First molecular subtyping and zoonotic significance of Blastocystis sp. in Dromedary (C. dromedarius) and Bactrian (C. bactrianus) camels in Iran: A molecular epidemiology and review of available literature.

BACKGROUND: Blastocystis sp. is a zoonotic protozoan parasite, and there is limited information about its molecular prevalence and subtypes (STs) distribution in camels globally, especially in Iran. OBJECTIVES: This study aimed to examine the prevalence, STs distribution, and zoonotic potential of Blastocystis sp. in one-humped and two-humped camels in Ardabil province, northwestern Iran. METHODS: A PCR-sequencing tool using the SSU rRNA gene was employed to examine the occurrence and genetic variation of Blastocystis sp. in 150 faecal samples from Bactrian (Camelus bactrianus, 50 samples) and Dromedary (Camelus dromedarius, 100 samples) camels in Ardabil province. RESULTS: The overall prevalence of Blastocystis sp. in camels was determined to be 12% (18/150) through microscopy and PCR analyses. Phylogenetically, this study identified three distinct zoonotic STs: ST7, ST10, and ST14. ST10 was the most prevalent, comprising 50% (9/18) of the isolated STs from camels. ST14 closely followed with 38.9% (7/18), while ST7 made up 11.1% (2/18) of the total STs. In brief, ST10, ST14, and ST7 represented 50% (7/14), 35.7% (5/14), and 14.3% (2/14) of the Blastocystis-positive cases in one-humped camels, respectively. Further, each of the ST10 and ST14 accounted for 50% (2/4) of the Blastocystis-positive samples in two-humped camels. An analysis of the available data reveals that out of the 37-44 identified Blastocystis STs, 15 (ST1-ST7, ST10, ST14, ST15, ST21, ST24, ST25, ST26, and ST30) have been reported in camels. The predominant STs observed are ST10 and ST14. Furthermore, among the 15 zoonotic STs (ST1-ST10, ST12-ST14, ST16, and ST23) of Blastocystis reported thus far, nine zoonotic STs (ST1-ST7, ST10, and ST14) have been found in camels. CONCLUSIONS: These findings indicate that camels serve as a proper reservoir for a diverse array of Blastocystis STs and thereby can play a significant role in the transmission of this protozoan infection to humans, animals, and water reservoirs.

Molecular prevalence and subtype distribution of Blastocystis spp. among children who have diarrheia or are asymptomatic in Wenzhou, Zhejiang Province, China.

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 < 0.001). Sequence analysis of the SSU rRNA gene identified five known Blastocystis spp. subtypes, ST1 (n = 12), ST2 (n = 5), ST3 (n = 35), ST4 (n = 12), and ST7 (n = 3). ST1 and ST3 were present in both diarrheic and asymptomatic children, while ST2, ST4, and ST7 were exclusive to diarrheic children. Intra-subtype genetic polymorphisms were identified, comprising four variations in ST1 (ST1-1 to ST1-4), five in ST3 (ST3-1 to ST3-5), two in ST4 (ST4-1 and ST4-2), and two in ST7 (ST7-1 and ST7-2). Notably, ST1-2 to ST1-4, ST3-3 to ST3-5, and ST7-1 and ST7-2 represent newly identified variations. The composition and genetic characteristics of subtypes among children in this region suggest various sources of infection, including human-to-human and animal-to-human transmission.

Title: Prévalence moléculaire et distribution des sous-types de Blastocystis spp. parmi les enfants diarrhéiques et asymptomatiques à Wenzhou, Province du Zhejiang, Chine. Abstract: Blastocystis sp., un parasite zoonotique important avec une distribution mondiale, était au centre de cette étude, qui visait à étudier sa prévalence et sa diversité génétique parmi les enfants diarrhéiques et asymptomatiques de Wenzhou, en Chine. Nous avons collecté 1 032 échantillons fécaux à l'hôpital pour enfants Yuying de Wenzhou, en Chine, dont 684 provenant d'enfants souffrant de diarrhée et 348 d'enfants asymptomatiques. L'ADN génomique extrait de ces échantillons a été utilisé pour détecter Blastocystis sp. par PCR, ciblant le gène de la petite sous-unité de l'ARN ribosomal. Par la suite, un arbre phylogénétique a été construit, en appliquant la méthode du maximum de vraisemblance. Blastocystis sp. a été détecté dans 67 (6,5 %) des échantillons fécaux. Le taux de prévalence de Blastocystis spp. chez les enfants diarrhéiques (8,8 % ; 60 / 684) était significativement plus élevé que chez les enfants asymptomatiques (2,0 % ; 7 / 348) (χ2 = 17,3, p < 0,001). L'analyse de la séquence du gène de l'ARNr SSU a identifié cinq sous-types de Blastocystis spp., ST1 (n = 12), ST2 (n = 5), ST3 (n = 35), ST4 (n = 12) et ST7 (n = 3). Les sous-types ST1 et ST3 étaient présents chez les enfants diarrhéiques et asymptomatiques, tandis que ST2, ST4 et ST7 étaient exclusifs aux enfants diarrhéiques. Des polymorphismes génétiques intra-sous-types ont été identifiés, comprenant quatre variations dans ST1 (ST1-1 à ST1-4), cinq dans ST3 (ST3-1 à ST3-5), deux dans ST4 (ST4-1 et ST4-2) et deux dans ST7 (ST7-1 et ST7-2). Notamment, ST1-2 à ST1-4, ST3-3 à ST3-5, ST7-1 et ST7-2 représentent des variations nouvellement identifiées. La composition et les caractéristiques génétiques des sous-types chez les enfants de cette région suggèrent diverses sources d'infection, notamment la transmission interhumaine et animale.

Subtypes of Blastocystis in Tibetan Antelope (Pantholops hodgsonii).

Blastocystis is a protist that is distributed in the gut tract of humans and animals. However, the reports about Blastocystis infection in Tibetan antelope are scarce. We collected 173 Tibetan antelope feces samples from Xinjiang, Qinghai and Xizang, and amplified the SSU rRNA gene of 600 bp region of Blastocystis in our research. Fifty-one samples in total were positive for Blastocystis, with all subtypes being ST31. The lowest prevalence of Blastocystis was observed in Xizang (2/20, 9.1%), followed by Qinghai (18/92, 16.4%), Xinjiang (31/61, 33.7%). The highest prevalence of Blastocystis in Tibetan antelope was detected during the summer was (19/30, 38.8%). This is the first research work regarding the Blastocystis subtypes ST31 in Tibetan antelope. Our research provides information for future researches on the distribution of this Blastocystis subtype and the control of Blastocystis infection.

Genetic characteristics of Blastocystis sp. in cattle from Hebei Province, China.

Blastocystis sp. is a protozoan parasite that infects the intestines of humans and animals, causing chronic diseases such as skin rashes, abdominal pain, and irritable bowel syndrome. A survey was conducted to determine the prevalence and genetic diversity of Blastocystis sp. infection in cattle, in Hebei Province, China. 2746 cattle fecal samples were collected from 11 cities in Hebei Province and analyzed using polymerase chain reaction targeting the Blastocystis sp. barcoding gene. MEGA, PhyloSuite, and PopART were used to analyze the subtype, sequence signature, pairwise genetic distance, and genetic diversity indices. The results showed that the Blastocystis sp. detection rate was 12.60% (346/2746). The infection rate in different herds was affected by region, age, breeding mode, and variety; that is, the infection rates in areas of southern Hebei, cattle under one year old, intensive raising, and dairy cattle were higher than the infection rates in northern Hebei, cattle over one year old, scatter feeding, and beef cattle. Seven Blastocystis subtypes were identified, namely, ST1, ST2, ST5, ST10, ST14, ST21, and ST26; ST10 was the dominant subtype, and ST14 was the second most common subtype. A total of 374 polymorphic and conserved sites were obtained, including 273 invariable (monomorphic) sites and 101 variable (polymorphic) sites, accounting for 27.01% of all nucleotides. The nucleotide diversity index (Pi) was 0.07749, and the haplotype (gene) diversity index (Hd) was 0.946. This study provides the first comprehensive information on the epidemiological situation of Blastocystis sp. infection in cattle from Hebei Province, China, and revealed rich genetic diversity of Blastocystis sp.

The involvement of cytokine gene polymorphism in determining the vulnerability to Blastocystis and Helicobacter pylori co-infection in the Egyptian population.

AIMS: The present study aimed to identify any potential association between IL-1ß and TNF-α gene polymorphism and the risk of Blastocystis infection as well as co-infection of Blastocystis with Helicobacter pylori (H.pylori). METHODOLOGY: A total of 314 stool samples were collected and examined microscopically for the detection of parasitic infection. DNA was extracted from all samples and utilized to identify Blastocystis molecularly. Positive samples were used for H. pylori detection by rapid tests and PCR. Moreover, we investigate polymorphism in the TNF-α gene at position -1031T/C, -308 G/A, and IL-1ß at position +3954C/T using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. RESULTS: Out of the 314 stool samples, Blastocystis was detected in 93 (29.6 %); among them, 54 (58.1 %) had a mixed infection of Blastocystis with H. pylori. The TT genotype of the IL-1ß gene at position +3954 was significantly higher in Blasocystis-infected patients than in uninfected patients (17.2% vs. 6.3 %, P = 0.02), which might be considered a risk factor (OR = 3.2; CI =1.21-8.52). The TNF-α at position -1031 TT genotype was significantly higher in Blastocystis-infected patients than uninfected patients (44.1% vs. 10.8 %, P< 0.0001). The T allele (OR= 2.67; CI=1.51-4.72, P = 0.0008) might be considered a risk factor. The TNF- α at position -308 AA genotype is higher in Blasocystis infected than uninfected (17.2% vs 7.2 %, P = 0.03). TNF-α -308 AA (OR = 2.72; CI = 1.08-6.89) and A allele (OR= 1.46; CI= 0.797-2.66) might be considered risk factors. The TNF- α at position -308 G/A showed that the GG is the most frequent genotype in Blastocystis with H. pylori-positive patients with a significant association (P = 0.004), as well as the G allele (P = 0.02). The G allele (OR=1.924; CI= 1.071-3.454) might be considered a risk factor for co-infection of Blastocystis and H. pylori. CONCLUSION: SNPs (-1031 T/C and -308 G/A) of the TNF-α and (+3954 C/T) of the IL-1ß may be a useful marker in the assessment of the risk of Blastocystis infection, and TNF-α at position -308 G/A) may be a predictor for co-infection of Blastocystis with H. pylori.

Molecular detection and characterization of Blastocystis in herbivore livestock species in Portugal.

Blastocystis is a ubiquitous intestinal protist in humans and animals worldwide. The traditional livestock free-roaming raising system in rural communities increases the risk of infection with contact with a wider range of pathogens transmitted via the faecal-oral route associated with that wildlife-livestock-human interface. However, no studies have been conducted to determine the occurrence and subtype distribution of Blastocystis in livestock in Portugal. Here, we collected 180 faecal samples from herbivore livestock (cattle, goats, horses, and sheep) in different regions of the country to investigate Blastocystis prevalence and subtype diversity using PCR and next-generation amplicon sequencing. Blastocystis was present in 40.6% (73/180; 95% CI: 33.31-48.11) of the samples (goats, 81.0%; sheep, 60.9%; cattle, 32.2%). None of the horse samples were Blastocystis-positive. Eighteen subtypes were detected (ST1-ST3, ST5-ST7, ST10, ST13, ST14, ST21, ST23-ST26, ST30, ST42-ST44). Mixed infections were detected in 97.3% of the Blastocystis-positive samples. Potentially zoonotic subtypes were identified in 75.0%, 96.4%, and 100% of the Blastocystis-positive specimens collected from cattle, sheep, and goats, respectively. These results demonstrate that cattle, sheep, and goats harbour a high diversity of Blastocystis subtypes in the study regions. Importantly, our data provide novel molecular evidence strongly suggesting that some Blastocystis STs/ST subgroups may have differential host specificity.

The clinical significance of Dientamoeba fragilis and Blastocystis in human stool-retrospective cohort study.

OBJECTIVES: The aim of this study was to assess the clinical significance of Dientamoeba fragilis (DF) and Blastocystis species (Bs) in human stool. METHODS: Observational study of patients ≥18 years, who were tested by stool multiplex PCR for bacteria and parasites between April 2019 and March 2022. Although DF and Bs are part of the PCR kit, these results are not routinely reported to the patient or the ordering physician. The main outcomes were the incidence of symptoms during 14 days before the referral to stool PCR test, and the incidence of several clinical outcomes during 60 days after the PCR test (symptoms, referrals to further evaluation, prescription of symptomatic, or antibiotic treatment). RESULTS: A total of 27 918 patients were tested by stool PCR during the 3 study years. A total of 6215 (22.3%) and 5337 (19.2%) were positive for DF and Bs, respectively. The incidence of symptoms before the test was similar in those positive for Bs or DF and those with all-negative PCR (adjusted OR and 95% CI of 0.87 [0.80-0.95] and 0.82 [0.76-0.88] for Bs and DF, respectively), whereas significantly higher (2.47 [2.23-2.73]) in those positive for the other multiplex PCR assay components. During the 60 days after the test, the prevalence of any of the outcomes was similar in those positive for Bs or DF and those with negative PCR (adjusted OR and 95% CI of 0.92 [0.83-1.02] and 0.89 [0.81-0.97] for symptoms, 0.84 [0.75-0.94] and 0.93 [0.85-1.01] for referrals, 0.88 [0.75-1.03] and 0.82 [0.71-0.94] for symptomatic treatment, and 0.88 [0.75-1.02] and 0.86 [0.75-0.98] for antibiotic treatment in the Bs and DF positive individuals, respectively). The PCR cycle threshold was not associated with any of the outcomes. DISCUSSION: Positive stool PCR for DF or Bs was not associated with any of the measured clinical outcomes.

Division of Blastocystis ST10 into three new subtypes: ST42-ST44.

The Blastocystis subtype ST10 has been recognized to contain a great deal of diversity at the sequence level, potentially indicating the presence of multiple new STs within the clade. However, the data needed to validate these new STs were not available. To help resolve this diversity, full-length small subunit (SSU) rRNA gene reference sequences were generated using Oxford Nanopore MinION long-read sequencing from 21 samples representing multiple domestic and wild hosts and geographic regions and covering the sequence diversity previously described using fragments of the SSU rRNA gene. Phylogenetic and pairwise distance analyses were used to compare full-length sequences of the SSU rRNA gene generated in this study with all other valid STs of Blastocystis. We present data supporting the division of ST10/ST23 cluster into five subtypes, ST10, ST23, and three new subtypes with the proposed ST designations of ST42, ST43, and ST44. As the host range of Blastocystis continues to expand with new subtypes and new hosts being frequently identified, the reference sequences provided in this study will assist in accurate sequence classification and help to clarify the epidemiology of this common intestinal microeukaryote.

Blastocystis genetic diversity in animal and human samples from different departments of Colombia using complete sequencing of the 18S rRNA gene (SSU rRNA) by Oxford Nanopore Technologies (ONT).

Blastocystis is an intestinal microeukaryote that has raised attention due to its wide distribution in animals and humans. The risk of zoonotic circulation primarily arises from close contact with infected animals. Therefore, the following study aimed to evaluate the diversity and frequency of Blastocystis subtypes in Colombian human and animal samples using complete sequencing of the 18S rRNA gene. For this purpose, 341 human stool samples and 277 animal fecal samples (from cattle, sheep, goat, pigs, cats, and dogs), were collected from different Colombian regions and analyzed using PCR-based detection and full-length 18S SSU rRNA gene Next-Generation Sequencing (NGS). Among the 618 samples from both hosts, humans and animals, the results revealed widespread Blastocystis frequency, with 48.09% (n = 164) in humans and 31.4% (n = 87) detection in animals. Dogs, cats, sheep, pigs, and wild animals tested positive, aligning with global prevalence patterns. Also, 29 human samples and 23 animal samples were sequenced using ONT technology from which 11 long-read unique sequences were generated and cluster with their compared reference sequences. The subtype distribution varied within hosts, detecting ST1 and ST3 in both human and animal samples. Subtypes ST5, ST10, ST14, ST15, ST21, ST24, ST25 and ST26 were limited to animals hosts, some of which are considered to have zoonotic potential. On the other hand, ST2 was found exclusively in human samples from Bolivar region. Mixed infections occurred in both animal and humans, 60.86% and 27.58% respectively. Moreover, to our knowledge, this is the first study in Colombia identifying ST15 in pigs and ST25 in sheep. The subtypes (STs) identified in this study indicate that certain animals may serve as reservoirs with the potential for zoonotic transmission. The identification of zoonotic subtypes highlights the use of Next Generation Sequencing as the depth and resolution of the sequences increases providing insights into STs of medical and veterinarian significance. It also reveals the coexistence of diverse subtypes among hosts. Further research is essential for understanding transmission dynamics, health implications, and detection strategies for Blastocystis occurrence in animals and humans, mainly associated to the role of animals as reservoirs and their close interaction with humans.

Prevalence of Blastocystis sp. in Morocco: Comparative assessment of three diagnostic methods and characterization of parasite forms in Jones' culture medium.

Blastocystosis is an infection caused by Blastocystis sp., which colonizes the digestive tract of various hosts, including humans, although its pathogenicity is debated. It is crucial to detect and distinguish the different forms of Blastocystis to understand better its impact on human health and its epidemiological evolution. This study evaluated three diagnostic methods on 105 stool samples: direct examination, culture in Jones' medium, and conventional PCR. PCR is considered the gold standard and revealed a high prevalence of Blastocystis (67.62%) compared to direct examination (20.95%) and culture in Jones' medium (51.43%). Although the sensitivity of direct examination and culture was 31% and 76.1%, respectively, their specificity was 100%. No significant risk factors were identified. A statistically significant association was observed between Blastocystis infection and abdominal pain. Microscopic analysis revealed various morphological forms. Molecular diagnosis is an essential tool to determine the true prevalence of Blastocystis, and studying the different forms of this microorganism will contribute to a better understanding of its biological cycle and, therefore, the impact of this emerging infection on human health.

Title: Prévalence de Blastocystis sp. au Maroc : évaluation comparative de trois méthodes de diagnostic et caractérisation des formes parasitaires en milieu de culture Jones. Abstract: La blastocystose est une infection causée par Blastocystis sp., qui colonise le tractus digestif de divers hôtes, y compris l'homme, bien que son pouvoir pathogène soit débattu. Il est crucial de détecter et de distinguer les différentes formes de Blastocystis pour mieux comprendre son impact sur la santé humaine et son évolution épidémiologique. Cette étude a évalué trois méthodes de diagnostic sur 105 échantillons de selles : l'examen direct, la culture en milieu de Jones et la PCR conventionnelle. La PCR, considérée comme méthode de référence, a révélé une prévalence élevée de Blastocystis (67,62 %) par rapport à l'examen direct (20,95 %) et à la culture en milieu de Jones (51,43 %). Bien que la sensibilité de l'examen direct et de la culture soit respectivement de 31 % et 76,1 %, leur spécificité était de 100 %. Aucun facteur de risque significatif n'a été identifié. Une association statistiquement significative a été observée entre l'infection à Blastocystis et les douleurs abdominales. L'analyse microscopique a révélé diverses formes morphologiques. Le diagnostic moléculaire est un outil essentiel pour déterminer la véritable prévalence de Blastocystis, et l'étude des différentes formes de ce microorganisme contribuera à une meilleure compréhension de son cycle biologique et, par conséquent de l'impact de cette infection émergente sur la santé humaine.

[Molecular detection and subtyping of Blastocystis sp. in pigs in Anhui Province].

OBJECTIVE: To investigate the prevalence and subtype distribution of Blastocystis sp. in pigs in Anhui Province. METHODS: A total of 500 stool samples were collected from large-scale pig farms in Bozhou, Anqing, Chuzhou, Hefei, Fuyang, and Lu'an cities in Anhui Province from October to December 2015. Blastocystis was detected in pig stool samples using a PCR assay based on the small subunit ribosomal RNA (SSU rRNA) gene, and positive samples were subjected to sequencing and sequence analysis. Blastocystis subtypes were characterized in the online PubMLST database, and verified using phylogenetic tree created with the neighbor-joining algorithm in the Meta software. RESULTS: The prevalence of Blastocystis infection was 43.2% (216/500) in pigs in 6 cities of Anhui Province, and all pig farms were tested positive for Blastocystis. There was a region-specific prevalence rate of Blastocystis (17.2% to 50.0%) (χ2 = 26.084, P < 0.01), and there was a significant difference in the prevalence of Blastocystis sp. among nursery pigs (39.6%), preweaned pigs (19.1%), and growing pigs (62.3%) (χ2 = 74.951, P < 0.01). Both online inquiry and phylogenetic analysis revealed ST1, ST3, and ST5 subtypes in pigs, with ST5 as the predominant subtype. CONCLUSIONS: The prevalence of Blastocystis sp. is high in pigs in Anhui Province, with three zoonotic subtypes identified, including ST1, ST3, and ST5.

Effects of Lactiplantibacillus plantarum and Lacticaseibacillus paracasei supplementation on the single-cell fecal parasitome in children with celiac disease autoimmunity: a randomized, double-blind placebo-controlled clinical trial.

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 < 0.03). Bacterial composition was influenced by the presence of the protists: positivity of Blastocystis was inversely associated with Akkermansia (at the levels of the genus as well as its family, order, class and phylum); P < 0.002), Faecalibacterium (P = 0.003) and Romboutsia (P = 0.029); positivity of D. fragilis was inversely associated with families Enterobacteriaceae (P = 0.016) and Coriobacteriaceae (P = 0.022) and genera Flavonifractor (P < 0.001), Faecalibacterium (P = 0.009), Lachnoclostridium (P = 0.029), Ruminococcus (P < 0.001) and Granulicatella (P = 0.018). CONCLUSIONS: The prevalence of single-cell protists is low in children with celiac disease autoimmunity. The colonization was stable regardless of the probiotic intervention and associated with increased diversity of the fecal bacteriome but inversely associated with some beneficial bacteria.

Prevalence and distribution of subtypes of Blastocystis in Asiatic brush-tailed porcupines (Atherurus macrourus), bamboo rats (Rhizomys pruinosus), and masked palm civets (Paguma larvata) farmed in Hainan, China.

Blastocystis sp. is an important gastrointestinal parasite with global distribution, prevalent in humans, farmed animals, and wildlife. Therefore, this study aimed to investigate the prevalence and genetic diversity of Blastocystis sp. in Asiatic brush-tailed porcupines (Atherurus macrourus), bamboo rats (Rhizomys pruinosus), and masked palm civets (Paguma larvata) in Hainan Province, China. A total of 900 fecal samples were collected from three farmed animal species including 257 porcupines, 360 rats, and 283 civets. Genomic DNA was extracted from each fecal sample and Blastocystis sp. was detected by PCR at the small subunit ribosomal RNA (SSU rRNA) gene. A phylogenetic tree was constructed using the maximum likelihood method. Blastocystis sp. was detected in 47 (5.2%) fecal samples: 12 (4.7%) Asiatic brush-tailed porcupines, 8 (2.2%) bamboo rats, and 27 (9.5%) masked palm civets. Three known Blastocystis sp. subtypes, including ST1, ST4, ST5, and one unnamed subtype (unST), were found in one, 19, 26, and one animal, respectively. Subtypes ST4 and unST were detected in porcupines, ST4 in rats, and ST1 and ST5 in civets. Our results suggest that the three farmed animal species reported in this study could serve as reservoirs for potentially zoonotic Blastocystis sp. subtypes and transmit this parasite to humans, other farmed animals, and wildlife.

Title: Prévalence et répartition des sous-types de Blastocystis chez les athérures à longue queue (Atherurus macrourus), les rats des bambous (Rhizomys pruinosus) et les civettes masquées (Paguma larvata) élevés en Chine dans le Hainan. Abstract: Blastocystis sp. est un parasite gastro-intestinal important avec une distribution mondiale, répandu chez les humains, les animaux d'élevage et la faune. Par conséquent, cette étude visait à étudier la prévalence et la diversité génétique de Blastocystis sp. chez les athérures à longue queue (Atherurus macrourus), les rats des bambous (Rhizomys pruinosus) et les civettes masquées (Paguma larvata) dans la province de Hainan, en Chine. Au total, 900 échantillons fécaux ont été collectés sur ces trois espèces animales d'élevage dont 257 athérures, 360 rats et 283 civettes. L'ADN génomique a été extrait de chaque échantillon fécal et Blastocystis sp. a été détecté par PCR au niveau du gène de la petite sous-unité de l'ARN ribosomal. Un arbre phylogénétique a été construit en utilisant la méthode du maximum de vraisemblance. Blastocystis sp. a été détecté dans 47 (5,2 %) échantillons fécaux : 12 (4,7 %) athérures, 8 (2,2 %) rats et 27 (9,5 %) civettes. Trois sous-types de Blastocystis sp., dont ST1, ST4, ST5 et un sous-type sans nom (unST), ont été trouvés respectivement chez 1, 19, 26 et 1 animal. Les sous-types ST4 et unST ont été détectés chez les athérures, ST4 chez les rats et ST1 et ST5 chez les civettes. Nos résultats suggèrent que les trois espèces animales d'élevage concernées par cette étude pourraient servir de réservoirs à des sous-types potentiellement zoonotiques de Blastocystis sp. et transmettre ce parasite aux humains, à d'autres animaux d'élevage et à la faune.

Development and application of recombinase polymerase amplification assay for rapid detection of Blastocystis sp.

Blastocystis sp. is a common parasite in the intestinal tract of humans and animals. The clinical diagnosis of Blastocystis sp. mainly depends on the microscopic observation of parasite, which can lead to false-negative results. An accurate and convenient diagnostic approach for Blastocystis sp. infection is crucial for effectively preventing and controlling blastocystosis. Herein, we developed a recombinase polymerase amplification (RPA) method for detecting Blastocystis sp. The results showed that the DNA amplification by RPA established in this study could be performed within 5 min at 37°C, with maximum band intensity observed at 30 min. The minimum detection limit of RPA was 100 fg µL−1, consistent with conventional polymerase chain reaction (cPCR). Furthermore, the RPA method exhibited no cross-reactivity with 7 other non-target pathogens in the intestinal tract. Next, the newly established RPA method was used to analyse 40 fecal samples collected clinically, and the detection results were consistent with cPCR. These results corroborate that the newly developed RPA method has good sensitivity and specificity and offers the advantage of short detection times, which can be harnessed for differential diagnosis and rapid detection of Blastocystis sp.

Prevalence of Blastocystis and Dientamoeba fragilis in diarrheal patients in Corum, Türkiye.

To investigate the prevalence of Blastocystis and Dientamoeba fragilis in diarrhea patients and healthy individuals in Corum, Türkiye, fecal samples from 92 diarrhea patients and 50 healthy individuals were collected and evaluated using direct microscopy and molecular methods to screen for bacteria, protozoa, and viruses. The prevalence of Blastocystis was 24.6% in total and more frequent in the healthy group (30.0%). The commonly detected STs (subtypes) were ST3 (40.0%) and ST2 (34.2%). The distribution of Blastocystis STs in the healthy and diarrheal groups did not show any difference in sex and age, but ST3 was detected more frequently in patients aged from 40 to 59 years (p < 0.05). Alleles 4 (8/12) and 2 (4/12) were present in ST1; 9 (3/5) and 12 (2/5) in ST2; 34 (9/14), 36 (3/14), and 38 (2/14) in ST3; and only allele 42 (2/2) in ST4. D. fragilis was present in 8.4% of the population. However, there was no statistically significant difference between the healthy and diarrheic groups (12.0% and 6.5%, respectively), neither with respect to age nor sex. Co-infection was 58.3% and was more frequent in healthy individuals (33.3%) than in diarrhea patients (25.0%). Blastocystis ST3 was the most common subtype detected, with D. fragilis at 33.3%. Salmonella, Shigella, or helminth eggs were not observed in all groups, but Entamoeba histolytica, Giardia intestinalis, Cryptosporidium, Rotavirus, Adenovirus, and Clostridium difficile toxin were found only in diarrhea patients. These findings support the hypothesis that Blastocystis and D. fragilis may be part of the healthy human gut microbiome.

Prevalence and genetic diversity of Blastocystis sp. among autochthonous and immigrant patients in Italy.

The prevalence of Blastocystis sp., its genetic diversity and the distribution of circulating subtypes (STs) were molecularly investigated in a cohort of autochthonous and immigrant patients with gastrointestinal symptoms hospitalized over the period February 2022-June 2023 at the Policlinico Ospedaliero-Universitario "Riuniti", Foggia, in Southern Italy. The population variables, including patient geographical origin, gender and age classes were reported. Out of the 927 investigated patients, 36 (3.9%) were positive for Blastocystis sp. A statistically significant association with African origin and age classes >18 years old was found. ST1 (allele 4), ST2 (alleles 9, 13), ST3 (alleles 34, 36) and ST4 (allele 92) were the subtypes detected with a different distribution between autochthonous and immigrant patients. Co-infections with enteric protozoa such as Giardia duodenalis and Dientamoeba fragilis, pathogenic bacteria as Clostridioides difficile, Campylobacter jejuni and Aeromonas sp. and viral infections such as Norovirus were found in 33% of cases. This is the first study of Blastocystis sp., its circulating subtypes and allele variability among patients with different geographical origin in an area of Southern Italy, in the Central Mediterranean, characterized by high immigrant pressure. These results provide baseline data to better investigate a potential interaction between Blastocystis sp. and other risk factors in patients with gastrointestinal symptoms.

Subtype Distribution of Blastocystis in Türkiye.

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.

Genetic characteristics of Blastocystis sp. ST3 at the genome level in the Chinese population.

The gut microbiota comprises the collective genomes of microbial symbionts and is composed of bacteria, fungi, viruses, and protists within the gastrointestinal tract of a host. Although the literature associated with gut microbiota is increasing, studies on eukaryotes in the human gut are just beginning to surface. Blastocystis is one of the most common intestinal parasites of humans and animals and is estimated to colonise more than 1 billion people on a global scale. However, the understanding of the genetic characteristics of Blastocystis subtype (ST) at the genome level and its relationship with other members of the gut microbiota is still limited. In this study, by surveying the prevalence and genome characteristics of Blastocystis sp. ST3 in a Chinese population (prevalence % = 6.09%), the association of Blastocystis sp. ST3 with region and time and the structure of the resident gut bacterial population was clarified. We identified novel sequences (50 mitochondrial and 41 genome sequences) and determined their genetic diversity amongst strains within Blastocystis sp. ST3 (4.14 SNPs/kb). Furthermore, we found that colonisation of Blastocystis was strongly associated with increased bacterial richness and higher abundance of several anaerobes. Finally, we performed time series sampling on two Blastocystis-positive individuals and confirmed that Blastocystis could exist continually in the human gut microbiota and persist for a long time, even for 4 years.

Detection and Molecular Characterization of Blastocystis Species in Polish Soldiers Stationed in the Republic of Kosovo.

Blastocystis species (sp.) is one of the less well-understood water- and foodborne protozoa of medical and veterinary importance linked to different gastrointestinal disorders. Soldiers participating in military missions are particularly vulnerable to infection with this protozoa. The present study used molecular methods to detect, identify, and subtype (ST) Blastocystis sp. in Polish soldiers stationed in the Republic of Kosovo. Fecal samples were collected from 192 soldiers on arrival and after four months of stay. After DNA extraction, the barcoding region of the small subunit ribosomal RNA (SSU-rRNA) gene was amplified and sequenced. The DNA of Blastocystis sp. was detected in six (3.13%) and thirty (15.16%) samples in the first and second batch, respectively. Sequencing analysis revealed infections with ST 2, 3, 4, and 7. There was no statistical association between Blastocystis sp. infection and the parasite's ST or the age or rank of soldiers. The results indicate that the visit to a new environment and prolonged stay in the area of military operation in Kosovo resulted in a significant increase in both Blastocystis sp. infections and ST diversity among surveyed soldiers. This shows the need to undertake appropriate countermeasures to reduce Blastocystis infections in the military environment abroad.

Dientamoeba fragilis associated with microbiome diversity changes in acute gastroenteritis patients.

This study examined the correlation between intestinal protozoans and the bacterial microbiome in faecal samples collected from 463 patients in New Zealand who were diagnosed with gastroenteritis. In comparison to traditional microscopic diagnosis methods, Multiplexed-tandem PCR proved to be more effective in detecting intestinal parasites. Among the identified protozoans, Blastocystis sp. and Dientamoeba fragilis were the most prevalent. Notably, D. fragilis was significantly associated with an increase in the alpha-diversity of host prokaryotic microbes. Although the exact role of Blastocystis sp. and D. fragilis as the primary cause of gastroenteritis remains debatable, our data indicates a substantial correlation between these protozoans and the prokaryote microbiome of their hosts, particularly when compared to other protists or patients with gastroenteritis but no detectable parasitic cause. These findings underscore the significance of comprehending the contributions of intestinal protozoans, specifically D. fragilis, to the development of gastroenteritis and their potential implications for disease management.