Frequency, spatial distribution, and genetic diversity of Blastocystis among referred individuals to a clinical laboratory: First report of subtype 9 in Brazil.

The enteric protist Blastocystis has a worldwide distribution, however its prevalence in the human population is still underestimated, especially in developing countries where proper diagnosis is not performed in the routine of clinical laboratories. In this study, we aimed to assess the frequency, genetic diversity, and spatial distribution of Blastocystis isolates detected in fecal samples referred to a clinical laboratory for routine examination in inner São Paulo State, Brazil. A total of 348 leftover stool samples available for disposal from female and male individuals with age ranging from 3 months to 88 years were analyzed by both microscopic examination and PCR/sequencing of the SSU rRNA gene. The overall frequency of Blastocystis sp. was 31% (108/348), including 20.1% (70/348) and 31% (108/348) by microscopic examination and PCR/sequencing, respectively. Significant association was found only between Blastocystis infection and age, since the highest rate of positive samples was detected among 5-9 years old individuals (p < 0.0001). In addition, spatial distribution revealed a wide distribution of the positive samples, however they were densely concentrated in more populated areas. Seven subtypes were identified, namely ST1 (40.7%), ST2 (9.2%), ST3 (45.3%), ST4 (0.9%), ST6 (1.8%), ST7 (0.9%) and ST9 (0.9%). The intra-subtype analysis revealed a total of 25 different alleles previously reported. Here, the findings lead us to highlight the following aspects: (1) the identification of a ST9 isolate is a relevant finding since it is considered a very rare subtype in human infections as well as this is the first report in Brazil; (2) the high frequency of Blastocystis in fecal samples submitted for examination in a clinical laboratory points to the need to consider its search in routine parasitological examinations, (3) the spatial distribution of Blastocystis infection was not homogeneous but concentrated in more populated areas where the access for population to diagnostic services in healthcare is likely to be easier and, (4) the genetic variability of Blastocystis isolates suggests exposure of inhabitants living in inner municipalities to different sources of contamination involving anthroponotic and zoonotic transmission pathways.

Molecular identification of hookworm species infecting free-roaming and owned dogs from an urban area in inner São Paulo State, Brazil.

Dogs are the most popular pet animals worldwide, but on the other hand, they are main hosts of pathogens potentially transmissible to humans. The aim of this study was to assess the occurrence of intestinal parasites in free- roaming and owned dogs in an urban area in southeastern Brazil and to identify the hookworm species infecting them. Faecal samples (80 from free-roaming and 53 from owned dogs) were examined for intestinal parasites using concentration methods. DNA extracted from hookworm microscopy-positive samples were tested by PCR targeting the ITS1-5.8S-ITS2 region and the amplicons retrieved were sequenced. Intestinal parasites were detected in 43.60% (58/133) of the dogs and hookworm infection was found at the highest prevalence rate (38.30%), followed by Toxocara canis (10.50%), Trichuris vulpis (2.25%), Giardia spp. (0.75%) and Cystoisospora spp. (0.75%). Out of the 51 samples positive for hookworm eggs, 26 (50.90%) were successfully amplified and sequenced. Single infections with Ancylostoma caninum and Ancylostoma braziliense were recorded in 18 (69.20%) and two (7.70%) isolates, respectively, and mixed infections were found in the remaining six samples (23.10%). Both species were found infecting free-roaming and owned animals, but A. caninum was more common. These findings highlight the public health relevance of dogs as reservoirs of zoonotic parasites, with emphasis on hookworm species commonly implicated in cutaneous larva migrans (CLM) in poor and deprived areas.

Prevalence and genetic characterization of Dientamoeba fragilis in asymptomatic children attending daycare centers.

In order to provide additional data on the prevalence and genetic diversity of Dientamoeba fragilis in human populations, we conducted a study in children from low-income communities in Sao Paulo State, Brazil. Fecal samples from daycare center attendees up to 6 years old (n=156) and staff members (n=18) were submitted to PCR and sequencing of D. fragilis as well as to microscopic examination for the presence of other intestinal parasites. All children assessed were asymptomatic and 10.3% (16/156) were positive for D. fragilis. No worker was found to be positive. An association between Dientamoeba and coinfection with other intestinal parasites was observed. Concerning the genetic diversity, 14 and only two isolates were genotype 1 and genotype 2, respectively. Our findings outline interesting aspects: (1) asymptomatic children as carriers of Dientamoeba in communities in which environmental conditions ensure parasite transmission and, (2) association between Dientamoeba infection in young children and coinfection with other enteric parasites, reinforcing its transmission via the fecal-oral route.

Genetic analysis of Giardia duodenalis isolates from children of low-income families living in an economically successful region in Southeastern Brazil.

Giardia duodenalis is one of the most important and widespread gastrointestinal parasites in the world. Despite its relevance as a causative agent of diarrhea, asymptomatic giardiasis occurs frequently, especially in low resources settings in which children are exposed to many risk factors. Based on microscopic examination and the polymerase chain reaction (PCR) amplification and sequencing of beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes, we assessed G. duodenalis occurrence and genetic diversity in isolates of children attending a daycare center and living in low income families, in an economically successful region. Considering both, microscopic examination and PCR/sequencing methods, the overall prevalence of Giardia infection was 51.4%, with the highest frequency in children aged 1-4 years old (p<0.05). Genotyping of 50 isolates revealed that the assemblage A was found in 60% of the samples (30/50), followed by the assemblage B in 38% (19/50) and 2% of mixed-assemblage infections (1/50). At the sub-assemblage level, isolates genotyped as A were AII and among isolates B, BIII and BIV were identified. Both assemblages A and B were detected in children of all age groups, however assemblage A was more prevalent. The detection of anthroponotic assemblages and sub-assemblages (AII, BIII and BIV) reinforces human-to-human transmission, mainly in children of all age groups when they have not yet received toilet training, making them more vulnerable to infection.

Diversity of Blastocystis subtypes in wild mammals from a zoo and two conservation units in southeastern Brazil.

The enteric protist Blastocystis is one of the most commonly parasite reported in humans and a variety of animal hosts worldwide. Regarding genetic diversity, at least 17 subtypes (STs) have been identified in mammals and birds, with eight of them (ST1-8) infecting both humans and animals. Recently, isolates from wild mammalian species have been genetically characterized, however data is still scarce, mainly in Latin America. Here, we aimed to verify the occurrence and genetic diversity of Blastocystis in captive wild mammals kept in one zoo and in two units of protection and conservation in southeastern Brazil. A total of 78 fecal samples (14 pooled and 64 individual samples) were recovered from 102 wild mammals of 35 species included in the following orders: Primates, Carnivora, Artiodactyla, Pilosa, Rodentia and Marsupialia. Zoo and units staff were invited to participated but only 16 fecal samples could be screened. Based on the sequence analyses of SSUrDNA gene, out of 29 PCR products from animal samples, 51.7% (15/29) were successfully sequenced and five Blastocystis subtypes were identified as follows: ST1 (2/15; 13.3%), ST2 (2/15; 13.3%), ST3 (4/15; 26.6%), ST5 (2/15; 13.3%) and ST8 (5/14; 33.3%). Only four isolates from humans were sequenced and identified as ST1 (2 isolates), ST2 and ST3. It was observed that Blastocystis infecting non-human primates belong to ST1 and ST2 and mainly to ST3 and ST8, artiodactyls ST5, carnivores ST1 and ST5 and rodents ST1. In addition, this present study reports some interesting findings: (1) 63% (12/19) of Blastocystis isolates from animals and employees belonged to the potentially zoonotic subtypes ST1-ST3; (2) most of these isolates displayed high identity with publicly available DNA sequences from non-human primates and humans, including primate handlers; (3) Blastocystis ST5 was found infecting the northern tiger cat, a native South American felid and one of the species facing a high risk of extinction in Brazil.

Blastocystis genetic diversity among children of low-income daycare center in Southeastern Brazil.

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.