Prevalence of Blastocystis in Patients Referred to Bushehr Medical Centers and Its Relationship with Urticaria.

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.

Chronic urticaria associated with Blastocystis hominis infection.

Urticaria is a skin rash with several etiologic factors, including infectious agents. Blastocystis hominis is an intestinal protozoan parasite that has been linked to urticaria and skin lesions. The aim of this work was to investigate the association between B. hominis infection and chronic urticaria. In a case-control study, stool samples were obtained from 94 patients with chronic urticaria as case group and 285 healthy individuals as control group. Urticaria activity score 7 (UAS7) was used to score the severity of urticaria, classified as mild, moderate and intense. All stool samples underwent routine stool examinations, as well as polymerase chain reaction (PCR) for the detection of B. hominis. Molecular detection was carried out using the small subunit ribosomal RNA (SSU-rRNA) gene and the parasite subtypes were determined by sequencing. The rate of B. hominis infection was 21.3% (20 out of 94) and 17.2% (49 out of 285) between the case and control groups, respectively (p = 0.463). Three subtypes of B. hominis, including ST-1, ST-2 and ST-3, were detected in the case and control groups (ST-1 = 30% vs. 8.3%, ST-2 = 40% vs. 25% and ST-3 = 30% vs. 66.6% in the case and control group, respectively), which was statistically significant (p = 0.00001). However, no statistical differences were found between the severity of the urticaria and the B. hominis subtypes (p = 0.533). This study revealed a higher prevalence (but not significant) of B. hominis infection among patients with urticaria than healthy individuals. However, the results did not find a significant association between the subtypes of B. hominis and the severity of urticaria.

Genetic diversity and host specificity of Blastocystis in reptiles, Eastern Thailand.

Blastocystis inhabits the digestive tracts of a diverse range of hosts. Transmission patterns, including host specificity, and the clinical and public health significance of Blastocystis in humans remain poorly understood. This study aimed to investigate the distribution and genetic diversity of Blastocystis in herbivorous and carnivorous reptiles in Eastern Thailand. A total of 501 faecal samples were collected from 363 iguanas, 79 bearded dragons, 50 tortoises, and nine snakes in an animal breeding farm in Chonburi Province, Eastern Thailand. Detection and differentiation of Blastocystis was based on amplification, sequencing, and phylogenetic analysis of specific small subunit (SSU) ribosomal RNA genes from faecal DNA extracted from the samples. Altogether 101/501 samples (20 %) were polymerase chain reaction (PCR) and sequencing-positive for Blastocystis, 90 (89 %) of which were from iguanas; the remaining positive samples were from African spurred tortoise (n=6), Bearded dragon (n=3), Leopard tortoise (n=1), and Red-footed tortoise (n=1). Phylogenetic analysis revealed that most of the Blastocystis sequences from iguanas were largely similar, and they were distinct from those of the tortoises. Subtype 17 was found in the three bearded dragons and likely reflected Blastocystis from prey animals. This is the largest survey of Blastocystis in reptiles to date. Remarkable differences in Blastocystis colonization rates and genetic diversity were observed between iguanas and other reptile orders, and what was considered Blastocystis colonization was only observed in herbivorous reptiles.

Molecular screening for enteric parasites and subtyping of Blastocystis sp. in haemodialysis patients in Slovakia.

INTRODUCTION AND OBJECTIVE: Intestinal parasitoses are important causes of morbidity and mortality, especially in immunocompromised individuals. In patients with chronic renal insufficiency (CRI), the accumulation of non-excreted metabolites leads to uraemia, which induces a state of immunodeficiency, increasing the incidence of infections. The aim of the study was molecular screening for enteric protozoa in patients with chronic renal insufficiency. MATERIAL AND METHODS: A total of 53 samples were collected in January 2023 from patients undergoing dialysis at Logman Ltd. Nephrodialysis Centre in Kosice, Slovakia. Samples were examined by polymerase chain reaction (PCR) for the presence of Cryptosporidium parvum / Cryptosporidium hominis, Giardia intestinalis, Microsporidia spp., and Blastocystis sp. RESULTS: From the 53 samples, the only pathogen identified by PCR was Blastocystis sp., in 13 patients (24.5 %). Sequence analyses confirmed that the most prevalent subtype (ST) among patients was ST 3 (n=9, 69.2%), followed by ST 1 (n=3, 23.1%) and ST 2 (n=1, 7.7%). CONCLUSIONS: Molecular methods for the detection of microscopic enteric parasites are not used as a first-line diagnostic method in Slovakia. In immunocompromised patients, diarrhoea can be caused not only by a chronic disease or therapy but can also be a result of an ongoing underdiagnosed infection. Early diagnosis leads to targeted therapy and subsequent partial improvement of the quality of life. This study also shows the first insights into Blastocystis sp. subtype distribution in humans in Slovakia.

Molecular investigation of Blastocystis sp. infections in wild rodents from the Inner Mongolian Autonomous Region and Liaoning province, China: High prevalence and dominance of ST4.

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.

Title: Enquête moléculaire sur les infections à Blastocystis chez des rongeurs sauvages de la région autonome de Mongolie intérieure et de la province du Liaoning, Chine : forte prévalence et dominance du sous-type ST4. Abstract: Les rongeurs sauvages sont des vecteurs clés de divers agents pathogènes humains, dont Blastocystis spp. Notre étude visait à évaluer la prévalence et les caractéristiques génétiques de Blastocystis chez les rongeurs sauvages de la région autonome de Mongolie intérieure et de la province chinoise du Liaoning. De novembre 2023 à février 2024, 486 rongeurs ont été capturés dans ces régions. Des matières fécales fraîches ont été collectées dans les intestins de chaque rongeur pour l'isolement de l'ADN et l'amplification par PCR du gène du cytochrome b des vertébrés (cytb) afin d'identifier les espèces de rongeurs. Par la suite, l'analyse PCR et le séquençage de la petite sous-unité partielle du gène de l'ARN ribosomal (ARNr) ont été utilisés pour détecter les Blastocystis dans tous les échantillons fécaux. Sur le total des échantillons, 27.4% (133/486) présentaient un résultat positif à Blastocystis. Les résultats ont révélé la présence de quatre espèces de rongeurs infectées par Blastocystis, 32.3% (63/195) chez Rattus norvegicus, 15.1% (16/106) chez Mus musculus, 20.2% (18/89) chez Apodemus agrarius et 37.5% (36/96) chez Cricetulus barabensis. L'analyse de séquence a confirmé l'existence de cinq sous-types de Blastocystis : ST1 (n = 4), ST2 (n = 2), ST4 (n = 125, le sous-type dominant), ST10 (n = 1) et un nouveau ST (n = 1). Les sous-types zoonotiques identifiés (ST1, ST2, ST4 et ST10) mettent en évidence le rôle possible joué par les rongeurs sauvages dans la transmission de Blastocystis à l'Homme, augmentant ainsi les risques d'infection humaine. Parallèlement, la découverte de nouvelles séquences fournit également de nouvelles informations sur la diversité génétique de ce parasite.

Recombinase polymerase amplification - lateral flow dipstick for rapid and visual detection of Blastocystis spp.

Blastocystis spp. is a ubiquitous protozoon in the intestinal tract of human and many animals. Microscopic examination is the main method of clinical diagnosis for Blastocystis spp., which is prone to false negative. A simple and rapid diagnosis of Blastocystis spp. infection is an important step to prevent and control blastocystosis. Here, a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay was developed for rapid visual detection of Blastocystis spp. DNA amplification could be performed within 18 min at 37°C. The minimum DNA detection limit was 1 pg/µL, and there was no cross-reactivity with 12 other non-target pathogens, which was consistent with the sensitivity of conventional PCR (cPCR). Furthermore, 56 fecal samples from the Third Affiliated Hospital of Xinxiang Medical University were tested using RPA and cPCR methods respectively, and the results were completely consistent. The results show that RPA-LFD method has high accuracy and visual results, which provides a new choice for the differential diagnosis and rapid field detection of Blastocystis spp.

Microscopic and molecular prevalence and associated risk factors with Toxocara and Blastocystis infection in dogs and cats in Mitidja, Algeria.

Domestic dogs and cats can serve as a source of environmental contamination with Toxocara spp. and Blastocystis spp., and this represents a neglected public and veterinary health problem. We assessed the microscopic and molecular prevalence of these species in a locality in Algeria and identified the associated risk factors. The faeces of 225 dogs and 78 cats were collected in Mitidja between March and July 2022. The samples were analysed by coproscopy and by polymerase chain reaction (PCR) targeting the Internal Transcribed Spacer 2 (ITS2) and Small Subunit Ribosomal (SSU-RNA) of T. canis and Blastocystis spp. respectively. The overall microscopic prevalence of Toxocara spp. in dogs and cats was 9.78 ± 1.98% and 12.82 ± 7.42%, respectively. The rate of Blastocystis spp. was 15.11 ± 2.39% and 15.38 ± 4.08% in dogs and cats, respectively while the molecular prevalence of T. canis in dogs was 4.89 ± 1.44% and in cats 1.28 ± 1.27%; the prevalence of Blastocystis spp. was 41.78 ± 3.29% and 34.62 ± 5.39% in dogs and cats, respectively. Phylogenetic and phylogeographic analyses identified the presence of the H1 subtype of T. canis in dogs, and the ST1 subtype of Blastocystis in dogs and cats. Dogs with clinical signs were more likely to be infected with T. canis (OR 6.039, P < 0.05) than healthy dogs. This study demonstrates that dogs and cats are carriers of Toxocara spp. and Blastocystis spp. and are therefore a source of environmental contamination. Veterinarians and human health professionals should work together to implement control strategies as part of a "One Health" approach to improving animal health and reducing the risk of transmission to humans.

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.

[Genetic Diversity of Blastocystis in Diarrheal Cases: Identification of Subtypes and Alleles]. / Ishalli Olgularda Blastocystis'in Genetik Çesitliligi: Alt tipler ve Alellerin Belirlenmesi.

Blastocystis spp. are the most common intestinal protozoan parasites detected in human stool samples. While identified long before today, its pathogenicity remains controversial. It is generally asymptomatic but in symptomatic cases, many gastrointestinal symptoms, especially diarrhea, have been associated with Blastocystis infection. In recent years, the relationship between the symptoms observed in cases and Blastocystis subtypes (ST) has been reported. The aim of this study was to detect Blastocystis in diarrheal cases admitted to the Aydin Adnan Menderes University Faculty of Medicine, Department of Parasitology Laboratory, to determine subtypes and allele diversity and to investigate its relationship with clinical symptoms. For this purpose, diarrheal stool samples of 200 cases were included in the study and their demographic characteristics (age, gender, residence) and clinical findings (abdominal pain, dyspepsia, nausea-vomiting, weakness, weight loss, anal itching, rash, urticaria) were recorded. Blastocystis was detected by direct microscope method (DM) and by molecular analyses which were performed with polymerase chain reaction (PCR). Subtype diversity was determined based on DNA sequence analysis by PCR targeting the Blastocystis ribosomal ribonucleic acid small subunit (SSU rRNA) gene. In addition, alleles related to Blastocystis subtypes were determined and statistically compared between all data and clinical findings. In the current study, Blastocystis was detected in 31 (15.5%) samples by DM and in 35 (17.5%) samples by PCR specific to the Blastocystis SSU rRNA gene among 200 diarrheal stool samples. No statistical difference was detected between Blastocystis and demographic characteristics. Dyspepsia and nausea-vomiting symptoms differed significantly in cases with Blastocystis compared to negative ones (p= 0.0025, p= 0.0498). Blastocystis subtype was detected in 33 samples by SSU rRNA sequence analysis, and the subtype distribution was ST1 (n= 10, 30.3%), ST2 (n= 4, 12.1%) and ST3 (n= 19, 57.6%). In the statistical evaluation between clinical findings and Blastocystis subtypes, a relationship was found between dyspepsia and Blastocystis ST3 (p= 0.0039). The allele diversity of Blastocystis subtypes was determined as allele 4 (10/10) in all ST1, allele 11 (2/4) and 12 (2/4) in ST2, allele 34 (14/19), 36 (4/19), and 38 (1/19) in ST3. In conclusion, our study provides important data on the molecular epidemiological characteristics of the Blastocystis by determining positivity, subtypes and alleles in diarrheal cases. Therefore, within the scope of the one health approach, comprehensive molecular epidemiological studies are required to determine the presence and genotypes of Blastocystis in human, animal and environmental samples.

Molecular detection and genotyping of Dientamoeba fragilis and Blastocystis sp. in housefly Musca domestica (Diptera: Muscidae): first report for Dientamoeba fragilis.

Dientamoeba fragilis and Blastocystis sp. are single-celled protozoan parasites of humans and animals. Although they are found in the intestines of healthy hosts, the pathogenicity of them is still unclear. To date, there is no report on D. fragilis and only two studies (without subtyping) on the occurrence of Blastocystis sp. in Musca domestica. In this study, fly samples were collected from livestock farms and their surroundings in the Kirsehir province (Central Anatolia Region) of Türkiye from May to August 2023. A total of 150 microscopically identified M. domestica samples were analyzed for the detection of D. fragilis and Blastocystis sp. molecularly. The overall prevalence of Blastocystis sp. and D. fragilis in M. domestica was determined to be 3.3% (5/150) and 8.0% (12/150), respectively. The SSU rRNA gene sequences of the isolates indicated genotype 1 of D. fragilis. Eleven isolates were identical and represented a single isolate (KAU-Dfrag1). BLAST analysis of KAU-Dfrag1 indicated identity with the isolates reported from humans, cattle, sheep, and budgerigars. The other isolate (KAU-Dfrag2) was polymorphic at two nucleotides from KAU-Dfrag1 and three nucleotides from known genotypes from GenBank and represented a variant of genotype 1. The Blastocystis sp. isolates were found to be identical and represent a single genotype (KAU-Blast1). BLAST analysis revealed that the KAU-Blast1 genotype belonged to the potentially zoonotic subtype 5 (ST5) and exhibited the highest genetic identity (ranging from 99.4 to 99.6%) with pigs, cattle, and sheep from different countries. Our study provides the first data on the molecular prevalence, epidemiology, and genotypic characterization of D. fragilis and Blastocystis sp. in M. domestica.

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.

Identification of Blastocystis spp. in Urban Rodents of Different Districts in Southwestern Iran: Subtype Distribution and Possible Zoonotic Potential.

PURPOSE: Rodents are one of the most abundant and diverse species of mammals and have recently been identified as carriers of numerous human pathogens. The current study was conducted to assess the prevalence, subtype (STs) distribution, and zoonotic potential of Blastocystis spp. in various species of rodents in Shiraz, southwestern Iran. METHODS: For this aim, a total of 120 fresh fecal samples were collected from Mus musculus (n = 40), Rattus norvegicus (n = 40), and Rattus rattus (n = 40) in various municipality districts of Shiraz (6 out of 10 districts) between February and November 2020. Upon detecting parasites using light microscopy, a DNA fragment of the Blastocystis SSU rDNA gene was amplified using conventional PCR. RESULTS: By employing direct wet mount examination, 8 out of 120 fecal samples (6.7%; 2 from house mice, 3 from black rats, and 3 from brown rats) tested positive. Similarly, 5% (2/40) of house mice, 7.5% (3/40) of black rats, and 7.5% (3/40) of brown rats tested positive using the molecular method. Phylogenetic analysis revealed that the Blastocystis infecting different rodent species in Shiraz belonged to two potentially zoonotic STs (ST1 and ST4). Accordingly, rodents should not be overlooked as potential reservoirs of zoonotic Blastocystis infections. Different sampled urban districts and their statistical association with reported prevalence rates were analyzed separately. CONCLUSION:  Overall, the issue of the frequency and ST distribution of Blastocystis in urban rodents of Iran is still open to question and for a proper understanding, wider and more comprehensive studies are needed.

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.

Detection of Blastocystis sp. and Dientamoeba fragilis using conventional and molecular methods in patients with celiac disease.

Blastocystis sp. and Dientamoeba fragilis are intestinal protists, which are common worldwide, but the pathogenic role of these organisms in gastrointestinal diseases is still controversial. This study aimed to investigate the frequency of Blastocystis sp. and D. fragilis in stool samples from adult patients with celiac disease (CD) by using conventional and molecular methods. A total of 75 patients with CD and 75 healthy individuals were included in this study. Fresh stool specimens collected from each individual were analyzed by conventional and molecular methods. The overall prevalence of Blastocystis sp. and D. fragilis was 41.3% (31/75) and 24% (18/75) in patients with CD, and 46.7% (35/75) and 13.3% (10/75) in healthy controls, respectively. There was no statistically significant difference in the prevalence of Blastocystis sp. and D. fragilis between CD patients and healthy individuals. Blastocystis sp. subtypes were identified in 20 CD and 16 control patients and the overall subtype distribution was observed as ST1 13.9%, ST2 30.6%, and ST3 55.6%. The prevalence of Blastocystis sp. and D. fragilis in adults with CD is similar to the prevalence of protozoa in healthy adults. In this study, the most prevalent Blastocystis subtype was ST3 and the most frequent allele was a34 in both CD patients and healthy individuals. No significant difference was found between the two groups in terms of the detection rates of Blastocystis sp. and D. fragilis, and it is thought that both protists may be colonisers of the intestinal microbiome.

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.

Irritable Bowel Syndrome Associated with Blastocystis hominis or Without Relationship to It? A Case-Control Study and Minireview.

BACKGROUND: Blastocystis hominis (B. hominis) is a protozoan parasite that has a worldwide distribution. Some studies have suggested a link between B. hominis and the development of irritable bowel syndrome (IBS). The objective of this study was to determine the prevalence of B. hominis in patients with IBS compared to healthy individuals. MATERIAL AND METHODS: A total of 65 stool samples from patients with IBS and 65 samples from healthy individuals in northern Iran were examined. The samples were tested using various methods including direct smear, formalin ether sedimentation and culture to detect the presence of B. hominis. Additionally, polymerase chain reaction (PCR) was performed on all culture-positive isolates to confirm the results and identify the genotype. RESULTS: B. hominis was detected in 15.38% of IBS patients and 9.2% of the healthy group. The culture in RPMI1640 was found to be better than the formalin ether and direct smear methods. Positive samples were confirmed using the molecular method. No significant difference was observed in the order of B. hominis infection between the two groups. CONCLUSIONS: The results of our study indicate that no significant difference was observed in the order of B. hominis infection between IBS patients and healthy groups. Therefore, further study is necessary to determine the potential pathogenic effects of this parasite and its role in causing IBS.

First Molecular Identification and Subtyping of Blastocystis sp. in the Most Consumed Edible Marine Fish of Iran: A Foodborne Concern.

PURPOSE: The presence of Blastocystis sp. is commonly observed in humans and different animals, displaying a wide range of genetic variations with the discovery of multiple subtypes (STs). However, the prevalence and distribution of these STs in edible marine fish and marine mammals remain uncertain. This study marks the first survey conducted in Iran and the second global molecular investigation to examine the occurrence and STs distribution of Blastocystis in various species of edible marine fish. METHODS: This study screened 200 fresh intestinal contents from 10 well-known fish species (Narrow-barred mackerel, Indo-pacific king mackerel, Tigertooth croaker, Silver pomfret, Black pomfret, Longtail tuna, John's snapper, Blackspotted croaker, Four-finger threadfin, and Javelin grunter) in southern Iran, caught in the Persian Gulf. All collected samples were evaluated by microscopy and SSU-PCR methods. RESULTS: Based on both microscopy and PCR, the overall prevalence of Blastocystis sp. in evaluated fish species was 2% (4/200). In brief, Blastocystis sp. was reported from Narrow-barred mackerel [10% (2/20)], Silver pomfret [5% (1/20)], and Tigertooth croaker [5% (1/20)]. Interestingly, among infected fish species three zoonotic STs (ST1, ST2, and ST7) were identified. ST2 was the most predominant ST [50% (2/4)], followed by ST1 and ST7, one sample each [5% (1/20)]. CONCLUSION: Overall, the prevalence and STs distribution of Blastocystis in edible marine fish along with the possibility of its zoonotic transmission are still open to question and require extensive and more detailed studies.

First molecular characterization of Blastocystis subtypes from domestic animals (sheep and cattle) and their animal-keepers in Ilam, western Iran: A zoonotic concern.

A total of 360 fecal samples were randomly collected from 150 cattle, 150 sheep, and 60 humans (30 people with close animal contact and 30 individuals without close animal contact) at 10 farms in Ilam, western Iran from June 2022 to August 2023. All samples were directly examined for Blastocystis by zinc sulfate flotation, followed by microscopic observation. Positive samples were further subtyped using conventional PCR and sequencing methods. A mean prevalence of 5.3% (16/300) was estimated for Blastocystis infection among examined animals, with 6% and 4.7% for cattle and sheep, respectively. Among the people who had close and non-close animal contact, 16.7% (5/30) and 3.3% (1/30) were infected with Blastocystis, respectively (p < 0.05). All 22 positive samples were successfully sequenced at the SSU rRNA locus. Accordingly, Blastocystis isolates infecting domestic animals in Ilam belonged to the four STs (ST1-ST3, and ST10). Of the 16 animal isolates, nine sequences (four ST10, three ST3, and two ST1) were related to cattle, and seven sequences (three ST10, two ST3, and two ST2) were isolated from sheep. Among the six human isolates, ST3 was the most predominant ST, followed by STs 1, 2, 6, and 7 (one case each). Of note, ST1-ST3 were isolated in various farms both from animals and their breeders, which indicates the possible circulation of these STs between animal and human populations.

Comparative molecular epidemiology, subtype distribution, and zoonotic potential of Blastocystis sp. in Equus animals (horses, donkeys, and mules) in northwestern Iran.

A total of 500 fecal samples were collected from Equus animals in six different cities (Ardabil, Namin, Nir, Meshginshahr, Germi, and Khalkhal) of Ardabil Province, northwestern Iran, with 200 samples from horses, 200 from donkeys, and 100 from mules. Of the horse samples, 100 were from racing horses under special monitoring and care, while the remaining 100 were from non-racing horses, including those used for herding or in rural areas. All fecal samples were examined for the presence of Blastocystis sp. using PCR amplification of the SSU rRNA gene's barcode region after DNA extraction. The molecular prevalence of Blastocystis infection in Equus animals was 7.6% (38/500). Blastocystis was more common in horses [11.5% (23/200)] than in donkeys [5.5% (11/200)] and mules [4% (4/100)] (P > 0.05). Compared to racing horses [3% (3/100)], non-racing/rural horses [20% (20/100)] exhibited a substantially higher prevalence of Blastocystis (P < 0.05). The prevalence of Blastocystis in diarrheal samples and younger animals was remarkably higher than in formed samples and older animals, respectively (P < 0.05). No significant difference in Blastocystis infection prevalence was found between the genders of examined animals (P > 0.05). In Equus animals, 38 Blastocystis isolates included eight STs: ST10 [31.6% (12/38)], ST1 [21.1% (8/38)], ST2 [15.8% (6/38)], ST3 [10.5% (4/38)], ST4 [7.9% (3/38)], ST7 [5.2% (2/38)], ST14 [5.2% (2/38)], and ST6 [2.6% (1/38)]. These results suggest that Equus animals act as a proper reservoir for numerous Blastocystis STs, consequently playing a crucial part in the spread of this protozoan infection to humans, animals, and water reservoirs.