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Abstract Objective This study aimed to investigate the prevalence and subtype distribution of the genus Blastocystis using high-resolution melting curve analysis (HRM), a novel molecular technique. Blastocystis sp., a unicellular, anaerobic, and zoonotic parasite, is commonly found in the digestive tracts of humans and animals. Transmission occurs via the fecal-oral route, with prevalence rates ranging from 20% in developed countries to over 60% in developing regions. Although the pathogenicity of Blastocystis remains debated, it has been detected in both symptomatic and asymptomatic individuals. Traditionally, direct microscopy with wet mounts is used for the rapid identification of Blastocystis in stool samples. However, molecular diagnostics have become essential for the accurate detection and differentiation of Blastocystis subtypes Materials and methods We collected 730 stool samples from humans and domestic animals. Initial screening for Blastocystis sp. was performed using direct microscopy. Negative samples were cultured in a two-phase culture medium and re-examined after 2–3 days. HRM analysis was then employed to identify and differentiate Blastocystis subtypes using specific primers via real-time PCR. Results Six subtypes were identified, with ST7 (30%) being the most prevalent, followed by ST3 (28%), ST2 (16%), ST1 (14%), ST5 (6%), and ST14 (6%). In humans, ST3 was the most common subtype, also found in poultry and sheep, whereas ST7 was mainly detected in domestic animals. Notably, ST1-ST3 were identified in domesticated animals, indicating a pattern of cross-species transmission. Conclusion ST3 (28%) and ST7 (30%) were the most prevalent subtypes across all samples, with distinct distributions in human and animal hosts. The HRM technique demonstrated efficiency and cost-effectiveness, providing a rapid and accurate method for Blastocystis subtype identification in developing countries, which can expedite diagnostic responses and reduce the need for sequencing. This supports the potential for HRM to improve epidemiological surveillance and understanding of cross-species transmission.

ObjectiveIrritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) are gastrointestinal disorders, which can be triggered by gut microbiota dysbiosis. The development of IBS-like symptoms has been linked to the overgrowth of Candida spp. In addition, the critical role of fungi has been highlighted in the pathogenesis of IBD. This study investigated the association between Blastocystis and selected yeasts in IBS and IBD patients.MethodsThis investigation is a cross-sectional study from 2022 to 2024, performed on 91 participants, including 20 healthy individuals, 27 patients with IBS, and 44 IBD patients [39 with ulcerative colitis (UC; 88.63%) and 5 (11.37%) Crohn’s disease (CD)], who were also categorized based on the presence of Blastocystis. Total DNA was extracted from stool samples, and the presence and quantity of yeasts including C. albicans, C. tropicalis, C. glabrata, C. parapsilosis, C. krusei, Geotrichum candidum, Rhodotorula spp., Cryptococcus neoformans, and Saccharomyces cerevisiae were evaluated by real-time PCR. Statistical tests were used to assess significant associations between variables.ResultsSaccharomyces cerevisiae and C. albicans were the most prevalent yeasts in all groups. Candida tropicalis and C. neoformans were identified in neither patients nor healthy subjects. The presence/absence of C. albicans was not significantly different between patients with IBD, IBS, and the control groups. This was similar for G. candidum. However, there was a difference in the presence of S. cerevisiae among patients, although it was insignificant (p-value = 0.077). There was a significant difference in the quantity of C. albicans between IBD (880.421 ± 2140.504), IBS (10.307 ± 15.206), and controls (2875.888 ± 8383.889) (p-value = 0.020). Specifically, the source of difference was seen between IBD patients and the control group (p-value = 0.005). In addition, considering the presence of Blastocystis, a statistically significant association was seen between the number of C. albicans and the sample groups (p-value = 0.013). The quantity of C. albicans was significantly different between IBS and IBD patients.ConclusionRegarding the presence of Blastocystis, the quantity of C. albicans and S. cerevisiae was increased and decreased in the studied groups, respectively. This is a preliminary study, and eukaryote–eukaryote association in IBS and IBD patients should be considered in further studies.

Background: General conditions in a prison may facilitate water- or food-borne infections. Methods: Detection of intestinal parasites was achieved in 471 male prison inmates by standard microscopic procedures on their stool samples. Positive samples were processed by PCR amplification of a 600-bp fragment of the Blastocystis SSU rRNA gene and partial sequences of the Giardia duodenalis bg genes. Identification of subtypes/genotypes was based on Sanger sequencing methods. Results: Blastocystis was found in 7.9 % (37/471) and G. duodenalis was found in 2.1 % (10/471). Out of the 37 Blastocystis positive samples, 54 % (20/37) were successfully subtyped, allowing the identification of the subtypes ST3 (50 %), ST1 (25 %), ST2 (15 %), ST4 (5 %) and ST6 (5 %). Out of 10 G. duodenalis positive samples, 50 % (5/10) were successfully genotyped, allowing the identification of genotypes A (80 %) and B (20 %). Conclusions: The predominance of ST3 within the prison inmates, together with its low intra-ST genetic variability, reflected inter-human transmission with spatial stability. The G. duodenalis distribution is not wide enough to consider the possibility of a generalized transmission via contaminated water or food. Personal hygiene practices among male prison inmates may be an important measure to prevent the transmission.

While the stramenopile Blastocystis, first discovered in 1911, is considered the most prevalent enteric protist in humans, its biology remains largely unexplored. Clinical studies have only recently begun investigating the role of Blastocystis in the gut and its relationship with the gut microbiome, and whether it plays a pathogenic role in human and animal health. Aiming to gather leading researchers in the field to encourage and stimulate cross-disciplinary dialogue while fostering long-term international collaborations, the Fourth International Blastocystis Conference was hosted from the 17th to the 19th of September 2024 in Heraklion (Crete, Greece). The event was mainly supported by the COST Action CA21105, “Blastocystis under One Health”, and the Microbiology Society. The multi- and interdisciplinary conference programme covered all aspects related to Blastocystis evolutionary biology and advances in omics, intestinal ecology (gut microbiome), clinical significance and association with disease, diagnosis and molecular characterisation, as well as epidemiology and One Health. The high-quality presentations discussed at the conference provided researchers with a synthesis of recent advancements, while key research questions, knowledge gaps, and future steps in Blastocystis research were identified. Herein, we aim to provide a thorough overview of the presentations at the congress. The COST Action CA21105, 'Blastocystis under One Health,' will build on the insights and collaborations fostered during the conference, promoting integrative research approaches, advancing our understanding of Blastocystis, and driving future efforts to translate these findings into improved public health strategies.

Intestinal protists in the gut microbiome are increasingly studied, but their basic epidemiology is not well understood. We explored the prevalence, genetic diversity, and potential zoonotic transmission of two protists colonizing the large intestine - Blastocystis sp. and Dientamoeba fragilis - in 37 species of non-human primates (NHPs) and their caregivers in six zoos in the Czech Republic. We analyzed 179 fecal samples (159 from NHPs, 20 from humans) by qPCR. Blastocystis sp. was detected in 54.7% (98/179) of samples, in 24 NHP species and in 57.2% of NHP samples (prevalence ranged between 36 and 80%), and in 35% of human samples (prevalence ranged between 0 and 67%). Using next generation amplicon sequencing, nine Blastocystis subtypes (ST1-ST5, ST7, ST8, and two novel subtypes) were identified. The two new Blastocystis subtypes (named ST47 and ST48) were described using Nanopore sequencing to produce full-length reference sequences of the small subunit ribosomal RNA gene. Some subtypes were shared between NHPs and their caregivers, suggesting potential zoonotic transmission. Mixed subtype colonization was frequently observed, with 52% of sequenced samples containing two or more subtypes. Dientamoeba was found only in NHPs with a prevalence of 6%. This study emphasizes the critical role of molecular diagnostics in epidemiological and transmission studies of these protists and calls for further research to better understand their impact on public health.

Abstract Background Blastocystis, a widely distributed zoonotic protozoan infecting both humans and numerous animals, remains poorly understood with its potential medical and veterinary significance. This study examined the molecular occurrence and genetic variation of Blastocystis in children and calves in Bangladesh to explore cross-species transmission and disease burden. Methods In total, 998 DNA samples were investigated, comprising 299 stool DNA from children and 699 fecal DNA from calves, using polymerase chain reaction and sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. Results This study detected Blastocystis in 5.35% of the children and 14.74% of the calves. While slight variations in occurrence rates were observed across different study variables, none were statistically significant. The occurrence was highest among children under 5 years and calves aged 1–3 months. Regarding breed, the Holstein Friesian cross and the Jersey cross exhibited higher rates of infection. Conversely, occurrences were lower among children and calves in Gazipur district. The remaining parameters displayed nearly equivalent percentages of Blastocystis. The subtypes identified in children included ST1, ST3, and ST4, with ST1 comprising 50% of them. ST3 and ST4 were also found in calves, alongside ST10 (55.34%) being the most prevalent. Other subtypes found in calves were ST14, ST21, and ST24–ST26. Conclusions This study found that Blastocystis is more common in calves than in children in Bangladesh, with genetic diversity of nine subtypes. The common occurrence of identical variants of two subtypes in both populations suggests potential zoonotic transmission, highlighting the necessity for further molecular investigations and comprehensive measures within the One Health framework to mitigate public health risks.

Abstract Blastocystis is the most common gastrointestinal protozoan parasite of humans and many vertebrates. This study was carried out to investigate the prevalence and determination subtype (ST) of Blastocystis in domestic ruminants of Shahrekord County, southwestern Iran. In this descriptive cross-sectional study, 330 ruminant fecal samples (107 cows, 115 sheep, and 108 goats) were evaluated by parasitological methods (direct wet mount microscopic examination and formalin-ether concentration), Giemsa staining, In vitro xenic culture (The modified Dobell and Laidlaw culture method), polymerase chain reaction, and sequencing from 2018 to 2019, then data were analyzed using SPSS software version 21. The overall Blastocystis positive in ruminants was 14.2% and the frequency of Blastocystis sp. in cattle, sheep, and goats were 0.93%, 17.4%, and 24.1% respectively. Molecular diagnosis techniques revealed that ruminants were infected with four STs (genotypes) of Blastocystis including ST5(21.3%), ST7(2.1%), ST10(17.1%) and ST14(57.4%). Also, the STs identified in cows were ST10, and the observed STs in sheep were ST5 (40%), ST7 (3%), ST10 (5%), ST14 (45%), and one unknown subspecies. Goats were infected by ST5 (7.7%), ST10 (23.1%), and ST14 (69.2%). In this study, ST14 was identified as the most common subtype of Blastocystis sp. that was not common between humans and livestock, meanwhile, ST5 and ST7 are common between humans and animals accounted 21.3% and 2.1% of the positive cases, respectively, and reinforces the hypothesis that ruminants are reservoirs of blastocystosis in humans.

Abstract Background Parasites Entamoeba spp., Enterocytozoon bieneusi and Blastocystis are prevalent pathogens causing gastrointestinal illnesses in animals and humans. Consequently, researches on their occurrence, distribution and hosts are crucial for the well-being of both animals and humans. Due to the confined spaces and frequent interaction between animals and humans, animal sanctuaries have emerged as potential reservoirs for these parasites. In this study, the wildlife sanctuary near the Huang Gorge of the Qinling Mountains in northwest China is chosen as an ideal site for parasite distribution research, considering its expansive stocking area and high biodiversity. Results We collected 191 fecal specimens from 37 distinct wildlife species and extracted genomic DNA. We identified these three parasites by amplifying specific gene regions and analyzed their characteristics and evolutionary relationships. All the parasites exhibited a high overall infection rate, reaching 90.05%. Among them, seven Entamoeba species were identified, accounting for a prevalence of 54.97%, with the highest infection observed in Entamoeba bovis. In total, 11 Enterocytozoon bieneusi genotypes were discovered, representing a prevalence of 35.08%, including three genotypes of human-pathogenic Group 1 and two novel genotypes (SXWZ and SXLG). Additionally, 13 Blastocystis subtypes were detected, showing a prevalence of 74.87% and encompassing eight zoonotic subtypes. All of the above suggests significant possibilities of parasite transmission between animals and humans. Conclusions This study investigated the occurrence and prevalence of three intestinal parasites, enhancing our understanding of their genetic diversity and host ranges in northwest China. Furthermore, the distribution of these parasites implies significant potential of zoonotic transmission, underscoring the imperative for ongoing surveillance and implementation of control measures. These efforts are essential to mitigate the risk of zoonotic disease outbreaks originating from wildlife sanctuary.

Objective: Recent studies determined that the amoeboid form of Blastocystis acts as a factor in stimulating the host’s immune responses and ultimately results in urticaria and other skin disorders. The present study was conducted in order to determine the prevalence of Blastocystis in people referred to Bushehr city health centers and the relationship of this parasite with urticaria. Methods: Fecal samples were collected from 180 males and females referred to Bushehr health centers and a questionnaire containing demographic information was completed for each person. Samples were examined by preparing direct smear (wet mount) and then formalin-detergent sedimentation techniques. Data were analyzed using SPSS 22.0 software and chi-square test. Results: The results showed that 11.1% of cases infected with Blastocystis and 55% of patients with Blastocystis had various gastrointestinal symptoms. Statistical analysis showed that there was no significant relationship between infection with some demographic factors such as sex, age, literacy level and residence, but this was significant with some clinical symptoms such as itching and urticaria. Conclusion: Despite the existence of conflicting information and many ambiguities about the Blastocystis, this emerging pathogen is very important in terms of causing allergic and skin disorders in sufferers, therefore, it is necessary that patients with urticaria be evaluated for Blastocystis along with other diagnostic procedures and physicians should request a test before any medical intervention. Thus, diagnosis and treatment of these people can play an important role in improving the health of society.

Los perros ofrecen múltiples beneficios en su relación con los humanos, pero también pueden ser portadores de parásitos zoonóticos que afectan la salud humana y animal. Las zoonosis representan alrededor del 58 % de todas las enfermedades infecciosas en los humanos. Se evaluó el parasitismo intestinal en perros con dueños en la ciudad de Barranquilla, Colombia, en los años 2016, 2017 y 2018. Se realizó un estudio descriptivo retrospectivo que incluyó 3,279 reportes de evaluación parasitológica de heces de perro. El 49.2 % de los perros presentó algún tipo de parásito intestinal. Los más frecuentes fueron los helmintos: Strongyloides sp. (9.6%), Toxocara canis (7.7 %) y Ancylostoma caninum (6.2 %); y los protozoarios Entamoeba spp. (10.0 %), Isospora spp. (6.9 %) y Giardia spp. (5.7 %). Un análisis de componentes principales de los perfiles parasitarios por año identificó diferencias significativas entre ellos. La presencia de parásitos intestinales de transmisión zoonótica en los perros domésticos analizados resalta la necesidad de establecer medidas correctivas y preventivas en el ámbito de la salud pública que permitan su control, ya que constituyen un riesgo significativo de enfermedad para los humanas y los perros.

Introduction. The multifactorial etiology of gastroenteritis emphasizes the need for different laboratory methods to identify or exclude infectious agents and evaluate the severity of diarrheal disease. Objective. To diagnose the infectious etiology in diarrheic children and to evaluate some fecal markers associated with intestinal integrity. Materials and methods. The study group comprised 45 children with diarrheal disease, tested for enteropathogens and malabsorption markers, and 76 children whose feces were used for fat evaluation by the traditional and acid steatocrit tests. Results. We observed acute diarrhea in 80% of the children and persistent diarrhea in 20%. Of the diarrheic individuals analyzed, 40% were positive for enteropathogens, with rotavirus (13.3%) and Giardia duodenalis (11.1%) the most frequently diagnosed. Among the infected patients, occult blood was more evident in those carrying pathogenic bacteria (40%) and enteroviruses (40%), while steatorrhea was observed in infections by the protozoa G. duodenalis (35.7%). Children with diarrhea excreted significantly more lipids in feces than non-diarrheic children, as determined by the traditional (p

Blastocystis is a common intestinal protist with a global distribution, frequently found in humans and various animals. Despite its prevalence, its role in human health remains debated, oscillating between being a harmless commensal and a potential pathogen. It has also been associated with gastrointestinal disorders such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). In Italy, the genetic and spatial diversity of Blastocystis remains understudied, despite the country’s diverse urbanized and environmental landscapes. This study investigates the haplotypic and spatial diversity of clinical isolates of Blastocystis across two different Italian regions, with an emphasis on subtype distribution and genetic variation. Using a network-based haplotype analysis, the study reveals a heterogeneous subtype distribution, with subtype ST4 (47.3%) being the most prevalent, followed by ST3 (20%), ST1 (16.4%), ST2 (12.7%), ST6 (1.8%) and ST7 (1.8%). The overall infection rate detected from symptomatic patients is 9.75%. Notably, ST4 shows limited haplotypic variation, suggesting a more stable population structure that is potentially linked to a human-adapted lineage. In contrast, ST1 and ST2 exhibit greater haplotypic diversity, likely due to ongoing zoonotic transmission. These findings enhance our understanding of the epidemiology of Blastocystis in Italy and underscore the need for further research on its pathogenic potential and transmission dynamics.

This issue of the Biomedical Journal highlights major advancements in drug delivery, including aptamer-functionalized liposomes and nanozymes. A new biomarker combination shows promise for improved diagnosis of idiopathic pulmonary fibrosis. Mesenchymal stem cells are suggested to mitigate inflammation in systemic lupus erythematosus, and a potential positive feedback loop driven by a prevalent mRNA modification is suggested to enhance NSCLC progression. Additional articles explore a pathological impact on autophagy leading to muscle dysfunction, the benefits of integrating an orphan drug with standard therapy for glioblastoma patients, and the influence of transcriptional super-enhancers in early-stage esophageal squamous cell carcinoma. Finally, this issue provides insights into the roles of different Blastocystis subtypes, and the use of laser light for treating infantile hemangioma.

IntroductionGlobally, rodents and shrew populations constitute crucial elements of diverse environments and animal communities. It is imperative to study their population dynamics to mitigate any potential negative impact on humans, as they can be involved in the transmission of critical zoonotic agents, such as Blastocystis. Therefore, this study aimed to identify the prevalence and genetic composition of Blastocystis in wild rodents and shrews residing in the Zhejiang provinces of China.MethodsA total of 652 wild rodents and and shrews were captured from three different regions in Zhejiang Province from April 1st to October 31, 2023. The DNA was isolated by collecting fresh feces from the intestines of each rodent or and shrew. Rodent and shrew species were examined by vertebrate cytochrome b (cytb) analysis and PCR amplification. Blastocystis was also found in all fecal samples using PCR analysis and sequencing of the partial small subunit of ribosomal RNA (SSU rRNA) gene.ResultsAmong all the samples, 6.6% (43/652) showed a positive result for Blastocystis. In the results, 6 species of rodent and shrew were identified with Blastocystis, including Apodemus agrarius (n = 36) (2.8%), Niviventer confucianus (n = 75) (17.3%), Rattus losea (n = 18) (5.6%), R. norvegicus (n = 155) (2.6%), R. tanezumi (n = 86) (3.5%), and Suncus murinus (n = 282) (7.4%). The existence of 6 Blastocystis subtypes, ST4 (n = 33), ST1 (4), ST7 (n = 3), ST2 (n = 1), ST3 (n = 1), and ST5 (n = 1), were confirmed by sequence analysis.DiscussionBased on the molecular data obtained, the wild rodents and shrews under investigation were found to be concurrently infected with zoonotic subtypes of Blastocystis, including ST1 to ST5 and ST7. This suggests that these animals could potentially pose a zoonotic threat to humans and other animals susceptible to Blastocystis infection.

IntroductionBlastocystis is one of the most critical intestinal protozoans in various hosts, including humans and mice. To determine the status of Blastocystis infection in wild rodents in China.MethodsA total of 344 faecal samples were collected from seven wild rodent species from three provinces, and the small subunit ribosomal RNA (SSU rRNA) genes of Blastocystis were amplified to determine their prevalence and subtypes.ResultsOf the 344 samples, 54 (15.70%) were detected as Blastocystis-positive. The prevalence of Blastocystis was 26.14% (40/153), 7.95% (7/88), and 6.80% (7/103) in wild rodents from Hunan Province, Yunnan Province, and Guangxi Province, respectively. The prevalence of Blastocystis in different wild rodent species varied from 0.00% (0/13) in Mus musculus to 40.00% (2/5) in Rattus rattus sladeni. The prevalence of Blastocystis in samples from the lake beach area (27.40%, 40/146) was significantly higher than in those from the mountain (6.80%, 7/103) and field regions (7.37%, 7/95). The prevalence in different seasons was 26.14% in summer (40/153), 7.95% in autumn (7/88), and 6.80% in winter (7/103). Moreover, a total of two Blastocystis subtypes were identified in the investigated wild rodents, including ST4 and ST5.DiscussionThe present study discovered the existence of Blastocystis infection in Rattus favipectus, Microtus fortis, Apodemus agrarius, Bandicota indica, Rattus rattus sladeni, and Rattus losea, expanding the host range of this parasite. The findings also demonstrate that wild rodents may be an important potential infection source for Blastocystis infection in humans and other animals.

Blastocystis is the most prevalent intestinal eukaryotic microorganism with significant impacts on both human and animal health. Despite extensive research, its pathogenicity remains controversial. The COST Action CA21105, "Blastocystis under One Health" (OneHealthBlastocystis), aims to bridge gaps in our understanding by fostering a multidisciplinary network. This initiative focuses on developing standardised diagnostic methodologies, establishing a comprehensive subtype and microbiome databank, and promoting capacity building through education and collaboration. The Action is structured into five working groups, each targeting specific aspects of Blastocystis research, including epidemiology, diagnostics, 'omics technologies, in vivo and in vitro investigations, and data dissemination. By integrating advances across medical, veterinary, public, and environmental health, this initiative seeks to harmonise diagnostics, improve public health policies, and foster innovative research, ultimately enhancing our understanding of Blastocystis and its role in health and disease. This collaborative effort is expected to lead to significant advancements and practical applications, benefiting the scientific community and public health.

Blastocystis is a parasitic protist that can infect humans and various domestic and wild animals. However, there is limited research on the prevalence of this parasite among rodents, particularly those living in pig farm settings. Therefore, to investigate the occurrence, molecular characterization, and zoonotic potential of Blastocystis among rodents within pig farm environments, we conducted an investigation of 227 rodents and shrews from 34 pig farms located in Henan, Shaanxi, and Shanxi provinces of China using nested PCR of the SSU rRNA gene of Blastocystis. The potential transmission and public health implications were also assessed from a One Health perspective. Blastocystis was detected in 86 (37.9%) fecal samples. The highest infection rate was observed among Ruttus norvegicus (73.7%, 42/58), followed by Ruttus tanezumi (30.1%, 41/136), and Mus musculus (12.0%, 3/25). However, it was not detected among individuals with Apodemus agrarius (n = 1) and Crocidura shantungensis (n = 7). Five known zoonotic Blastocystis subtypes (ST1–ST5) were identified, with ST4 (51.2%, 44/86) and ST5 (40.7%, 35/86) being the predominant ones, followed by ST1 (3.5%, 3/86), ST3 (3.5%, 3/86), and ST2 (1.2%, 1/86). ST4 was prevalent among R. norvegicus (83.3%, 35/42), while ST5 dominated R. tanezumi (70.7%, 29/41). Furthermore, ST5 exhibited the widest distribution at pig farm level, accounting for 65.0% (13/20) of Blastocystis-positive pig farms. This investigation presents the first documented Blastocystis infection in R. tanezumi and M. musculus, highlighting the predominant presence of the zoonotic ST5 subtype in rodents for the first time. The results demonstrate that sympatric rodents can serve as natural reservoirs for Blastocystis and play a role in its transmission. These findings provide information on the dynamics of rodent transmission and emphasize the potential public health threat posed by zoonotic Blastocystis subtypes spillover from pig farms.

Non-human primates (NHPs) host a variety of helminth and protist parasites that are able to cause infection in humans. Gastrointestinal parasites in NHPs living in two zoological gardens of Northern Italy were studied. An total of 96 faecal pools were collected from 26 groups of NHPs. The mini-Flotac method was applied to fecal samples to detect gastrointestinal helminthiases, while the detection of the protists Cryptosporidium spp., Blastocystis sp. and Giardia duodenalis was performed by targeting SSU rRNA through nested PCR and real-time PCR; they were further studied by sequencing the same gene for Blastocystis and βgiardine and triosephosphate isomerase (TPI) genes for Giardia. Twenty-two out of the 96 examined fecal pools (22.9%) were positive for one or more helminth species, including Hymenolepis diminuta, Trichurid, Capillariid and Strongylid eggs. All samples were negative for Cryptosporidium spp., while 16/26 (61.5%) animals were positive for G. duodenalis in the real-time PCR; the sequences obtained assigned them all to sub-assemblage BIV. Blastocystis sp. was detected in 22/26 of the NHPs (84.6%); molecular analyses attributed the isolates to ST 4, allele 92. Analyses of the feces of sympatric rats revealed the presence of the same allele, as well as of Hymenolepis diminuta eggs, raising concern about their role as parasite reservoirs in the facilities.

Blastocystis is an anaerobic parasite that colonizes the intestinal tract of humans and animals. When it was first discovered, Blastocystis was considered to be a normal flora with beneficial effects on human health, such as maintaining gut hemostasis and improving intestinal barrier integrity. Later, with increasing research on Blastocystis, reports showed that Blastocystis sp. is associated with gastrointestinal disorders, colorectal cancer (CRC), and neurological disorders. The association between Blastocystis sp. and CRC has been confirmed in several countries. Blastocystis sp. can mediate CRC via similar mechanisms to CRC-associated bacteria, including infection-mediated inflammation, increased oxidative stress, induced gut dysbiosis, and damage to intestinal integrity, leading to a leaky gut. IL-8 is the main inflammatory cytokine released from epithelial cells and can promote CRC development. The causal association of Blastocystis sp. with other diseases needs further investigation. In this review, we have provided an update on Blastocystis sp. and summarized the debate about the beneficial and harmful effects of this parasite. We have also highlighted the possible mechanisms of Blastocystis-mediated CRC.

Abstract Background Lactiplantibacillus plantarum HEAL9 and Lacticaseibacillus paracasei 8700:2 positively affect the fecal bacteriome in children with celiac disease autoimmunity after 6 months of supplementation. The aim of the present investigation was to study the effects of Lactiplantibacillus plantarum HEAL9 and Lacticaseibacillus paracasei 8700:2 on the single-cell parasitome, with a primary focus on Blastocystis. Methods Stool samples were collected from 78 Swedish children with celiac disease autoimmunity participating in a randomized, double-blind, placebo-controlled clinical trial to either receive a mixture of supplementation with L. plantarum HEAL9 and L. paracasei 8700:2 (n = 38) or placebo (n = 40). A total of 227 stool samples collected at baseline and after 3 and 6 months of intervention, respectively, were retrospectively analyzed for Blastocystis by quantitative real-time PCR and subtyped by massively parallel amplicon sequencing. Other single-cell parasites were detected by untargeted 18S rDNA amplicon sequencing and verified by real-time PCR. The relation between the parasites and the bacteriome community was characterized by using 16S rDNA profiling of the V3-V4 region. Results Three different single-cell protists were identified, of which the highest prevalence was found for Dientamoeba fragilis (23.1%, 18/78 children), followed by Blastocystis (15.4%, 12/78) and Entamoeba spp. (2.6%, 2/78). The quantity of the protists was stable over time and not affected by probiotic intervention (P = 0.14 for Blastocystis, P = 0.10 for D. fragilis). The positivity of the protists was associated with increased bacteriome diversity (measured by multiple indices, P

Abstract Irritable bowel syndrome (IBS) is a prevalent gastrointestinal (GI) tract disorder. Although the main reason for IBS is not clear, the interaction between intestinal microorganisms and the gut barrier seems to play an important role in pathogenesis of IBS. The current study aimed to investigate the effect of Blastocystis on the gut microbiota profile and the circulation levels of microRNA (mir)-16 of IBS patients compared to healthy subjects. Stool and blood samples were collected from 80 participants including 40 samples from each IBS and healthy group. Upon DNA extraction from stool samples, barcoding region and quantitative real-time PCR were analyzed to investigate Blastocystis and the microbiota profile, respectively. RNA was extracted from serum samples of included subjects and the expression of mir-16 was evaluated using stem-loop protocol and qreal-time PCR. Significant changes between IBS patients and healthy controls was observed in Firmicutes, Actinobacteria, Faecalibacterium, and Alistipes. In IBS patients, the relative abundance of Bifidobacteria was directly correlated with the presence of Blastocystis, while Alistipes was decreased with Blastocystis. Lactobacillus was significantly increased in Blastocystis carriers. In healthy subjects, the relative abundance of Bifidobacteria was decreased, but Alistipes was increased in Blastocystis carriers. The changes in the Firmicutes/Bacteroidetes ratio was not significant in different groups. The relative expression of mir-16 in Blastocystis-negative IBS patients and healthy carriers was significantly overexpressed compared to control group. The presence of Blastocystis, decreased the relative expression of mir-16 in IBS patients compared to Blastocystis-negative IBS patients. The present study revealed that Blastocystis has the ability to change the abundance of some phyla/genera of bacteria in IBS and healthy subjects. Moreover, Blastocystis seems to modulate the relative expression of microRNAs to control the gut atmosphere, apply its pathogenicity, and provide a favor niche for its colonization.

Blastocystis is an anaerobic protozoan with global importance because of infecting a variety of hosts and having high prevalence in many countries. Blastocystis isolates display remarkable genetic differences, and many subtypes (STs) have currently been defined based on polymorphism in SSU rRNA coding gene. Each 25 subtype may have different characteristics such as pathogenicity, host specificity, and structural variations. Most current research on Blastocystis has focused on these differences and molecular epidemiology. This review aimed to provide a summary of Blastocystis subtype distribution in Türkiye. Regarding human samples, 16 manuscripts were found in the literature, which presented 783 Blastocystis isolates from 9 cities in Türkiye. The most common subtype was ST3 (47.9%), the others were ST1 30 (17.5%), ST2 (14.7%), ST4 (4%), and ST5-ST7 (15.9%). There were few studies on animal hosts and environmental samples. The faecal samples from rats, farm, and pet animals were examined for Blastocystis subtypes and ST1, ST3, ST4-ST7, ST10, and ST12-ST14 were reported. In addition, two studies reported Blastocystis ST1 and ST3 subtypes in environmental water samples. In conclusion, the review of available literature showed that a systematic understanding of the subtype distribution of 35 Blastocystis in Türkiye is still lacking. Most of the studies were performed in a limited number of cities, animal hosts, and environmental samples, therefore, more studies from different provinces are needed in forthcoming research. The majority studies were performed in a limited number of provinces, animal species and very few environmental samples, so in the future; there is a need of novel studies that evaluate more samples from different provinces.

Blastocystis sp. is a common widely distributed gut protozoan, with water transmission identified as one of its transmission routes. This study aimed to investigate the effect of chlorine, ultraviolet (UV)-C, and microwave (MW) treatments on the in vitro viability of cysts of Blastocystis sp. Purified Blastocystis sp. cysts were molecularly subtyped. Viable cysts were subjected to different free chlorine concentrations (1, 2, and 4 ppm), different doses of UV-C (5.13, 10.26, 20.52, and 40.47 mJ/cm2), and MW irradiation times (10, 15, 30, and 45 s). Viability reduction percentage, log10 inactivation, and micrometre-based optical microscopy examined cyst number and appearance after each disinfection trial. The three disinfectants’ efficacy and application conditions were assessed. The analysed isolates of Blastocystis cysts were subtype 3, possessed varying sizes and shapes, but two identical genomes. The cysts of Blastocystis sp. were resistant to chlorine at all doses and exposure durations tested. UV-C at a dose of 40 mJ/cm2 and MW treatment for 15 s were able to completely disinfect the cysts. The MW was the most effective disinfectant against Blastocystis cysts based on all evaluated factors. MW irradiation is the most efficient water treatment method for eradicating Blastocystis cysts in an easy and safe manner. HIGHLIGHTS Blastocystis is a waterborne protozoan with transmissible infective cyst form.; There is a paucity of studies on water treatment methods to inactivate the cysts.; Cysts were collected, purified, subtyped, and sequenced for in vitro treatment.; Chlorine, UV-C, and microwave were assessed for their effect on cysts' viability.; Microwave was the most effective method for eradicating cysts of Blastocystis.;

Stramenopiles form a clade of diverse eukaryotic organisms, including multicellular algae, the fish and plant pathogenic oomycetes, such as the potato blight Phytophthora, and the human intestinal protozoan Blastocystis. In most eukaryotes, glycolysis is a strictly cytosolic metabolic pathway that converts glucose to pyruvate, resulting in the production of NADH and ATP (Adenosine triphosphate). In contrast, stramenopiles have a branched glycolysis in which the enzymes of the pay-off phase are located in both the cytosol and the mitochondrial matrix. Here, we identify a mitochondrial carrier in Blastocystis that can transport glycolytic intermediates, such as dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, across the mitochondrial inner membrane, linking the cytosolic and mitochondrial branches of glycolysis. Comparative analyses with the phylogenetically related human mitochondrial oxoglutarate carrier (SLC25A11) and dicarboxylate carrier (SLC25A10) show that the glycolytic intermediate carrier has lost its ability to transport the canonical substrates malate and oxoglutarate. Blastocystis lacks several key components of oxidative phosphorylation required for the generation of mitochondrial ATP, such as complexes III and IV, ATP synthase, and ADP/ATP carriers. The presence of the glycolytic pay-off phase in the mitochondrial matrix generates ATP, which powers energy-requiring processes, such as macromolecular synthesis, as well as NADH, used by mitochondrial complex I to generate a proton motive force to drive the import of proteins and molecules. Given its unique substrate specificity and central role in carbon and energy metabolism, the carrier for glycolytic intermediates identified here represents a specific drug and pesticide target against stramenopile pathogens, which are of great economic importance.

Blastocystis is a genus of intestinal stramenopiles that infect vertebrates, and may cause disease of the alimentary tract. Currently, at least 40 genotypes (“subtypes”) of Blastocystis are recognised worldwide based on sequence data for the small subunit of the nuclear ribosomal RNA (SSU-rRNA) gene. Despite the numerous studies of Blastocystis worldwide, very few studies have explored Blastocystis in wild animals, particularly in Australia. Here, we used a PCR-based next generation sequencing (NGS)–phylogenetic approach to genetically characterise and classify Blastocystis variants from selected wildlife in the Australian state of Victoria. In total, 1658 faecal samples were collected from nine host species, including eastern grey kangaroo, swamp wallaby, common wombat, deer, European rabbit, canines and emu. Genomic DNA was extracted from these samples, a 500 bp region of the SSU-rRNA gene amplified by polymerase chain reaction (PCR) and, then, a subset of samples sequenced using Illumina technology. Primary PCR detected Blastocystis in 482 of the 1658 samples (29%), with the highest percentage in fallow deer (63%). Subsequent, Illumina-based sequencing of a subset of 356 samples revealed 55 distinct amplicon sequence variants (ASVs) representing seven currently-recognised subtypes (STs) [ST13 (prominent in marsupials), ST10, ST14, ST21, ST23, ST24 and ST25 (prominent in deer)] and two novel STs (ST45 and ST46) in marsupials. Mixed infections of different STs were observed in macropods, deer, emu and canids (fox, feral dog or dingo), but no infection was detected in rabbits or wombats. This study reveals marked genetic diversity within Blastocystis in a small number of species of wild animals in Australia, suggesting complexity in the genetic composition and transmission patterns of members of the genus Blastocystis in this country.

Blastocystis sp. is a gastrointestinal protozoan commonly encountered in humans and animals. Specificity to certain hosts may be associated with 38 known subtypes (STs) and 8 nonmammalian and avian STs (NMASTs). This can be determined by analyzing ST–host associations, ST–allele data, genetic variability analyses, and fixation index (FST) with sufficient data present. Thus, newly acquired and previously published data on Blastocystis sp. STs and NMASTs from the Philippines were compiled to determine the following: (1) ST–host associations, (2) ST–allele diversity per ST in certain hosts/sources, (3) intrasubtype diversity of certain STs found in different hosts using genetic variability analysis, and (4) comparison of similarities between specific ST populations to determine if these are the same circulating populations using FST. A total of 448 samples subtyped using both sequence-tagged site primers and the 600-bp barcoding region of the Blastocystis sp. SSU rRNA gene were analyzed in this study. Patterns of association for the Philippine samples were similar to those from neighboring Southeast Asian countries and around the world: ST1–ST4 were found in humans but ST3 was the most common, ST5 were found in pigs, and ST6 and ST7 were found in poultry. Blastocystis sp. from humans are mostly the same ST alleles (ST3 allele 34 and ST1 allele 4) while 3–5 ST alleles were found in the most common STs in pigs, macaques, and poultry. Also, ST1, ST3, ST5, and NMAST I are undergoing population expansion according to genetic variability analyses through possible addition of new alleles based on ST–allele diversity. Moreover, FST shows the same circulating population of ST1 in humans, pigs, and water indicating a possible waterborne route of cross-transmission. In contrast, ST3 found in humans possibly come from the same circulating population and is genetically distinct from those in nonhuman sources.

Abstract Blastocystis is a common microeukaryotic intestinal parasite in humans and other animal hosts globally. However, no large-scale longitudinal study has ever been conducted for Blastocystis. To understand patterns of infection prevalence and subtype diversity and their relationship with host age, we have conducted the most comprehensive longitudinal study of Blastocystis infection ever performed. Dairy calves from a herd located in Maryland, USA, were followed from birth through 24 months of age, and 990 individual fecal samples from 30 calves were collected over the study period, representing three age groups (pre-weaned, post-weaned, and heifer). All samples were screened for Blastocystis via PCR, and subtype determination was performed using next-generation amplicon sequencing. Associations between age group and infection status were assessed using logistic regression analyses. Blastocystis infection prevalence increased with time, significant associations were observed between age groups and infection risk, and a cumulative prevalence of 100% was observed among the study population during the 24-month period. Thirteen previously reported subtypes (ST1-6, ST10, ST14, ST21, ST23–26) and one potentially novel subtype were observed. Diversity within ST10 supports the need for division of the subtype into new subtype designations. Associations between subtype and age group were explored, and relationships between subtypes and infection chronicity are described. While subtype diversity increased with age in the study population, distinct patterns of individual subtype prevalence and chronicity were observed, supporting the importance of subtype discrimination in studies of host infection and disease. The data from this study represent a significant advance in our understanding of Blastocystis infection dynamics within a single host population over time and can be used to inform future studies of Blastocystis epidemiology in both humans and other animal hosts. Graphical Abstract

Introducción: la ausencia de estudios en perillas de puertas como vectores pasivos en la transmisión de parásitos entéricos en Venezuela y particularmente en el estado Lara, motivó el presente estudio. Metodología: se examinaron las perillas de 20 puertas, para cada puerta se frotó la perilla interna y externa tres veces (cada una) con un hisopo estéril impregnado con solución salina isotónica 0,85% (SSI), resuspendiéndolo en un tubo con 5 ml de SSI, el cual se centrifugó a 3000 rpm por 10 minutos, para luego separar el sobrenadante e inmediatamente montar el sedimento entre lámina y laminilla (SSI y lugol) para seguidamente observar al microscopio. Resultados: el 80% de las muestras analizadas estuvieron contaminadas, consiguiendo 13 taxones de enteroparásitos, a saber: Endolimax nana (70% del total de muestras examinadas), Blastocystis sp (50%), Giardia lamblia (20%), Cryptosporidium sp (20%), Entamoeba histolytica/E. dispar (10%), Entamoeba coli (10%), Chilomastix mesnili (5%), Entamoeba hartmanni (5%), Iodamoeba butschlii (5%), Cystoisospora belli (5%), Ascaris lumbricoides (15%), Hymenolepis nana (5%) y Trichuris trichiura (5%). Discusión: la diversidad enteroparasitaria observada y su abundancia, se corresponden con lo reportado en estudios de prevalencia parasitaria humana del estado Lara, igualmente se relaciona con contaminaciones señaladas en billetes, intercomunicadores y pasamanos en Barquisimeto. Conclusión: la ausencia del correcto y frecuente aseo en las perillas, favorece potencialmente la transmisión de enteroparásitos en miembros de esta comunidad educativa, lo cual quizás, se podría extrapolar a otras instituciones en condiciones parecidas.

Abstract Background Blastocystis is a common intestinal protist with a wide range of hosts. Thus far, 38 subtypes have been identified. In recent years, wild animals have been confronted with habitat fragmentation as well as an increasing risk of zoonotic disease transmission due to human disturbance. Only limited data are available on Blastocystis infection and subtype distribution in wild rhesus macaques in China. The aim of the present study was to investigate the prevalence and genetic diversity of Blastocystis in nine wild rhesus macaque populations in China. Methods A total of 276 faecal samples were collected from five high-altitude populations (high-altitude group [HAG]; 2800–4100 m a.s.l.) and four low-altitude populations (low-altitude group [LAG]; 5–1000 m a.s.l) of rhesus macaques. PCR-based analysis, using a new primer pair for the amplification of a 1690-bp sequence of the small subunit ribosomal RNA (SSU rRNA) gene, was used for prevalence and genetic diversity analysis. Result Analysis of faecal samples revealed that Blastocystis infection was common in rhesus macaques, with an infection positivity rate of 80.1% (n = 221/276 samples). There was no significant difference (P = 0.121) in positivity rate between the LAG (84.3%) and HAG (76.8%). Overall, 33 haplotypes were obtained and classified into four subtypes (STs), of which three were potentially zoonotic subtypes (ST1, 29.7%; ST2, 16.7%; ST3, 31.9%) and one that was first identified in this study and named ST39 (12.0%). The STs were distributed differently among the rhesus macaque populations, except for ST3, which was found in all populations. Phylogenetic analyses revealed two major divergent clades of ST3 for the HAG and LAG. Genetic diversity analysis showed a high genetic diversity of ST3 (haplotype diversity: 0.846; nucleotide diversity: 0.014) in the rhesus macaques, but a high genetic differentiation (FST > 0.25) and a low gene flow (Nm = 0.09) between the HAG and LAG. Conclusion Our study, which is the first investigation on Blastocystis infection in multiple wild rhesus macaque populations in China, indicates a potential risk of zoonotic transmission of Blastocystis in the study areas. Blastocystis ST3 showed high genetic diversity in wild rhesus macaques and significant genetic differentiation between the HAG and LAG. Our results provide fundamental information on the genetic diversity and prevalence of Blastocystis in wild rhesus macaque populations. Graphical Abstract

Blastocystis spp. is a common anaerobic intestinal parasite infecting humans and a diverse range of animals. The aim of the study was to compare different diagnostic methods for the detection of Blastocystis and survey the occurrence of its subtypes in farm animals, namely sheep, cows and camels, in Al-Ain, United Arab Emirates.

Background: Blastocystis is a genus of intestinal, anaerobic protozoan parasites that can be isolated from humans, animals, and the environment. We aimed to determine the distribution of Blastocystis and subtypes (STs) using stool samples obtained from healthy volunteers at collection centers in South Korea. Methods: A total of 478 stool samples from volunteers were collected at five collection centers throughout South Korea. The presence of Blastocystis was determined using PCR based on the small subunit (SSU) rRNA gene, and Blastocystis STs were confirmed through sequencing of the SSU rRNA gene. Results: Molecular analysis revealed the presence of Blastocystis in 27 (5.6%) of the enrolled participants. Two STs were identified: ST3 (66.7%) and ST1 (33.3%). The positive rates of Blastocystis varied by geographical region, ranging from 1.2%–12.0%. ST3 was the predominant subtype in all centers except one, where only ST1 was isolated. Phylogenic analysis showed clustering based on ST, but no significant differences were found among the regions. There was no association between Blastocystis colonization and either age or sex of the participants. Conclusions: The results of this multicenter study demonstrated colonization by Blastocystis, mainly ST3, in the gastrointestinal tracts of asymptomatic individuals in South Korea.

Abstract Background Blastocystis is one of the most common eukaryotic microorganisms colonizing the intestines of both humans and animals, but the conditions under which it may be a pathogen are unclear. Methods To study the genomic characteristics of circulating subtypes (ST) in Colombia, we established nine xenic cultures from Blastocystis isolated from human fecal samples, we identified 10 different subtypes, since one sample had a mixed infection. Thus, the genomes of the subtypes ST1 (n = 3), ST2 (n = 1), ST3 (n = 2), ST6 (n = 1), ST7 (n = 1), and ST8 (n = 2) were sequenced using Illumina and Oxford Nanopore Technologies (ONT). Results Analyses of these draft nuclear genomes indicated remarkable diversity in terms of genome size and guanine-cytosine (GC) content among the compared STs. Illumina sequencing-only draft genomes contained 824 to 2077 scaffolds, with total genome size ranging from 12 to 13.2 Mb and N50 values ranging from 10,585 to 29,404 base pairs (bp). The genome of one ST1 isolate was sequenced using ONT. This assembly was more contiguous, with a size of 20 million base pairs (Mb) spread over 116 scaffolds, and an N50 of 248,997 bp. Conclusion This work represents one of the few large-scale comparative genomic analyses of Blastocystis isolates, providing an additional glimpse into its genomic diversity. Graphical Abstract

Background and Aim: Blastocystis sp. is a common intestinal protozoan that can infect humans and a wide range of animals such as mammals, birds, reptiles, and arthropods. The prevalence of Blastocystis species is relatively high in developing countries and has been reported as the most common parasite in Iran. In recent years, various studies have shown the importance of its pathogenicity in humans. Blastocystis sp. infection is associated with skin lesions and a variety of gastrointestinal disorders such as diarrhea and irritable bowel syndrome (IBS). The aim of this study was to evaluate the prevalence of Blastocystis sp. in children with cancer in Sananadaj City by PCR method. Materials and Methods: To determine prevalence rate of Blastocystis sp infection in children with cancer, 93 fecal samples from available cancer patients were collected from the hospitals in Sananadaj City on the basis of census method from 2019 to 2021. The samples were analyzed by molecular method (PCR) using primers in the barcoding region of 18 rRNA gene for Blastocystis sp. amplification. Results: The results of PCR showed 15 cases (16.1%) had 600 bp fragment for Blastocystis sp.Statistical analysis showed that Blastocystis sp.had no significant correlation with infection and residential place (Pvalue = 0. 48), age (Pvalue = 0.88) and contact to animals (Pvalue =0.83). Conclusion: According to the results of this study, Blastocystis sp. is prevalent in cancer patients in Sananadaj city. These data can play an important role in the development of studies and research on Blastocystis sp. infections in cancer patients. In general, determination of the prevalence rate of the infection and identification of the different types of Blastocystis sp. in children with cancer and reporting these data to regional and national health officials can be beneficial in order to adopt a more appropriate treatment strategy and provide health strategies to control this infection.

Abstract Background Blastocystis sp. is one of the most common colonisers of the intestinal tract that demonstrate strong interaction with accompanying gut bacteria. Previously, the protozoan isolated from individuals with irritable bowel syndrome (IBS) showed altered phenotypic features suggesting that it can be triggered to become pathogenic. Previous studies reported altered gut microbiota and high prevalence of Blastocystis sp. in schizophrenia patients. However, the phenotypic characteristics of Blastocystis sp. isolated from individuals with SZ have yet to be described. Methods In this study, faecal samples from 50 patients with severe schizophrenia (SZ) and 100 non-schizophrenic (NS) individuals were screened for Blastocystis sp. infection. Positive isolates were subjected to genotypic and phenotypic characterization. Results We found that 12 out of 50 (24%) SZ and 5 out of 100 (5%) NS individuals were detected Blastocystis sp. positive using both in vitro culture and PCR method with no significant association to age and gender. Out of the 15 sequenced isolates, ST3 was the most prevalent subtype (66.7%) followed by ST1 (20%) and ST6 (13.3%). The isolates from SZ individuals demonstrated significant slower growth rate (34.9 ± 15.6 h) and larger range of cell diameter (3.3–140 µm). We detected higher amoebic forms and metronidazole resistance among SZ isolates with variation in cell surface glycoprotein where 98% of cells from SZ showed consistent medium to high binding affinity (+ 2 to + 3) to Concavalin A staining compared to NS isolates that demonstrated only 76% high lectin (+ 3) binding affinity. Cysteine and serine protease levels were predominantly found among SZ isolates. We also demonstrate the presence of metalloprotease in Blastocystis sp. especially among NS isolates. Introduction of solubilised antigens from SZ isolates increased the cell proliferation of HCT116 cells by two fold when compared to NS isolates. Conclusion Our findings demonstrated Blastocystis sp. isolated from SZ individuals showed variation in phenotype specifically in morphology and drug resistance. The findings indicate that the gut environment (SZ and NS) and treatment of SZ could have influenced the phenotype of Blastocystis sp. Graphical Abstract

Abstract Background Blastocystis is a common protistan parasite inhabiting the gastrointestinal tract of humans and animals. While there are increasing reports characterizing the associations between Blastocystis and the gut microbiome in healthy individuals, only a few studies have investigated the relationships between Blastocystis and the gut microbiota in diarrheal patients. Methods The effects of a specific subtype (ST7) of Blastocystis on the composition of gut microbiota in diarrheal patients were investigated using 16S ribosomal RNA (rRNA) gene sequencing and bioinformatic analyses. Results Compared with diarrheal patients without Blastocystis, diarrheal patients infected with Blastocystis ST7 exhibited lower bacterial diversity. Beta diversity analysis revealed significant differences in bacterial community structure between ST7-infected and Blastocystis-free patients. The proportion of Enterobacteriaceae and Escherichia-Shigella were significantly enriched in ST7-infected patients. In contrast, the abundance of Bacteroides and Parabacteroides were more prevalent in Blastocystis-free patients. Conclusions The results of this study revealed, for the first time, that infection with Blastocystis ST7 is associated with lower bacterial diversity and altered microbial structure in diarrheal patients. Our study on clinical diarrheal patients is also the first to reinforce the notion that ST7 is a pathogenic subtype of Blastocystis. Graphical Abstract

Objective:This study aimed to evaluate the distribution of intestinal parasites in refugee and native patients who applied to a territory hospital in Turkey.Methods:A total of 17911 patients who were admitted to our hospital between January 2018 and January 2019 were evaluated retrospectively in terms of intestinal parasites. The patients’ stool samples were investigated for the existence of intestinal parasites by direct wet mount preparation, formalin ether concentration technique and cellophane tape method. The data obtained were compared between patient groups according to the examination method.Results:The overall prevalence of E. vermicularis in refugee children was found twice higher than that in native patients and the most common symptom was abdominal pain in these patients. Intestinal parasite detection rates were significantly higher in the stool concentration method than in the direct wet mount examination. Cutaneous complaints and protein energy malnutrition/growth retardation were the most common clinical conditions besides gastrointestinal symptoms in patients with intestinal parasitosis.Conclusion:In our study, the prevalence of Blastocystis sp. in refugees was found to be higher than in the normal population. Intestinal parasitic infections should be investigated with proper diagnostic methods especially in children with PEM/GR and cutaneous symptoms in addition to gastrointestinal problems.

Aim: Helicobacter pylori and intestinal parasitic infections are commonly seen, especially in areas with low socioeconomic status and poor hygiene conditions. H. pylori and Giardia duodenalis can be commonly found in patients with upper gastrointestinal system complaints. It is thought that the urease activity of H. pylori may help intestinal parasites pass into the intestines without being affected by the acidic environment of the stomach. In this study, it was aimed to investigate the association of H. pylori and intestinal parasites (IP) in patients with gastrointestinal system complaints. Material and Method: A total of 408 patients, who were admitted to our hospital with gastrointestinal complaints between 2018 and 2020 and whose H. pylori rapid antigen test was studied simultaneously with intestinal parasite examination in the stool, were evaluated retrospectively. Results: Out of 408 patients whose stool samples were examined, one or more intestinal parasites were detected in 80 (19.6%), and H. pylori antigen test was positive in 65 (15.9%). While there was no statistically significant difference between H. pylori positivity and age groups, the rate of IP detection was found to be significantly higher in children aged 6-18 years. The most prevalent IP was Blastocystis sp. in 74 (18.1%) patients. Intestinal parasite and H. pylori antigen co-positivity in stool samples was detected in eight patients and it was not found statistically significant. Conclusion: H. pylori and intestinal parasites are common all over the world. The relationship between H. pylori and IP is still controversial, and more studies that are comprehensive are needed to understand the association of H. pylori and IP, especially in patients with upper gastrointestinal system complaints.

Blastocystis is a usual intestinal protist that always found in humans and various animals. Currently, the prevalence of Blastocystis in the migratory whooper swan (Cygnus cygnus) is unknown. In our research, we aimed to determine the occurrence, prevalence, subtype distribution and dynamic transmission mechanisms of Blastocystis in the migratory whooper swan in China. We also assessed the zoonotic potential of Blastocystis isolates, as well as possible routes of transmission and impact of this organism on One Health perspective. Fecal samples (n = 770) were collected from whooper swans inhabiting the Sanmenxia Swan Lake National Urban Wetland Park, China. The overall prevalence of Blastocystis was 11.6% (89/770). We identified 9 subtypes of Blastocystis sp., including 5 zoonotic subtypes [ST1 (Cakir et al., 2019 (8)), ST4 (Selma and Karanis, 2011 (4)), ST5 (Stensvold et al., 2009 (1)), ST6 (Fare et al., 2019 (5)) and ST7(58)] and 3 host-specific subtypes [ST10 (Zhao et al., 2018 (7)), ST14 (Tan et al., 2010 (2)), ST23 (Wang et al., 2018 (3)), and ST25 (Stensvold et al., 2009 (1))]. Subtypes ST4, ST5, ST6, ST10, ST14, ST23, and ST25 were first identified in the whooper swan. Among these subtypes, ST23 and ST25 were identified in birds for the first time, indicating that these subtypes are expanding their host range. So far, this is the first research reporting on the prevalence and subtypes distribution of Blastocystis in the migratory whooper swan in China. The findings obtained in this study will provide new insights into the genetic diversity and transmission routes of Blastocystis, and the possible public health concerns posed by this organism.

Abstract Background Blastocystis is a common intestinal protozoa found in animal and human fecal samples, with over 1 billion individuals infected worldwide. Since domestication, dogs and cats have had a close bond with humans. However, their close proximity poses a potential health risk since they may harbor several zoonotic agents. A global estimate of Blastocystis infection and subtype (ST) distribution in dogs and cats would therefore be of great health importance to humans. Methods We performed a comprehensive systematic search of four English-language databases (PubMed, Scopus, Google Scholar, Web of Science) for relevant articles up to 8 November 2021. The random-effects model was used to make pooled estimates with confidence intervals (CIs). Results In total, we identified 49 publications that met our inclusion criteria and subsequently analyzed the 65 datasets in these articles, of which 23 and 42 datasets were on cats and dogs, respectively. Among the 2934 cats included in the 23 datasets, which involved 16 countries, the prevalence rate of Blastocystis infection was 9.3% (95% CI 5.3–15.9%). The prevalence of Blastocystis infection was slightly lower [7%, 95% CI 4.7–10.4%) among the 7946 dogs included in the 42 datasets, involving 23 countries. The sensitivity analysis showed that no remarkable variation in the estimates upon the stepwise removal of each dataset. Higher ST diversity was found among the examined dogs (ST1-8, ST10, ST23, ST24) than among cats (ST1-4, ST10, ST14). Among dogs, ST3 was the most frequent ST (41.3%), followed by ST2 (39.3%), ST1 (30.9%), ST4 (13.4%), ST8 (12.7%), ST10 (11%) and ST5 (8.1%). Also among dogs, each of ST6, ST7, ST23 and ST24 was observed in only one study. Of the ST found in the cats examined, ST4 (29.5%), followed by ST10 (22.5%), ST1 (19.8%) and ST3 (17.6%) were the most common. A single study also reported the presence of both ST2 and ST14 in cats. With respect to zoonotic Blastocystis STs (ST1–ST9 and ST12), eight were reported from dogs (ST1-ST8) and four were isolated from cats (ST1–ST4), showing the implication of dog and cats in zoonotic transmission. Conclusions Taken together, our results show that elucidation of the true epidemiology and ST distribution of Blastocystis in dogs and cats demands more comprehensive studies, particularly in the negelected regions of the world. Graphical Abstract

Background: Blastocystis is an extracellular and immobile protozoan known as the most common protozoan found in the human gastrointestinal tract and a wide range of animals. Although it takes a long time to identify it, its role in the disease and health of people is controversial. Parasite protease is one of the important factors in eliciting symptoms. Thus, this study investigated protease activity in several subtypes.Methods: Stool samples were collected from 600 patients referred to Isfahan laboratory centers. The samples were examined microscopically and the blastocysts positive samples were cultured in medium. The blastocysts were purified using the ficoll method. PCR and sequencing were performed to identify the subtypes. Gelatin/SDS-PAGE method was used to evaluate protease activity.Findings: A total of 34 samples was positive in culture and PCR. Of these, 18 samples were sequenced, and 4 subtypes 1-3 and 6 were isolated. Subtype 2 was more common. In the Gelatin/ SDS-PAGE method, a large difference in the size of the bands resulting from protease activity was evident in different subtypes. 20 kDa band was observed in subtypes 1-3. In Subtype 6, the band pattern was completely different from other subtypes.Conclusion: Differences in protease band size were observed in different subtypes, which could probably be the reason for the difference in protease activity and pathogenicity in different subtypes. The present study obtained more reliable results using the ficoll method.

Blastocystis spp. are among the few enteric parasites with a prevalence that can reach up to approximately 80% in communities of developing countries. This systematic review updates and summarizes available literature on the molecular prevalence and subtype distribution of Blastocystis spp. in Latin American people. This work follows the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. The literature revised covers from 1 January 2015 to 6 October 2023 in seven different scientific databases, and the material was selected through inclusion and exclusion criteria. According to data found in the 36 selected articles, the prevalence of Blastocystis spp. in Latin America ranged between 5.8% (Bolivian rural communities) and 94.0% (Colombian general public). Generally, genomic DNA was extracted from approximately 200 mg fecal sediments using commercial kits, such as the QIAamp Stool Mini Kit (QIAGEN, Hilden, Germany) or the Norgen Stool DNA Isolation Kit (Norgen Biotek Corporation, Thorold, ON, Canada). Subtype-specific primers (such as the couple of primers BhRDr–RD5) developed from unique sequences of the SSU rRNA gene were applied to Blastocystis subtyping. Ten specific subtypes (STs) were found as well as various mixed infections, and the most circulating Blastocystis STs were in the order ST3, ST1, ST2, and ST4. The most recent data about Blastocystis spp. molecular epidemiology and the STs in communities of Latin America are limited to studies from specific countries. Novel scientific data from the other countries are required to obtain a complete picture and truly understand the distribution and prevalence of Blastocystis spp. and the STs.

Blastocystis is the most common gastrointestinal protist found in humans and animals. Although the clinical significance of Blastocystis remains unclear, the organism is increasingly being viewed as a commensal member of the gut microbiome. However, its impact on the microbiome is still being debated. It is unclear whether Blastocystis promotes a healthy gut and microbiome directly or whether it is more likely to colonize and persist in a healthy gut environment. In healthy people, Blastocystis is frequently associated with increased bacterial diversity and significant differences in the gut microbiome. Based on current knowledge, it is not possible to determine whether differences in the gut microbiome are the cause or result of Blastocystis colonization. Although it is possible that some aspects of this eukaryote’s role in the intestinal microbiome remain unknown and that its effects vary, possibly due to subtype and intra-subtype variations and immune modulation, more research is needed to characterize these mechanisms in greater detail. This review covers recent findings on the effects of Blastocystis in the gut microbiome and immune modulation, its impact on the microbiome in autoimmune diseases, whether Blastocystis has a role like bacteria in the gut–brain axis, and its relationship with probiotics.

Blastocystis sp., a significant zoonotic parasite with a global distribution, was the focus of this study, which aimed to investigate its prevalence and genetic diversity among diarrheic and asymptomatic children in Wenzhou, China. We collected 1,032 fecal samples from Yuying Children’s Hospital, Wenzhou, China, comprising 684 from children with diarrhea and 348 from asymptomatic children. Genomic DNA extracted from these samples was used to detect Blastocystis spp. by PCR, targeting the small subunit ribosomal RNA gene. Subsequently, a phylogenetic tree was constructed, applying the maximum likelihood method. Blastocystis spp. were detected in 67 (6.5%) of the fecal samples. The prevalence rate of Blastocystis spp. in diarrheic children (8.8%; 60/684) was significantly higher than that in asymptomatic children (2.0%; 7/348) (χ 2 = 17.3, p

Wild rodents are key carriers of various human pathogens, including Blastocystis spp. Our study aimed to assess the prevalence and genetic characteristics of Blastocystis among wild rodents in the Inner Mongolian Autonomous Region and Liaoning Province of China. From November 2023 to February 2024, 486 rodents were captured in these regions. Fresh feces were collected from the intestines of each rodent for the isolation of DNA and PCR amplification of the vertebrate cytochrome b (cytb) gene to identify rodent species. Subsequently, PCR analysis and sequencing of the partial small subunit of the ribosomal RNA (rRNA) gene were utilized to detect Blastocystis in all fecal samples. Of the total samples, 27.4% (133/486) were found to be Blastocystis positive. The results revealed the presence of four species of rodents infected with Blastocystis, 32.3% (63/195) in Rattus norvegicus, 15.1% (16/106) in Mus musculus, 20.2% (18/89) in Apodemus agrarius, and 37.5% (36/96) in Cricetulus barabensis. Sequence analysis confirmed the existence of five Blastocystis subtypes: ST1 (n = 4), ST2 (n = 2), the ST4 (n = 125, the dominant subtype), ST10 (n = 1), and a novel ST (n = 1). The identified zoonotic subtypes (ST1, ST2, ST4, and ST10) highlight the possible role played by wild rodents in the transmission of Blastocystis to humans, thereby elevating the chances of human infection. Meanwhile, the discovery of novel sequences also provides new insights into the genetic diversity of this parasite.

Abstract Background Blastocystis ST4 is a common protistan parasite of the gastrointestinal tract of humans and a wide range of animals. While it has been suggested that colonization with ST4 is associated with healthy gut microbiota, how ST4 influences the gut microbiota remains poorly studied. This study aimed to examine the interactions between ST4 and several intestinal bacteria using in vitro co-culture systems, and to further investigate the mechanism of interaction and its effect on the epithelial barrier integrity of HT-29 cells. Methods Seven intestinal bacteria Bacteroides fragilis, Bifidobacterium longum, Bacillus subtilis, Bacteroides vulgatus, Escherichia coli, Enterococcus faecalis, and Lactobacillus brevis were co-cultured with Blastocystis ST4 in vitro. Flow cytometry and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to determine the role of reactive oxygen species (ROS) and bacteria oxidoreductase genes, respectively, in response to Blastocystis co-incubation. Transepithelial electrical resistance (TEER) and flux assays were performed to assess the effect of microbiota representatives on the integrity of the intestinal epithelial barrier. Results Co-incubation with Blastocystis ST4 showed a beneficial influence on most intestinal bacteria, while ST4 significantly inhibited the growth of B. vulgatus, a common pathogen in the genus Bacteroides. The decrease in B. vulgatus when co-incubated with Blastocystis ST4 was associated with high levels of ROS and the upregulation of oxidative stress-related genes. Furthermore, co-incubation with Blastocystis ST4 was able to protect the intestinal epithelial barrier from damage by B. vulgatus. Conclusions This study demonstrated, for the first time, that Blastocystis ST4 has beneficial effects on intestinal commensal bacteria in vitro, and can inhibit the growth of pathogenic B. vulgatus. Combined with previous microbiome research on ST4, our data suggest that ST4 may be a beneficial commensal. Graphical Abstract

Abstract Background Blastocystis is an anaerobic unicellular protist frequently detected in the gastrointestinal tracts of humans and animals worldwide. However, the prevalence and subtype distribution of Blastocystis in the coypu (Myocastor coypus) population have not been reported so far. The aim of this study was to determine the prevalence, genetic characteristics, and zoonotic potential of Blastocystis isolates detected in coypus in China. Results A total of 308 fecal samples were collected from coypus in seven regions across China and subsequently examined. Blastocystis was detected in 44 (14.3%) specimens by nested PCR amplification of the small subunit ribosomal rRNA (SSU rRNA) gene. Further DNA sequencing and phylogenetic analyses resulted in the identification of two zoonotic known subtypes, ST4 and ST5, and an unknown subtype. ST4 was the most predominant subtype observed in the samples. ST5 infections were only observed in three coypus. Factors that were associated with prevalence of Blastocystis included age, geographical region and subtype. Interestingly, this is the first report about a potentially novel subtype infecting coypus. Conclusions This is the first comprehensive report of Blastocystis in M. coypus across a wide geographic range of China. A moderate degree of genetic divergence was observed. The presence of zoonotic subtypes in farmed M. coypus suggests that these animals have the potential to transmit blastocystosis to both humans and domestic animals. These findings provide a better understanding of the genetic diversity of Blastocystis in rodents and contribute towards the establishment of efficient blastocystosis control strategies in the investigated areas. Graphical abstract

Abstract Background Blastocystis is a typical anaerobic colon protist in humans with controversial pathogenicity and has relation with alterations in the intestinal microbiota composition (dysbiosis), whose eventual indicator is the Firmicutes/Bacteroidetes ratio (F/B ratio); this indicator is also linked to complications such as diabetes, obesity, or inflammatory bowel disease. The present study investigated the prevalence of Blastocystis and its association with Firmicutes/Bacteroidetes ratio in healthy and metabolic diseased subjects. Methods Fecal and blood samples were collected consecutively from 200 healthy subjects and 84 subjects with metabolic disease; Blastocystis and its most frequent subtypes were identified by end-point PCR and the two most representative phyla of the intestinal microbiota Firmicutes and Bacteroidetes by real-time PCR. Results The prevalence of Blastocystis in healthy subjects was 47.0, and 65.48% in subjects with metabolic disease; the most prevalent subtype in the total population was ST3 (28.38%), followed by ST1 (14.86%), ST4, ST5, and ST7 (each one of them with 14.19% respectively), and finally ST2 (8.78%). The low F/B ratio was associated with the prevalence of Blastocystis in the two cohorts FACSA (OR = 3.78 p

Blastocystis cf. Blastocystis hominis is the most common unicellular parasite found in human and animal intestines. Little is known about the life cycle, transmission and mechanisms of pathogenesis for this parasite. The aim of this study was to obtain new data on the genetic diversity of Blastocystis in selected species of mammals found in the Białowieża Primeval Forest (BPF), Poland, the best preserved lowland forest in temperate Europe. 113 faecal samples were collected in the period 2018–2020 from seven species of wild mammals occurring within the Polish part of BPF and its surroundings. Blastocystis was detected by molecular amplification and sequencing the small subunit rRNA gene. The overall prevalence of Blastocystis in animals was 8.9%. A larger number of stool samples (90) were collected from European bison and only for this species was it possible to calculate the prevalence of infection (5.6%). The isolates obtained from European bison were classified as ST1, ST3, ST5, ST7. Blastocystis was also detected in the grey wolf (Canis lupus) (ST5), wild boar (Sus scrofa) (ST5) and red deer (Cervus elaphus) (ST1). In conclusion, for the first time we have demonstrated the presence of Blastocystis in wild European bison. Preliminary studies have shown that Blastocystis is present in at least 4 species of wild mammals in the BPF area and that it exhibits great genetic diversity.

Background: Recent theories on the possible interactions between the intestinal parasites and COVID-19 have stated that these co-infections may cause immune imbalance and further complications in the affected patients. Until now, there is no data about Blastocystis subtypes as an intestinal parasite in COVID-19 patients. Therefore, the present work was done to evaluate the molecular prevalence of Blastocystis spp. and related risk factors in Iranian patients with COVID-19. Method: Stool samples were gathered from 200 COVID-19 patients and 200 control, being matched regarding age, gender and residence. Then, stool samples were surveyed by parasitological methods, including direct slide smear and formalin-ether concentration. In the following, PCR and sequencing were used to detect Blastocystis spp. and their subtypes. Results: The frequency of Blastocystis spp. in patients with COVID-19 (7.5%; 15/200 by molecular method vs. 6%; 12/200 by microscopy method) was slightly higher than in individuals without COVID-19 (4.5%; 9/200 by molecular method vs. 4%; 8/200 by microscopy method), this difference was not statistically significant (P value = 0.57 for molecular method vs. P value = 0.81 for microscopy method). Regarding associated factors for Blastocystis spp., we found significant differences regarding the residence (rural), loose and watery stool with diarrhea, and duration of treatment (6 weeks

ABSTRACT: Background: Irritable bowel syndrome (IBS) is a prevalent lifestyle-associated ailment linked to the gut microbiota that significantly influences patients' quality of life. A notable correlation exists between Blastocystis infections and susceptibility to IBS, with infected individuals exhibiting an increased likelihood of developing the condition. Despite promising results from using probiotics to modulate the gut microbiota and manage IBS, the precise mechanisms and potential risks remain unclear. Objective: This review aims to explore the therapeutic potential of probiotics, particularly Saccharomyces boulardii, in the management of IBS, highlighting the role of the gut microbiota and the gut–brain axis in IBS pathophysiology. Methods: A comprehensive literature survey was conducted to examine the association between gut microbiota and IBS, the role of probiotics in managing IBS, the mechanisms of their action, and the potential risks associated with their long-term use. Additionally, this study addresses the influence of Blastocystis infections on IBS susceptibility and evaluates various ongoing clinical trials investigating probiotic use for IBS. Results: S boulardii, a yeast species with probiotic properties, has demonstrated effectiveness in both the treatment and prophylaxis of IBS. Its administration is associated with a decrease in the proinflammatory cytokine interleukin 8 and an increase in the anti-inflammatory cytokine interleukin 10. Probiotics appear to function by inhibiting the growth of pathogenic microorganisms and regulating neurotransmitter activity, influencing the gut–brain axis. However, selecting appropriate probiotic strains and dosing regimens is crucial because of potential adverse effects, such as infections and allergic reactions. Conclusions: Probiotics, specifically S boulardii, offer a promising avenue for IBS management by modulating gut microbiota. However, further research is necessary to delineate the precise mechanisms of action, optimal strains, dosing regimens for IBS treatment, and potential risks associated with long-term use. A comprehensive approach incorporating probiotics, a low-FODMAP diet, and cognitive-behavioral therapy may provide effective management of IBS symptoms.