Use of laboratory methodologies to confirm an outbreak of acute diarrheal disease caused by multiple pathogens / Uso de metodologias laboratoriais para confirmação de surto de doença diarreica aguda causada por múltiplos patógenos

The aim of this study was to investigate an outbreak caused by protozoa, which occurred in a municipality in the Brazil southern region. The investigations were carried out analyzing 47 fresh stool samples and 26 water samples by parasitological and molecular methods, as well as, direct immunofluorescence. After the filtrations of water samples and purification of stool samples, the concentrates were evaluated microscopically for presence of parasites. Molecular analyses were performed by polymerase chain reaction (PCR) for DNA detection of Giardia spp., Cryptosporidium parvum, C. hominis and Cyclospora cayetanensis. Out of 26 water samples, 30.8% (8/26) had waterborne protozoa and C. cayetanensis was the most prevalent (15.5%). Out of the 47 stool samples, 23.4% (11/47) were infected with C. cayetanensis and Giardia spp. The results showed that backwash water samples from filters of the Water Treatment Station were contaminated with C. cayetanensis, C. hominis and Giardia spp., suggesting the contamination of water sources with human waste brought by sewage. These results show the importance of protozoa investigation in water and stool samples by laboratory methodologies principally in outbreaks causing acute diarrheal disease (AU).

O objetivo do presente estudo foi investigar um surto causado por protozoários, ocorrido em um município da região sul do Brasil. As investigações foram realizadas analisando 47 amostras de fezes frescas e 26 amostras de água por métodos parasitológicos, moleculares e de imunofluorscência direta. Após as filtrações das amostras de água e purificação das amostras de fezes, os concentrados foram avaliados microscopicamente a procura de parasitas. A seguir, foram analisadas, pela reação em cadeia da polimerase (PCR), a detecção de DNA de Giardia spp., Cryptosporidium parvum, C. hominis e Cyclospora cayetanensis. Das 26 amostras de água, 30,8% (8/26) apresentaram protozoários de veiculação hídrica, sendo que, C. cayetanensis foi o mais prevalente (15,5%). Das 47 amostras de fezes, 23,4% (11/47) estavam infectadas por C. cayetanensis e Giardia spp. Os resultados mostraram que as águas de retrolavagem dos filtros da Estação de Tratamento de Água estavam contaminadas com C. cayetanensis, C. hominis e Giardia spp. sugerindo a contaminação dos mananciais com dejetos humanos trazidos pelo esgoto. Estes resultados mostram a importância da investigação de protozoários em água e fezes por metodologias laboratoriais, principalmente em surtos que causam doença diarreica aguda (AU).

Caracterización físico-química y microbiológica del agua de un municipio del departamento de Boyacá (Colombia) / Physical Chemical and Microbiological Characterization of Water of a Municipality of the Department of Boyacá-Colombia / Caracterização físico-química e microbiológica da água de um município do departamento de Boyacá (Colômbia)

Introducción: El agua salubre y fácilmente accesible es importante para la salud pública, si se utiliza para beber, uso doméstico, producir alimentos o fines recreativos. El agua contaminada y el saneamiento deficiente están re-lacionados con la transmisión de enfermedades. Objetivo: Evaluar las características físico-químicas y microbiológicas de la red de distribución del acueducto urbano de un municipio de Boyacá. Materiales y métodos: Estudio cuantitativo descriptivo transversal. Se tomaron muestras de agua en 13 puntos, 7 concertados y materializados de la red de distribución, uno en la bocatoma de entrada de la planta, dos pozos y tres nacimientos, de donde se abastece la población. Se realizaron análisis físicos, químicos y microbiológicos de cada una de las muestras. Resultados: En las muestras sin tratamiento hubo concentraciones altas de unidades platino, cobalto y turbiedad. El cloro residual libre en dos muestras de agua tratada estaba por debajo de los límites establecidos. Se detecta-ron microorganismos heterótrofos y coliformes en las muestras de agua obtenidas de fuentes de abastecimiento. En dos puntos se encontraron quistes de Giardia y ooquistes de Cryptosporidium. De las 13 muestras estudiadas, tres arrojaron un índice de riesgo por calidad del agua inviable sanitariamente. Conclusiones: Se evidenció que el agua se encuentra en un nivel de riesgo inviable sanitariamente, lo cual demuestra que el sistema de tratamiento es insuficiente para garantizar el suministro de agua apta para el consumo humano.

Introduction: Safe and easily accessible water is important to public health, whether it is used for drinking, domestic use, food production, or recreational purposes. Contaminated water and poor sanitation are linked to disease transmission. Objective: Evaluate the physical chemical and microbiological characteristics of the distribution network of the urban aqueduct of a municipality of Boyacá. Materials and methods: Cross-sectional descriptive quantitative study, water samples were taken at thirteen points, seven points agreed and materialized from the distribution network, one at the entrance of the plant, one in a deep well and in three births, where it is supplied the population. Phy-sical, chemical and microbiological analyzes were performed. Results: The untreated samples showed high levels of cobalt platinum units and turbidity. The free residual chlorine in two samples of treated water was below the established limits. Heterotrophic and Coliform microorganisms were detected in water samples obtained from sources of supply. At two points, Giardia cysts and Cryptosporidium oocysts were found. Of the Thirteen samples studied, three showed a Risk Index for sanitary water quality. Conclusions: It is evident that the water is at a level of sanitary unfeasible risk, which demonstrates that the treatment system is not sufficient to guarantee the supply of water suitable for human consumption.

Introdução: Água segura e de fácil acesso é importante para a saúde pública, seja ela usada para beber, uso doméstico, produção de alimentos ou para fins recreativos. A água contaminada e o sa-neamento precário estão ligados à transmissão de doenças. Objetivos: Avaliar as características físico-químicas e microbiológicas da rede de distribuição do aque-duto urbano de um município de Boyacá. Materiais e métodos: Estudo quantitativo descritivo transversal. Foram coletadas amostras de água em 13 pontos, 7 concertados e materializados da rede de distribuição, um na entrada da planta, dois poços e três nascentes, dos quais a população é abastecida. Foram realizadas analises físicas, químicas e microbiológicas em cada uma das amostras. Resultados: Nas amostras não tratadas havia concentrações altas de unidades platina, cobalto e turbidez. O cloro residual livre em duas amostras de água tratada estava abaixo dos limites estabele-cidos. Microrganismos heterótrofos e coliformes foram detectados em amostras de água obtidas de fontes de abastecimento. Os cistos de Giardia e os oocistos de Cryptosporidium foram encontrados em dois pontos. Das 13 amostras estudadas, três apresentaram um índice de risco para a qualidade não higiénica da água. Conclusões: Ficou evidente que a água está em um nível de risco sanitário inviável, o que mostra que o sistema de tratamento é insuficiente para garantir o abastecimento da água adequada para o consumo humano.

Factores asociados con la presencia de endoparásitos y ectoparásitos en perros domiciliados de la zona metropolitana de Toluca, México / Factors associated with endoparasites and ectoparasites in domiciled dogs in the metropolitan area of Toluca, México

Resumen | Introducción. Los endoparásitos y ectoparásitos en perros son de distribución mundial. La estrecha relación entre los perros y el hombre implica un riesgo de transmisión de parasitosis zoonóticas, por lo cual es necesario conocer las especies que parasitan a los perros de esta zona y determinar los factores asociados. Objetivos. Estimar la prevalencia de endoparásitos y ectoparásitos, identificarlos en perros domiciliados de la zona metropolitana de Toluca, México, y determinar la prevalencia de Dipyilidium caninum en pulgas del género Ctenocephalides spp. Materiales y métodos. Se recolectaron muestras de 402 perros que fueron llevados a consulta en cuatro hospitales de referencia de Toluca. En el diagnóstico de endoparásitos, se utilizaron las técnicas coproparasitoscópicas de frotis directo, flotación y sedimentación; además, se recolectaron ectoparásitos para su identificación taxonómica. Por último, la detección de D. caninum en pulgas se hizo mediante la reacción en cadena de la polimerasa (PCR). Resultados. El 37,2 % de los perros resultó positivo para endoparásitos. Los géneros o especies identificados fueron Toxocara spp., Giardia spp., Ancylostoma spp., Cystoisospora spp., D. caninum, Taenia spp. y Trichuris vulpis. Se determinó una prevalencia de ectoparásitos de 13,13 %. Se identificaron pulgas de las especies Ctenocephalides felis y C. canis, en tanto que solo un animal presentó parasitosis por Rhipicephalus sanguineus y otro por Trichodectes canis. La prevalencia de D. caninum en pulgas fue del 9,5 %. Conclusión. La prevalencia de endoparásitos fue de 37,2 % y, la de ectoparásitos, de 13,1 %. Por primera vez en México se hizo un análisis de endoparásitos y ectoparásitos en una misma población de perros, así como el diagnóstico molecular de D. caninum.

Abstract | Introduction: Endoparasites and ectoparasites in dogs are of global distribution. The close relationship between dogs and man implies a risk for the transmission of zoonotic parasites. Therefore, it is necessary to determine the parasites hosted by dogs in specific areas and the factors associated with their presence. Objectives: To identify and to estimate the prevalence of endoparasites and ectoparasites in domiciled dogs in the Metropolitan area of Toluca, México, and the prevalence of D. caninum in fleas of the genus Ctenocephalides spp. Materials and methods: We collected samples from 402 domiciled dogs in four reference hospitals in the area in Toluca. We diagnosed endoparasites using direct smear, flotation, and sedimentation techniques and we performed the taxonomic identification of ectoparasites. Finally, the molecular diagnosis of D. caninum in fleas was made using the polymerase chain reaction technique (PCR). Results: A total of 37.2% of dogs were positive for endoparasites; the genera or species identified were Toxocara spp., Giardia spp., Ancylostoma spp., Cystoisospora spp., D. caninum, Taenia spp., and Trichuris vulpis; the prevalence of ectoparasites was 13.13%. We identified fleas of the species Ctenocephalides felis, Ctenocephalides canis; only one animal was parasitized with Rhipicephalus sanguineus and another one with Trichodectes canis; the prevalence of D. caninum in fleas was 9.5%. Conclusion: The prevalence of endoparasites was 37.2% while that of ectoparasites was 13.1%; this is the first analysis of endoparasites and ectoparasites conducted in the same population of dogs in México together with the molecular diagnosis of D. caninum in fleas.

Quantitative Microbial Risk Assessment of / 生物医学与环境科学（英文）

We aimed to assess the risks of

Occurrence and molecular characterization of Giardia duodenalis from naturally infected dogs in the municipality of Santa Maria, Rio Grande do Sul, Brazil / Ocorrência e caracterização molecular de Giardia duodenalis em cães naturalmente infectados no município de Santa Maria, Rio Grande do Sul, Brasil

Giardiasis is an important and prevalent zoonosis in dogs and humans caused by Giardia spp. The close relationship between pets and humans has physical, emotional and social benefits. The dogs have an important role in Giardia duodenalis cycle and transmission. This study aimed to verify the occurrence of the parasite in dogs from Central Region, in Santa Maria, Rio Grande do Sul State, Brazil, from April to October 2018. Dog feces (230) were submitted to Faust coproparasitological and molecular analyses. The positive samples in the nested-PCR (β-giardin gene) were sent for DNA sequencing and phylogenetic analyses (Neighbor-Joining). The occurrence of G. duodenalis, was 5.6% (13/230) and 4.3% (10/230) detected by coproparasitological technique and nested-PCR, respectively. There was no difference in the sensitivity of the tests used. From the faecal samples analyzed, there were no differences among the variables: diagnostic techniques, local, sex, and age of the animals (p>0.05). Only in the stool examination methodology a difference was observed between the ages (p<0.05). G. duodenalis assemblages were C and D, frequently reported in dogs. The close relationship between dogs and people may allow co-infections of circulating parasites in the population, including Giardia spp. and increasing the risk of transmission of zoonotic agents.(AU)

A giardíase é uma zoonose importante e prevalente em cães e humanos, sendo causada por Giardia spp. A estreita relação entre animais de estimação e seres humanos traz benefícios físicos, emocionais e sociais. Os cães têm um papel importante no ciclo e transmissão de Giardia duodenalis. Este estudo teve como objetivo verificar a ocorrência do parasita em cães da Região Central, em Santa Maria, RS, Brasil, de abril a outubro de 2018. As fezes de cães (230) foram submetidas a técnica coproparasitológica de Faust e análises moleculares. As amostras positivas no nested-PCR (gene β-giardin) foram enviadas para sequenciamento de DNA e posterior análise filogenética (Neighbor-Joining). A ocorrência de G. duodenalis foi de 5,6% (13/230) e 4,3% (10/230) detectados pela técnica coproparasitológica e nested-PCR, respectivamente. Não houve diferença na sensibilidade dos testes utilizados. Das amostras fecais analisadas, não houve diferenças entre as variáveis: técnicas de diagnóstico, local, sexo e idade dos animais (p>0,05). Somente na metodologia de exame de fezes observou-se diferença entre as idades (p<0,05). As assemblages de G. duodenalis encontradas foram C e D, frequentemente relatadas em cães. A estreita relação entre cães e pessoas pode permitir co-infecções de parasitas circulantes na população, incluindo Giardia spp. e aumentando o risco de transmissão de agentes zoonóticos.(AU)

Caracterização molecular de cistos de Giardia spp. e de oocistos de Cryptosporidium spp. recuperados de amostras de água de reúso / Molecular characterization of Giardia spp. and Cryptosporidium spp. recovered from recycled water samples

O aumento da demanda de recursos hídricos causado pelo crescimento da população mundial, pela necessidade de produção de alimentos e utilização na indústria é uma realidade. Como enfrentamento a este cenário de segurança hídrica, existe uma crescente tendência mundial de uso de águas recicladas para fins que demandam águas de menor qualidade, poupando assim recursos hídricos e o meio ambiente. O presente estudo teve como objetivo identificar as espécies Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium meleagridis e Giardia duodenalis (em seus assemblages A e B) nas águas de reúso provenientes das duas ETEs localizadas na cidade de São Paulo. Para a identificação das espécies Cryptosporidium parvum, Cryptosporidium meleagridis e Cryptosporidium hominis foram realizadas análise por regiões do gene 18S rRNA, com os primers descritos por ARAUJO (2018), onde foram detectadas em 5,76% (3/52) das amostras de água de reúso analisadas pelo método real-time PCR (qPCR). A espécie Giardia duodenalis foi identificada através de um fragmento do gene Gdh, com os primers descritos por (CACCIO et al, 2008) e foi detectada em 11,53% (6/52) das amostras de água de reúso analisadas. Após a identificação das amostras via qPCR, foi realizado o nested PCR para que os fragmentos de DNA identificados pudessem ser sequenciados, onde confirmamos a presença das espécies de Cryptosporidium spp. anteriormente citadas, porém as amostras de Giardia spp. não apresentaram resultados positivos no sequenciamento, não sendo possível a identificação dos assemblages. Este trabalho subsidia, com dados de presença de Giardia spp. e Cryptosporidium spp. nas águas de reúso produzida por duas ETEs da cidade de São Paulo, a elaboração de uma regulamentação adequada para o uso deste recurso em ambiente urbano com o intuito de se proteger a saúde da população exposta, em especial das populações mais vulneráveis e de se reduzir o risco de infecção pelo contato ocupacional.

The increasing demand for water resources caused by the growth of the world's population, the food production and use in industry is a reality. With this water crisis scenario, there is a growing worldwide trend towards the use of recycled water for purposes that demand lower water quality, thus saving water resources and the environment. In a previous study, we identified the presence of Giardia spp. and Cryptosporidium spp. in reuse waters from two wastewater treatment plants (WWTPs) in the city of São Paulo (called stations "A" and "B") using the USEPA 1693/2014 method. The present study aimed to identify the species Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium meleagridis and Giardia duodenalis (in their assemblages A and B) in the reuse waters from the WWTPs in the city of São Paulo. Different molecular analyzes were carried out to identify the species found in the WWTPs. The species Cryptosporidium parvum, Cryptosporidium meleagridis and Cryptosporidium hominis were identified by regions of their 18S rRNA gene, with the primers described by ARAUJO (2018), and were detected in 5.76% (3/52) of the reuse water samples analyzed by real-time PCR (qPCR) method. The species Giardia duodenalis was identified through a fragment of the Gdh gene, with the primers described by (CACCIO et al., 2008) was detected in 11.53% (6/52) of the analyzed water samples. After identifying the samples via qPCR, the nested PCR was performed so that the identified DNA fragments could be sequenced, where we confirmed the presence of the previously mentioned Cryptosporidium spp., however the Giardia spp. samples did not show positive results in the sequencing, being not possible the identification of assemblages. This work subsidizes, with data on the presence of Giardia spp. and Cryptosporidium spp. in the two studied WWTPs in the city of São Paulo, the elaboration of an adequate regulation for the use of this resource in an urban environment, protecting the health of the exposed population, especially the most vulnerable populations and reducing the risk of infection by occupational use.

Uso de secnidazol y homeopatía para el control de giardiasis en perros / Use of secnidazole and homeopathy for giardiasis control in dogs

RESUMEN Los perros con infecciones por Giardia tienen diarrea que persiste durante días, incluso después del tratamiento. Manejamos la giardiasis canina usando dos enfoques distintos: tratamiento con el secnidazol antiprotozoario solo; y secnidazol más un producto homeopático indicado para el control de la diarrea en perros. Tratamiento I: Diez perros beagle mostraron signos clínicos de diarrea severa. Sus heces revelaron una gran cantidad de quistes compatibles con Giardia sp. Por lo tanto, tratamos a los perros con secnidazol (10 mg/kg, dosis única) por vía oral. Cinco días después del tratamiento, todos los perros fueron negativos para Giardia spp. Tratamiento II: Tres meses después, los mismos diez Beagles nuevamente presentaron diarrea. Se recogieron las heces, revelando Giardia sp. en todos los perros. Esta vez, los tratamos con un antiprotozoo (secnidazol, dosis de 10 mg/kg) combinado con un medicamento homeopático comercial para el control de la diarrea (dosis de 3 g/animal a intervalos de 12 h). Descubrimos que se necesitaban de tres a cinco dosis homeopáticas para lograr puntajes fecales normales en todos los perros. Después de 5 días de tratamiento, las muestras de heces tuvieron una consistencia normal y fueron negativas para Giardia. Este es el primer registro de uso de secnidazol para el control de la giardiasis canina asociado con homeopáticos para evitar la diarrea.

ABSTRACT Dogs with Giardia infections have diarrhea that persists for days, even after treatment. We managed canine giardiasis using two distinct approaches: treatment with the antiprotozoan secnidazole alone; and secnidazole plus a homeopathic product that is indicated for control of diarrhea in dogs. Treatment I: Ten beagle dogs showed clinical signs of severe diarrhea. Their feces revealed large numbers of cysts consistent with Giardia sp. Therefore, we treated the dogs with secnidazole (10 mg/kg, single dose) given orally. Five days after treatment, all dogs were negative for Giardia spp. Treatment II: Three months later, the same ten Beagles again presented with diarrhea. Feces were collected, revealing Giardia sp. in all dogs. This time, we treated them with an antiprotozoan (secnidazole, 10 mg/kg dose) combined with a commercial homeopathic drug for diarrhea control (3 g dose/animal at 12 h interval). We found that three to five homeopathic doses were needed to achieve normal fecal scores in all dogs. After 5 days of treatment, stool samples had normal consistency and were negative for Giardia. This is the first record of using secnidazole to control canine giardiasis associated with homeopathic drugs to prevent diarrhea.

Diagnóstico de especies de Giardia por comparación de dos pruebas inmunoabsorbentes ligadas a enzimas / Diagnosis of Giardia Species by Comparison of Two Enzyme-Linked Immuno-Sorbent Tests

Background: There was some trade produced enzyme-immuno-sorbent assays tests to diagnose antigens of Giardia spin stool specimens, like Rida Quick Giardia and Serazym ELISA Giardia® tests. In this study, we evaluated the performance of two commercially available EIA kits for detecting Giardia antigens. Methods: A total of 96 stool specimens were collected from patients who attended for different Primary Health Care centers in Al-Qurna sector / Basrah Health Directorate, Ministry of Health/ Environment, Basrah, Iraq at period from 2nd of March 2018 to 28th of September 2018. All specimens were examined by conventional methods of microscopic examination, and by both enzyme-immuno-sorbent assays tests. Results: Serazym ELISA Giardia® had a sensitivity of 90.1%, a specificity of 100%, and an accuracy of 91.7%. The Rida Quick Giardia® showed a sensitivity of 79%, a specificity of 100%, and an accuracy of 82.3%. Serazym ELISA Giardia® showed 10% discrepancy better than Rida Quick Giardia® which showed 8%, this was of a statistically significant difference (P<0.05). Conclusion: Antigen detection by EIA has been established as a valuable tool to make parasite stool diagnostics more effective. Serazym ELISA Giardia® is more reliable than Rida Quick Giardia

Epidemiology of Giardia duodenalis assemblages in Brazil: there is still a long way to go

Giardia duodenalis infection is distributed worldwide and can achieve prevalence around 60%, especially in developing countries. This protozoan is divided into eight assemblages, in which A and B have high zoonotic potential, whereas C to H are host-specific. This scenario is changing as molecular studies progress, highlighting that knowledge on host-specificity still has a long way to go. Understanding the players involved in transmission routes enables rational designs of control strategies. Considering the high prevalence of giardiasis, this review aims to gather together the data on available studies on the distribution of G. duodenalis assemblages in Brazil until September 2020.

Cryptosporidium spp. e Giardia spp. em carnívoros silvestres do Estado do Pará ­ Brasil / Cryptosporidium spp. and Giardia spp. in wild carnivorous animals the State of Pará ­ Brazil

O presente estudo investigou a ocorrência de Cryptosporidium spp. e Giardia spp. em diferentes espécimes silvestres da ordem Carnívora de vida livre e de cativeiro procedentes de municípios do Estado do Pará. Coletou-se amostras fecais de 37 animais distintos (quatro de vida livre e 33 de cativeiro). Para pesquisa de Cryptosporidium spp. e Giardia spp. foram utilizados métodos microscópicos (direto e Kinyoun) e imunológico (RIDA®QUICK Cryptosporidium/Giardia/Entamoeba Combi - N1722). Do total de amostras, 24,32% (9/37) foram positivas, correspondendo a 5,4% (2/37) para Cryptosporidium spp. e 18,91% (7/37) para Giardia spp., respectivamente. Nenhum animal apresentou infecção concomitante para os agentes. Cryptosporidium spp. e Giardia spp., são protozoários zoonóticos que representam um emergente problema de saúde pública. Esses parasitos podem apresentar elevada frequência em regiões em que as condições de saneamento básico são precárias, promovendo surtos de diarreia em animais domésticos, silvestres e no homen. Mamíferos silvestres, como os carnívoros, são susceptíveis à contaminação por enteroparasitas presentes tanto no habitat natural como em cativeiro. Portanto, a pesquisa comprova a presença desses protozoários em carnívoros silvestres, tanto mantidos em criatórios como nos de vida livre no Estado do Pará, considerando-se que esses animais podem atuar como fontes de infecção para o homem, para outros animais e para o meio ambiente.

The presente survey has had the purpose to investigate the occurrance of Cryptosporidium spp. and Giardia spp. in free and under captivity carnivorous wild animals, from several counties in the State of Pará. Samples of feces from 37 distinct animals (four in their natural habitat and 33 raised in captivity). For the research of Cryptosporidium spp. and Giardia spp. microscopic immunological, direct and Kinyoun methods were used (RIDA®QUICK Cryptosporidium/Giardia/Entamoeba Combi - N1722). The samples gathered from wild animals have resulted in 24,32% of positive infecction on the rate of (9/37), being. 5,4% (2/37) positive to Cryptosporidiumspp. and 18,91% (7/37) positive to Giardia spp., what shows that no amimals had both infections at the same time. Cryptosporidium spp. and Giardia spp., are zoonotic enteroparasites that have been taking place as an emmerging problem to public health. Theese species of protozoa may reach high levels of frequency in regions where the basic sanitation conditions are precarious, promoting outbraks of diarrhea to men, wild and domestic animals. Wild mammals, as the carnivorous, are susceptible to contamination by enteroparasites, being present at their natural habitat or captivity. So, the reserach strenghtens the real presence of these protozoas in wild carnivorous in both conditions of life, free or under captivity, in the State of Pará, making us consider the possibility that the cited animals may be natural reservoirs for infections, not only to men but to other animals and also to environment.

Cryptosporidium spp. e Giardia spp. em carnívoros silvestres do Estado do Pará Brasil / Cryptosporidium spp. and Giardia spp. in wild carnivorous animalsthe State of Pará – Brazil

O presente estudo investigou a ocorrência de Cryptosporidium spp. e Giardia spp. em diferentes espécimes silvestres da ordem Carnívora de vida livre e de cativeiro procedentes de municípios do Estado do Pará. Coletou-se amostras fecais de 37 animais distintos (quatro de vida livre e 33 de cativeiro). Para pesquisa de Cryptosporidium spp. e Giardia spp. foram utilizados métodos microscópicos (direto e Kinyoun) e imunológico (RIDA®QUICK Cryptosporidium/Giardia/Entamoeba Combi - N1722). Do total de amostras, 24,32% (9/37) foram positivas, correspondendo a 5,4% (2/37) para Cryptosporidium spp. e 18,91% (7/37) para Giardia spp., respectivamente. Nenhum animal apresentou infecção concomitante para os agentes. Cryptosporidium spp. e Giardia spp., são protozoários zoonóticos que representam um emergente problema de saúde pública. Esses parasitos podem apresentar elevada frequência em regiões em que as condições de saneamento básico são precárias, promovendo surtos de diarreia em animais domésticos, silvestres e no homen. Mamíferos silvestres, como os carnívoros, são susceptíveis à contaminação por enteroparasitas presentes tanto no habitat natural como em cativeiro. Portanto, a pesquisa comprova a presença desses protozoários em carnívoros silvestres, tanto mantidos em criatórios como nos de vida livre no Estado do Pará, considerando-se que esses animais podem atuar como fontes de infecção para o homem, para outros animais e para o meio ambiente.

The present survey has had the purpose to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in free and under captivity carnivorous wild animals, from several counties in the State of Pará. Samples of feces from 37 distinct animals (four in their natural habitat and 33 raised in captivity). For the research of Cryptosporidium spp. and Giardia spp. microscopic immunological, direct and Kinyoun methods were used (RIDA®QUICK Cryptosporidium/Giardia/Entamoeba Combi - N1722). The samples gathered from wild animals have resulted in 24,32% of positive infection on the rate of (9/37), being. 5,4% (2/37) positive to Cryptosporidium spp. and 18,91% (7/37) positive to Giardia spp., what shows that no animals had both infections at the same time. Cryptosporidium spp. and Giardia spp., are zoonotic enteroparasites that have been taking place as an emerging problem to public health. These species of protozoa may reach high levels of frequency in regions where the basic sanitation conditions are precarious, promoting outbreaks of diarrhea to men, wild and domestic animals. Wild mammals, as the carnivorous, are susceptible to contamination by enteroparasites, being present at their natural habitat or captivity. So, the research strengthens the real presence of these protozoas in wild carnivorous in both conditions of life, free or under captivity, in the State of Pará, making us consider the possibility that the cited animals may be natural reservoirs for infections, not only to men but to other animals and also to environment.

Comparación de métodos de extracción de ADN de Giardia spp. medidos por PCR convencional / Comparison of methods of DNA extraction from Giardia spp. measured by conventional PCR

RESUMEN Objetivos . Comparar diferentes métodos de extracción de ADN a partir de quistes y trofozoítos de Giardia spp. mediante la técnica de reacción en cadena de la polimerasa (PCR) convencional. Materiales y métodos. Se aislaron quistes de Giardia spp. a partir de 65 muestras coprológicas procedentes de hospitales de referencia nacional, obteniéndose una carga promedio de 5x104 parásitos. Asimismo, se cultivaron trofozoítos de Giardia intestinalis (ATCC® 30957™) obteniéndose una carga parasitaria de 5x106. Se compararon once métodos de extracción para quistes y seis para trofozoítos. La concentración y pureza del ADN extraído se determinó por espectrofotometría y el rendimiento de la extracción se evaluó mediante la amplificación de los genes beta giardina (bg) y glutamato deshidrogenasa (gdh) por PCR semi-anidada. Resultados. Se observó que el método I mostró la mayor concentración de ADN a partir de quistes (12,24 ng/µL), pureza (1,4) y mejor rendimiento (100% amplificación bg, 60% gdh) en comparación con los otros métodos evaluados. En el caso de los trofozoítos el método que no tuvo pretratamientos presentó la mayor concentración de ADN, pureza y rendimiento (26,56 ng/µL; 1,85; 100% amplificación bg y gdh). Conclusiones. Los pretratamientos mecánicos, de choque térmico y enzimáticos son necesarios para la ruptura de la pared quística de Giardia spp., siendo el marcador molecular bg de mayor rendimiento para detección de ADN de quistes. Los trofozoítos no requieren pretratamientos para lograr resultados satisfactorios. Se cuenta con una metodología reproducible para la extracción de ADN de Giardia spp. a partir de cualquier estadio evolutivo.

ABSTRACT Objectives. To compare different methods of DNA extraction from cysts and trophozoites of Giardia spp. using the conventional polymerase chain reaction (PCR) technique. Materials and Methods. Cysts of Giardia spp. were isolated from 65 coprological samples from national reference hospitals, obtaining an average load of 5x104 parasites. In addition, Giardia intestinalis trophozoites (ATCC® 30957™) were cultured obtaining a 5x106 parasitic load. Eleven extraction methods for cysts and six for trophozoites were compared. The concentration and purity of the extracted DNA were determined by spectrophotometry and the extraction yield was assessed by amplification of the ß-giardin (bg) and glutamate dehydrogenase (gdh) genes with a semi nested PCR assay. Results. It was observed that method 1 showed the highest concentration of DNA from cysts (12.24 ng/µL), purity (1.4) and best performance (bg: 100% amplification; gdh: 60% amplification) compared to the other methods evaluated. In the case of trophozoites, the method without pre treatment showed the highest level of DNA concentration, purity, and yield (26.56 ng/µL; 1.85; 100% amplification of bg and gdh, respectively). Conclusions . Mechanical, thermal shock, and enzymatic pre-treatments are necessary for the rupture of the cystic wall of Giardia spp. making it the highest-yielding bg molecular marker for detecting cyst DNA. Trophozoites do not require pre-treatment to achieve satisfactory results. A reproducible methodology for the extraction of DNA from Giardia spp. from any evolutionary stage is available.

Prevalencia de parásitos intestinales en niños de 2-5 años en hogares comunitarios de Cartagena de Indias, Colombia / Presence of intestinal parasites in children 2-5 years of age in community homes in Cartagena de Indias, Colombia

RESUMEN Las enteroparasitosis constituyen un importante problema de salud pública a nivel global, afectando principalmente a la población infantil de los países tropicales. Nuestro objetivo fue determinar la prevalencia de parásitos intestinales en niños de 2 a 5 años en 17 hogares comunitarios del Instituto Colombiano de Bienestar Familiar (ICBF) en la Zona Industrial y Bahía de Cartagena de Indias. Para ello, se realizó un estudio descriptivo de corte transversal; se analizaron muestras mediante coprología de 207 niños de ambos sexos. No hubo diferencias significativas asociadas al sexo y edad (p>0,05). La frecuencia de enteroparasitos en la población infantil de 2 a 5 años fue de un 70,5%, donde el 59,5%, presentó una sola especie de parásito, el 34,2% dos y 6,1% tres y cuatro especies. Los principales protistas (86,3%) encontrados fueron Blastocystis hominis (60,2%) y Giardia lamblia (24,6%). En el grupo de Helmintos (13,7%) se destacaron Ascaris lumbricoides (9,6%) e Hymenolepis nana (6,2%). Las enteroparasitosis continúan como un importante problema de salud pública en condiciones del trópico Colombiano, especialmente en población infantil de zonas vulnerables de Cartagena - Colombia. Evidenciando la necesidad de revisar las políticas dirigidas a reducir la frecuencia del parasitismo intestinal.

ABSTRACT Intestinal parasites constitute an important public health problem at a global level, mainly affecting the pediatric population of tropical countries. The objective of this study was to determine the prevalence of intestinal parasites in children 2 to 5 years of age in 17 community homes of the Colombian Institute of Family Welfare (ICBF) in the Industrial Zone and Cartagena de Indias Bay, Colombia. A descriptive cross-sectional study was carried out among 207 children of both sexes. Samples were analyzed by coprology. There were no significant differences associated with sex and age (p>0.05). The frequency of enteroparasites in children aged 2 to 5 was 70.5 %, where 59.5% presented a single species of parasite, 34.2% two and 6.1% three and four species. The main protists (86.3%) found were Blastocystis hominis (60.2%) and Giardia lamblia (24.6%). In the group of Helminths (13.7%), Ascaris lumbricoides (9.6%) and Hymenolepis nana (6.2%) were observed. Intestinal parasites continue to be an important public health problem in the Colombian tropics, especially among children in vulnerable areas of Cartagena, Colombia. There is a need to review policies aimed at reducing the frequency of intestinal parasitism.

Evaluación de métodos de concentración y purificación de Giardia spp. a partir de muestras coprológicas / Evaluation of methods of concentration and purification of Giardia spp. from coprological samples

RESUMEN El objetivo del estudio fue comparar diferentes métodos de concentración para recuperar la mayor cantidad de quistes de Giardia spp. a partir de muestras coprológicas. Se analizaron 100 muestras procedentes de hospitales de referencia nacional y se aplicaron cuatro métodos parasitológicos: concentración por sedimentación espontánea en tubo (TSET), Faust, gradiente de sucrosa de una fase y gradiente de sucrosa de dos fases. Se encontró que el método de gradiente de sucrosa de dos fases alcanzó resultados significativamente mejores en concentración de quistes (121 903 quistes/ml) y cantidad de detritos (6%), en comparación con los métodos de Faust (35 355 quistes/ml), concentración por sedimentación espontánea en tubo (20,145 quistes/ml) y gradiente de sucrosa de una fase (18 702 quistes/ml). Se concluye que el método más eficaz para la concentración y purificación de quistes de Giardia spp. a partir de muestras coprológicas es el método de gradiente de sucrosa de dos fases, lo que facilitaría los cultivos in vitro de Giardia spp.

ABSTRACT The aim of this study was to compare different methods of concentration to recover the largest number of Giardia spp. cysts from coprological samples. One hundred (100) samples from national reference hospitals were analyzed and four parasitological methods were applied: spontaneous tube sedimentation concentration (TSET), Faust, single-phase sucrose gradient, and two-phase sucrose gradient. The two-phase sucrose gradient method was found to achieve significantly better results in cyst concentration (121,903 cysts/ml) and amount of debris (6%), compared to Faust methods (35,355 cysts/ml), spontaneous tube sedimentation concentration (20,145 cysts/ml), and single-phase sucrose gradient (18,702 cysts/ml). It is concluded that the most effective method for the concentration and purification of Giardia spp. cysts from coprological samples is the two-phase sucrose gradient method, which would facilitate in vitro culture of Giardia spp.

Surveillance of Giardia and Cryptosporidium in sewage from an urban area in Brazil / Monitoramento de Giardia e Cryptosporidium em esgoto de uma área urbana no Brasil

Abstract Cryptosporidium and Giardia are protozoan parasites that cause diarrhea in humans and animals. Molecular characterization of these pathogens in sewage may provide insight on their occurrence and prevalence in Brazil. This study aimed to investigate the presence of Giardia and Cryptosporidium in raw and treated sewage from Londrina, Paraná, Brazil. Samples were collected every two weeks during a year. Samples were concentrated, then DNA was extracted and subjected to a nested PCR targeting the Giardia 18S rRNA gene and the Cryptosporidium 18S rRNA gene. Species of Cryptosporidium were characterized by restriction fragment length polymorphism (RFLP). All raw sewage and 76% of the treated sewage were positive for Giardia; 84% of raw sewage samples and 8% of treated sewage were positive for Cryptosporidium. C. muris, C. hominis, C. baileyi, C. parvum and C. suis were detected in 100%, 19%, 9%, 9% and 4% of raw sewage, respectively. C. muris was the only species found in treated sewage. Multiple species of Cryptosporidium were present in 19.04% of the raw sewage. Treated sewage water can pose a threat to human health. The speciation of Cryptosporidium revealed the presence of non-common zoonotic species as C. suis and C. muris.

Resumo Cryptosporidium e Giardia são protozoários causadores de diarreia em animais e humanos. A caracterização molecular destes protozoários em esgoto pode prover dados ainda desconhecidos da ocorrência de espécies. O objetivo do presente estudo foi monitorar a ocorrência de Giardia e espécies de Cryptosporidium em esgoto bruto e tratado em uma estação de tratamento de esgoto (ETE) de Londrina, Paraná. Amostras de esgoto bruto e tratado foram coletadas no período de um ano, com periodicidade quinzenal. A ocorrência destes protozoários foi caracterizada por meio de concentração das amostras e posterior extração de DNA seguida de nested-PCR para amplificação de fragmentos dos genes 18S rRNA de Giardia e 18S rRNA de Cryptosporidium. A caracterização das espécies de Cryptosporidium foi realizada por meio de análise por polimorfismo de comprimento do fragmento de restrição (RFLP) dos produtos obtidos. Foram coletadas no total 25 amostras de cada, esgoto bruto e esgoto tratado. Para Giardia, todas as amostras de esgoto bruto e 76% das de esgoto tratado foram positivas. Cryptosporidium esteve presente em 84% das amostras de esgoto bruto e em 8% do tratado. No esgoto tratado foi encontrado apenas C. muris, já nas amostras de esgoto bruto foram encontradas cinco espécies: C. muris, C. hominis, C. baileyi, C. suis e C. parvum em 100%, 19%, 9%, 9% e 4%, respectivamente. A presença de espécies mistas foi observada em 19,04% das amostras. A presença de Giardia e Cryptosporidium em esgoto tratado pode pôr em risco a saúde humana. A discriminação de espécies de Cryptosporidium revelou a presença de espécies zoonóticas incomuns como C. suis e C. muris.

Survey of bovine norovirus infections from diarrheic calves in South Korea, 2015–2017 / 대한수의학회지

This study examined complex infections with various enteropathogens and the genetic diversity of bovine norovirus (BNoV) in 932 fecal samples from diarrheic calves in South Korea. Overall, seventeen (1.8%) of the samples tested positive for BNoV following RT-PCR examination. All BNoV-positive samples were co-infected with other intestinal pathogens, including bovine Rotavirus, Giardia, Cryptosporidium, and Escherichia coli. The genetic diversity of the BNoVs shared high nucleotide identity (98.1–99.5%) and amino acid homology (93.5–98.1%) with genotype 2 BNoV (GIII.2) strains. In conclusion, BNoV infections with GIII genotypes were detected in complex infections of diarrheic calves in South Korea.

Causative agents and epidemiology of diarrhea in Korean native calves

Calf diarrhea caused by infectious agents is associated with economic losses in the cattle industry. The purpose of this study was to identify the causative agents and epidemiological characteristics of diarrhea in Korean native calves (KNC). In total, 207 diarrheal KNC aged less than 7 months were investigated. Fecal samples collected from the rectum were examined for causative agents using polymerase chain reaction (PCR) or real-time PCR and the number of oocysts were counted. Fourteen causative agents were detected from 164 of the 207 diarrheal KNC. Rotavirus was the most common agent (34.8%), followed by Eimeria spp. (31.7%), Escherichia coli (22.0%), Giardia spp. (14.0%), Clostridium difficile (9.8%), bovine viral diarrhea virus (8.5%), coronavirus (7.9%), Cryptosporidium spp. (7.3%), torovirus (6.7%), parvovirus (5.5%), norovirus (4.9%), kobuvirus (1.8%), adenovirus (1.2%), and Salmonella spp. (0.6%). About 95 (57.9%) of 164 calves were infected with a single causative agent and 42.1% were infected by multiple agents. No significant difference was observed in mortality between calves infected with a single agent and multiple agents. The occurrence of diarrhea caused by rotavirus, Eimeria spp., kobuvirus, and Giardia spp. was significantly different based on onset age, and the prevalence of diarrhea caused by rotavirus or C. difficile was significantly different between seasons. This study help the understanding of KNC diarrhea for the development of an effective strategy for disease prevention and control, especially in Eastern provinces of South Korea.

Molecular Prevalence and Genotypes of Cryptosporidium parvum and Giardia duodenalis in Patients with Acute Diarrhea in Korea, 2013–2016

Cryptosporidium parvum and Giardia duodenalis are the main diarrhea-causing parasitic pathogens; however, their prevalence in Korea is unknown. Here, we conducted a survey to determine the prevalence and genotype distribution of these 2 pathogens causing acute diarrhea in 8,571 patients hospitalized in 17 Regional Institute of Health Environment sites in Korea, during 2013–2016. C. parvum and G. duodenalis were detected and genotyped by nested PCR, and the isolate were molecularly characterized by sequencing the glycoprotein 60 (Gp60) and β-giardin genes, respectively. The overall prevalence of C. parvum and G. duodenalis was 0.37% (n=32) and 0.55% (n=47), respectively, and both pathogens were more prevalent in children under 9 years old. Molecular epidemiological analysis showed that the C. parvum isolates belonged to the IIa family and were subtyped as IIaA13G2R1, IIaA14G2R1, IIaA15G2R1, and IIaA18G3R1. Analysis of the β-giardin gene fragment from G. duodenalis showed that all positive strains belong to assemblage A. This is the first report on the molecular epidemiology and subtyping of C. parvum and G. duodenalis in such a large number of diarrheal patients in Korea. These results highlight the need for continuous monitoring of these zoonotic pathogens and provide a basis for implementing control and prevention strategies. Further, the results might be useful for epidemiological investigation of the source of outbreak.

Increased Innate Lymphoid Cell 3 and IL-17 Production in Mouse Lamina Propria Stimulated with Giardia lamblia

Innate lymphoid cells (ILCs) are key players during an immune response at the mucosal surfaces, such as lung, skin, and gastrointestinal tract. Giardia lamblia is an extracellular protozoan pathogen that inhabits the human small intestine. In this study, ILCs prepared from the lamina propria of mouse small intestine were incubated with G. lamblia trophozoites. Transcriptional changes in G. lamblia-exposed ILCs resulted in identification of activation of several immune pathways. Secretion of interleukin (IL)-17A, IL-17F, IL-1β, and interferon-γ was increased, whereas levels of IL-13, IL-5, and IL-22, was maintained or reduced upon exposure to G. lamblia. Goup 3 ILC (ILC3) was found to be dominant amongst the ILCs, and increased significantly upon co-cultivation with G. lamblia trophozoites. Oral inoculation of G. lamblia trophozoites into mice resulted in their presence in the small intestine, of which, the highest number of parasites was detected at the 5 days-post infection. Increased ILC3 was observed amongst the ILC population at the 5 days-post infection. These findings indicate that ILC3 from the lamina propria secretes IL-17 in response to G. lamblia, leading to the intestinal pathology observed in giardiasis.

RNA-sequencing Profiles of Cell Cycle–Related Genes Upregulated during the G2-Phase in Giardia lamblia

To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1/S- or G2-phase by treatment with nocodazole and aphidicolin were prepared from the synchronized cell cultures. RNA-sequencing analysis of the 2 stages of Giardia cell cycle identified several cell cycle genes that were up-regulated at the G2-phase. Transcriptome analysis of cells in 2 distinct cell cycle stages of G. lamblia confirmed previously reported components of cell cycle (PcnA, cyclin B, and CDK) and identified additional cell cycle components (NEKs, Mad2, spindle pole protein, and CDC14A). This result indicates that the cell cycle machinery operates in this protozoan, one of the earliest diverging eukaryotic lineages.