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.

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Blastocystis, an unicellular anaerobic eukaryote, is known to be a very common intestinal parasite found in humans and animals fecal samples worldwide. Currently, there is an increasing interest to yield insights into its prevalence and diversity in human populations living in poor and deprived areas. In this study, we describe the prevalence and genetic variability of Blastocystis isolates obtained from daycare center attendees aged 0 to 6years and staff, as well as some children family members and their dogs in a low-income community in São Paulo State, Brazil. A total of 181 stool samples (123 from daycare children, 14 from workers, 44 from household members and 20 from dogs) were submitted to DNA extraction, tested by polymerase chain reaction (PCR) targeting the SSUrDNA gene and the amplicons retrieved were sequenced. The prevalence of Blastocystis was 40.7% (50/123) in children, 28.6% (4/14) in workers and 50% (22/44) in household members. No dog was found positive. Of the 76 PCR products generated, 57 were successfully sequenced. Four subtypes were identified and the most common were ST1 (54.4%) and ST3 (33.3%), followed by ST2 (7.0%) and ST7 (5.3%). The intra-subtype analysis revealed a total of 10 different alleles previously reported. No statistically significant correlation was observed between subtypes and sociodemographic variables analyzed. Here, the following findings must be highlighted: (1) predominance of subtypes 1 and 3, a pattern that has been observed in many populations worldwide; (2) absence of ST4, a common subtype in Europe but rarely detected in South America's human populations and, (3) human infection with ST7, a subtype primarily found in birds but occasionally seen in human infections, raising the possibility of zoonotic transmission.

Giardia duodenalis is a common intestinal parasite infecting children attending daycare centres. This study aimed to verify Giardia occurrence and the genotypes of isolates infecting children aged 0-6 years and workers at a daycare centre in the state of São Paulo, Brazil. The families of children who tested positive for Giardia, were asked to provide stool samples from household members and their dogs. Samples (123 children, 14 centre employees, 44 household members, 19 children after treatment, and 20 dogs) were examined for intestinal parasites using concentration methods. DNA extracted from all samples was submitted for polymerase chain reaction (PCR) testing and the amplicons generated were used for multilocus sequence typing of beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. Giardia was detected in 15·9% and 28·6% of the 220 samples by microscopy and PCR, respectively. Analysis of sequences retrieved from 29 isolates revealed both assemblages A (31%) and B (69%). Sub-assemblages AII, BIII and BIV were identified and the alignment of the bg, gdh and tpi sequences revealed the presence of some single nucleotide polymorphisms, especially in assemblage B sequences. The higher predominance of assemblage B and the identification of the AII type support the view that anthroponotic transmission appears to be an important route of transmission in environments that concentrate children at an age when poor hygiene practices make them more vulnerable to such infection.

To study the frequency of Blastocystis hominis among healthy individuals, feces were collected from 153 children and 20 staff members of some municipal day-care centers. Three separate stool specimens of each individual were processed by Lutz and Faust methods. From 173 studied individuals, 60 (34.7%) showed B. hominis, frequently in association with other intestinal parasites and/or commensals. B. hominis was found mainly in adults and children between 36 and 72 months old. All positive cases were detected only by Lutz method and the use of three stool specimens increased the positivity of the parasitological diagnostic.

There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in pathogenesis of giardiasis. This report describes a preliminary characterization of the proteolytic activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). The protease activity of E/S products in conditioned medium by trophozoites of each strain was analyzed using substrate (gelatin and collagen) impregnated SDS-PAGE and hemoglobin assay. The protease characterization was based on inhibition assays including synthetic inhibitors. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted degradation of the substrates and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibition assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases although the presence of serine proteases was also indicated, mainly in the hydrolysis of hemoglobin.

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Considering that the number of day-care centers for pre-school-age children has expanded rapidly in developing countries, and that these institutions presenting conditions that facilitate the transmission of many enteric agents, a parasitological survey was carried out in three municipal day-cares from Botucatu: two in the urban area (one in downtown area and the other one in the city periphery area) and the third in the rural area. Three separate stool specimens were collected from 147 children ranging from 0 to 72 months old and 20 staff members. Each stool specimen was processed by Lutz and zinc sulfate flotation methods. The frequency of giardiasis observed among children of downtown, periphery and rural day-cares was 69.6%, 52.7% and 69.6%, respectively. Only one employee was positive for G. lamblia. The examination of three stool specimens increased the positivity for G. lamblia: from the ninety three final positive examinations, 24 (25.5%) and 8 (8.5%) were positives only after examination of the second and third samples, respectively. Others intestinal organisms like Ascaris lumbricoides (20.4%), Trichuris trichiura (19.0%). Hymenolepis nana (8.8%), Entamoeba coli (22.4%) and Blastocystis hominis (32.0%) were frequently found in the children. There was no significant association among localization of the day-cares, sex of the children and the levels of G. lamblia infection. According to the age, G. lamblia was found mainly in children between 12 to 47 months old.