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.

[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.

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 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.

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.

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 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.

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.

Blastocystis: view from atop the gut-brain iceberg.

Blastocystis is a common intestinal parasite that has been linked to gut pathology in humans. In this article, we highlight recent publications that offer insight into how these organisms can influence human cognition and the gut microbiome. We also suggest a potential mechanism of action by which this might occur.

The Significance of Opportunistic Parasitosis and Blastocystosis in Patients with Gastric Cancer: a Study with Control Group.

Objective: The aim of this study was to determine the prevalence of opportunistic parasites and Blastocystis spp. in patients with gastric cancer (CA) and to determine the significance of these parasite. Methods: The patient group and the control group were composed of 100 people each. The stool samples were examined under the microscope for intestinal parasites with the native-Lugol method. Then, samples were multiplied by formol-ethyl acetate method and stained with modified acid-fast method. Results: Intestinal parasite positivity was indicated in 14% of the gastric CA, and 2% of the healthy individuals (p=0.001). Blastocystis spp. (p=0.009) was identified in 11%, Cryptosporidium spp. was identified in 4%, G. intestinalis was identified in 2%, and C. cayetanensis was identified in 1% of the patient group. There were significant differences between the intestinal parasite positivity (p=0.012), abundant Blastocystis spp. positivity (p=0.041) and all Blastocystis spp. positivity (p=0.037) in patient and control groups. Most of the patients who were positive for parasites had diarrhea. Conclusion: Based findings, it was concluded that it would be beneficial to evaluate gastric CA patients, especially those with diarrhea, for intestinal parasites.

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.

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.

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.

Metataxomics reveals Blastocystis subtypes mixed infections in Colombian children.

Blastocystis sp., is an intestinal protist with a broad host range and a high prevalence in human populations worldwide, even in developed Western countries. The publication of conflicting evidence has divided the scientific community about the pathogenic role of this parasite. Even though, genetic studies on Blastocystis sp. revealed associations between genotypes and different pathogenic profiles. Conventionally, the detection of this parasite is based on microscopic or PCR methods, which offer meager or null performance in detecting mixed infections. In this work, we applied a metataxonomic NGS approach targeting the V4 region of the eukaryotic SSU-rRNA gene and classical phylogenetic methods. This approach allowed us to detect Blastocystis sp. in stool samples from infected children living in an urban setting in the city of Medellin attending the same daycare center. Phylogenetic analysis identified the subtypes present in the children as ST1, ST2, and ST3. Besides, mixed infections of subtypes ST1 + ST3 were spotted in 16% of the analyzed stool samples.

Distribution of Blastocystis subtypes isolated from various animal hosts in Thailand.

Blastocystis is a parasitic protist of a variety of hosts, including humans. Mapping the distribution of Blastocystis and its genetic variants across different host species can help us understand the epidemiology of this organism and its role in health and disease. This study aimed to identify subtypes of Blastocystis detected in different animal hosts in Thailand. A total of 825 fecal samples belonging to 18 vertebrate orders, 36 families, 68 genera, and 80 species were collected. Of these, 111 specimens were Blastocystis-positive by culture. Seventy-nine samples were subjected to small subunit (SSU) ribosomal DNA amplification by PCR, and reliable subtype data were obtained for 61 specimens. At least 14 subtypes (ST), namely ST1 to ST10, ST14/ST24/ST25 complex, ST23, ST26, and ST29 were detected. In addition, Blastocystis was found in tortoises. ST1 (3.2%) and ST5 (11.5%) were found in pigs, ST2 (1.6%) and ST3 (3.2%) in non-human primates, ST4 (14.7%) in rodents and ruminants, ST6 (4.9%), ST7 (30%), ST9 (1.6%), and ST29 (1.6%) in birds, ST8 (6.6%) in Green peafowl and East Asian Porcupine, and ST10 (4.9%), ST14/ST24/ST25 (9.8%), ST23 (1.6%) and ST26 (1.6%) in ruminants. The sequence recovered from the elongated tortoises (Indotestudo elongata) (3.2%) was phylogenetically placed within the reptilian cluster of Blastocystis, for which no subtype system is available yet. Of note, we did not obtain Blastocystis sequences from any of the many canids and felids sampled in the study, and our data are in support of host specificity of Blastocystis, according to both colonization and subtype distribution.

IMPACT OF BLASTOCYSTIS HOMINIS INFECTION ON IMMUNOLOGICAL PARAMETERS IN PATIENTS WITH DIARRHEA: A CROSS-SECTIONAL STUDY.

Blastocystis parasites are common intestinal parasites found in various hosts, including humans. This study involved two groups: the patient group, consisting of 220 samples, and the control group, consisting of 100 samples. The participants' age range was 4-40 years, and the samples were obtained from Al-Kadhimiya Teaching Hospital and Al-Shaheed Mohammed Baqir Al-Hakeem Hospital in Baghdad, Iraq. Stool samples were examined under a light microscope using Lugal's Iodine Stain and direct wet smears. The age group did not show a significant difference (P≥0.05) in patients with diarrhea infected with the Blastocystis hominis parasite compared to the control group. Furthermore, males had a higher infection rate (58.00%) compared to females (42.00%), and this rate was statistically significant (P≤0.05). The aim of this study was to evaluate the effect of Blastocystis hominis infection on the levels of certain immunological parameters. The results of the ELISA technique for immunological examinations revealed a significant increase (P≤0.01) in the levels of IL-10 and IL-17 in the serum of patients with diarrhea infected with the Blastocystis hominis parasite compared to the control group. The immunological tests also showed a significant increase (P≤0.01) in IgG, IgM antibodies, and IgA levels in patients with diarrhea infected with Blastocystis hominis parasite compared to the control group. These findings suggest that Blastocystis infection may influence immunological responses.

Blastocystis infection among diarrhea outpatients in Ningbo, Southeast China: A potential zoonotic health threat.

BACKGROUND: Blastocystis is one of the important zoonotic parasites which can infect humans and various animals worldwide and has become a growing global public health concern. The study aims to obtain the data of Blastocystis infection and the information of the genetic characteristic. METHODS: In the present study, 489 fecal samples were collected from diarrhea outpatients in Ningbo, Zhejiang province, and were examined the presence of Blastocystis by polymerase chain reaction combined with sequencing. RESULTS: A total of 10 samples (2.04%, 10/489) were positive for Blastocystis with no significant difference among sex and age groups, respectively. Eight samples were successfully sequenced, and five zoonotic ST3 and three zoonotic ST1 with two new sequences were identified. CONCLUSIONS: We first demonstrated the occurrence of Blastocystis infection in diarrhea outpatients in Ningbo, with two zoonotic subtypes (ST1 and ST3) and two new sequences being characterized. Meanwhile, mixed infection of Blastocystis and E. bieneusi was found which indicates the importance of investigation of multiple parasites. Finally, more extensive studies will be needed to better understand the transmission of Blastocystis at human-animal-environment interface and provide evidence for the development of one health strategies for the prevention and control of such diseases.

A longitudinal study of Blastocystis in dairy calves from birth through 24 months demonstrates dynamic shifts in infection rates and subtype prevalence and diversity by age.

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.

The role and influence of bacteria on the growth of Blastocystis sp. subtype 3.

Blastocystis sp. is an enteric protistan parasite that affects individuals worldwide with gastrointestinal symptoms such as abdominal discomfort, diarrhea, and flatulence. However, its pathogenicity is controversial due to its presence among asymptomatic individuals. Blastocystis sp. subtype 3 (ST3) is the most prevalent subtype among humans that have been associated with irritable bowel syndrome (IBS), Crohn's disease, ulcerative colitis, and colorectal cancer. Axenization of the parasite has been shown to impede its growth thus revealing the importance of accompanying bacteria in ensuring Blastocystis sp. survival. This study aims to identify the influence of accompanying bacteria on the growth of Blastocystis sp. ST3. Blastocystis sp. cultures were treated with Meropenem, Vancomycin, and Amoxicillin-Clavulanic acid (Augmentin). Bacteria-containing supernatant of antibiotic-treated and control cultures were isolated and identified through 16 s rRNA sequencing. Morphological changes of antibiotic-treated Blastocystis sp. ST3 were also observed. The cultures treated with meropenem and augmentin exhibited opposing effects with reduced growth of isolates from symptomatic patients and a significant increase in asymptomatic isolates. Whereas, vancomycin-treated cultures had no difference in the growth of Blastocystis sp. ST3 isolates from symptomatic and asymptomatic patients. Isolates from symptomatic and asymtomatic patients had 6 and 2 distinct bacterial species identified with Proteus mirabilis as the common bacteria among both types of isolates. Morphologically, Blastocystis sp. ST3 cultures exposed to meropenem and augmentin demonstrated an increase in pre-cystic forms. These findings demonstrate the effects of accompanying bacteria on the growth of Blastocystis sp. ST3 that could translate into clinical manifestations observed among Blastocystis sp.-infected patients.

Prevalence and associated risk factors of intestinal parasites among schoolchildren in Ecuador, with emphasis on the molecular diversity of Giardia duodenalis, Blastocystis sp. and Enterocytozoon bieneusi.

BACKGROUND: Intestinal helminths, including Soil-Transmitted Helminth (STH), and Gastrointestinal Protist (GP) infections are major contributors to the global burden of disease, particularly in low-income countries such Ecuador. Their epidemiology in these settings is largely unknown. METHODOLOGY: This prospective cross-sectional study investigates the carriage of intestinal helminths, including STH, and GP in asymptomatic schoolchildren (3-11 years) in the Chimborazo and Guayas provinces, Ecuador. Single stool samples (n = 372) and epidemiological questionnaires on demographics and potential risk factors were collected from participating schoolchildren. Conventional microscopy examination was used as screening method, and molecular (PCR and Sanger sequencing) assays were used to further investigate the epidemiology of some GP. A multivariate logistic regression analysis was used to evaluate the strength of the association of suspected risk factors with the presence of helminths and GP. PRINCIPAL FINDINGS: At least one intestinal parasite species was observed by microscopy in 63.2% (235/372) of the participating schoolchildren. Enterobius vermicularis (16.7%, 62/372; 95% CI: 13.0-20.9) and Blastocystis sp. (39.2%, 146/372; 95% CI: 34.2-44.2) were the most prevalent among helminths and GP, respectively. Assemblages A (50.0%), B (37.5%) and A+B (12.5%) were detected within Giardia duodenalis and ST3 (28.6%), ST1 and ST2 (26.2% each), and ST4 (14.3%) within Blastocystis sp. Three genotypes, two known (A: 66.7%; KB-1: 16.7%) and a novel (HhEcEb1, 16.7%) were identified within Enterocytozoon bieneusi. Municipality of origin, household overcrowding, and poor sanitation and personal hygiene habits were risk factors for childhood intestinal parasites colonization. CONCLUSIONS/SIGNIFICANCE: Despite massive government drug administration programs, STH and GP infection remain a public health concern in paediatric populations living in poor-resource settings. Molecular analytical methods are required to better understand the epidemiology of these intestinal parasites. This study provides novel information on the occurrence of Blastocystis sp. and E. bieneusi genetic variants circulating in Ecuadorian human populations.