Survey in Intermediate Hosts (Rodents)

The rodent survey has been ongoing since spring 1993. The study period assessed in this article is spring 1993 to spring 1998. A. terrestris and Microtus arvalis were caught as described (7) by using standardized 100-m trap lines to obtain density estimates (8). Each trapped animal was assessed for biologic variables and E. multilocularis infection status as described (2,7). Liver parts were preserved in 70% ethanol and 4% buffered paraformaldehyde after microscopy examination to detect metacestode lesions. All preserved lesions were subsequently assessed by immunohistochemistry or polymerase chain reaction (PCR) (2).

Survey in Definitive Hosts (Dogs and Cats)

Three local veterinarians participated in the survey by distributing information and diagnostic fecal containers to dog and cat owners. This part of the study took place from June 1996 to October 1997. Fecal samples were examined by the following methods: Taeniid egg detection by microscopy following a flotation enrichment (9); identification of isolated E. multilocularis eggs by PCR (10); and detection of E. multilocularis antigens by sandwich-enzyme-linked immunosorbent assay (ELISA) (9). Owners of E. multilocularis-positive dogs or cats obtained detailed information and support by their veterinarians for treating E. multilocularis-infected animals and taking the necessary safety precautions, which included a recommendation to inform their physicians about previous exposure risk and to have all household members undergo serologic testing (procedures described in the next section).

Survey in Humans

The seroepidemiologic survey was done in collaboration with the Blood Transfusion Centre, Cantonal Hospital, Fribourg. Serum samples were obtained from 2,943 blood donors in October 1996 to August 1997. The blood donation collection area included 38 villages located in the periphery of the initial rodent study area. In Switzerland, blood donation is voluntary and not compensated financially. Blood donation statistics have shown that a constant rate of 15% of the population (independent of geographic area) participates in blood donation (men four times and women three times a year). The sampling strategy was designed by the blood donation center to obtain all registered donors of the study area and avoid double donations by the same person. An identical sampling procedure and strategy had been used earlier, which also had covered approximately 15% of the study area (6). Informed consent was obtained from blood donors. All sera were stored at -30°C until tested. Em2-ELISA was performed as described, including the respective data management processing (2,6). For complementary serologic investigations, the serum samples were also tested in parallel for reactivity with an E. granulosus hydatid fluid antigen (EgHF) ELISA (11). If Em2-serologic test results were positive, a second blood sample was obtained through the responsible physician to rule out errors in handling the first sample and confirm the first serologic test result. If the result was confirmed, hepatic ultrasonography was performed in a regional imaging center. In cases where sonography showed hepatic lesions, the patient was referred to the University Hospital in Bern for further assessment by computed tomography (CT).

Statistical Analyses

Comparative statistical analyses of the present seroepidemiologic data and those of a previous study (6) were done with SAS v.6.12 and Fisher's exact test (2-tail); p values were <0.005, unless otherwise stated.

E. multilocularis in Definitive Hosts (Dogs and Cats)

The prevalence of intestinal E. multilocularis infection in dogs and cats living near the area under investigation are provided in Table 2. The definitive prevalence of 7% for dogs and 3% for cats may be underestimated as the veterinarians involved in the collection of samples informed regional cat and dog owners about the possibility of preventing infection by monthly administration of praziquantel (5 mg per kg body weight). Bias was suggested by the fact that most E. multilocularis-positive dogs were detected at the beginning of this study in 1996.

E. multilocularis in Humans

Figure

Figure. . Abortive alveolar echinococcosis detected in a 44-year-old blood donor in Switzerland. A, ultrasonography of the liver demonstrates the presence of a small echodense lesion (arrow). B, a liver computed tomography...

Sera from 2,943 healthy blood donors were tested by Em2-ELISA (Table 3). All six Em2-positive blood donors had a relatively high anti-Em2 antibody concentration (>15 antibody units, AU) (11). The donors were referred to their physicians for hepatic imaging analyses by ultrasonography. Four of the six donors agreed to this procedure; in three of the four, no lesions could be detected by the initial ultrasonography. The procedure was repeated approximately 6 months and 24 months after immunodiagnosis and then annually. No lesions have been detected in these three donors; one, however, had a small echodense lesion of a few centimeters in diameter in the right liver lobe (Figure, A). The donor was referred to the outpatient clinic of the University Hospital in Bern for an abdominal CT, which confirmed the presence of a small hypodense lesion assumed to be fully calcified (Figure, B). No other pathologic changes of the liver were observed. As the morphologic features of this lesion matched the criteria for nonviable, so-called "died-out" or "abortive" lesions (1,12,13), the case was classified in this category. Further CT investigations using contrast enhancement detected a very small hypodense area in the periphery of the calcified herd, which could have harbored a putatively still viable parasitic zone (Figure, C). The patient was reexamined by CT at the same time intervals as described above for the other seropositive donors. No changes in size and morphologic features of the calcified lesion and the peripheral hypodense zone have been demonstrated. The patient received no treatment at any time. The inert lesion was definitively rated as abortive in 1999.

In parallel, all sera were serologically tested by EgHF-ELISA to compare data with those from a former study performed in Switzerland in 1985-86 (2). Of 2,943 sera, 33 were seropositive in this EgHF ELISA (including the six Em2-positive sera described above). Ten EgHF ELISA-positive (but Em2-negative) persons were arbitrarily selected for a subsequent hepatic ultrasonography investigation. Nine persons had negative ultrasonography results. One person, however, had a typical hepatic E. granulosus hydatid cyst of 5 cm in diameter and was referred to his physician for further clinical testing and treatment.

Retrospective Statistical Comparative Analyses

Seroprevalence and clinical findings obtained in the present study (Table 3) were directly compared with those obtained in a previous study, which had used similar immunodiagnostic tools and blood donor population (6). The key data used from this previous study originated from 17,166 blood donors, six Em2-ELISA-positive (two of these with hepatic lesions by CT). Subsequent surgery confirmed the presence of active E. multilocularis metacestodes. Of 5,166 blood donors additionally tested with EgHF-ELISA, 16 Em2-ELISA-negative donors had EgHF seropositivity. Echinococcosis tests and standard laboratory tools were the same in both studies (11): Diagnostic sensitivity of the Em2-ELISA was 95%, and specificity was 100%. Diagnostic sensitivity of the EgHF-ELISA was 96%, and specificity was 97%. By these key data and the 2-tailed Fisher's exact test, a significant increase of seroprevalence from 1986 to 1996-97 became apparent for both the Em2-ELISA (p <0.005) and the EgHF-ELISA (p <0.01). Conversely, clinical findings (CT hepatic lesions compatible with E. multilocularis infection) were not significantly different between the two studies (p = 0.378).