Unveiling the incidences and trends of the neglected zoonosis cystic echinococcosis in Europe: a systematic review from the MEmE project.

The neglected zoonosis cystic echinococcosis affects mainly pastoral and rural communities in both low-income and upper-middle-income countries. In Europe, it should be regarded as an orphan and rare disease. Although human cystic echinococcosis is a notifiable parasitic infectious disease in most European countries, in practice it is largely under-reported by national health systems. To fill this gap, we extracted data on the number, incidence, and trend of human cases in Europe through a systematic review approach, using both the scientific and grey literature and accounting for the period of publication from 1997 to 2021. The highest number of possible human cases at the national level was calculated from various data sources to generate a descriptive model of human cystic echinococcosis in Europe. We identified 64 745 human cystic echinococcosis cases from 40 European countries. The mean annual incidence from 1997 to 2020 throughout Europe was 0·64 cases per 100 000 people and in EU member states was 0·50 cases per 100 000 people. Based on incidence rates and trends detected in this study, the current epicentre of cystic echinococcosis in Europe is in the southeastern European countries, whereas historical endemic European Mediterranean countries have recorded a decrease in the number of cases over the time.

Co-Circulation of Leishmania Parasites and Phleboviruses in a Population of Sand Flies Collected in the South of Portugal.

In the Old World, phlebotomine sand flies from the genus Phlebotomus are implicated in the transmission of Leishmania spp. parasites (Kinetoplastida: Trypanosomatidae) and viruses belonging to the genus Phlebovirus (Bunyavirales: Phenuiviridae). Two of the five sand fly species known to occur in Portugal, Phlebotomus perniciosus and Ph. ariasi, the former being the most ubiquitous, are recognized vectors of Leishmania infantum, which causes visceral leishmaniasis, the most prevalent form of leishmaniasis in the country. Phlebotomus perniciosus is also the vector of the neurotropic Toscana virus, which can cause aseptic meningitis. Entomological surveillance is essential to provide fundamental data about the presence of vectors and the pathogens they can carry. As such, and given the lack of data in Portugal, an entomological survey took place in the Algarve, the southernmost region of the country, from May to October 2018. Polymerase chain reaction assays were performed in order to detect the presence of the above-mentioned pathogens in sand fly pools. Not only were both Leishmania parasites and phleboviruses detected during this study, but more importantly, it was the first time their co-circulation was verified in the same sand fly population collected in Portugal.

Parallel Propagation of Toxoplasma gondii In Vivo, In Vitro and in Alternate Model: Towards Less Dependence on the Mice Model.

Toxoplasma gondii is an obligate intracellular protozoan. In pregnant women, it can lead to severe birth defects or intrauterine death of the fetus. Most of what is currently know on cell biology of T. gondii comes from studies relying on the RH strain propagated in mice. According to the recommendations concerning the animal welfare, we assayed in vitro/in vivo procedures to replace, or at least reduce, the demanding animal model for strain propagation. We evaluated the genetic and phenotypic stability of the RH strain throughout its parallel continuous propagation in mice, in human foreskin fibroblasts (HFF) and in an alternate fashion of these two procedures. We also assessed the virulence impact on the RH strain after different periods of its long-term propagation strictly in cells. The RH strain completely lost its virulence after long-term passage in HFF. Nevertheless, we obtained a successful outcome with the alternate passaging of the parasite in HFF and in mice as this approach enabled T. gondii to maintain the evaluated phenotypic properties, mainly its virulence potential. Also, no genetic changes were observed in genes known to be highly polymorphic or involved in pathoadaptation. In conclusion, the alternate model seems to be a feasible method for T. gondii propagation and maintenance, strongly impacting the number of sacrificed mice.

First Report of Echinococcus ortleppi in Free-Living Wild Boar (Sus scrofa) from Portugal.

Cystic echinococcosis (CE) is a zoonosis that is prevalent worldwide. It is considered endemic in Portugal but few studies have been performed on Echinococcus granulosus sensu lato and their hosts. In this study, CE cysts are reported for the first time in a free-living wild boar (Sus scrofa) in Portugal. The presence of the metacestodes in the liver of the wild boar was identified by morphological features, microscopic examination and molecular analysis. The sequencing of part of the DNA nuclear ribosomal internal transcribed spacer-1 (ITS-1) region revealed a G5 genotype that presently corresponds to Echinococcus ortleppi. This is the first report of E. ortleppi in Portugal and to the best of the authors' knowledge, in Europe. These results suggest that wild boar may be a host of CE, namely, crossing the livestock-wildlife interface, which has important public health implications. Wildlife reservoirs must be taken into account as CE hosts and surveillance of game as well as health education for hunters should be implemented using a One Health approach, with implementation of feasible and tailor-made control strategies, namely, proper elimination of byproducts in the field.

Travel-associated human trichinellosis in Portugal.

Trichinellosis is an emerging or re-emerging foodborne parasitic zoonotic disease caused by nematodes of the genus Trichinella. It represents a global food safety problem and a public health hazard. This case report aims to describe the first case of human trichinellosis in Portugal since the creation of the European Union surveillance system. Infection by Trichinella spp. is a rare cause of hypereosinophilia and hospital admission, but it can cause high morbidity. Clinical detailed history is crucial to obtain a correct diagnosis.

PCR Detection of Toxoplasma gondii in European Wild Rabbit (Oryctolagus cuniculus) from Portugal.

Wildlife plays an important role in the epidemiological cycle of Toxoplasma gondii. The European wild rabbit (Oryctolagus cuniculus) can be a source of infection to wild and domestic hosts, including human beings. Additionally, as an herbivorous animal, the European wild rabbit may also be a sentinel of environmental contamination with T. gondii and, consequently, an indicator of the potential transmission of this parasite. The purpose of the present work was to detect T. gondii DNA in European wild rabbit from central Portugal, as well as the possible implications for public health. Heart and diaphragm samples were obtained from 28 rabbits hunted in central Portugal. Nested PCR separately amplified the 5' and 3' ends of the surface antigen 2 (SAG2) gene. T. gondii DNA was detected in 19 out of the 28 sampled animals, resulting in a prevalence of 67.9%. These results show that T. gondii infection occurs in European wild rabbit and therefore may pose a potential risk for humans if consumed as raw or undercooked meat. Measures should be taken in order to prevent infection by this zoonotic parasite and for the conservation of wildlife. To the best of our knowledge, this is the first study performed by means of PCR on T. gondii in European wild rabbit meat samples.

Occurrence of larval anisakids in horse mackerel (Trachurus trachurus) caught in Portuguese waters.

The third-stage larvae (L3) of the Anisakidae family are parasitic nematodes with zoonotic impact and are frequently encountered in the organs and musculature of various fish intended for human consumption. Since Anisakis simplex (s.s.) and A. pegreffii are the major aetiological agents of human disease, this study aims to combine the morphological and molecular data on the recovered anisakid larvae to contribute to a simplified morphological distinction of those species and conducted a survey of anisakid larvae infection in horse mackerel (Trachurus trachurus). Here, 116 horse mackerel caught in Portuguese waters were analysed for the presence of L3 of anisakids, and 3148 larvae were collected, of which only 30% were retrieved during visual inspection. As such, visual inspection does not appear to be very effective in anisakid detection. A prevalence of 84.5% of infected fish was found, and the mean intensity and mean abundance were 32.1 and 27.1 parasites per fish, respectively. The morphological and molecular analyses of 196 L3 randomly chosen from the total sample of parasites demonstrated the presence of L3 of mostly Anisakis spp., with only one L3 of Hysterothylacium aduncum. Relative frequencies of 62.9% for A. pegreffii and 37.1% for A. simplex (s.s.) were obtained. The morphometry differences between these two sibling species were evaluated, and the results demonstrated significant differences between the length of the ventriculus and the length of the oesophagus. Precisely, A. simplex (s.s.) has a longer oesophagus and ventriculus than A. pegreffii. As such, these differences may be used to distinguish the two species through morphological analysis.

Towards a rapid sequencing-based molecular surveillance and mosaicism investigation of Toxoplasma gondii.

Advances in molecular epidemiology of Toxoplasma gondii are hampered by technical and cost-associated hurdles underlying the acquisition of genomic data from parasites. In order to implement an enhanced genotyping approach for molecular surveillance of T. gondii, we applied a multi-locus amplicon-based sequencing strategy to samples associated with human infection. This approach, targeting genome-dispersed polymorphic loci potentially involved in adaptation and virulence, genetically discriminated almost all 68 studied strains and revealed a scenario of marked genomic mosaicism. Two-thirds (n = 43) of all strains were classified as recombinant, although recombination seemed to be linked to the classical archetypal lineage. While 92% of the Sag2 archetype I strains revealed genetic mosaicism, only 45% of Sag2 archetype II strains were identified as recombinant. Contrarily to the virulence-associated archetype I, most type II strains (regardless of their recombination background) were non-virulent in mouse. Besides Sag2, some of the newly studied loci (namely the type I/I-like alleles of Sag1, B17, PK1, and Sag3 and type III/III-like alleles of TgM-A) constitute promising candidates to rapidly infer T. gondii mouse virulence. Our successful attempt to capture microsatellite length variation launches good perspectives for the straightforward transition from the laborious intensive historical method to more informative next-generation sequencing (NGS)/bioinformatics-based methodologies. Overall, while T. gondii whole-genome sequencing will be hardly feasible in most laboratories, this study shows that a discrete loci panel has the potential to improve the molecular epidemiology of T. gondii towards a better monitoring of circulating genotypes with clinical importance.

Molecular and virulence characterization of Toxoplasma gondii strains isolated from humans in Portugal.

Toxoplasma gondii is an apicomplexan parasite responsible for toxoplasmosis which infects all warm-blooded vertebrates, including mammals and birds. The majority of studies conducted in Europe have revealed that more than 80 % of strains isolated from human infections belong to genotype II, whereas genotypes I and III are responsible for a small number of cases. Atypical and recombinant strains are generally associated with more severe infections. In Portugal, there is a lack of data concerning genetic diversity as the classical typing studies in humans have never been performed. We aimed to determine the Sag2 and microsatellite-based (TUB2, TgM-A, W35, B17, B18) genotypes of T. gondii isolated from humans in Portugal, as well as to study their virulence in mice. We analyzed 48 strains from congenital and acquired toxoplasmosis collected during the last two decades. Sag2-based genotyping of T. gondii was achieved in all 48 strains where 35 (73 %) were classified as type II and 13 (27 %) were type I. The multilocus PCR of five microsatellites allowed the classification of 10 strains (21 %) as recombinant strains that had been previously identified as type II or I by Sag2 typing. Seven out of the 48 strains, including three type I, three recombinant, and one type I, were virulent in mice. This study constitutes the first evidence of recombinant strains circulating in Portugal in humans from congenital infection, highlighting the need for a better evaluation of these strains as their phenotype is still barely understood.

Toxoplasma gondii seroprevalence in the Portuguese population: comparison of three cross-sectional studies spanning three decades.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan infecting up to one-third of the world's population, constituting a life threat if transmitted from mother to child during pregnancy. In Portugal, there is a lack of knowledge of the current epidemiological situation, as the unique toxoplasmosis National Serological Survey was performed in 1979/1980. METHODS: We studied the seroprevalence trends in the Portuguese general population over the past 3 decades, by assessing chronological spread cross-sectional studies, with special focus on women of childbearing age, by age group, region and gender. RESULTS: The T. gondii overall seroprevalence decreased from 47% in 1979/1980 to 22% (95% CI 20% to 24%) in 2013. Generally, we observed that the prevalence of T. gondii IgG increased significantly with age and it decreased over time, both in the general population and in the childbearing women (18% prevalence in 2013). CONCLUSIONS: The scenario observed for the latter indicates that more than 80% of childbearing women are susceptible to primary infection yielding a risk of congenital toxoplasmosis and respective sequelae. Since there is no vaccine to prevent human toxoplasmosis, the improvement of primary prevention constitutes a major tool to avoid infection in such susceptible groups.

Genotyping Characterization of Toxoplasma gondii in Cattle, Sheep, Goats and Swine from the North of Portugal.

BACKGROUND: Epidemiological investigations on Toxoplasma gondii infection have found a significant association between human toxoplasmosis and consumption of raw or undercooked meat. The present study aimed to characterize genotypes of T. gondii in 20 cattle, 40 sheep, 15 goats and 16 pigs from the North of Portugal. METHODS: Nested PCR amplified the surface antigen 2 (SAG2) gene. Sequencing analysis was performed in order to assess the prevalence of SAG2 type strains (I, II and III). RESULTS: Three and 4 strains of SAG2 type II were identified in heart samples of cattle and sheep, respectively. Three SAG2 type II strains were detected in brain, diaphragm and heart of 3 pigs. Three strains detected in heart samples of 3 goats belonged to SAG2 types I or II; with the same result being observed in heart samples from 2 sheep and in 2 brain and 1 heart samples from 3 pigs. CONCLUSION: SAG2 type II has been shown for the first time to infect cattle in North of Portugal. In addition, SAG2 type II has also been confirmed as the predominant strain in sheep and pigs in the same region. This is the first molecular report of T. gondii in goats from Portugal.

In vitro isolation and seroprevalence of Toxoplasma gondii in stray cats and pigeons in Lisbon, Portugal.

Oral contamination with Toxoplasma gondii oocysts shed by cats into the environment has been linked to severe outbreaks of human toxoplasmosis. Pigeons (Columba livia) are highly susceptible to oral infection with oocysts and indirectly indicate soil contamination, since they feed from the ground. A seroprevalence study was performed on cats and pigeons captured in the city of Lisbon. Serum samples collected from 1507 pigeons captured at 64 feeding sites and 423 stray cats were screened for antibodies anti-T. gondii using a commercial direct agglutination test. Seroprevalence in pigeons was 2.6% (39/1507) (95% CI: 1.9-3.5%) and 37.5% (24/64) of pigeon flocks sampled showed to be infected with T. gondii. The proportion of infected pigeons within seropositive flocks ranged between 4.8% and 21.1%. Among cats, seroprevalence was 44.2% (187/423) (95% CI: 39.5-49.1%). Isolation of T. gondii from animal tissues was attempted by in vitro assay. Inoculation of brain homogenates from 20 pigeons and 56 cats into Vero cell cultures allowed isolation of T. gondii from 13 pigeons (65%) and 15 cats (26.8%). Inoculation of muscle homogenates (heart and limbs) prepared by acid-peptic digestion from a subset of 15 cats resulted in the recovery of T. gondii from 10 cats (66.7%).