Latent class models for Echinococcus multilocularis diagnosis in foxes in Switzerland in the absence of a gold standard

Background: In Europe the principal definitive host for Echinococcus multilocularis, causing alveolar echinococcosis in humans, is the red fox (Vulpes vulpes). Obtaining reliable estimates of the prevalence of E. multilocularis andrelevant risk factors for infection in foxes can be difficult if diagnostic tests with unknown test accuracies are used. Latent-class analysis can be used to obtain estimates of diagnostic test sensitivities and specificities in the absence of a perfect gold standard. Samples from 300 foxes in Switzerland were assessed by four different diagnostic tests including necropsy followed by sedimentation and counting technique (SCT), an egg-PCR, a monoclonal and a polyclonal copro-antigen ELISA. Information on sex, age and presence of other cestode species was assessed as potential covariates in the Bayesian latent class models. Different Bayesian latent-class models were run, considering dichotomized test results and, additionally, continuous readings resulting in empirical ROC curves. Results: The model without covariates estimated a true parasite prevalence of 59.5% (95% CI: 43.1–66.4%). SCT, assuming a specificity of 100%, performed best among the four tests with a sensitivity of 88.5% (95% CI: 82.7–93.4%). The egg-PCR showed a specificity of 93.4% (95% CI: 87.3–99.1%), although its sensitivity of 54.8% was found moderately low (95% CI: 48.5–61.0%). Relatively higher sensitivity (63.2%, 95% CI: 55.3–70.8%) and specificity (70.0%, 95% CI: 60.1–79.4%) were estimated for the monoclonal ELISA compared to the polyclonal ELISA with a sensitivity and specificity of 56.0% (95% CI: 48.0–63.9%) and 65.9% (95% CI: 55.8–75.6%), respectively. In the Bayesian models, adult foxes were found to be less likely infected than juveniles. Foxes with a concomitant cestode infection had double the odds of an E. multilocularis infection. ROC curves following a Bayesian approach enabled the empirical determination of the best cut-off point. While varying the cut-offs of both ELISAs, sensitivity and specificity of the egg-PCR and SCT remained constant in the Bayesian latent class models. Conclusions: Adoption of a Bayesian latent class approach helps to overcome the absence of a perfectly accurate diagnostic test and gives a more reliable indication of the test performance and the impact of covariates on the prevalence adjusted for diagnostic uncertainty

In vivo viability of Echinococcus multilocularis eggs in a rodent model after different thermo-treatments

Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a serious and emerging zoonotic disease in many parts of the northern hemisphere. Humans but also primates and other accidental hosts can acquire the infection by the ingestion of eggs excreted by the carnivore definitive hosts, e.g. after hand contact with egg-contaminated environments or by consumption of contaminated food or beverages. The goal of this study was to develop a sensitive in vivo method to determine the viability of E. multilocularis eggs and to establish suitable conditions (optimal temperature, exposure time and humidity) for their (prophylactic) inactivation. The sensitivity of a rodent model was evaluated and, conclusively, C57Bl/6 mice were most susceptible to subcutaneous inoculation of small numbers of sodium hypochlorite-resistant oncospheres, even more than to oral inoculation of mature eggs. In the second part of the study, various combinations of exposure temperature (between 45 °C and 80 °C), times (between 30 min and 180 min) and relative humidity (70% vs. suspended in water) were tested. After heat treatment in an incubator, the sodium hypochlorite resistance test was used to assess in vitro egg viability at the time of inoculation. Subsequently, the infectivity of the oncospheres was evaluatedby subcutaneous inoculation in mice. Eggs exposed to increasing temperatures were more resistant to heat if suspended in water as compared to eggs exposed on a filter paper at 70% relative humidity. As survival of eggs in water droplets on the vegetables cannot be excluded, further experiments were performed with eggs suspended in water only. Eggs were infectious after heat exposure at 65 °C for up to 120 min, however, no echinococcosis developed after treatment of the eggs at 65 °C for 180 min or at 70, 75 and 80 °C for 7.5, 15 or 30 min

Detection of taeniid (Taenia spp., Echinococcus spp.) eggs contaminating vegetables and fruits sold in European markets and the risk for metacestode infections in captive primates

Due to frequent cases of alveolar echinococcosis (AE) in captive primates in Europe, 141 samples of food, which consisting of vegetables and fruits, were investigated for contamination with egg-DNA of taeniids. Each sample consisted of at least 40 heads of lettuce as well as various vegetables and fruits. The samples were purchased at different times of the year: either from September to November (autumn), originating from greenhouses or fields in the Basel region in the North of Switzerland, or in April and May (spring) when fruit and vegetables are sourced from throughout Europe from various wholesalers. Each sample was washed, and the washing water sieved through mesh apertures of 50 μm and 21 μm, respectively. The debris, including taeniid eggs, collected on the 21 μm sieve were investigated by a multiplex PCR-analysis followed by direct sequencing. In 17 (18%) of the 95 samples collected in autumn, taeniid-DNA was detected (Taenia hydatigena in four, Taenia ovis in three, Taenia polyacantha in two and Hydatigera (Taenia) taeniaeformis in five cases). Similarly, in 13 (28%) of the 46 samples collected during spring taeniid-DNA was detected (Echinococcus granulosus s.l. in two, Taenia crassiceps in one, T. hydatigena in two, Taenia multiceps/Taenia serialis in two, Taenia saginata in one and H. taeniaeformis in five cases). Although DNA of Echinococcus multilocularis was not found specifically in this study, the detection of other fox taeniids reveals that vegetables and fruit fed to the primates at the Zoo Basel at different times of the year and from different origin are contaminated with carnivore's faeces and therefore act as a potential source of AE infections

Detection of Echinococcus granulosus and Echinococcus ortleppi in Bhutan

In this pilot study, fecal samples were collected from community dogs around slaughterhouses and from the city of Thimphu (n = 138) as well as from carnivores in the forest area around a farm in Bhutan (n = 28). Samples were analyzed microscopically for the presence of taeniid eggs by the floatation and sieving method. Further molecular analyses of 20 samples of community dogs positive for taeniid eggs confirmed 10 Echinococcus granulosus sensu lato and one Taenia hydatigena case. From 14 environmental fecal samples from the forest area positive for taeniid eggs, one contained E. granulosus s.l., six T. hydatigena and one Taenia taeniaeformis DNA. In the remaining samples considered positive for taeniid eggs, no molecular confirmation could be achieved. Additionally, Echinococcus cysts were collected from locally slaughtered cattle and imported cattle organs. Seven Echinococcus cysts (one fertile) from the local animals and 35 (four fertile) from imported cattle organs were confirmed as E. granulosus (G1–3) by PCR/sequencing. One Echinococcus cyst each from a local animal and from an imported cattle organ (both fertile) were confirmed to be Echinococcus ortleppi (G5). Sterile Echinococcus cysts were also collected from local yaks (n = 10), and all revealed to be E. granulosus (G1–G3). Hospital records of cystic echinococcosis in humans and the presence of Echinococcus spp. in dogs and ungulates indicate the existence of local transmission for both E. ortleppi and E. granulosus in Bhutan

Successful intestinal Echinococcus multilocularis oncosphere invasion and subsequent hepatic metacestode establishment in resistant RccHan™:WIST rats after pharmacological immunosuppression

Susceptibility/resistance to larval Echinococcus multilocularis infection varies greatly depending on host species and strains. Whereas several mice strains and non-human primates are highly susceptible to alveolar echinococcosis, rats and most of humans are considered as more resistant. In this study, we aimed to elucidate factors responsible for host resistance in rats (Experiments A-D). (A) The parasite establishment was not observed in immunocompetent Wistar rats orally inoculated with sodium hypochlorite resistant eggs with/without pig bile, or activated/non-activated oncospheres (NAO). Peritoneal inoculation with NAO or metacestode tissue allowed the parasite establishment in rats. (B) T-cell-deficient athymic nude rats showed complete resistance against the metacestode establishment after oral inoculation with parasite eggs. This finding suggests that T-cell-independent parasite clearance occurred in the animals during early phase of the parasite invasion. Finally, Wistar rats that received pharmacological immunosuppression using either dexamethasone (DMS) alone or methotrexate (MTX) i.p. alone or a combination of these compounds were orally inoculated with the parasite's eggs. As a result (D), successful establishment of metacestode with protoscoleces was observed in all 3 rats treated with DMS (s.c.) alone or in all 6 rats treated with DMS (s.c.) plus MTX but not in 8 rats with MTX alone, suggesting that factors affected by DMS treatment are responsible to regulate the parasite invasion and establishment

The occurrence of taeniids of wolves in Liguria (northern Italy)

Canids are definitive hosts of Taenia and Echinococcus species, which infect a variety of mammals as intermediate or accidental hosts including humans. Parasite transmission is based on domestic, semi-domestic and wildlife cycles; however, little is known of the epidemiological significance of wild large definitive hosts such as the wolf. In this study, 179 scats of wolves (Canis lupus italicus) collected throughout the Italian region of Liguria were analyzed for the detection of taeniid infection. Taeniid egg isolation was performed using a sieving/flotation technique, and the species level was identified by PCR (gene target: 12S rRNA and nad 1) followed by sequence analyses. Based on sequence homologies of ≥99%, Taenia hydatigena was identified in 19.6%, Taenia krabbei in 4.5%, Taenia ovis in 2.2%, Taenia crassiceps in 0.6%, Hydatigera taeniaeformis in 0.6% and Echinococcus granulosus in 5.6% of the samples. According to these results, Canis lupus italicus can be considered as involved in the wild (including cervids and rodents) and semi-domestic cycles (including sheep and goats) of taeniids in this area

First case of peritoneal cystic echinococcosis in a domestic cat caused by Echinococcus granulosus sensu stricto (genotype 1) associated to feline immunodeficiency virus infection

A new cystic echinococcosis case in a cat in Uruguay is reported herein. The cat was taken to a veterinary clinic in Rocha city, Uruguay, due to dyspnea, constipation and abdominal enlargement. During surgery a large quantity of cysts was retrieved from the abdominal cavity. The cysts were morphologically studied and confirmed as Echinococcus granulosus sensu stricto (genotype 1) by molecular tools using cytochrome oxidase submit 1 and small subunit ribosomal RNA gene as target genes. Moreover, for the first time a coinfection with feline immunodeficiency virus (FIV) was detected. FIV-induced immunosuppression could be a determining factor in the development of cystic echinococcosis in cats

Red foxes (Vulpes vulpes) and wild dogs (dingoes (Canis lupus dingo) and dingo/domestic dog hybrids), as sylvatic hosts for Australian Taenia hydatigena and Taenia ovis

Foxes (n = 499), shot during vertebrate pest control programs, were collected in various sites in the Australian Capital Territory (ACT), New South Wales (NSW) and Western Australia (WA). Wild dogs (dingoes (Canis lupus dingo) and their hybrids with domestic dogs) (n = 52) captured also as part of vertebrate pest control programs were collected from several sites in the ACT and NSW. The intestine from each fox and wild dog was collected, and all Taenia tapeworms identified morphologically were collected and identified to species based on the DNA sequence of the small subunit of the mitochondrial ribosomal RNA (rrnS) gene. Taenia species were recovered from 6.0% of the ACT/NSW foxes, 5.1% of WA foxes and 46.1% of ACT/NSW wild dogs. Taenia ovis was recovered from two foxes, 1/80 from Jugiong, NSW and 1/102 from Katanning, WA. We confirm from rrnS sequences the presence of T. ovis in cysts from hearts and diaphragms and T aenia hydatigena in cysts from livers of sheep in Australia. T. ovis was not recovered from any of the wild dogs examined but T. hydatigena were recovered from 4(8.3%) wild dogs and a single fox. With foxes identified as a definitive host for T. ovis in Australia, new control strategies to stop transmission of T. ovis to sheep need to be adopted