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

We aimed to assess the risks of

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 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.

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.

Utilidad de los registros sanitarios locales para vincular la tasa de incidencia de diarreas con la calidad del agua de consumo / Usefulness of local health reports to link the incidence rate of diarrhea with the quality of drinking water

En este trabajo se realizó un relevamiento de los informes del centro de salud ubicado en Vaqueros (Salta, Argentina) durante un período de 8 meses. Al mismo tiempo se determinó la concentración de Escherichia coli y de quistes de Giardia spp. en muestras procedentes de cuatro fuentes de agua diferentes. No se encontró una relación estadísticamente significativa entre la calidad del agua y los casos de diarrea. Sin embargo, mediante un cálculo de odds ratios, se pudo determinar que uno de los sistemas de potabilización estudiados actúa como factor de protección contra diarreas. El presente trabajo brinda información útil para la planificación de medidas preventivas por parte del sistema de salud local.

In this study, we analyzed the reports of the health care center located in Vaqueros (Salta, Argentina) over an 8-month period. Moreover, we determined the concentration of Escherichia coli and Giardia spp. cysts in samples from four different drinking water sources. A statistical relationship between water quality and cases of diarrhea could not be found. However, using an odds ratio calculation, it was possible to determine that one of the studied drinking water systems acts as a protection factor in cases of diarrhea. The present work provides useful information for planning preventive measures by the local health system.

Contamination by parasites of zoonotic importance in fecal samples from Florianópolis Beaches, Santa Catarina State, Brazil / Contaminação por parasitas de importância zoonótica em amostras fecais nas praias de Florianópolis, SC, Brasil

Helminths and protozoa are examples of endoparasites that, during their biological cycles, can alternate between free-life stages and parasitic stages in the environment. Pets, such as dogs and cats, live together with men and play an important role in the society; however, these animals may carry a large number of parasites which, besides showing direct pathogenicity to the host, represent risks to the human health, as is the case of parasitic zoonoses. Public areas like parks, gardens, squares and beaches may offer risks to human users since these environments may contain feces deposited by parasitized dogs. The present study verified the occurrence of helminth eggs and/or protozoan cysts or oocysts in fecal samples collected from sand strips at the beaches Armação do Pântano do Sul, Campeche and Morro das Pedras, in the south of Florianópolis Island, Santa Catarina State (SC), during the summer period. Pet feces were collected in the 2016/2017 summer season, from December 2016 to February 2017. In the Laboratory of Animal Parasitology (CCA-UFSC), fecal samples were processed based on Willis-Mollay flotation technique and Hoffman sedimentation technique. Samples from all three analyzed beaches were positive for the presence of parasites and fecal material was found along the seashore, sites where people walk freely and barefoot. Of 104 fecal samples collected, 45 (43.27%) were positive for one or more parasites. The greatest prevalence was found in Campeche Beach, which had 72.22% positive samples. Ancylostomids were most prevalent, indicating that cutaneous larva migrans is likely to occur in humans. Other parasites such as Trichuris vulpis, Toxocara spp. and Giardia spp. were also detected in coproparasitological analyses. Based on the results, it was concluded that contamination of the beaches in Florianópolis Island, SC, constitutes a public health problem due to the possibility of zoonosis transmission. These results reinforce the importance of establishing health programs for parasite control and educational programs for elucidating the risks of transmission of these zoonoses to the population, as an attempt to reduce environmental contamination risks at beaches of seaside resorts.(AU)

Helmintos e protozoários são exemplos de endoparasitas que, durante seus ciclos biológicos, podem alternar fases de vida livre e de vida parasitária no ambiente. Animais de companhia, como cães e gatos, possuem convívio direto com o homem e desempenham importante papel na sociedade, porém estes animais podem possuir muitos parasitas próprios que, além da patogenicidade direta ao hospedeiro, representam riscos à saúde humana, no caso das zoonoses parasitárias. A utilização de áreas públicas como parques, jardins, praças e praias por estes animais, pode oferecer riscos aos seus frequentadores humanos, uma vez que pode ocorrer a deposição de fezes nesses ambientes por cães parasitados. O presente estudo investigou a ocorrência de ovos de helmintos e/ou cistos ou oocistos de protozoários em amostras de fezes coletadas nas faixas de areia das praias da Armação do Pântano do Sul, Campeche e Morro das Pedras, no sul da ilha de Florianópolis, Estado de Santa Cararina-SC, Brasil, durante o período de verão. As coletas de amostras de fezes de animais domésticos foram realizadas na temporada de verão 2016/2017, a partir de dezembro de 2016 até fevereiro de 2017. No Laboratório de Parasitologia Animal (CCA-UFSC), as amostras de fezes foram processadas pela técnica de flutuação de Willis-Mollay e pela técnica de sedimentação de Hoffman. Amostras colhidas nas três praias analisadas mostraram-se positivas para a presença de parasitos. Material fecal foi observado ao longo de toda orla, locais em que as pessoas caminham livremente e sem calçados. De um total de 104 amostras de fezes coletadas nas praias, 45 (43,27%) estavam positivas a um ou mais parasitas. A praia do Campeche foi a que apresentou maior prevalência, com 72,22% de amostras positivas. Os ancilostomídeos, responsáveis pela larva migrans cutânea em humanos, foram os mais prevalentes. Outros parasitas como Trichuris vulpis, Toxocara spp. e Giardia spp. também foram detectados nas análises coproparasitológicas. Com base nos resultados obtidos, concluímos que a contaminação das praias na Ilha de Florianópolis, SC, constitui um problema de saúde pública, devido à possibilidade de transmissão de zoonoses. Estes resultados reforçam a importância da implantação de programas sanitários de controle de parasitas e programas de educação, esclarecendo os riscos de transmissão destas zoonoses à população, a fim de reduzir os riscos de contaminação ambiental nas praias de balneários.(AU)

Occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system, Paraná, Southern Brazil / Ocorrência de Cryptosporidium spp. e Giardia spp. em uma estação pública de tratamento de água, Paraná, Sul do Brasil

The purpose of this study was to investigate the occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system. Samples of raw and treated water were collected and concentrated using the membrane filtration technique. Direct Immunofluorescence Test was performed on the samples. DNA extraction using a commercial kit was performed and the DNA extracted was submitted to a nested-PCR reaction (n-PCR) and sequencing. In the immunofluorescence, 2/24 (8.33%) samples of raw water were positive for Giardia spp.. In n-PCR and sequencing, 2/24 (8.33%) samples of raw water were positive for Giardia spp., and 2/24 (8.33%) samples were positive for Cryptosporidium spp.. The sequencing showed Cryptosporidium parvum and Giardia duodenalis DNA. In raw water, there was moderate correlation among turbidity, color and Cryptosporidium spp. and between turbidity and Giardia spp.. The presence of these protozoans in the water indicates the need for monitoring for water-treatment companies.

O objetivo deste estudo foi investigar a ocorrência de Cryptosporidium spp. e Giardia spp. em um sistema público de tratamento de água. Amostras de água bruta e tratada foram coletadas e concentradas, utilizando-se a técnica de filtração em membranas. Foi realizada a técnica de Imunofluorescência Direta nas amostras. A extração de DNA foi realizada, utilizando-se um kit comercial, e o DNA extraído foi submetido a uma reação de nested-PCR (n-PCR). Na imunofluorescência, 2/24 (8,33%) amostras de água bruta foram positivas para Giardiaspp.. Na n-PCR, 2/24 (8,33%) amostras de água bruta foram positivas para Giardia spp., e 2/24 (8,33%) amostras foram positivas para Cryptosporidium spp.. O sequenciamento demonstrou DNA de Cryptosporidium parvum e de Giardia duodenalis. Na água bruta, houve correlação moderada entre turbidez, cor e Cryptosporidium spp. e entre a turbidez e Giardia spp.. A presença desses protozoários na água indica a necessidade de monitoramento pelas empresas de tratamento de água.

Formas parasitarias de importancia zoonótica, encontradas en heces de perros recolectadas desde plazas y parques públicos de la ciudad de Los Ángeles, Región del Bío Bío, Chile / Parasites of zoonotic importance in dog feces collected in parks and public squares of the city of Los Angeles, Bío-Bío, Chile

Introduction: The contamination of public squares and parks with dog feces poses a risk to the population, since it may contain parasitic elements of zoonotic importance. Aim: To identify human pathogenic parasites in samples of dog feces collected from parks and public squares. Methods: 452 fecal dog samples collected from 65 squares and public parks were analyzed using the technique of Burrows. Results: 60% (39/65) of the samples contained some parasitic forms with a zoonotic potential. Parasitic taxa with zoonotic risk were Toxocara sp., Ancylostoma sp., Dipylidium caninum, Giardia sp., Taenia sp., Toxascaris sp., Strongyloides sp., and Uncinaria sp. Conclusion: The detected parasites present a risk to human health, so it seems necessary to implement health education activities in the community, develop deworming plans, and control the canine overpopulation.

Introducción: La contaminación de plazas y parques con heces de perros representa un riesgo para la población, ya que éstas pueden contener elementos o estadios parasitarios de importancia zoonótica. Objetivo: Identificar formas parasitarias de posible riesgo para el ser humano, en muestras de heces caninas recolectadas desde el suelo de parques y plazas públicas. Material y Métodos: Se recolectaron 452 muestras fecales de perros (Canis lupus familiaris) en 65 plazas y parques públicos, las que fueron analizadas utilizando la técnica de Burrows. Resultados: Se obtuvo que 60% (39/65) de las muestras analizadas se encontraban contaminadas con alguna forma parasitaria con potencial zoonótico. Las taxas parasitarias con riesgo zoonótico diagnosticadas fueron Toxocara sp., Ancylostoma sp., Dipylidium caninum, Giardia sp., Taenia sp., Toxascaris sp., Strongyloides sp. y Uncinaria sp. Conclusión: La prevalencia de parásitos de importancia zoonótica representa un riesgo para la salud humana, por lo que se hace necesario implementar acciones de educación sanitaria a la comunidad, elaborando planes de desparasitación y control de la sobrepoblación canina.

Water from different sources used for the irrigation of vegetables to be marketed: research on Cryptosporidiumspp, giardiaspp, and Coliforms in Parana, Brazil / Água de diferentes fontes utilizadas na irrigação de hortaliças comercializadas: pesquisa de Cryptosporidiumspp, giardiaspp, e coliformes, Paraná, Brasil

SUMMARYThe aim of this work was to compare, from a parasitological ( Cryptosporidiumspp. and Giardia duodenalis), bacteriological (total and thermotolerants coliforms) and physicochemical perspective, water sources used for drinking and irrigation of vegetables intended to be sold for human consumption. From January 2010 to May 2011, samples of different water sources from vegetable producing properties were collected; 100 liters for parasitological analysis, 200 mL for bacteriological analysis, and five liters for physicochemical analysis. Water samples were filtered under vacuum with a kit containing a cellulose acetate membrane filter, 1.2 µm (Millipore(r), Barueri, SP, Brazil). The material retained on the membrane was mechanically extracted and analyzed by direct immunofluorescence (Merifluor(r)kit). From 20 rural properties investigated, 10 had artesian wells (40 samples), 10 had common wells (40 samples), and one had a mine (four samples), the latter contaminated by Cryptosporidiumspp. In samples from artesian wells, 90 to 130 meters depth, 42.5% were positive for total coliforms and 5.0% were identified to have abnormal coloration. From the samples of common wells, 14 to 37 meters depth, 87.5% were contaminated with total coliforms, 82.5% were positive for thermotolerant coliforms, and 12.5% had color abnormalities. We did not detect the presence of Giardiaspp. or Cryptosporidiumspp. in artesian and common wells. The use of artesian or common wells is an important step in the control of the spreading of zoonoses, particularly Cryptosporidiumspp. and Giardiaspp., as well as artesian wells for coliform control in local production of vegetables to be marketed.

RESUMOO objetivo do estudo foi investigar fontes de água utilizadas para consumo e irrigação de hortaliças a serem comercializadas sob o aspecto parasitológico ( Cryptosporidiumspp. e Giardia duodenalis), bacteriológico (coliformes totais e termotolerantes) e físico-químico. De janeiro de 2010 a maio de 2011 foram coletadas amostras de água de diferentes fontes de abastecimento de propriedades produtoras de hortaliças; 100 litros para análise parasitológica, 200 mL para bacteriológica e cinco litros para análise físico-química. As amostras de água foram filtradas a vácuo com um kit de filtragem contendo uma membrana de acetato de celulose, 1,2 µm, (Millipore(r), Barueri, São Paulo, Brasil). O material retido na membrana foi extraído mecanicamente e analisado por imunofluorescência direta (kit Merifluor(r)). De 20 propriedades rurais, 10 tinham poços artesianos (40 amostras), 10 semi-artesianos (40 amostras) e um possuía uma mina (quatro amostras). Esta última contaminada por Cryptosporidiumspp. Das amostras de poços artesianos com 90 a 130 metros de profundidade, 42,5% foram positivas para coliformes totais e 5,0% apresentavam coloração alterada. Em amostras de poços semi-artesianos com 14 a 37 m de profundidade, 87,5% apresentaram coliformes totais, 82,5% termotolerantes, e 12,5% destas amostras tinham alteração de cor. Não foi detectada a presença de Giardiaspp. e Cryptosporidiumspp. em poços artesianos ou semi-artesianos. A utilização de poços artesianos ou semi artesianos constitui importante medida no controle da disseminação de zoonoses, principalmente Cryptosporidiumspp. e Giardiaspp., assim como de poços artesianos para o controle de coliformes, em locais de produção de hortaliças irrigadas, a serem comercializadas.

Estudo longitudinal da infecção por enteropatógenos em bezerros neonatos, com diarreia, sob diferentes estratégias de aleitamento / Longitudinal study of infection by enteropathogens in newborn calves with diarrhea under different feeding strategies

Foram utilizados 17 bezerros, recém nascidos, da raça Holandesa, com o objetivo de avaliar a influência do volume de sucedâneo nos principais patógenos causadores de diarreia neonatal. [...] Foram coletadas amostras de sangue dos bezerros com cinco dias de idade para dosagem da proteína total. A média da proteína total foi 6,33 e 6,21g/dL nos grupos 1 e 2 respectivamente. O grupo 2 apresentou tendência (p<0,1) de maior consumo de sucedâneo no período avaliado. A quantidade de sucedâneo oferecida aos animais não influenciou a incidência de diarreia e sua etiologia, ou seja, não foi observada diferença (p>0,05) na frequência das amostras positivas para cada agente entre os grupos. A frequência dos enteropatógenos nas amostras foi de 100 e 75 por cento para Cryptosporidium spp.; 28,5 e 43,7 por cento para Salmonella spp.; 28,5 e 15,6 por cento para patotipos de E. coli; 3,5 e 6,2 por cento para Rotavírus e 10,7 e 9,4 por cento para Giardia sp. nos grupos 1 e 2 respectivamente. Foram encontrados os sorotipos de Salmonella infantis e muenster. Os patotipos de E. coli isolados foram classificados como E. coli enterohemorrágica, enteropatogênica, enterotoxigênica e produtoras de toxinas Shiga 1 e 2. Foi observada associação entre o Cryptosporidium spp. e os patotipos de E. coli em 30 por cento das amostras do grupo 1 e Cryptosporidium spp. e Salmonella spp. em 45,5 por cento no grupo 2. Os resultados do presente trabalho demonstraram que o fornecimento de diferentes volumes de sucedâneo não apresentou influência sobre a incidência e etiologia da diarreia neonatal. A avaliação longitudinal dos enteropatógenos durante o período de patência da diarreia demonstrou que a associação entre eles ocorre a partir do primeiro dia da doença e destacou a importância da infecção pelo Cryptosporidium spp. agente encontrado em todos os momentos e animais.

Seventeen Holstein newborn calves were used with the objective of evaluating the influence of milk replacer volume in the pattern of pathogens causing neonatal diarrhea. […] Were collected blood samples from calves with five days of age for determination of total protein. The average total protein was 6.33 and 6.21g/dL in groups 1 and 2, respectively. The group 2 tended (p<0.1) higher consumption of milk replacer during the study period. The volume of milk replacer did not influenced the incidence of diarrhea and the frequency of positive samples for each etiologic agent between the two groups (p>0.05). Also, there was no difference (p>0.05) on the pattern of the frequency of positive samples for evaluated pathogens. The frequency of pathogens in the samples was 100 and 75 percent for Cryptosporidium, 28.5 and 43.7 percent for Salmonella spp., 28.5 and 15.6 percent for E. coli pathotypes, 3.5 and 6.2 percent for Rotavirus and 10.7 and 9.4 percent for Giardia in groups 1 and 2, respectively. Serotypes of Salmonella infantis and muenster were found. The isolated pathotypes of E. coli isolates were classified as Escherichia coli enteropathogenic, enterotoxigenic and Shiga-toxin-producing 1 and 2. Associations between Cryptosporidium spp. and E. coli pathotypes, and between Cryptosporidium spp. and Salmonella spp. were found in 30 percent of the samples in group 1 and in 45.5 percent in group 2, respectively. Our results showed that the different volumes of milk replacer did not influence the incidence and etiology of neonatal diahrrea. Longitudinal evaluation of enteropathogens during patency demonstrated that the association between the etiologic agents starts from the first day of disease. This result highlighted the great importance of the infection by Cryptosporidium spp. which was present in every moments and animals evaluated.

MOLECULAR TYPING OF Giardia duodenalis ISOLATES FROM NONHUMAN PRIMATES HOUSED IN A BRAZILIAN ZOO / Genotipagem de isolados de Giardia duodenalis de primatas não humanos mantidos em zoológico do Brasil

Giardia infections in captive nonhuman primates (NHP) housed at a Brazilian zoo were investigated in order to address their zoonotic potential. Fresh fecal samples were collected from the floors of 22 enclosures where 47 primates of 18 different species were housed. The diagnosis of intestinal parasites after concentration by sedimentation and flotation methods revealed the following parasites and their frequencies: Giardia (18%); Entamoeba spp. (18%); Endolimax nana (4.5%); Iodamoeba spp. (4.5%); Oxyurid (4.5%) and Strongylid (4.5%). Genomic DNA extracted from all samples was processed by PCR methods in order to amplify fragments of gdh and tpi genes of Giardia. Amplicons were obtained from samples of Ateles belzebuth, Alouatta caraya, Alouatta fusca and Alouatta seniculus. Clear sequences were only obtained for the isolates from Ateles belzebuth (BA1), Alouatta fusca (BA2) and Alouatta caraya (BA3). According to the phenetic analyses of these sequences, all were classified as assemblage A. For the tpi gene, all three isolates were grouped into sub-assemblage AII (BA1, BA2 and BA3) whereas for the gdh gene, only BA3 was sub-assemblage AII, and the BA1 and BA2 were sub-assemblage AI. Considering the zoonotic potential of the assemblage A, and that the animals of the present study show no clinical signs of infection, the data obtained here stresses that regular coproparasitological surveys are necessary to implement preventive measures and safeguard the health of the captive animals, of their caretakers and of people visiting the zoological gardens.

A pesquisa de infecções por Giardia e a caracterização genotípica deste protozoário foi realizada em primatas não humanos (PNH) mantidos em Zoológico a fim de avaliar o seu potencial zoonótico. As amostras dos animais consistiram de fezes colhidas do piso de 22 baias onde eram mantidos 47 primatas de 18 diferentes espécies. Exames coproparasitológicos foram realizados pelos métodos de concentração por sedimentação e centrífugo-flutuação e revelaram a presença dos seguintes parasitas e suas respectivas frequências: Giardia (18%); Entamoeba spp. (18%); Endolimax nana (4.5%); Iodamoeba spp. (4.5%); oxiurídeos (4.5%) e estrongilídeos (4.5%). O DNA extraído de todas as amostras fecais foi submetido à técnica de PCR para a amplificação dos genes gdh e tpi de Giardia, porém, só foram obtidos amplicons das quatro amostras positivas provenientes de Ateles belzebuth, Alouatta caraya, Alouatta fusca and Alouatta seniculus. O seqüenciamento dos fragmentos amplificados foi possível apenas para as amostras oriundas de Ateles belzebuth (BA1), Alouatta fusca (BA2) e Alouatta caraya (BA3), cuja análise fenética de ambos os genes revelou pertencerem ao genótipo A. As análises das sequências de tpi revelaram que todas as amostras pertencem ao subgenótipo AII. No que se refere ao gene gdh as análises revelaram uma amostra pertencente ao subgenótipo AII (BA3) e duas ao subgenótipo A1 (BA1 e BA2). Considerando o potencial zoonótico do genótipo A e o fato de que os animais não apresentavam sintomas de infecção, os dados do presente trabalho salientam a importância de se realizar, periodicamente, exames coproparasitológicos dos animais de zoológico, para implementação de medidas preventivas para resguardar a saúde dos animais em cativeiro, a de seus tratadores e dos visitantes de parques zoológicos.

Detecção Molecular de Giardia spp. em amostras de esgoto bruto provenientes do Estado de São Paulo e da cidade de Lima, Peru / Molecular detection of Giardia spp. in samples of raw wastewater from the State of São Paulo and the city of Lima, Peru

Giardia intestinalis é um dos principais protozoários flagelados causadores de diarreia em humanos e animais, sendo um micro-organismo re-emergente, responsável por vários surtos de veiculação hídrica em nível mundial, razão pela qual tem merecido atenção das autoridades de Saúde Pública, e deve ser avaliado e monitorado, principalmente devido ao seu impacto negativo na qualidade do abastecimento público, em particular através do esgoto. Através de técnicas de biologia molecular é possível caracterizar e genotipar cistos presentes no esgoto, e identificar a circulação dos agrupamentos A e B, patogênicos para o homem. Neste estudo foram avaliadas amostras de esgoto bruto de cidades cosmopolitas da América Latina, no Estado de São Paulo, e em Lima, Peru. Objetivos Detectar através da técnica de reação em cadeia pela polimerase (PCR) a presença de G. intestinalis e os agrupamentos A e B, de importância para a saúde humana, em amostras de esgoto bruto provenientes do Estado de São Paulo, Brasil e de Lima, no Peru. Material e Métodos Um total de 18 amostras de esgoto bruto provenientes de portos, aeroportos e estações rodoviárias, com alto trânsito de pessoas de cinco municípios do estado de São Paulo foram coletadas pela técnica de Moore. Adicionalmente, 10 amostras provenientes de dois bairros (urbano e semi-urbano) na entrada da ETE de Carapongo, Lima, no Peru foram coletadas e concentradas por centrifugação. O DNA genômico foi extraído utilizando kit comercial...

Giardia intestinalis is on of the major flagellated protozoans that cause diarrhea in humans and animals, and a re-emerging microorganism, responsible for several waterborne outbreaks worldwide, therefore it has received attention from public health authorities, and should be evaluated and monitored mainly due to their negative impact on the quality of public supply, in particular through the sewage. Through molecular biology techniques it is possible to characterize and to genotype cysts present in wastewater, and to identify the circulation of Assemblages A and B, that are pathogenic to humans. In this study were evaluated samples of raw wastewater from cosmopolitan cities in Latin America, in the State of São Paulo and in Lima, Peru. Objectives Detect, through the technique of polymerase chain reaction (PCR), the presence of G. intestinalis and its Assemblages A and B, of importance to human health, in samples of raw wastewater from the State of São Paulo, Brazil and Lima, Peru. Material and Methods A total of 18 samples of raw wastewater from harbors, airports and bus stations, with high traffic of people from five municipalities of the state of São Paulo were collected by the technique of Moore. In addition, 10 samples from two districts (urban and semi-urban) at the entrance of the WWTP Carapongo, Lima, Peru, were collected and subjected to centrifugation. Genomic DNA was extracted by using a commercial kit (QIAamp DNA Stool Mini Kit ® - Qiagen, USA)...

Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay) were analysed using a semi-nested polymerase chain reaction-restriction fragment length polymorphism assay that targets the glutamate dehydrogenase (gdh) gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61%) were classified as assemblage B (26 as BIII and 16 as BIV), 22 (32%) as assemblage A (3 as AI and 19 as AII) and five (7%) as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A.

Prevalência da infecção por Helicobacter pylori e de parasitoses intestinais em crianças do Parque Indígena do Xingu / Prevalence of Helicobacter pylori infection and intestinal parasitosis in children of the Xingu Indian Reservation

OBJETIVO: Avaliar a prevalência da infecção por Helicobacter pylori e sua associação com parasitoses intestinais em crianças da comunidade indígena do Parque Indígena do Xingu. MÉTODOS: Foram incluídas 245 crianças indígenas entre 2 e 9 anos, de seis aldeias da região do rio Xingu, afluente do Amazonas. H. pylori foi detectado pelo teste respiratório com ureia-13C. Foram coletadas amostras de ar expirado, em jejum e 30 minutos após a ingestão de 50 mg de ureia-13C diluída em 100 mL de água aromatizada com suco de maracujá. Foram coletadas amostras de fezes de 202/245 (82,4 por cento) crianças para exame protoparasitológico. RESULTADOS: A prevalência do H. pylori foi de 73,5 por cento. Foi observada associação significativa do H. pylori com maior idade entre as diferentes aldeias e etnias. Resultaram positivas para a presença de parasitas 97,5 por cento (198/202) das amostras de fezes, sem associação com a infecção por H. pylori. Encontrou-se, na análise multivariada, uma relação entre a infecção por giárdia e o H. pylori. As etnias Kisêjê [odds ratio (OR) = 3,36] e Kaibi (OR = 4,00), e as aldeias Tuiararé (OR = 8,10), Ngojwere (OR = 4,10), Capivara (OR = 4,88), Diauarum (OR = 1,85) e Pavuru (OR = 1,40) foram fatores de risco para a infecção por H. pylori. CONCLUSÕES: Foi encontrada alta prevalência de H. pylori e de parasitose intestinal em crianças nas comunidades presentemente investigadas. No entanto, houve diferença significativa na prevalência do H. pylori entre as diversas aldeias estudadas. Verificou-se associação entre a presença de giárdia e a infecção por H. pylori.

OBJECTIVE: To evaluate the prevalence of Helicobacter pylori infection and its association with intestinal parasitoses in children from indigenous communities of the Xingu Indian Reservation, in Brazil. METHODS: A total of 245 Native Brazilian children between 2 and 9 years of age, from six villages of the Xingu River region, a tributary of the Amazon River, were assessed. H. pylori was detected using the 13C-urea breath test. Breath samples were collected at baseline and 30 minutes after ingestion of 50 mg of 13C-urea diluted with 100 mL of water flavored with passion fruit juice and sweetener. Stool samples were collected for the stool ova and parasites exam for 202/245 (82.4 percent) children. RESULTS: The overall prevalence of H. pylori was 73.5 percent. A significant association of H. pylori with increased age was observed among the different villages and ethnic groups. Positive results for the presence of parasites - 97.5 percent (198/202) - from the stool samples collected showed no association with H. pylori. Giardia showed an association with H. pylori in the multivariate analysis. Risk factors for H. pylori infection were observed in Kisêjê and Kaibi ethnic groups (OR [odds ratio] = 3.36 and 4.00, respectively), as well as in Tuiararé, Ngojwere, Capivara, Diauarum, and Pavuru villages (OR = 8.10, 4.10, 4.88, 1.85, and 1.40, respectively). CONCLUSIONS: H. pylori infection is highly prevalent in these communities, as well as intestinal parasitoses. However, there were significant differences in the prevalence of H. pylori among the diverse villages studied. Giardia was closely associated with H. pylori infection.

Giardiasis as zoonosis: between proof of principle and paradigm in the Northwestern region of São Paulo State, Brazil

OBJECTIVE: In order to evaluate the potential zoonotic transmission of Giardia duodenalis, isolates from humans and dogs in the Northwestern region of the São Paulo State, Brazil were characterized based on the β-giardin gene. METHODS: The samples were analyzed by sequencing of the Nested-PCR products. RESULTS: The A1 and A2 subgenotypes were detected in human and dogs. Cysts of assemblage B, C and D have not been found in any isolates studied. CONCLUSIONS: These results are consistent with the view that giardiasis in the largest endemic region of the Brazil should not be seen as a single entity.

Giardia duodenalis: genotypic comparison between a human and a canine isolates / Giardia duodenalis: comparação genotípica entre isolados humano e canino

INTRODUCTION: Evidence suggests that giardiasis is a zoonotic disease. The present work aimed to evaluate the genetic identity of Giardia duodenalis isolated from human and dog fecal samples from Belo Horizonte. METHODS: Human and dog fecal samples were cultured for isolation of G. duodenalis. To determine the genotype of the isolates, primers that amplify a specific region in rRNA of the protozoan were used. RESULTS: Two G. duodenalis isolates were obtained, which belong to the subgroup A genotype. CONCLUSIONS: These findings suggest that the transmission of giardiasis follows a zoonotic pattern.

INTRODUÇÃO: Evidências sugerem que a giardíase é uma doença zoonótica. O presente trabalho tem como objetivo avaliar a identidade genética da Giardia duodenalis isolada de fezes humanas e de cães de Belo Horizonte. MÉTODOS: Amostras de fezes humanas e de cães foram cultivadas para isolamento de G. duodenalis. Para determinação do genótipo dos isolados, foram usados oligonuclotídeos que amplificam regiões específicas do gene para rRNA. RESULTADOS: Dois isolados de G. duodenalis foram obtidos, os quais apresentaram o genótipo do sub-grupo A. CONCLUSÕES: Estes dados sugerem que a transmissão da giardíase segue um padrão zoonótico.

Controle de qualidade analítica dos métodos utilizados para a detecção de protozoários patogênicos em amostras de água / Control of the analytic quality of the methods used for the detection of pathogenic protozoans in water samples

As espécies de protozoários patogênicos de veiculação hídrica Giardia spp. e Cryptosporidium spp. foram responsáveis por cerca 90% dos surtos de gastroenterite ocorridos nos últimos 25 anos, em vários países. Critérios de avaliação das etapas referentes à detecção destes agentes em amostras de água, tais como: precisão inicial, inoculação nas amostras em água bruta e, diagnóstico e localização de erros estão abordados neste trabalho. Estes critérios devem ser correntemente aplicados e constantemente avaliados pelos laboratórios, a fim de assegurar a confiabilidade dos resultados obtidos durante um monitoramento e, assim, fornecer subsídios para uma avaliação do risco de surtos.

The waterborne pathogenic protozoan species Giardia spp. and Cryptosporidium spp. have been responsible for the majority (90%) of the gastroenteritis outbreaks that have occurred in the last 25 years in various countries. This paper discusses evaluation criteria pertaining to the steps involved in the detection of these microorganisms in water samples, such as: initial precision, inoculation in environmental samples, diagnosis and error localization. These criteria should be often applied and evaluated by laboratories in order to assure the reliability of the results obtained in a program of monitoring, thus aiding in the evaluation of the risks of outbreaks.

Cryptosporidium and giardia in water in Alexandria: detection and evaluation of viability by flow cytometry and different stains

Cryptosporidium oocysts and Giardia cysts have become ubiquitous in surface waters worldwide. The number and extent of outbreaks of waterborne diseases indicate a significant risk for their possible transmission by drinking-water. Since many Egyptian cities depend on surface water as their main source of drinking water, knowledge of the prevalence of waterborne protozoa in water resources is important. The present study was designed to use flow cytometry to detect Cryptosporidium oocysts and Giardia cysts in water samples in Alexandria city in comparison with the standard staining techniques. Testing the viability of the encountered parasites was also carried out comparing flow cytometry and trypan blue vital stain. Thirty water samples were collected from water tanks from different districts of Alexandria city. Samples were subjected to staining techniques and flow cytometry. Stains used were modified Zeihl-Neelsen [MZN], safranin methylene blue [SMeB], modified trichrome, fluorescent stains [phenol auramine and acridine orange]. Viability was evaluated comparing trypan blue stain and flow cytometry using 4'-6-Diamidino-2-phenylindole immunostain [DAPI]. Flow cytometry proved to be much more sensitive than staining techniques with a sensitivity of 100% for both Cryptosporidium oocysts [30 samples] and Giardia cysts [11 samples]. Following flow cytometry, the fluorescent phenol auramine stain had the greatest sensitivity of 94.74% and 80% [18 and 4 samples, respectively]. The percent of live parasites present in each sample was always significantly higher by DAPI than trypan blue stain. The results of the present study clearly demonstrate that incorporation of flow cytometry can improve sensitivity of detection of Giardia cysts and Cryptosporidium oocysts in water samples. Although it is more expensive than the other staining methods, it is rapid, simple and accurate in estimating the quantity and viability of the parasites in each sample. Thus, flow cytometry can be recommended for detection of protozoa in water

Protozoarios en aguas superficiales y muestras fecales de individuos de poblaciones rurales del municipio Montes, estado Sucre, Venezuela / Protozoans in superficial waters and faecal samples of individuals of rural populations of the Montes municipality, Sucre state, Venezuela

En el estado Sucre, el Río Manzanares se ve amenazado por actividades domésticas, agrícolas e industriales originadas por el hombre, convirtiéndose en factor de riesgo ambiental para sus habitantes. En este sentido se planteó evaluar la presencia de protozoarios en aguas superficiales de afluentes del Río Manzanares (Río Orinoco, Quebrada Seca, Río San Juan), municipio Montes, estado Sucre, Venezuela, así como también el análisis de muestras fecales de los habitantes de poblados aledaños. Se recolectaron muestras de aguas superficiales de los afluentes en estudio y fecales entre mayo 2006-abril 2007. Las muestras de aguas superficiales se procesaron con sedimentación por centrifugación, floculación y tinciones de Kinyoun y tricrómica; las muestras fecales se sometieron a examen directo con solución salina fisiológica y lugol, Ritchie modificado y las coloraciones antes mencionadas. Los protozoarios observados con mayor frecuencia en las aguas superficiales en los afluentes fueron: Amebas, Blastocystis sp., Endolimax sp., Chilomastix sp. y Giardia sp. Mientras que Blastocystis hominis, Endolimax nana y Entaomeba coli fueron los de mayor frecuencia observada en las muestras fecales. Los habitantes de Orinoco La Peña resultaron ser los más afectados por las infecciones parasitarias (77,60%), seguido de Río San Juan con 46,63%, y Quebrada Seca con 39,49%. La presencia de protozoarios patógenos y no patógenos en las aguas superficiales demuestra la contaminación fecal de los afluentes evaluados, por lo que representa un foco de infección permanente para los individuos que viven en las cercanías de estas aguas, esto se refleja por la observación de los mismos parásitos en ambas muestras.

In Sucre state, the Manzanares river is threatened by domestic, agricultural and industrial activities, becoming an environmental risk factor for its inhabitants. In this sense, the presence of protozoans in superficial waters of tributaries of the Manzanares river (Orinoco river, Quebrada Seca, San Juan river), Montes municipality, Sucre state, as well as the analysis of faecal samples from inhabitants of towns bordering these tributaries were evaluated. We collected faecal and water samples from may 2006 through april 2007. The superficial water samples were processed after centrifugation by the direct examination and floculation, using lugol, modified Kinyoun and trichromic colorations. Fecal samples where analyzed by direct examination with physiological saline solution and the modified Ritchie concentration method and using the other colorations techniques above mentioned. The most frequently observed protozoans in superficial waters in the three tributaries were: Amoebas, Blastocystis sp, Endolimax sp., Chilomastix sp. and Giardia sp. Whereas in faecal samples, Blastocystis hominis, Endolimax nana and Entaomeba coli had the greatest frequencies in the three communities. The inhabitants of Orinoco La Peña turned out to be most susceptible to these parasitic infections (77.60%), followed by San Juan River (46.63%) and Quebrada Seca (39.49%). The presence of pathogenic and nonpathogenic protozoans in superficial waters demonstrates the faecal contamination of the tributaries, representing a constant focus of infection for their inhabitants, inferred by the observation of the same species in both types of samples.