First genetic characterization of human cystic echinococcosis in Uruguay.

Cystic echinococcosis is endemic and hyperendemic in Uruguay. The objective of this study was to determine the species and genotype of Echinococcus granulosus sensu lato in symptomatic patients with cystic echinococcosis who underwent surgery, together with the location and stage of the cysts. The study included 13 patients aged between 6 and 57 years old. Samples of cysts from these cases were analyzed using DNA extraction, polymerase chain reaction amplification and sequencing. The results revealed the presence of E. granulosus sensu stricto in all cases, with 12 samples belonging to the G1 genotype and 1 to the G3 genotype, suggesting that disease persistence might be related to the dog-sheep cycle. However, other intermediate hosts, such as cattle, could also be involved. Cysts were most frequently found in the liver, followed by muscle and other sites (e.g. pulmonary, vertebral, pelvic and cardiac); and stage CE1 was most frequently found, followed by CE2 and CE3b. Three cases occurred in children or adolescents, suggesting an active parasite cycle in at least some areas of the country. Since there is considerable diversity of E. granulosus sensu lato species and genotypes in South America, it is important to continue the present study in order to draw stronger epidemiological conclusions.

La equinococosis quística es endémica e hiperendémica en Uruguay. El objetivo de este estudio fue determinar la especie y el genotipo de Echinococcus granulosus sensu lato en pacientes sintomáticos con equinococosis quística que fueron sometidos a cirugía, además de la localización y estadio de los quistes. En el estudio participaron 13 pacientes de entre 6 y 57 años. Las muestras de quistes de estos casos se analizaron mediante la extracción de ADN, la amplificación de la reacción en cadena de la polimerasa y la secuenciación. Los resultados revelaron la presencia de E. granulosus sensu stricto en todos los casos, con 12 muestras pertenecientes al genotipo G1 y 1 muestra de genotipo G3, lo que sugiere que la persistencia de la enfermedad podría estar relacionada con el ciclo perro/ovinos. Sin embargo, otros huéspedes intermedios, como el ganado, también podrían estar involucrados. Los quistes se encontraron con mayor frecuencia en el hígado, seguido por los músculos y otros sitios (por ejemplo, quistes pulmonares, vertebrales, pélvicos y cardíacos); y el estadio más frecuente fue CE1, seguido por CE2 y CE3b. Tres de los casos ocurrieron en niños o adolescentes, lo que sugiere un ciclo activo de parásitos al menos en algunas áreas del país. Dado que existe una considerable diversidad de especies y genotipos de E. granulosus sensu lato en América del Sur, es importante continuar con este estudio para extraer conclusiones epidemiológicas más sólidas.

A equinococose cística é endêmica e hiperendêmica no Uruguai. O objetivo do estudo foi determinar a espécie e o genótipo do Echinococcus granulosus sensu lato em pacientes sintomáticos com equinococose cística que foram submetidos a cirurgia, juntamente com a localização e o estágio dos cistos. O estudo incluiu 13 pacientes entre 6 e 57 anos de idade. As amostras de cistos foram analisadas utilizando extração do DNA, amplificação pela reação em cadeia da polimerase e sequenciamento. Os resultados revelaram a presença de E. granulosus sensu stricto em todos os casos, com 12 amostras pertencentes ao genótipo G1 e uma ao genótipo G3, sugerindo que a persistência da doença pode estar relacionada ao ciclo cão-ovelha. Entretanto, outros hospedeiros intermediários como gado também poderiam estar envolvidos. Os cistos foram mais frequentemente encontrados no fígado, seguido por músculos e outros locais (por exemplo, pulmão, vértebras, pelve e coração), e o estágio mais frequentemente encontrado foi o CE1, seguido por CE2 e CE3b. Três casos ocorreram em crianças ou adolescentes, o que sugere um ciclo parasitário ativo em pelo menos algumas áreas do país. Devido à considerável diversidade de espécies e genótipos de E. granulosus sensu lato na América do Sul, é importante dar continuidade do presente estudo a fim de se chegar a conclusões epidemiológicas mais sólidas.

Development of a low-cost copro-LAMP assay for simultaneous copro-detection of Toxocara canis and Toxocara cati.

Toxocariasis is a zoonotic disease caused mainly by Toxocara canis and Toxocara cati and diagnosis in dogs and cats is an important tool for its control. For this reason, a new coprological loop-mediated isothermal amplification (LAMP) assay was developed for the simultaneous detection of these species. The primer set was designed on a region of the mitochondrial cox-1 gene. Amplification conditions were evaluated using a temperature gradient (52°C to 68°C), different incubation times (15­120 min), and different concentrations of malachite green dye (0.004­0.4% w/v). The analytical sensitivity was evaluated with serial dilutions of genomic DNA from T. canis and T. cati adult worms, and with serial dilutions of DNA extracted from feces using a low-cost in-house method. The specificity was evaluated using genomic DNA from Canis lupus familiaris, Felis catus, Escherichia coli, Toxascaris leonina, Ancylostoma caninum, Echinococcus granulosus sensu stricto and Taenia hydatigena. The LAMP assay applied to environmental fecal samples from an endemic area showed an analytical sensitivity of 10­100 fg of genomic DNA and 10−5 serial dilutions of DNA extracted from feces using the low-cost in-house method; with a specificity of 100%. Additionally, the total development of the assay was carried out in a basic laboratory and per-reaction reagent cost decreased by ~80%. This new, low-cost tool can help identify the most common agents of toxocariasis in endemic areas in order to manage prevention strategies without having to rely on a laboratory with sophisticated equipment.

First report of cystic echinococcosis caused by Echinococcus granulosus sensu stricto/G1 in Felis catus from the Patagonian region of Argentina.

Two domestic cats from the Patagonia rural area in Argentina were found to be naturally infected with Echinococcus granulosus sensu stricto/G1 genotype; so far, the only species/genotype of E. granulosus sensu lato complex described to infect domestic cats. The felines developed abdominal disseminated larval disease; the diagnosis was performed by ultrasound, exploratory laparotomy, and molecular techniques. These results indicate that cystic echinococcosis must be considered for differential diagnosis of felines with abdominal distension and/or observation of vesicles through ultrasound, from endemic areas. Even though cats and dogs are carnivores, differences in digestive physiology and immunological characteristics between them could allow the development of larval or adult worm parasites. Domestic cats with cystic echinococcosis show to be environmentally infected with E. granulosus s. s./G1 eggs.

Cystic echinococcosis in South America: systematic review of species and genotypes of Echinococcus granulosus sensu lato in humans and natural domestic hosts.

OBJECTIVE: To systematically review publications on Echinococcus granulosus sensu lato species/genotypes reported in domestic intermediate and definitive hosts in South America and in human cases worldwide, taking into account those articles where DNA sequencing was performed; and to analyse the density of each type of livestock that can act as intermediate host, and features of medical importance such as cyst organ location. METHODS: Literature search in numerous databases. We included only articles where samples were genotyped by sequencing since to date it is the most accurate method to unambiguously identify all E. granulosus s. l. genotypes. Also, we report new E. granulosus s. l. samples from Argentina and Uruguay analysed by sequencing of cox1 gene. RESULTS: In South America, five countries have cystic echinococcosis cases for which sequencing data are available: Argentina, Brazil, Chile, Peru and Uruguay, adding up 1534 cases. E. granulosus s. s. (G1) accounts for most of the global burden of human and livestock cases. Also, E. canadensis (G6) plays a significant role in human cystic echinococcosis. Likewise, worldwide analysis of human cases showed that 72.9% are caused by E. granulosus s. s. (G1) and 12.2% and 9.6% by E. canadensis G6 and G7, respectively. CONCLUSIONS: E. granulosus s. s. (G1) accounts for most of the global burden followed by E. canadensis (G6 and G7) in South America and worldwide. This information should be taken into account to suit local cystic echinococcosis control and prevention programmes according to each molecular epidemiological situation.

Comparison of three LAMP protocols for the simultaneous detection of DNA from species that produce cystic echinococcosis.

Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by species of the Echinococcus granulosus sensu lato complex. Different types of canids may act as definitive hosts by eating raw viscera infected with fertile hydatid cysts. The intermediate host (mainly ungulates) and humans acquire the infection through the fecal oral route (i.e. egg ingestion). Globally, more than 1 million people are affected by CE, causing a loss of 1-3 million disability-adjusted life years (DALYs) and a financial burden of US$ 3 billion annually. Loop mediated isothermal amplification (LAMP) protocols promise to be a useful tool to detect DNA, providing a low cost and thermocycle-free methodology. Given that surveillance for CE can be performed in feces from canids or other environmental matrixes contaminated with eggs, the characteristics of a LAMP protocol would favor implementation in endemic areas with basic resources. Herein, we compared three LAMP protocols for the simultaneous detection of E. granulosus s.l. species that cause CE. This comparation was carried with DNA obtained from different stages of E. granulosus s.l. Two of these are newly developed protocols that showed good analytical sensitivity and specificity. In both cases, the use of malachite green dye to directly visualize the test result was possible. From these two new LAMP protocols, one had better values for the detection of DNA from different types of E. granulosus s.l. DNA samples. Therefore, through this study, we provide a low-cost new tool for DNA detection of E. granulosus s.l. in poorly equipped laboratories from endemic areas.

Environmental Toxocara spp. presence in crowded squares and public parks from San Juan Province, Argentina: A call for a "One Health" approach.

Introduction: Canine soil-transmitted helminth (cSTH) parasites need specific environmental conditions to complete their life cycle. Toxocara canis and T. cati are the most important zoonotic cSTH, since they are the causal agents of human toxocariasis. Canine STHs are dispersed in feces from infected domestic and wildlife canines. In this study, the presence of STH in canine feces was evaluated in 34 crowded public parks and squares from San Juan Province (Argentina). Methods: Fecal samples were collected during different seasons in 2021-2022 and analyzed by standard coprological methods, including Sheather and Willis flotation and Telemann sedimentation. InfoStat 2020, OpenEpi V. 3.01 and R and RStudio® were used for statistical analysis and QGIS 3.16.10 for mapping. Results: From a total of 1,121 samples collected, 100 (8.9%) were positive for at least one intestinal parasite (IP) and three cSTH species were detected: Toxocara spp., Toxascaris leonina and Trichuris vulpis. The most prevalent cSTH species was T. vulpis (64/1121; 0.057%), while the least prevalent was Toxocara spp. (19/1121; 0.017%). The detection of Toxocara spp. eggs was significantly different depending on the season. The geo-spatial variation of each cSTH per season is described. Discussion: This is the first study in San Juan Province to identify environmental contamination of cSTHs in public areas. The specific localization of areas with the presence of cSTH eggs could provide information to guide strategies to reduce the cSTH infection burden in dogs and promote serological screening of the human population for Toxocara spp. Given the zoonotic nature of Toxocara spp. We hope this information will help to reinforce activities of control programs, focusing on the "One Health" approach.

Development of a New LAMP Assay for the Detection of Ancylostoma caninum DNA (Copro-LAMPAc) in Dog Fecal Samples.

Ancylostoma caninum is a zoonotic nematode which is able to affect animals and humans. Diagnosis in the definitive host and environmental detection are key to prevent its dissemination and achieve control. Herein, a new coprological LAMP method for the detection of A. caninum (Copro-LAMPAc) DNA was developed. DNA extraction was performed using a low-cost method and a fragment of the cox-1 gene was used for primer design. The analytical sensitivity, evaluated with serial dilutions of genomic DNA from A. caninum adult worms, was 100 fg. A specificity of 100% was obtained using genomic DNA from the host and other pathogens. The Copro-LAMPAc was evaluated using environmental canine fecal samples. When compared with gold standard optical microscopy in epidemiological studies, it proved to be more sensitive. This new LAMP assay can provide an alternative protocol for screening and identification of A. caninum for epidemiological studies in endemic areas.

Development of a copro-LAMP assay for detection of several species of Echinococcus granulosus sensu lato complex.

Cystic echinococcosis represents a significant problem in human and animal health and constitutes one of the most severe Neglected Tropical Diseases prioritized by the World Health Organization. The etiological agent is the complex Echinococcus granulosus sensu lato (s. l.), composed of several species/genotypes. Diagnosis in the definitive host and molecular epidemiology studies are important points for cystic echinococcosis control. Here we developed a new copro-LAMP assay, LAMP EGSL, for diagnosis in the definitive host for simultaneous detection of Echinococcus granulosus sensu stricto (s. s.), Echinococcus ortleppi, and Echinococcus canadensis species. Also, the analytical sensitivity, specificity and plausibility of performance in a rural context of a previously reported species-specific LAMP reaction, was evaluated. Both reactions showed high analytical sensitivity values (10 fg-100 fg DNA) and did not show cross reaction with DNA from host or other helminthic parasites. LAMP EGSL was performed with samples from an endemic area. In addition, the alkaline hydrolysis of one E. granulosus s. s. adult parasite followed by specific LAMP to E. granulosus s. s. was performed in a laboratory with low resources from another cystic echinococcosis endemic area. The results obtained suggest that LAMP EGSL represents a potential tool for canine diagnosis that could be useful for cystic echinococcosis control programs. In addition, we showed that LAMP reaction for E. granulous s. s., E. ortleppi and E. canadensis specific detection, could be useful for molecular epidemiology studies applicable to the definitive host. Both reactions were performed in endemic, rural areas without sophisticated equipment.

First genetic characterization of human cystic echinococcosis in Uruguay

[ABSTRACT]. Cystic echinococcosis is endemic and hyperendemic in Uruguay. The objective of this study was to deter- mine the species and genotype of Echinococcus granulosus sensu lato in symptomatic patients with cystic echinococcosis who underwent surgery, together with the location and stage of the cysts. The study included 13 patients aged between 6 and 57 years old. Samples of cysts from these cases were analyzed using DNA extraction, polymerase chain reaction amplification and sequencing. The results revealed the presence of E. granulosus sensu stricto in all cases, with 12 samples belonging to the G1 genotype and 1 to the G3 genotype, suggesting that disease persistence might be related to the dog–sheep cycle. However, other inter- mediate hosts, such as cattle, could also be involved. Cysts were most frequently found in the liver, followed by muscle and other sites (e.g. pulmonary, vertebral, pelvic and cardiac); and stage CE1 was most frequently found, followed by CE2 and CE3b. Three cases occurred in children or adolescents, suggesting an active par- asite cycle in at least some areas of the country. Since there is considerable diversity of E. granulosus sensu lato species and genotypes in South America, it is important to continue the present study in order to draw stronger epidemiological conclusions.

[RESUMEN]. La equinococosis quística es endémica e hiperendémica en Uruguay. El objetivo de este estudio fue deter- minar la especie y el genotipo de Echinococcus granulosus sensu lato en pacientes sintomáticos con equinococosis quística que fueron sometidos a cirugía, además de la localización y estadio de los quistes. En el estudio participaron 13 pacientes de entre 6 y 57 años. Las muestras de quistes de estos casos se analizaron mediante la extracción de ADN, la amplificación de la reacción en cadena de la polimerasa y la secuenciación. Los resultados revelaron la presencia de E. granulosus sensu stricto en todos los casos, con 12 muestras pertenecientes al genotipo G1 y 1 muestra de genotipo G3, lo que sugiere que la persistencia de la enfermedad podría estar relacionada con el ciclo perro/ovinos. Sin embargo, otros huéspedes intermedios, como el ganado, también podrían estar involucrados. Los quistes se encontraron con mayor frecuencia en el hígado, seguido por los músculos y otros sitios (por ejemplo, quistes pulmonares, vertebrales, pélvicos y cardíacos); y el estadio más frecuente fue CE1, seguido por CE2 y CE3b. Tres de los casos ocurrieron en niños o adolescentes, lo que sugiere un ciclo activo de parásitos al menos en algunas áreas del país. Dado que existe una considerable diversidad de especies y genotipos de E. granulosus sensu lato en América del Sur, es importante continuar con este estudio para extraer conclusiones epidemiológicas más sólidas.

[RESUMO]. A equinococose cística é endêmica e hiperendêmica no Uruguai. O objetivo do estudo foi determinar a espécie e o genótipo do Echinococcus granulosus sensu lato em pacientes sintomáticos com equinococose cística que foram submetidos a cirurgia, juntamente com a localização e o estágio dos cistos. O estudo incluiu 13 pacientes entre 6 e 57 anos de idade. As amostras de cistos foram analisadas utilizando extração do DNA, amplificação pela reação em cadeia da polimerase e sequenciamento. Os resultados revelaram a presença de E. granulosus sensu stricto em todos os casos, com 12 amostras pertencentes ao genótipo G1 e uma ao genótipo G3, sugerindo que a persistência da doença pode estar relacionada ao ciclo cão-ovelha. Entretanto, outros hospedeiros intermediários como gado também poderiam estar envolvidos. Os cistos foram mais frequentemente encontrados no fígado, seguido por músculos e outros locais (por exemplo, pulmão, vértebras, pelve e coração), e o estágio mais frequentemente encontrado foi o CE1, seguido por CE2 e CE3b. Três casos ocorreram em crianças ou adolescentes, o que sugere um ciclo parasitário ativo em pelo menos algumas áreas do país. Devido à considerável diversidade de espécies e genótipos de E. granulosus sensu lato na América do Sul, é importante dar continuidade do presente estudo a fim de se chegar a conclusões epidemiológicas mais sólidas.

First genetic characterization of human cystic echinococcosis in Uruguay / Primera caracterización genética de la equinococosis quística humana en Uruguay / Primeira caracterização genética da equinococose cística humana no Uruguai

ABSTRACT Cystic echinococcosis is endemic and hyperendemic in Uruguay. The objective of this study was to determine the species and genotype of Echinococcus granulosus sensu lato in symptomatic patients with cystic echinococcosis who underwent surgery, together with the location and stage of the cysts. The study included 13 patients aged between 6 and 57 years old. Samples of cysts from these cases were analyzed using DNA extraction, polymerase chain reaction amplification and sequencing. The results revealed the presence of E. granulosus sensu stricto in all cases, with 12 samples belonging to the G1 genotype and 1 to the G3 genotype, suggesting that disease persistence might be related to the dog-sheep cycle. However, other intermediate hosts, such as cattle, could also be involved. Cysts were most frequently found in the liver, followed by muscle and other sites (e.g. pulmonary, vertebral, pelvic and cardiac); and stage CE1 was most frequently found, followed by CE2 and CE3b. Three cases occurred in children or adolescents, suggesting an active parasite cycle in at least some areas of the country. Since there is considerable diversity of E. granulosus sensu lato species and genotypes in South America, it is important to continue the present study in order to draw stronger epidemiological conclusions.

RESUMEN La equinococosis quística es endémica e hiperendémica en Uruguay. El objetivo de este estudio fue determinar la especie y el genotipo de Echinococcus granulosus sensu lato en pacientes sintomáticos con equinococosis quística que fueron sometidos a cirugía, además de la localización y estadio de los quistes. En el estudio participaron 13 pacientes de entre 6 y 57 años. Las muestras de quistes de estos casos se analizaron mediante la extracción de ADN, la amplificación de la reacción en cadena de la polimerasa y la secuenciación. Los resultados revelaron la presencia de E. granulosus sensu stricto en todos los casos, con 12 muestras pertenecientes al genotipo G1 y 1 muestra de genotipo G3, lo que sugiere que la persistencia de la enfermedad podría estar relacionada con el ciclo perro/ovinos. Sin embargo, otros huéspedes intermedios, como el ganado, también podrían estar involucrados. Los quistes se encontraron con mayor frecuencia en el hígado, seguido por los músculos y otros sitios (por ejemplo, quistes pulmonares, vertebrales, pélvicos y cardíacos); y el estadio más frecuente fue CE1, seguido por CE2 y CE3b. Tres de los casos ocurrieron en niños o adolescentes, lo que sugiere un ciclo activo de parásitos al menos en algunas áreas del país. Dado que existe una considerable diversidad de especies y genotipos de E. granulosus sensu lato en América del Sur, es importante continuar con este estudio para extraer conclusiones epidemiológicas más sólidas.

RESUMO A equinococose cística é endêmica e hiperendêmica no Uruguai. O objetivo do estudo foi determinar a espécie e o genótipo do Echinococcus granulosus sensu lato em pacientes sintomáticos com equinococose cística que foram submetidos a cirurgia, juntamente com a localização e o estágio dos cistos. O estudo incluiu 13 pacientes entre 6 e 57 anos de idade. As amostras de cistos foram analisadas utilizando extração do DNA, amplificação pela reação em cadeia da polimerase e sequenciamento. Os resultados revelaram a presença de E. granulosus sensu stricto em todos os casos, com 12 amostras pertencentes ao genótipo G1 e uma ao genótipo G3, sugerindo que a persistência da doença pode estar relacionada ao ciclo cão-ovelha. Entretanto, outros hospedeiros intermediários como gado também poderiam estar envolvidos. Os cistos foram mais frequentemente encontrados no fígado, seguido por músculos e outros locais (por exemplo, pulmão, vértebras, pelve e coração), e o estágio mais frequentemente encontrado foi o CE1, seguido por CE2 e CE3b. Três casos ocorreram em crianças ou adolescentes, o que sugere um ciclo parasitário ativo em pelo menos algumas áreas do país. Devido à considerável diversidade de espécies e genótipos de E. granulosus sensu lato na América do Sul, é importante dar continuidade do presente estudo a fim de se chegar a conclusões epidemiológicas mais sólidas.

Echinococcus oligarthrus in the subtropical region of Argentina: First integration of morphological and molecular analyses determines two distinct populations.

Echinococcosis is a parasitic zoonosis that is considered as a neglected disease by the World Health Organization. The species Echinococcus oligarthrus is one of the causative agents of Neotropical echinococcosis, which is a poorly understood disease that requires a complex medical examination, may threaten human life, and is frequently associated with a low socioeconomic status. Morphological and genetic diversity in E. oligarthrus remains unknown. The aim of this work is to identify and characterize E. oligarthrus infections in sylvatic animals from the Upper Paraná Atlantic Forest in the province of Misiones, Argentina, by following an integrative approach that links morphological, genetic and ecological aspects. This study demonstrates, for the first time, one of the complete life cycles of E. oligarthrus in an important ecoregion. The Upper Paraná Atlantic Forest constitutes the largest remnant continuous forest of the Atlantic Forest, representing 7% of the world's biodiversity. This is the first molecular determination of E. oligarthrus in Argentina. In addition, the agouti (Dasyprocta azarae), the ocelot (Leopardus pardalis) and the puma (Puma concolor) were identified as sylvatic hosts of Neotropical echinococcosis caused by E. oligarthrus. Mitochondrial and nuclear molecular marker analyses showed a high genetic diversity in E. oligarthrus. Moreover, the genetic distance found among E. oligarthrus isolates is higher than the one observed among Echinococcus granulosus genotypes, which clearly indicates that there are at least two different E. oligarthrus populations in Argentina. This study provides valuable information to understand the underlying conditions that favour the maintenance of E. oligarthrus in sylvatic cycles and to evaluate its zoonotic significance for devising preventive measures for human and animal wellbeing.