts; target organs for hydatid cysts are mainly the liver and the lungs. 5, Bovine lung harboring hydatid cysts. Cross section(5a) shows the cyst, containing brood capsules with protoscolices. The fluid-filled cyst is surrounded by an outer laminated layer and containsan inner germinal membrane budding into protoscolices containing brood capsules. Multiple detached and sedimented brood capsules formthe so-called hydatid sand. Segmentation of hydatid cysts forms so-called daughter cysts (not shown). Courtesy of the Institute ofParasitology, University of Zurich.

decreased to 10 to 14%, most likely because of markedimprovements in diagnosis, surgery, and chemotherapy (8).

IMMUNOLOGY

Definitive Hosts

The primary site of host-parasite interaction between theadult-stage echinococcus and its carnivorous host is themucosa of the gastrointestinal tract. For many years, it waswidely held that adult cestodes were non- or poorly immu-nogenic (67). Thus, little information has been elaborated onthe specific immunology of adult Echinococcus infections indefinitive host animals. The structures of the adult Echino-coccus worm that interact with the intestinal immune systemare the scolex, the integument, and all molecules excreted orsecreted by the tapeworm. For E. granulosus, there isexperimental evidence for induction of an adult-stage-spe-cific humoral immune response (44, 70). The induction of alocal immune response, however, does not necessarily imply

functionally protective interactions. Acquired protective im-munity to experimental E. granulosus infections in dogs hasbeen reported (46), but instead of showing a continuousdecline in susceptibility, each dog remained susceptible to anumber of infections and then became less susceptible.Movsesijan et al. used 1,000 to 2,500 irradiated E. granulo-sus protoscolices for oral immunization to demonstratesubsequent induction of protective immunity against chal-lenge infections (93).Recent evidence suggests not only that new strategies

aimed at the vaccination of definitive hosts will have to accu-rately and specifically elucidate potential immunological modesof protective responses but also that new (especially recombi-nant DNA) technologies will have to be developed for vaccineantigen production, administration, and presentation to theintestinal immune system. A very promising technologicalapproach in this respect, for instance, is gene expression inbiocarriers such as live attenuated Salmonella spp., which mayprove ideal for delivering the recombinant parasite antigens tothe correct anatomical site in the definitive host (56).

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4a

FIG. 2. Life cycle of E. multilocularis. 1, The red fox is the most important definitive host, occasionally replaced by dogs (la) and cats(lb). 2, Proglottid, containing tapeworm eggs. 3, Egg. 4, Rodents are the main intermediate hosts. Humans (4a) can become infectedaccidentally as intermediate hosts; the main target organ for the metacestode is the liver. 5, Rodent liver harboring the parasitic metacestodelesion. Cross section (Sa) shows the presence of vesicles containing protoscolices. Single vesicles (Sb) are surrounded by an outer laminatedlayer and contain an inner germinal membrane budding into protoscolices containing brood capsules. Courtesy of the Institute of Parasitology,University of Zurich.

Regarding peripheral humoral immune responses in dogswith adult-stage E. granulosus infections, various authorswere able to demonstrate parasite-specific serum antibodieswith diagnostic potential (44, 69, 92, 118, 137). Similar to E.granulosus, adult E. multilocularis is assumed to induce ahumoral immune response in definitive hosts such as foxes.Thus, serum antibodies against an E. multilocularis-specificEm2 antigen have been shown to be of practical value forseroepidemiology in fox populations (50).

Intermediate Hosts and Humans

Information on the immune response to migrating andsubsequently established oncospheres and their develop-ment to the Echinococcus metacestode in humans is sparse.The diagnosis of echinococcosis is generally based on a fullydeveloped and still proliferating metacestode, which hasalready induced and potentially influenced an immune re-

sponse of the host (reviewed in reference 62). Cellular andhumoral immune responses in humans, in contrast to thosein experimentally infected animals, can vary enormously, asevidenced, e.g., by the different patterns of parasite antigensin different patients and courses of disease (43, 82). Thesedisparities are likely related to human and/or parasite geneticdiversity, which is unlike the uniform genetic background ofmost experimental animals (122).

Investigations of cell-mediated immune response in casesof murine cystic echinococcosis have revealed polyclonalB-cell activation (25), a marked drop of mean T-cell percent-age (134) but increase in suppressor cell activity (104), directsplenic T-lymphocyte cytotoxicity to the metacestode (135),and impairment of the host defense potential by the forma-tion of anti-human leukocyte antigen-reactive host antibod-ies (6). Several researchers have suggested that the host'scapacity for developing a parasite-specific cellular responseable to eliminate the parasite may be modulated by parasite-

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252 GOTTSTEIN

1OFIG. 3. E. granulosus. Cross section through a human liver containing hydatid cysts (hydatid fluid removed). Courtesy of the Institute of

Parasitology, University of Zurich.

derived effector substances (9, 36, 82). Local immune mod-ulation by the parasite has been shown to enhance suscep-tibility to mycobacterial infections close to the site ofparasite lesions (39).With regard to alveolar echinococcosis, most human pa-

tients develop parasite-specific serum antibodies, includingall isotypes of immunoglobulins; very few patients fail todemonstrate a humoral immune response (52, 60). Antibod-ies are thought to be involved in immunopathological mech-anisms responsible for the occasional chronic granulomatouscourse of the disease, including immune complex-associatedmembranous nephropathy (98) and histopathologicalchanges related to the incidence of amyloid and immunecomplex deposits in the liver, as was found in severalAlaskan patients (2).Most patients show a specific response of peripheral blood

mononuclear cells to in vitro stimulation with parasite anti-gen (17, 54). Information addressing potential immunologicalevents at the site of host-parasite interplay has shown thatthe periparasitic granuloma in regressive courses of diseaseis mainly composed of macrophages, myofibroblasts, Tcells, and a large number of CD4+ lymphocytes (131).Patients with proliferative metacestodes have increasednumbers of CD8+ cells.Murine models of alveolar echinococcosis have shown

that a preexisting larval infection can prevent or suppress thedevelopment of a secondary infection (83), but once aprimary infection is established in susceptible laboratoryrodents, the initial E. multilocularis metacestode appearswell protected from the host immune response. It grows andmetastasizes despite a marked lymphoproliferative activityin the B- and T-cell areas of lymphoid tissues.

Parasite-specific antibodies alone are unable to control

parasite growth, and host tissue infiltration may be duepartly to complement-neutralizing factors released by themetacestode that cause complement depletion at the host-parasite interface (61) or partly to the inactivation of C3 as itenters the metacestode tissue (72). Undoubtedly, T lympho-cytes play the main role in the immunological control of E.multilocularis infection. Activated macrophages (13) andneutrophils (3, 4) were suggested as key factors in the attackof E. multiloculanis metacestode cells. Fragmentary infor-mation on cell-mediated immunity in different host cellpopulations is restricted to more general aspects (16, 79).However, analyses of the actual effector functions of differ-ent lymphokines and lymphocyte populations or subsetshave not yet provided key findings for understanding thedifferent forms of progression or regression in alveolarechinococcosis.

IMMUNODIAGNOSIS

The clinical signs and symptoms in hepatic cystic oralveolar echinococcosis resemble those of hepatic carci-noma, cirrhosis, or other liver diseases. Noninvasive imag-ing techniques are primarily applied and can be combinedwith immunodiagnostic procedures (90). The role of immu-nodiagnosis is to confirm clinical findings or give diagnostichelp by providing detailed information on parasite or hostpeculiarities (e.g., species differentiation in radiologicallyunclear cases and determination of the patient's immunestatus, etc.). Immunodiagnosis of echinococcosis has beencomprehensively reviewed in various articles (80, 103, 110,111).



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FIG. 4. E. multilocularis. Cross section through a human liver with alveolar echinococcosis. The parasitic metacestode tissue ischaracterized by some central necrotic cavities. Courtesy of the Institute of Parasitology, University of Zurich.

Antibody Detection in Humans with Echinococcosis forClinical Diagnosis, Seroepidemiology, and

Posttreatment Follow-Up

For primary serological diagnosis and for support ofclinical diagnosis of echinococcosis, the selection of a par-ticular immunodiagnostic test involves consideration of thediagnostic operating characteristics of the technique and thepurpose for which it will be used. The diagnostic sensitivityand specificity of the tests used most frequently vary accord-ing to the (i) nature, purity, and quality of the antigen, (ii)nature of the patient's immunoglobulins (isotypes, etc.)specified in the test, and (iii) sensitivity of the selectedtechnology.

Until a few years ago, most serological tests for immuno-diagnosis of both cystic and alveolar echinococcosis em-ployed E. granulosus antigens because they could be ob-tained easily and because, in a very early study, E.granulosus hydatid fluid had appeared to be a better diag-nostic reagent than antigens prepared from E. multilocularis(96). Cystic echinococcosis also occurs more frequently thanalveolar echinococcosis and thus has initiated more investi-gations in the development of homologous test systems. E.granulosus hydatid fluid antigen, which was generally avail-able, was reported to be diagnostically relatively sensitive(75 to 94%) in the indirect hemagglutination test (11, 64, 113).One of the most specific immunodiagnostic approaches for

cystic echinococcosis (E. granulosus) relies on the demon-stration of serum antibodies precipitating an antigen calledantigen 5 (19) by immunoelectrophoresis or similar tech-niques. Experimental studies indicated that antibodies toantigen 5 are among the first detectable after infection (19,23, 142). Diagnostic sensitivity for hepatic cystic echinococ-

cosis has been reported to vary between 50 and 80% (seereferences in reference 110). Antibodies to antigen 5 alsooccur in the sera of human patients with neurocysticercosis(125) and alveolar echinococcosis (127), and comparativestudies showed that only 58% of Swiss patients with alveolarechinococcosis had antibodies to antigen 5 compared with74% of patients with cystic echinococcosis (60).

Subsequently, the antigenic components of E. granulosusresponsible for the arc-5 phenomenon were investigated byvarious approaches that used monoclonal antibodies orimmunoblotting or both (20, 35, 82, 119) (Fig. 5). Further-more, preliminary results had indicated some potential forusing purified antigen 5 in highly sensitive techniques such asthe enzyme-linked immunosorbent assay (ELISA) (65). Fa-con et al. cloned an E. granulosus gene encoding an antigen5 component and suggested recombinant antigen 5 as theimmunodiagnostic reagent (40). However, despite the use-fulness of antigen 5 for various immunodiagnostic applica-tions, there is some evidence of a lack of species specificityand of problems of diagnostic sensitivity due to the absenceof anti-antigen 5 antibodies in some patients. Consequently,multiple attempts to further characterize E. granulosusantigenic components and to identify corresponding frac-tions or molecules with optimal diagnostic characteristicswere investigated. Shepherd and MacManus (117) initiallyfound a low-molecular-weight subunit of antigen B (97) to bespecies specific, but this could not be confirmed subse-quently (82). Resolution of E. granulosus hydatid cyst fluidby sodium dodecyl sulfate-polyacrylamide gel electrophore-sis resulted in the immunoblot finding of a genus-specificdiagnostic component with an apparent molecular mass of 8kDa (86). Further evaluation of this agent will be needed to

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254 GO1TSTEIN

IEP5i mmunoelectrophoresis

DD5double diffusion

J

CIE5e&ectrosyneresi3

FIG. 5. Immunoprecipitation patterns (including especially arc-5) demonstrated by three different techniques, immunoelectrophoresis,double diffusion test, and electrosyneresis, run in parallel with the same antigen (hydatid cyst fluid from cattle of Swiss origin) and the samesera (from one patient with cystic and one patient with alveolar echinococcosis). Arrows point to the identity reaction site of arc-5. Courtesyof the Institute of Parasitology, University of Zurich.

demonstrate its immunodiagnostic suitability. Another ap-proach to improve immunodiagnostic characteristics of E.granulosus serological tools was chromatographic fraction-ation (5) of hydatid cyst fluid. The resulting purified 20-kDaantigen, which resembled antigen B, was first thought to bespecies specific for E. granulosus and was suggested forfurther evaluation of immunodiagnostic potential.

In summary, the presently recommended strategy forimmunodiagnosis of cystic echinococcosis is to rely on a

diagnostically sensitive test such as an ELISA, employinghydatid fluid antigen. Positive test results, which depend on

the geographical origin of the patient and the implied prob-lems of cross-reactivity due to potential infection with otherparasite species, must subsequently be confirmed by testsdemonstrating antibody activity against antigen 5 or otherspecific antigenic components, as discussed above.

Alternatively, E. multilocularis metacestode tissue was

used as a source of immunodiagnostic Echinococcus anti-gens. Gottstein et al. used affinity chromatography to isolateantigens shared by E. granulosus and E. multiloculans(called the Eml fraction) from crude extracts of E. multiloc-ularis metacestode tissue (51). This Eml antigen was used asa reagent for the immunodiagnosis of both cystic and alve-olar echinococcosis. It became obvious that, for the diagno-sis of alveolar echinococcosis, homologous E. multilocularismetacestode antigens were superior to heterologous E. gran-ulosus antigens, especially with regard to specificity. Similarfindings were described by other groups and have beenreviewed by Schantz and Gottstein (110). When crude E.multilocularis antigens are used, however, nonspecific andcross-reactions are the cause of difficulties similar to thosewell known with E. granulosus antigens. Subsequent re-

search has thus addressed the question of purifying highlyspecific antigens from E. multilocularis. The first docu-mented attempt used affinity chromatographic procedures toimmunosorb cross-reactive antigenic components from a

crude E. multilocularis metacestode antigen solution (51).The resulting fractions (Eml and Em2 antigens) were suc-

cessfully used in an ELISA to correctly differentiate 95% ofpatients with cystic echinococcosis from patients with alve-olar echinococcosis (57). In subsequent studies (31, 49), theantigenic component of the Em2 antigen fraction was puri-fied and characterized by immunochemical means, and a

monoclonal antibody was raised against the Em2 antigen.Attempts to differentiate both forms of echinococcosis

serologically have also been undertaken by others (10, 75)but without full documentation of the immunodiagnosticproperties of the antigens in question. Furuya et al. used

Western blotting (immunoblotting) to analyze banding pat-terns of serum antibodies from Japanese echinococcosispatients. They reported specific serum antibody activityagainst 55- and 60-kDa E. multilocularis antigens (43). Morerecently, recombinant DNA technology has been used tosynthesize E. granulosus and E. multilocularis antigens.These will be discussed below.Another approach to improving the value of immunodiag-

nostic properties was the individual analysis of antibodyclasses with respect to parasite antigens. Parasite-specificimmunoglobulin E (IgE) has attracted particular attentionbecause of its well-known importance in helminthic diseasessuch as those caused by E. granulosus (1, 18, 34, 87, 99, 136)and E. multilocularis (52, 67, 132).

In addition to problems of diagnostic sensitivity andspecificity due to cross-reactions, there is accumulatingevidence of false-positive antibody reactions not related toinfections with heterologous helminth species. Such false-positive antibody reactions have been related to malignan-cies (30), the presence of anti-P1 antibodies (14), and livercirrhosis (66).

Early diagnosis of persons with asymptomatic echinococ-cosis is considered a prerequisite for efficient managementand treatment of the disease (71). Consequently, serologicalscreening has been offered to populations and communitiesin many areas. Techniques used for areas in which E.granulosus is endemic have been reviewed by Schantz andGottstein (110). The tests most widely used were based ondetection of arc-5 (reviewed in reference 110). They demon-strated appropriate specificity in that most arc-5-positivepersons could subsequently be shown to harbor hydatidcysts (21, 126). However, many studies have clearly dem-onstrated the limits of using serology alone in epidemiology,especially in areas where seropositivity is low among pa-tients with cystic echinococcosis, such as the Turkanadistrict in Kenya (28). Recent studies therefore employedwhat can presently be considered the optimal epidemiologi-cal tool: ultrasound examination for abdominal cysticechinococcosis combined, if possible, with immunodiagno-sis (22, 85, 91, 106).The use of E. granulosus antigens for seroepidemiological

investigations in areas in which E. multiloculans is endemicpermitted the identification of clinical cases (108, 113, 138).More recent findings in similar studies, however, suggestedsignificant improvement not only of diagnostic sensitivitybut also of specificity by the application of crude andsubsequently purified E. multilocularis antigens.A first direct comparison between homologous purified E.

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multilocularis Em2 antigen (49) and E. granulosus hydatidfluid antigen was performed by Gottstein et al. in a large-scale serosurvey in Switzerland. This survey showed that E.granulosus antigen exhibits a relatively high degree of falsepositivity (53). Similar findings were reported from Alaskanstudies (77). In general, the epidemiological situation of lowprevalence for both cystic and alveolar echinococcosis re-quires high diagnostic sensitivity and high species specific-ity, so that positive and negative predictive values result injustifiable clinical investigation of seropositive individuals.

Seroepidemiological studies in Alaska have shown thatEm2 ELISA detected not only asymptomatic cases of hu-man alveolar echinococcosis serologically negative by othertechniques (58) but also unique cases in which the metaces-tode had died out at an apparently early stage of infection(102). This finding of spontaneous rejection of the infectionmay provide valuable information for future research in theimmunology of E. multilocularis infection. Another ap-proach used to circumvent specificity problems in seroepi-demiology was Western blotting (42), which shows thesuitability of immunoblotting to confirm clinical cases ofalveolar echinococcosis.

Serological tests used for postoperative monitoring ofpatients with cystic echinococcosis have been reviewed bySchantz and Gottstein (110). More recent studies haveemphasized determination of parasite-specific antibody iso-types (99), but the small contribution of serology to moni-toring the course of disease has been discussed and implic-itly requires that serology be combined with an instrumentalor imaging examination in order to provide an accurateprognosis.

Surgically treated cases of alveolar echinococcosis areusually treated postsurgically with chemotherapy. As onlycomplete surgical removal of the entire parasite lesion offersa prospect of cure, the accurate assessment of the success ofthe resection is an urgent requirement in the clinical moni-toring of patients. Serological tests have generally demon-strated a decrease in the concentration of parasite-specificserum antibody after successful surgery (113). The prelimi-nary use of Em2 ELISA indicated that anti-Em2 antibodyconcentrations declined dramatically within months after asuccessful radical operation (77). These findings were con-firmed in subsequent larger studies (59).An exceptional immunological situation is encountered

with patients undergoing orthotopic liver transplantation (47,89). Serum antibodies were artifactually reduced or elimi-nated by abundant blood transfusions coupled with immu-nosuppressive therapy. Patients with remaining residual fociof extrahepatic parasite tissue had extremely high recur-rence rates due to immunosuppression and interruption ofchemotherapy with antiparasitic benzimidazoles (15). Suchrecurrences were generally accompanied by the reappear-ance of anti-Echinococcus serum antibodies.

Patients with alveolar echinococcosis who received onlychemotherapeutical treatment were very difficult to monitorby classical serological means (8, 60, 74, 77, 113). In general,specific antibody concentrations decreased in chemothera-peutically treated patients with regressive forms of disease,whereas specific antibody concentrations in sera of patientswith nonresectable lesions and/or palliative surgery and aprogressive course of disease remained elevated or in-creased. To date, the need for clinicians to have a clearpredictive interpretation of serology with regard to progres-sive or regressive disease has not been met by classicserological methods.

Parasite-specific antibody isotypes may correlate better

with clinical findings than do results of classic serologicaltests, especially with regard to IgA and IgE (52). Vuitton etal. reported transient changes in parasite-specific serum IgAand IgM antibodies in patients after chemotherapy withflubendazole (133) and suggested that basophil-bound IgEcould be correlated specifically with positive or negativeresponses to therapy (132).

Antigen Detection in Patients with Echinococcosis

Tests for the determination of circulating immune com-plexes in patients with cystic echinococcosis have beenreviewed by Schantz and Gottstein (110). Soluble E. granu-losus antigens circulating in patient sera have been detectedin 33 to 85% of serum samples from patients with cysticechinococcosis (27, 48). Generally, it was suggested that theidentification of circulating parasite antigens with their po-tential for immune complex formation in the sera of patientsmight be useful for monitoring the disease and might reflectmore reliably than antibody titers the viability and biologicalactivities of parasites in the host (38). Surprisingly, circulat-ing E. multilocularis antigens have been neglected in thisrespect.

Lymphoproliferative Responses in Patientswith Echinococcosis

Skin tests and basophil degranulation tests have beenextensively discussed and reviewed by Schantz andGottstein (110). Aside from these tests, remarkable develop-ments in basic cellular immunology have attracted the atten-tion of parasite immunologists to parasite-specific host cel-lular immune responses and have implied cytokine interplayat the site of parasitic lesions as well as at their peripheries(33). In these studies, the in vitro lymphoproliferative re-sponse to E. granulosus antigen stimulation was assessed in40 patients with cystic echinococcosis (120). There was nocorrelation between serological and lymphoproliferative re-sults. The diagnostic sensitivity of positive test reactionswas 75% for both serology and lymphocyte proliferation.Finding seronegative patients with positive proliferationassay results and seropositive patients with negative prolif-eration assay results suggested that the lymphocyte-specificimmunoassays should be used as diagnostic tests.With regard to E. multilocularis, the relevance of cellular

immune responses and reactions is suggested by the impor-tant granulomatous infiltration surrounding E. multiloculanislesions in infected human livers (130). The in vitro determi-nation of lymphocyte proliferation to stimulation with E.multilocularis antigens has been proposed as a diagnosticalternative to antibody detection in patients with alveolarechinococcosis (17). The same study addressed the param-eters of E. multiloculanis-specific cellular immune responseduring a 2- to 4-year period of mebendazole treatment. Aprogressive decrease in the capacity to respond to parasite-specific lymphocyte stimulation was observed in most of thepatients with regressive disease. On the other hand, anincrease of stimulation indices was usually shown to beassociated with a progression of the liver lesion. Gottstein etal. showed that the in vitro lymphoproliferative response toE. multilocularis antigen stimulation was very high in curedpatients who had radical surgery or in patients with inactivelesions, but the response was significantly lower in patientswho had had partial or no surgical resection (54). Distinctdifferences in the parasite-specific humoral and cellularimmune status of patients with self-limited infections and

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other patient groups with different courses of alveolarechinococcosis may provide insight into potentially protec-tive immune mechanisms.

Immunodiagnosis in Definitive Hosts

Definitive carnivore hosts are usually examined for infec-tions with intestinal stages of Echinococcus spp. by eitherexamination of purged fecal samples for tapeworms orparasitological examination of small intestines after nec-ropsy. These techniques exhibit some problems of diagnos-tic sensitivity in cases of infection with low numbers ofworms. Antibody detection, therefore, has been experimen-tally investigated as an alternative for the diagnosis of E.granulosus infections in dogs (69, 70). Anti-E. granulosusserum antibodies were detected by ELISA by 2 to 3 weeksafter experimental infection of dogs with E. granulosus.Cross-reactions were low with respect to serum antibodiesfrom dogs experimentally infected with Taenia hydatigenaand T. pisifornis. Unfortunately, when serological tests forassessing E. granulosus infections were evaluated underfield conditions in dogs shot in northwestern Turkana, wherethe parasite is hyperendemic, diagnoses of currently infecteddogs were not reliable (68).

Gasser et al. demonstrated stage-specific antioncospheralhumoral immune responses, which strongly suggested thatoncospheres from Echinococcus eggs actually hatch in theintestines of the specific definitive hosts (44). Identicalmechanisms are assumed to occur for E. multilocularis (50).In this respect, serum antibodies against the metacestodestage-specific Em2 antigen could be demonstrated in dogsand foxes infected with adult-stage E. multilocularis. Thecorresponding Em2 ELISA was evaluated for assessing foxpopulations with E. multiloculanis infection. The speciesspecificity of the test was demonstrated by the absence ofcross-reactions with antibodies from carnivores infectedwith intestinal or tissue-dwelling non-Echinococcus cestodesor nematodes. There was experimental evidence that anti-Em2 immunoglobulin synthesis was induced during a pos-toncospheral development of E. multilocularis following aninfection of the definitive host with its own viable E. multi-locularis eggs (31). The value of the test consequentlyrelated to (i) the reliable identification of fox populationswith or without E. multilocularis infections and (ii) theestimation of the prevalence of infection within the foxpopulations by extrapolation. Thus, the test may be valuablein sequentially assessing the dynamics of prevalence in areasundergoing control campaigns (50).

Alternatives to the coprological or serological diagnosis ofadult-stage Echinococcus infections in definitive hosts havebeen proposed, for example, the antibody sandwich-ELISA(32). Affinity-purified polyclonal antibodies raised againstexcretory and secretory antigens of adult-stage tapewormswere used to develop an ELISA that permitted the detectionof coproantigens from E. granulosus and E. multilocularis indog or fox fecal samples. The absence of cross-reactionswith antigens related to most infections with other cestodesor nematodes proved to be genus specific. Conversely, thesensitivity was relatively low in that only hosts with a highintestinal burden of tapeworms (>1,000 Echinococcusworms per animal) reacted positively.Another immunological diagnostic approach has been

developed to identify the generic origin of taeniid eggs byusing monoclonal antioncosphere antibodies (27). Alterna-tive molecular diagnostic approaches such as the detectionof parasite-specific DNA fragments originating from either

parasite eggs or cells of adult tapeworms are rapidly attract-ing attention, especially with the development of highlysensitive techniques such as the polymerase chain reaction(PCR). Such techniques are discussed below.

MOLECULAR DIAGNOSIS

Recombinant Echinococcus AntigensTechniques in molecular biology exhibit great potential as

tools for the synthesis of defined protein antigens. Obtaininga sufficient supply of diagnostically sensitive and specificEchinococcus antigens has always been a problem whenclassic immunochemical methods are used. Cloning andexpressing Echinococcus genes in suitable vectors maycircumvent these problems (81). The logistical approach isgenerally based on the construction of a cDNA expressionlibrary that uses mRNA obtained from the appropriateparasite stage. Libraries are subsequently screened with(polyclonal or monoclonal) antibodies or lymphocytes inorder to identify bacterial clones producing recombinantantigens bearing relevant B- or T-cell epitopes. Finally,efficient production of recombinant antigens in an appropri-ate biological form requires appropriate gene expressionsystems.A cDNA library derived from E. granulosus protoscolex

mRNA was established by using the Escherichia coli expres-sion vector Agtll and was successfully screened for clonessynthesizing antigens suitable for serodiagnosing intestinaladult-stage infections in dogs (45). The subsequent geneexpression system selected for the production of diagnosticantigens resulted in the synthesis of glutathione S-transfer-ase fusion proteins. One of the fusion proteins investigatedhad demonstrated high specificity (100%), although its diag-nostic sensitivity was low. Screening an identical librarywith sera from patients with cystic echinococcosis resultedin the finding of several clones with production of diagnos-tically highly sensitive fusion proteins (81). The diagnosti-cally misleading reactivity of patient antibodies with gluta-thione S-transferase from the fusion proteins might becircumvented by using a modified gene expression system(pGEX2), which allows cleavage of the recombinant parasitepolypeptide from the glutathione S-transferase (121). Thecloning of an E. granulosus gene encoding an antigen 5component has already been mentioned above (40).The first published E. multilocularis cDNA library was

constructed by Vogel et al. (128), who also used the Esche-richia coli expression vector Agtll. An identified E. multi-locularis species-specific recombinant clone, 11/3, demon-strated optimal immunodiagnostic characteristics, asassayed by Western blotting. Poor bacterial expression andlack of an appropriate purification protocol hampered theapplication of the respective antigen in routine diagnosis.This problem was solved by shortening the initial 1.0-kbcDNA sequence encoding for the antigen 11/3 to a 0.6-kbfragment and then subcloning it into plasmid vectorpAR3038 (94). The resulting increased bacterial productionallowed efficient biochemical purification of the recombinantantigen II/3-10. In the pAR3038 vector, the recombinantantigen II/3-10 was synthesized as a polypeptide fused to ashort (11-amino-acid) N-terminal peptide of bacteriophageT7 origin. This short phage peptide was shown to be immu-nologically irrelevant and thus needed no further modifica-tion prior to investigation by ELISA. A preliminary diagnos-tic evaluation of the recombinant antigen II/3-10 by ELISAresulted in operating characteristics suitable for immunodi-

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agnosis of alveolar echinococcosis in humans. The produc-tion of other recombinant E. multilocularis antigens withimmunodiagnostic potential has been reported subsequently(41, 63). Recent comparative experimental results haveshown sequence homology between the recombinant antigen11/3 described by Vogel et al. (128) and antigen EmlOdescribed by Frosch et al. (41).

In expression systems such as those given above for theproduction of E. granulosus or E. multiloculanis recombi-nant antigens, the products usually accumulate within thebacterial cell as a soluble protein or as insoluble precipitates.Consequently, alternative systems such as the insertion ofEchinococcus genes into the mglB gene of plasmid pVB2have been proposed (115). Resulting fusion proteins arelinked to the mglB-encoded periplasmic galactose-bindingprotein, which is excreted into the periplasmic space (95).Recombinant Echinococcus antigens fused to the galactose-binding protein could be conveniently purified in solubleform from a bacterial cell culture supernatant by an osmoticshock procedure. The purified recombinant antigen consti-tuted >50% of total cellular protein and could be applieddirectly in ELISA. In conclusion, the molecular cloning ofEchinococcus genes encoding epitopes with immunodiag-nostic potential is promising, not only because the produc-tion of antigens in large amounts is facilitated but alsobecause these techniques offer the advantage of producingserological reagents of standardized quality.

DNA Hybridization Techniques and PCR

Molecular biological techniques have evolved rapidly,resulting in technical innovations with potential applicationsto diagnostic parasitology. The identification of parasitespecies- or even stage-specific nucleic acid sequences hasresulted in the development of DNA probes useful forhybridization to DNA from diagnostic samples. To date, thistechnology has limited value in that its application focusesmainly on the characterization of Echinococcus isolates orstrains, thus providing epidemiological rather than clinicalinformation.A variety of DNA probes have been developed and used

by several groups to characterize, identify, or group differentE. granulosus (84, 105, 141) or E. multilocularis (129) strainsor isolates. Apart from the restricted availability of specificdiagnostic probes, one major problem is the limited sensitiv-ity of hybridization and labeling techniques. Current hybrid-ization techniques do not allow identification of single tae-niid eggs. The possibility of differentiating single cestodeeggs at the species level represents an important goal inparasite diagnosis (88). These technical limitations can nowbe essentially eliminated by an extraordinary new tool, thePCR (107). Diagnostic PCR depends on the availability ofappropriate target nucleic acid sequences that flank regionsof interest, which help in the design of synthetic oligonucle-otide primers. By using Taq polymerase (obtained from thethermophilic aquatic bacterial species Thermus aquaticus),which is stable up to DNA denaturing temperatures of 95°C,a millionfold cyclic amplification of target DNA sequencescan be obtained. Furthermore, sensitivity can be enhancedby additional techniques such as reamplification with inter-nal primers or Southern dot hybridization labeled withnucleotide probes. On the basis of an E. multilocularis DNAprobe, pALl (129), the respective nucleic acid sequence wasanalyzed in order to obtain oligonucleotide primers suitablefor use in PCR amplification of specific target sequencesfrom diagnostic Echinococcus genomic DNA (55). Two

designed E. multilocularis oligonucleotides, BG1 and BG2,defined a 2.6-kbp fragment in the genome of E. multilocu-laris. A PCR study including 14 independent E. multilocu-laris isolates (originating from Switzerland, Alaska, Canada,France, Germany, and Japan) and various other cestodesrevealed that the 2.6-kb PCR product was amplified from thegenomic DNA of all E. multilocularis isolates but no othercestode species. Another E. multilocularis primer set, BG1and BG3, defined a 0.3-kbp fragment that resulted in ampli-fication of a genus-specific PCR product, i.e., from E.multilocularis, E. granulosus, and E. vogeli genomic DNAsonly. The diagnostic sensitivity of the E. multilocularis PCRwas evaluated experimentally and approached 2.5 pg oftemplate DNA, which corresponds approximately to theDNA content of one single Echinococcus egg (105). Thediagnostic application of the E. multilocularis PCR puta-tively addressed the identification of fine-needle biopsymaterial obtained from patients with liver lesions of un-known etiology, the rapid and easy identification of E.multilocularis liver lesions from rodents in epidemiologicalstudies, and, perhaps the most promising and importantapproach, the demonstration and identification of adult-stageparasite tissue, DNA, or eggs in samples derived from feces,small intestines, or anal swabs of definitive carnivore hosts.

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