Genetic study of Toxoplasma gondii strains isolated from humans and animals

Toxoplasma gondii is responsible for toxoplasmosis, a zoonosis spread worldwide. This PhD thesis aimed to provide a more comprehensive overview of the genetic characteristics and virulence of T. gondii strains isolated from humans and animals in Portugal. We enrolled T. gondii strains collected from intermediary (farm, urban and sylvatic animals) and the definitive (cat) hosts, as well as from human toxoplasmosis. In general, we applied Sag2 and microsatellites classical genotyping, next-generation sequencing and inoculation in mice. As major results, we highlight: i) the majority of isolated strains were avirulent in mice bioassay, where virulent strains belonged almost exclusively to Sag2 type I; ii) type II strains were the most identified type in strains from human toxoplasmosis as well as from all animal species, followed by type I strains; iii) this is the first report that shows type II T. gondii strains isolated from cattle, boars, foxes and hares in north of Portugal and recombinant strains isolated both from infected cats and pigeons; iv) the development of a rapid multi-loci-based NGS scheme, which allowed evaluating multi loci polymorphism and genomic mosaicism simultaneously. This schema is now implemented as the surveillance tool in the National Reference Laboratory of Parasitic and Fungal Infections (URSZ-INSA) to perform molecular surveillance of T. gondii; v) with this extended typing scheme, we identified a high rate (63 %) of recombinant strains isolated from humans, which had been previously identified as type I and II, as well as new putative genetic markers of virulence. Overall, this PhD shed some light on the genetic and virulence diversity of T. gondii strains circulating in Portugal and revealed a surprising scenario of rampant genomic exchange in this parasite. These data highlight the need for further extended genomic studies to better understand the genotype/phenotype associations in this important human pathogen and thus contribute to better infection control measures

Malaria: a retrospective study of clinical suspected infection cases by Plasmodium sp. between 2010 and 2014

[PT] A malária é uma infeção parasitária causada por um protozoário intracelular obrigatório (género Plasmodium), cuja transmissão é assegurada quando uma fémea de mosquito género Anopheles infetada efetua a sua refeição de sangue num indivíduo. Estão descritas mais de 150 espécies mas apenas 5 infetam o Homem: P. falciparum, P. vivax, P. malariae, P. ovale e mais recentemente, P. knowlesi, uma espécie que causa malária em macacos no sudeste asiático. É a doença parasitária mais frequentes para o Homem, encontrando-se infetada cerca de 5% da população mundial, sendo o P. falciparum o agente mais disseminado, responsável por mais de 80% dos casos mundiais e o que apresenta uma patogenia mais agressiva. O objetivo deste estudo foi descrever as características demográficas dos casos de malária, em doentes com quadro clinico compatível e cujo diagnóstico laboratorial foi confirmado. As metodologias de diagnóstico utilizadas foram, serológicas (IFI), moleculares (PCR em tempo real, adaptada de Swan et al.) e a pesquisa direta em gota espessa e esfregaço. Foram analisadas 471 amostras, 6 das quais provenientes do surto de ébola (África Ocidental, 2014). Detetaram-se 55 (11,7%) casos positivos. Todos os casos positivos de malária são importados. A maioria dos casos ocorreu em viajantes, indivíduos adultos. Pelo facto da transmissão local continuar a ser possível na Europa e pela quantidade de viagens efetuadas entre a Europa e os países endémicos, salienta-se a necessidade de vigilância e prevenção desta parasitose.[EN] Malaria is a human disease caused by a protozoan infection of red blood cells transmitted by the bite of a female anopheline mosquito. There are described more than 150 species of Plasmodium but only five infected humans, P. falciparum, P. vivax, P. malariae, P. ovale and more recently P. knowlesi, a specie that causes malaria in monkeys in Southeast Asia. Malaria is the most important parasitic disease of man; approximately 5% of the world’s population is infected. P. falciparum is the most widespread specie responsible for over 80% of cases worldwide and which has a more aggressive pathogenesis. The aim of the study was to describe the demographic characteristics of malaria cases, in patients with clinical symptoms compatible with malaria and whose laboratory diagnosis was confirmed. The methods of diagnosis used were: serological (IIF), molecular (real-time PCR, adapted Swan et al.) and direct visualization of thick and thin blood smears. We analyzed 471 specimens, 6 of which belonging to Ebola outbreak (West Africa, 2014). Fifty-five (11.7%) were positive cases. All positive cases of malaria are imported. Most cases occurred in adults travellers. Since local transmission continues to be possible in Europe and the big number of trips between Europe and endemic countries are a reality underlines the need of surveillance and prevention of this disease

Malaria: laboratory confirmation of clinical suspected cases of Plasmodium sp. between 2010-2017

A malária é uma infeção parasitária causada por um protozoário intracelular obrigatório (género Plasmodium), cuja transmissão é assegurada quando uma fémea de mosquito género Anopheles infetada efetua a sua refeição de sangue num indivíduo. Estão descritas mais de 150 espécies mas apenas 5 infetam o Homem: P. falciparum, P. vivax, P. malariae, P. ovale e mais recentemente, P. knowlesi, uma espécie que causa malária em macacos no sudeste asiático. É a doença parasitária mais frequente para o Homem, encontrando-se infetada cerca de 5% da população mundial, sendo o P. falciparum o agente mais disseminado, responsável por mais de 80% dos casos mundiais e o que apresenta uma patogenia mais agressiva. O objetivo deste estudo foi descrever as características demográficas dos casos de malária, em doentes com quadro clinico compatível e cujo diagnóstico laboratorial foi confirmado. As metodologias de diagnóstico utilizadas foram, serológicas (IFI), moleculares (PCR em tempo real, adaptada de Swan et al.) e a pesquisa direta em gota espessa e esfregaço. Foram analisadas 700 amostras, 15 das quais provenientes do sur to de ébola (África Ocidental, 2014/2015). Detetaram-se 103 (14,7%) casos positivos. Todos os casos positivos de malária são impor tados. A maioria dos casos ocorreu em viajantes, indivíduos adultos. Pelo facto da transmissão local continuar a ser possível na Europa e pela quantidade de viagens efetuadas entre a Europa e os países endémicos, salienta-se a necessidade de vigilância e prevenção desta parasitose.Malaria is a human disease caused by a protozoan infection of red blood cells transmitted by the bite of a female anopheline mosquito. There are described more than 150 species of Plasmodium but only five infected humans, P. falciparum, P. vivax, P. malariae, P. ovale and more recently P. knowlesi, a specie that causes malaria in monkeys in Southeast Asia. Malaria is the most impor tant parasitic disease of man; approximately 5% of the world’s population is infected. P. falciparum is the most widespread specie responsible for over 80% of cases worldwide and which has a more aggressive pathogenesis. The aim of the study was to describe the demographic characteristics of malaria cases, in patients with clinical symptoms compatible with malaria and whose laboratory diagnosis was confirmed. The methods of diagnosis used were: serological (IIF), molecular (real-time PCR, adapted Swan et al.) and direct visualization of thick and thin blood smears. We analyzed 700 specimens, 15 of which belonging to Ebola outbreak (West Africa, 2014/2015). One hundred and three (14.7%) were positive cases. All positive cases of malaria are impor ted. Most cases occurred in adults travellers. Since local transmission continues to be possible in Europe and the big number of trips between Europe and endemic countries are a reality underlines the need of sur veillance and prevention of this disease.info:eu-repo/semantics/publishedVersio

Toxoplasmose: diagnóstico laboratorial de casos clínicos suspeitos de infeção entre 2009 e 2013

Objetivo: Este estudo tem como objetivo descrever as características demográficas de doentes com quadro clínico suspeito de toxoplasmose, cujo diagnóstico laboratorial foi confirmado no Laboratório Nacional de Referência de Infeções Parasitárias e Fúngicas do Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA) entre janeiro de 2009 e dezembro de 2013

Genetic and virulence characterization of Toxoplasma gondii strains isolated from pigeons in Lisbon region

habitat with cats and humans, bands are observed in recreational areas such as urban parks, playgrounds and parks. The interaction between cats, birds and human population is quite evident favoring the fecal-oral transmission of T. gondii between the definitive host and intermediate hosts, in the urban cycle of the parasite. The results of the inoculation in vivo of the brain homogenates showed pigeon isolation rates (58.5%) significantly higher when compared with previous studies, including the preliminary study in 2006 that the isolation rate in mice was 39,1% (9/23) (Waap 2008) and another that was not achieved any isolation in mouse (Godoi 2010). The genotypic analysis revealed a majority of strains of type II, which is consistent with what has been described in Portugal, the rest of Europe and the USA (Ajzenberg 2005, Fazaeli 2000, Honoré 2000, Howe 1997, Waap 2008) . We also isolated strains of type III and type I. The identification of type III strains in animals have been reported by other authors, but the type I have been rarely found in animals has not been previously described in Portugal except in a preliminary study of our team at the 2008 (Waap 2008). The type I strains are usually associated with high virulence in laboratory mice, leading to death within days. This strain was identified by molecular biology and has not been isolated in vivo. The difficulty in isolation of strain may be related to the small number of cysts of the type I strains can develop, these type strains are considered low cystogenic. Genetic characterization of strains of T. gondii is far from its terminus, more sequences of different genes should be studied to help the understanding of the molecular epidemiology and genetic characterization of T. gondii, a relevant parasite for which these data are lacking. The combination of data from humans and animals, through the use of high resolution genetic characterization should improve our perceptive of T. gondii, which will be ultimately beneficial for the control of T. gondii transmission

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.info:eu-repo/semantics/publishedVersio

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

This article belongs to the Special Issue Leishmaniasis: Vector-Host-Pathogen Interactions in Health and Disease.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.This research was partially supported by the Portuguese Foundation for Science and Technology (FCT) through the project: “Phleboviruses in Portugal—vectors, pathogenesis and co-infections” (PTDC/DTP-SAP/0859/2014).info:eu-repo/semantics/publishedVersio

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.The Portuguese National Institute of Health funded this study.info:eu-repo/semantics/publishedVersio