Nilson Branco

Molecular characterization of intestinal protozoa in two poor communities in the State of São Paulo, Brazil.

BackgroundSeveral species of protozoa cause acute or chronic gastroenteritis in humans, worldwide. The burden of disease is particularly high among children living in developing areas of the world, where transmission is favored by lower hygienic standards and scarce availability of safe water. However, asymptomatic infection and polyparasitism are also commonly observed in poor settings. Here, we investigated the prevalence of intestinal protozoa in two small fishing villages, Porto Said (PS) and Santa Maria da Serra (SM), situated along the river Tietê in the State of São Paolo, Brazil. The villages lack basic public infrastructure and services, such as roads, public water supply, electricity and public health services.MethodsMultiple fecal samples were collected from 88 individuals in PS and from 38 individuals in SM, who were asymptomatic at the time of sampling and had no recent history of diarrheal disease. To gain insights into potential transmission routes, 49 dog fecal samples (38 from PS and 11 from SM) and 28 river water samples were also collected. All samples were tested by microscopy and PCR was used to genotype Giardia duodenalis, Blastocystis sp., Dientamoeba fragilis and Cryptosporidium spp.ResultsBy molecular methods, the most common human parasite was Blastocystis sp. (prevalence, 45% in PS and 71% in SM), followed by D. fragilis (13.6% in PS, and 18.4% in SM) and G. duodenalis (18.2% in PS and 7.9% in SM); Cryptosporidium spp. were not detected. Sequence analysis revealed large genetic variation among Blastocystis samples, with subtypes (STs) 1 and 3 being predominant, and with the notable absence of ST4. Among G. duodenalis samples, assemblages A and B were detected in humans, whereas assemblages A, C and D were found in dogs. Finally, all D. fragilis samples from humans were genotype 1. A single dog was found infected with Cryptosporidium canis. River water samples were negative for the investigated parasites.ConclusionsThis study showed a high carriage of intestinal parasites in asymptomatic individuals from two poor Brazilian villages, and highlighted a large genetic variability of Blastocystis spp. and G. duodenalis.

First report of Cryptosporidium spp. oocysts in oysters (Crassostrea rhizophorae) and cockles (Tivela mactroides) in Brazil.

The consumption of oysters and cockles, which are usually eaten raw or lightly-cooked, can cause outbreaks of human diseases, especially if these shellfish are harvested from polluted areas. In Brazil data about the occurrence of pathogens, like hepatitis A virus, in shellfish have been reported but research on natural contamination for pathogenic protozoa is still non-existent. Cryptosporidium oocyst contamination of oysters (Crassostrea rhizophorae) and cockles (Tivela mactroides) was evaluated during two different periods in a coastal area from São Paulo, Brazil. From June to November 2005, and from July to December 2006, 180 mollusks were harvested for tissue examination. The gills and gastrointestinal tract (n = 36 pools) were carefully extracted from the animals and homogenized in a tissue homogenizer by adding surfactant Tween 80 (0.1%). Immunofluorescence assays were performed and Cryptosporidium oocysts were detected in 50.0% of gill pools of cockles and 10.0% of gill pools of oysters. In order to evaluate seawater quality in shellfish growing areas, total levels of thermotolerant coliforms, Escherichia coli and enterococci were determined. This is the first time that Cryptosporidium oocysts were found in shellfish from the coastal region of Brazil, and to the best of our knowledge it is also the first report in Latin America and the case might be of public health importance, reflecting the extension of the contamination on seafood, requiring a need for quality control standards.

A Parasitological Survey of Natural Water Springs and Inhabitants of a Tourist City in Southeastern Brazil

The goals of this study were to investigate the occurrence of Cryptosporidium oocysts and Giardia cysts in main springs of Campos do Jordão, an important tourist city, in Brazil and to gather the largest amount of parasitological data from autochthonous population that live in rural areas of this city. The membrane filtration technique followed by direct immunofluorescence assay was employed for concentration and visualization of waterborne protozoa. In the period between June 2003 and May 2004, the presence of at least one pathogenic protozoa was detected in 25.0% (3/12) of the springs studied, with mean concentrations ranging from 0.2 to 0.3 Cryptosporidium sp. oocysts and 0.07 to 0.1 Giardia sp. cysts/L. The coproparasitological investigation conducted in dwellers from two rural communities from this city revealed that 49.2% (91/185) of people had intestinal parasites. Among pathogenic protozoa, Cryptosporidium was the most prevalent species (8.1%) followed by Giardia duodenalis (5.9%), Entamoeba histolytica/Entamoeba dispar (2.7%), and Blastocystis hominis (2.2%). The most prevalent geohelminths were Ascaris lumbricoides (14.9%) and Trichuris trichiura (9.7%). This study demonstrated the contamination and the distribution of intestinal parasites, especially Cryptosporidium and Giardia species, in different springs of an important tourist city in Brazil, highlighting the need of monitoring natural water sources. The high prevalence of intestinal parasitosis detected in some specific populations of this city may function as a link of transmission of different intestinal parasitosis due to soil and water contamination, contributing to the maintenance of parasite life cycles. Therefore, the inclusion of consistent public health interventions with measures that include the protection of springs, the installation of minimum health infrastructure, and primary education of the population are widely necessary, aiming the control and prevention of parasite infections.

Enterocytozoon bieneusi detected by molecular methods in raw sewage and treated effluent from a combined system in Brazil

BACKGROUND Enterocytozoon bieneusi are the most common microsporidia associated with different clinical manifestations such as diarrhoea, respiratory tract inflammation and acalculous cholecystitis, especially in immunocompromised patients. Infection usually occurs by ingestion of food and water contaminated with spores, but can also result from direct contact with spores through broken skin, eye lesions, and sexual transmission, depending on the microsporidian species. Although there are reports of E. bieneusi found in humans and animals in Brazil, there are no published studies of environmental samples examined by molecular methods. OBJECTIVES The purpose of this study was to verify the presence of E. bieneusi in raw sewage and treated effluent from a combined system by molecular methods. METHODS Raw sewage and treated effluent samples collected from a combined system were analysed for the presence of E. bieneusi using the internal transcriber spacer (ITS) region of E. bieneusi by nested polymerase chain reaction. FINDINGS The analysis revealed E. bieneusi presence and a novel genotype (EbRB) in one raw sewage sample and one treated effluent. MAIN CONCLUSIONS The presence of E. bieneusi in final effluent indicates that the combined system may not remove microsporidian spores. This study is the first report of E. bieneusi in environmental samples in Brazil.

Avaliação da performance de metodologias de detecção de Cryptosporidium spp. e Giardia spp. em água destinada ao consumo humano, para o atendimento às demandas da Vigilância em Saúde Ambiental no Brasil

Objective: this study aimed to evaluate three different concentration protocols of cysts and oocysts in raw water samples in Brazilian rivers. Methods: the protocols studied were chemical precipitation, filtration in membranes of 47mm of diameter versus nominal porosity of 3μm and filtration using Filta-Max® system. The raw water samples collected from rivers in the states of Sao Paulo and Minas Gerais were analyzed after being seeded with Color-Seed®. The results of initial precision using filtration in membranes and recovery of Color-Seed® organisms were compared to the acceptance criteria established by the United States Environmental Protection Agency (USEPA). Results: the study of initial precision to Giardia using filtration in membranes and recovery of Color-Seed® met the USEPA criteria; for Cryptosporidium only the Filta-Max® achieved the established criteria. Conclusion: Filta-Max® system showed to be the only method that achieved all the performance criteria for identification of Giardia and Cryptosporidium in water.

Fluorescence decay of dyed protozoa: differences between stressed and non-stressed cysts

Several series of tests have shown that fresh, intact samples of Giardia duodenalis and Cryptosporidium parvum (oo)cysts are not marked by fluorescent probes such as carboxyfluorcein-succinimidyl-diacetate-ester (CFDA-SE), C12-resazurin and SYTOX® Green, probably because of their robust cell walls. These dyes fail to indicate the viability of such protozoa and allow negative responses to be recorded from living and infectious samples. Cryptosporidium parvum showed stronger isolation from chemicals, with living oocysts remaining unstained by the probe for up to 90 days after extraction. However, in further fluorescence decay (FD) experiments run with G. duodenalis samples stained using CFDA-SE (comprising living, non-stressed but aged cysts, heat-killed samples and UV-C-stressed samples) each showed a different FD decay profile, here studied in seven series of tests of five replicates each. The FD profiles were fitted by double-exponential decay kinetics, with the decay constant k2 being five times higher than k1. This FD procedure is fast and can be easily reproduced in 10 steps, taking ~ 1 h of laboratory work for already purified samples.

Label-Free Darkfield-Based Technique to Assist in the Detection of Giardia Cysts

Giardia is a pathogenic protozoan that has caused giardiasis outbreaks worldwide, and this water-borne disease may occur due to faults in water supply and treatment systems. To support surveillance and control programs, the monitoring of this parasite is essential in water samples. Although Giardia cyst detection is usually based on standard light microscopy, the resulting low contrasted cysts together with a wide array of particles of comparable size to the target protozoan demands a high level of observer skill and experience, as well as a long time to process the individual samples. The visualization of this protozoan can be significantly improved by using staining procedures, e.g., Lugol’s iodine in brightfield or fluorescence-based methods such as fluorescence-isothiocyanate (FITC) and 4′,6-diamidino-2-phenylindole (DAPI). However, the significant costs make fluorescence microscopy difficult to be performed in less-developed countries. Accordingly, the present study evaluated the effectiveness of inexpensive darkfield (DF) setups to acquire higher contrasted images of unstained cyst samples as a direct and rapid method for monitoring protozoa. Two low-cost custom-made DF illumination modes, transmitted and reflected, were evaluated on a standard light microscope. Examination of purified Giardia cyst suspensions with both DF setups revealed a direct correlation between morphological appearance and uptake of DAPI. Images captured under transmitted illumination showed higher contrast and sharpness when compared to the reflected images. DF microscopy might provide a simple, direct, and inexpensive method for observing Giardia cysts, which shows basic aspects of their intracellular structure, although the applicability of the method to raw water concentrates remains to be demonstrated.

Segurança Alimentar: Detecção de protozoários potencialmente patogênicos (Giardia spp. e Cryptosporidium spp.) em hortaliças de produção orgânica e convencional

Resumo Giardiose e Criptosporidiose são doenças que causam grande problemas para a saúde pública, afetando principalmente as crianças, nas quais ocasionam a gastroenterite; a transmissão desses protozoários ocorre principalmente atraves de água e alimentos contaminados com cistos de Giardia sp. e oocistos de Cryptosporidium sp. O objetivo do estudo é avaliar a presença desses parasitas em amostras de rúcula e alface vendidos comercialmente na região de Campinas, de modo a avaliar as diferenças entre o cultivo orgânico e convencional, porém ainda não existe uma metodologia padronizada para detecção desses parasitas em vegetais frescos no Brasil, e devido a isso, testes de eficiência de recuperação dessas formas de resistência parasitária foram realizadas a partir de contaminação das hortaliças em laboratório e lavagem com glicina 1M, onde o líquido obtido foi sedimentado e concentrado por centrifugação e em seguida, analisado através de método de imunofluorescência.

Toxoplasma-SPECIFIC IgG SUBCLASS ANTIBODY RESPONSE IN CEREBROSPINAL FLUID SAMPLES FROM PATIENTS WITH CEREBRAL TOXOPLASMOSIS.

SUMMARY Cerebral toxoplasmosis can be highly debilitating and occasionally fatal in persons with immune system deficiencies. In this study, we evaluated the Toxoplasma gondii-specific IgG subclass antibody response in 19 cerebrospinal fluid (CSF) samples from patients with cerebral toxoplasmosis who had a positive IgG anti-T. gondiiELISA standardized with a cyst antigen preparation. There were no significant differences between the rates of positivity and the antibody concentrations (arithmetic means of the ELISA absorbances, MEA) for IgG1 and IgG2, but the rates of positivity and MEA values for these two IgG subclasses were significantly higher than those for IgG3 and IgG4. The marked IgG2 response in CSF from patients with cerebral toxoplasmosis merits further investigation.