Blastocystis is a protist of controversial pathogenicity inhabiting the gut of humans and other animals. Despite a century of intense study, understanding of the epidemiology of Blastocystis remains fragmentary. Here, we aimed to explore its prevalence, stability of colonisation and association with various factors in a rural elementary school in northern Thailand. One hundred and forty faecal samples were collected from 104 children at two time points (tp) 105 days apart. For tp2, samples were also obtained from 15 animals residing on campus and seven water locations. Prevalence in children was 67% at tp1 and 89% at tp2, 63% in chickens, 86% in pigs, and 57% in water. Ten STs were identified, two of which were shared between humans and animals, one between animals and water, and three between humans and water. Eighteen children (out of 36) carried the same ST over both time points, indicating stable colonisation. Presence of Blastocystis (or ST) was not associated with body mass index, ethnicity, birth delivery mode, or milk source as an infant. This study advances understanding of Blastocystis prevalence in an understudied age group, the role of the environment in transmission, and the ability of specific STs to stably colonise children.

Parasites have the power to impose significant regulatory pressures on host populations, making evolutionary patterns of host switching by parasites salient to a range of contemporary ecological issues. However, relatively little is known about the colonization of new hosts by parasitic, commensal and mutualistic eukaryotes of metazoans. As ubiquitous symbionts of coelomate animals, Blastocystis spp. represent excellent candidate organisms for the study of evolutionary patterns of host switching by protists. Here, we apply a big-data phylogenetic approach using archival sequence data to assess the relative roles of several host-associated traits in shaping the evolutionary history of the Blastocystis species-complex within an ecological framework. Patterns of host usage were principally determined by geographic location and shared environments of hosts, suggesting that weight of exposure (i.e. propagule pressure) represents the primary force for colonization of new hosts within the Blastocystis species-complex. While Blastocystis lineages showed a propensity to recolonize the same host taxa, these taxa were often evolutionarily unrelated, suggesting that historical contingency and retention of previous adaptions by the parasite were more important to host switching than host phylogeny. Ultimately, our findings highlight the ability of ecological theory (i.e. ‘ecological fitting’) to explain host switching and host specificity within the Blastocystis species-complex.

This epidemiological study assesses the occurrence of enteric parasites in 4303 patients attended at two public hospitals in Ankara (Turkey) during 2018–2019. Microscopy was used as a screening test. Giardia duodenalis was also identified using a commercial ELISA for the detection of parasite-specific coproantigens. Giardia-positive samples by microscopy/ELISA were confirmed by real-time PCR and characterized using a multilocus genotyping scheme. Blastocystis sp. was genotyped in a sample subset. Blastocystis sp. (11.1%, 95% CI 11.4‒14.8%) and G. duodenalis (1.56%, 95% CI 1.22‒1.96) were the most prevalent pathogens found. Cryptosporidium spp., Entamoeba histolytica and intestinal helminths were only sporadically (<0.5%) found. For G. duodenalis, sequence (n = 30) analyses revealed the presence of sub-assemblages AII (23.3%), discordant AII/AIII (23.3%) and mixed AII + AIII (6.7%) within assemblage A, and BIII (10.0%), BIV (3.3%) and discordant BIII/BIV (23.3%) within assemblage B. Two additional sequences (6.7%) were assigned to the latter assemblage but sub-assemblage information was unknown. No associations between G. duodenalis assemblages/sub-assemblages and sociodemographic and clinical variables could be demonstrated. For Blastocystis sp., sequence (n = 6) analyses identified subtypes ST1, ST2 and ST3 at equal proportions. This is the first molecular characterization of G. duodenalis based on MLG conducted in Turkey to date.

Blastocystis spp. is the most frequent infectious unicellular, luminal parasite in all species of animals and humans. It has been linked to diarrhoea and irritable bowel syndrome. Saccharomyces boulardii (Sb) is a widely used probiotic that previously showed efficacy against several intestinal pathogens. The aim of this study was to investigate the therapeutic role of Sb on Blastocystis spp. Methods: Five groups of Blastocystis subtype-3 infected rats were treated with either live Sb alone, metronidazole (MTZ) alone, Sb extract, both Sb and MTZ, or placebo-treated besides the noninfected control group. Assessment of treatment effectiveness was done by study of parasitological cure rate, histopathological effect and analysis of the colonic mucosal level of mRNAs expressions for the proinflammatory cytokines interleukin-6 (IL-6), IL-8, tumour necrosis factor alpha (TNF-α) and Inducible nitric oxide synthase (iNOS) by real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). Results showed that live Sb significantly improved the histological characteristics and decreased the cytokines and iNOS in the colonic mucosa. Co-administration of live Sb together with MTZ gave a better effect than other treatments and had early efficacy and revealed a 100% reduction of the parasite stages from both the stool and intestinal wash fluid.

Research into the gut microbiota of human infants is necessary in order to better understand how inter-species interactions and ecological succession shape the diversity of the gut microbiota, and in turn, how the specific composition of the gut microbiota impacts on host health both during infancy and in later years. Blastocystis is a ubiquitous intestinal protist that has been linked to a number of intestinal and extra-intestinal diseases. However, emerging data show that asymptomatic carriage is common and that Blastocystis is prevalent in the healthy adult gut microbiota. Nonetheless, little is known about the prevalence and diversity of this microorganism in the healthy infant gut, including when and how individuals become colonized by Blastocystis. Here, we surveyed the prevalence and diversity of Blastocystis in an infant population (n = 59) from an industrialized country (Ireland) using Blastocystis-specific primers at three or more time-points up to 24 months old. Only three infants were positive for Blastocystis (prevalence = 5%) and this was only noted for samples collected at month 24. This rate is comparatively low relative to previously reported prevalence rates in the contemporaneous adult population. These data suggest that infants in Westernized countries that are successfully colonized by Blastocystis most likely acquire this microorganism via horizontal transfer.

Blastocystis is an anaerobic protist, commonly inhabiting the intestinal tract of both humans and other animals. Blastocystis is extremely diverse comprising 17 genetically distinct subtypes in mammals and birds. Pathogenicity of this enteric microbe is currently disputed and knowledge regarding its distribution, diversity and zoonotic potential is fragmentary. Most research has focused on Blastocystis from primates, while sampling from other animals remains limited. Herein, we investigated the prevalence and distribution of Blastocystis in animals held within a conservation park in South East England. A total of 118 samples were collected from 27 vertebrate species. The barcoding region of the small-subunit ribosomal RNA was used for molecular identification and subtyping. Forty one per cent of the species were sequence positive for Blastocystis indicating a high prevalence and wide distribution among the animals in the park. Six subtypes were identified, one of which is potentially novel. Moreover, the majority of animals were asymptomatic carriers, suggesting that Blastocystis is not pathogenic in animals. This study provides a thorough investigation of Blastocystis prevalence within a wildlife park in the UK and can be used as a platform for further investigations on the distribution of other eukaryotic gut microbes.

Blastocystis sp. is a protozoan commonly found in human and animal stool samples. Several pathogenic and zoonotic aspects of this organism are still unknown. The aim of the present study was to investigate Blastocystis subtypes (STs) in samples from patients of the Hospital das Clínicas of the Faculdade de Medicina at the Universidade de São Paulo (HC-FMUSP), Brazil. Blastocystis sp.-positive stool samples diagnosed at the Section of Parasitology of the Central Laboratory (HC-FMUSP) were used for DNA isolation. Polymerase chain reaction (PCR) was performed using specific primers targeting the small-subunit rRNA gene. Direct DNA sequencing of the PCR products was performed and the DNA sequences were then aligned and compared with other sequences obtained from the GenBank database. Phylogenetic analysis was used to identify STs and determine the phylogenetic relationships between the sequences. Four STs were identified: ST1 (22·5%), ST2 (12·5%), ST3 (60%) and ST6 (5%). In conclusion, ST3 was the most prevalent ST among the human isolates followed by ST1. The present study is one of the few providing STs data from the human population in South America. Determining ST prevalence in human samples may contribute to the monitoring of Blastocystis sp. infection transmission in endemic regions.

Blastocystis spp. pathogenic potential remains unclear as these anaerobic parasitic protozoa are frequently isolated from stools of both symptomatic and asymptomatic subjects. In silico analysis of the whole genome sequence of Blastocystis subtype 7 revealed the presence of numerous proteolytic enzymes including cysteine proteases predicted to be secreted. To assess the potential impact of proteases on intestinal cells and gut function, we focused our study on two cysteine proteases, a legumain and a cathepsin B, which were previously identified in Blastocystis subtype 7 culture supernatants. Both cysteine proteases were produced as active recombinant proteins. Activation of the recombinant legumain was shown to be autocatalytic and triggered by acidic pH, whereas proteolytic activity of the recombinant cathepsin B was only recorded after co-incubation with the legumain. We then measured the diffusion of 4-kDa FITC-labelled dextran across Caco-2 cell monolayers following exposition to either Blastocystis culture supernatants or each recombinant protease. Both Blastocystis culture supernatants and recombinant activated cathepsin B induced an increase of Caco-2 cell monolayer permeability, and this effect was significantly inhibited by E-64, a specific cysteine protease inhibitor. Our results suggest that cathepsin B might play a role in pathogenesis of Blastocystis by increasing intestinal cell permeability.

Blastocystis spp. are common anaerobic intestinal protozoa found in both human and animals. They are characterized by a high genetic diversity with at least 17 subtypes (STs) that have been described on the basis of a 600 bp ‘barcoding region’ from the 18S rDNA gene. However, analysis of the recently sequenced genome of a Blastocystis ST7 isolate (strain B) revealed the presence of multiple variable copies of the 18S rDNA gene, with 17 completely assembled copies. Comparison of the barcoding region from these 17 copies allowed us to classify the 18S rDNA sequences into 6 clusters, each cluster containing identical sequences. Surprisingly, 4 of these clusters had the highest homology with 18S rDNA sequences from 2 other Blastocystis ST7 isolates referred as QQ98-4 and H. These results suggest that the 18S rDNA gene is not the marker of choice to discriminate between strains within STs. In the present study, we identified a single-copy subtyping rDNA marker in the genome of the mitochondria-like organelles (MLOs). Using a partial sequence of the MLO rDNA, we successfully subtyped 66 isolates from both human and animals belonging to Blastocystis ST1 to ST10. Our results also indicate that this mitochondrial marker could be useful to detect co-infections by different isolates of a same ST.

Blastocystis SSU-rDNA sequence data from 317 captive and free-living non-human primates (NHPs) representing 30 genera of apes, Old and New World (OW and NW) monkeys and prosimians were analysed to investigate subtype (ST) and allele distribution among hosts. Excluding 20 mixed ST infections, 27% of the sequences belonged to ST1, 22% to ST2, 34% to ST3, 1% to ST4, 4% to ST5, 11% to ST8, <1% to ST13 and 1% to ST15. The study confirmed cryptic host specificity of ST1 and ST3; conversely, considerable overlap in ST2 alleles exists among humans and NHPs. Subtype distribution in humans and NHPs differs mainly in that ST4 is rarely reported in NHPs while ST5 and ST8 are both unusual in humans. This may be due to host specificity and/or the apparent geographically restricted range of some subtypes. While the distribution of ST1, ST2 and ST3 was independent of NHP group or geographical association, ST5 was seen only in apes and OW monkeys and ST8 primarily in arboreal NHPs and only in species native to Asia or South America.

Blastocystis infection has a worldwide distribution especially among the disadvantaged population and immunocompromised subjects. This study was carried out to determine the prevalence and the association of Blastocystis infection with the socio-economic characteristics among 300 primary schoolchildren, living in rural communities in Lipis and Raub districts of Pahang state, Malaysia. Stool samples were collected and examined for the presence of Blastocystis using direct smear microscopy after in vitro cultivation in Jones' medium. The overall prevalence of Blastocystis infection was found to be as high as 25·7%. The prevalence was significantly higher among children with gastrointestinal symptoms as compared to asymptomatic children (x2=4·246; P=0·039). Univariate and multivariate analyses showed that absence of a piped water supply (OR=3·13; 95% CI=1·78, 5·46; P<0·001) and low levels of mothers’ education (OR=3·41; 95% CI=1·62, 7·18; P<0·01) were the significant predictors of Blastocystis infection. In conclusion, Blastocystis is prevalent among rural children and the important factors that determine the infection were the sources of drinking water and mothers' educational level. Interventions with provision of clean water supply and health education especially to mothers are required.

Parasitic diseases are endemic in parts of the tropics, but there is no convincing evidence that their prevalence or incidence is increasing due to the HIV epidemic. Available scientific data on parasitic infections in patients with the Acquired Immunodeficiency Syndrome (AIDS) suggests a predominance of Pneumocystis carinii, Toxoplasma gondii and Cryptosporidium spp. For reasons which are unclear, parasitic infections such as Plasmodium falciparum, Strongyloides stercoralis and Entamoeba histolytica, where cell-mediated immune responses are also thought to be significant, do not appear to be opportunists of importance. It is being increasingly recognized that chemotherapy for parasitic diseases has a host-dependent component, although scientific data on this subject remain scanty. The management of opportunistic parasitic infections in patients infected with HIV is dogged by failures and relapses, aptly illustrating the notion of the relationship between chemotherapy and the immune response. This review discusses the immunity and chemotherapy of opportunistic parasite infections in patients infected with the Human Immunodeficiency Virus (HIV).

In vitro propagation followed by PCR, and a PCR-based method capable of the direct detection of Blastocystis in faeces were utilized to detect Blastocystis from various hosts in Australia, including primates and their handlers from the Perth Zoo. In addition, Blastocystis isolates from dogs and humans living in a localized endemic community in Thailand were also characterized genetically. PCR-based detection directly from faeces was shown to be more sensitive compared with in vitro culture for the detection of Blastocystis. Moreover, phylogenetic analysis of Blastocystis isolates amplified utilizing in vitro techniques prior to PCR revealed that this method favoured the preferential amplification of Blastocystis subtype 5 over subtype 1. This study is the first to provide molecular-based evidence supporting the zoonotic potential of Blastocystis in dogs, possums and primates in a natural setting.

Clonal cultures of Histomonas meleagridis, Tetratrichomonas gallinarum and a Blastocystis sp. were established for the first time. Single microbes were successfully isolated from a mixture of micro-organisms obtained from caecal contents of turkeys, using a micromanipulation approach. The cloned parasites were propagated in vitro and maintained through continuous passages multiplying to high numbers. Identification of the protists was done by morphological investigation identifying various forms of each parasite. PCR and partial sequencing of the small subunit rRNA were used to confirm clonality and to determine the relationship of the cloned parasites with known protozoan parasites. The clonal cultures established by this technique will be useful to gain more insight into the biological repertoire of the organisms. In addition, refined infection experiments in different poultry species can now be performed to elucidate the pathological pathways of the respective protozoa.

Blastocystis is a common single-celled enteric parasite found in a large variety of hosts. Recent molecular analysis supports the concept that this eukaryotic organism is a stramenopile most closely related to Proteromonas lacertae, a parasite of reptiles. In this study, the internal transcribed spacer region, partial small subunit rRNA and large subunit rRNA genes from 7 Blastocystis isolates (5 human, 1 pig and 1 sheep), and a Proteromonas lacertae isolate were amplified by PCR, cloned and sequenced. Blastocystis was found to be a typical eukaryote with both ITS1 and ITS2 regions present. Phylogenetic analysis based on the entire PCR amplicon revealed that the Blastocystis isolates did not segregate according to host or geographic origin. The highest sequence identities with the conserved Blastocystis 5·8S rDNA sequence were with the stramenopiles Fibrocapsa japonica, Chattonella marina, Cylindrotheca closterium and Hyphochytrium catenoides. The most parsimonious tree based on the 5·8S rDNA sequence from P. lacertae, 11 other stramenopiles, 2 fungi, 3 algae and 3 alveolates showed Blastocystis positioned within the stramenopiles, with P. lacertae as its closest relative. This work therefore supports the hypothesis that Blastocystis is most closely related to P. lacertae, and that it should be regarded as an unusual stramenopile.

Genes encoding small subunit ribosomal RNA (SSUrRNA) of 16 Blastocystis isolates from humans and other animals were amplified by the polymerase chain reaction, and the corresponding fragments were cloned and sequenced. Alignment of these sequences with the previously reported ones indicated the presence of 7 different sequence patterns in the highly variable regions of the small subunit ribosomal RNA. Phylogenetic reconstruction analysis using Proteromonas lacertae as the outgroup clearly demonstrated that the 7 groups with the different sequence patterns are separated to form independent clades, 5 of which consisted of the Blastocystis isolates from both humans (B. hominis) and other animals. The presence of 3 higher order clades was also clearly supported in the phylogenetic tree. However, a relationship among the 4 groups including these 3 higher order clades was not settled with statistical confidence. The remarkable heterogeneity of small subunit ribosomal RNAs among different Blastocystis isolates found in this study confirmed, with sequence-based evidence, that these organisms are genetically highly divergent in spite of their morphological identity. The highly variable small subunit ribosomal RNA regions among the distinct groups will provide useful information for the development of group-specific diagnostic primers.

Although programmed cell death (PCD) has been associated with multicellular organisms, there have been more reports of its presence in some protozoans. Our study shows the existence of PCD in an intestinal protozoan, Blastocystis hominis. Light and electron microscopy, biochemical and flow cytometry studies showed apoptosis-like death in B. hominis cells exposed to a cytotoxic monoclonal antibody (MAb 1D5). B. hominis cells displayed key morphological and biochemical features of apoptosis, namely, nuclear condensation and in situ fragmentation, reduced cytoplasmic volume, some externalization of phosphatidylserine and maintenance of plasma membrane integrity. No oligonucleosomal DNA laddering was observed in gel electrophoresis. This study supports earlier observations that the cellular machinery that is required to carry out PCD may have existed before the advent of multicellularity. Our study also ascribes a novel function for the B. hominis central vacuole in apoptosis; it acts as a repository where apoptotic bodies are stored before being released into the extracellular space.

The elongation factor-1 alpha (EF-1α) is a highly conserved ubiquitous protein that is involved in translation and is desirable for use in phylogenetic studies on Blastocystis, an enigmatic intestinal parasite with a contentious taxonomic position. In the present study, a PCR product (BEα) that codes for a major part of the coding region of the EF-1α protein was amplified. Genome walking experiments together with cloning were implemented to elucidate the 5′ and 3′ ends of the EF-1α gene of the human isolate, Blastocystis hominis C. The genomic organization and the potential transcription factor binding sites of the 5′ end of B. hominis C EF-1α were identified. A comparative study on the deduced amino acid sequences of BEα of 13 Blastocystis isolates from various hosts was done to evaluate the phylogenetic relationship among the speciesThe nucleotide sequence data reported in this paper are available in the GenBank database with the accession numbers as shown in Table 1.. A phylogenetic reconstruction analysis with other eukaryotic EF-1α sequences was carried out to trace the phylogenetic position of Blastocystis among eukaryotic organisms.