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.

[Progress of researches on Blastocystis hominis infection among patients with inflammatory bowel disease and irritable bowel syndrome].

Blastocystis is a common unicellular intestinal protozoa in humans and animals, and the most common clinical manifestations of infections include abdominal pain and diarrhea. Based on the sequence of the small-subunit ribosomal RNA (SSU rRNA) gene, 28 subtypes of B. hominis (ST1 to ST17, ST21 and ST23 to ST32) have been characterized. Previous studies have demonstrated that B. hominis infection is strongly associated with inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) and other intestinal diseases, which threatens the health and quality of life among patients with B. hominis infection and is considered as an important public health problem. This review summarizes the progress of researches on B. hominis infection among IBD and IBS patients during the past 20 years, so as to provide insights into management of blastocystosis in China.

Phylogenetic tree of Blastocystis hominis in Iraqi children in Salah AL-Deen province, Iraq.

Blastocystis hominis is an intestinal protozoan that inhabits the large intestine of humans and a wide range of animals. Blastocystis species has a worldwide distribution. The current study aimed to determine the prevalence and the genetic variety of Blastocystis sp. in Iraqi children in Salah AL-Deen province, Iraq. 150 faecal samples were collected from children (5-10 years old) who attended the Salah AL-Deen hospital during the period from March to November 2020. The results revealed that 33.3% of children (50 out of 150) were found infected with Blastocystis sp., when the polymerase chain reaction (PCR) was used. The presence of ST3 gene was at a band of 526 bp where this gene was observed in 11 samples out of 50 samples. The results also showed significant differences in the prevalence rate between rural and urban regions; between symptomatic and asymptomatic children, and between children who contacted domestic animals and those who did not contact animals (P<0.05). No significant differences in the prevalence rate were between different age groups (P>0.05). Regarding the genetic variation in subtype 3(ST3) revealed in phylogenetic tree analysis, there were three variations (transversion, deletion, and transition) which were detected through the sequence alignment, also the similarity was 97% with the sequences of Blastocystis sp. registered in GenBank. The Iraqi ST3 isolate was registered with ID: OL410286 in GenBank.

MOLECULAR STUDY OF BLASTOCYSTIS HOMINIS ISOLATED FROM DIFFERENT REGIONS OF DIYALA GOVERNORATE.

OBJECTIVE: The aim: To detect the infection rate of Blastocystis hominis in children less than 10 years old with diarrhea in Diyalaby polymerase chain reaction (PCR) method, to determine the subtype of Blastocystis hominis by sequencing the product of the positive result, and to determine the association between Blastocystis hominis infection and different factors such as gender, age, the level of mother education and the presence or absence animals in their houses. PATIENTS AND METHODS: Materials and methods: A cross-sectional study was conducted on children with diarrhea at Al-Batool Teaching Hospital in Diyala governorate, during the period from November 2020 to April 2021, a total of 100 children 55 males and 45 females, then, stool samples were collected and examined by conventional polymerase chain reaction. RESULTS: Results: The rate of infection with the parasite Blastocystis hominis was 8%, 8 out of 100. The infection was higher among females 62.5% than to males 37.5%, while the positive result was higher in the age group less than two years 75%, the highest percentage occur with patient whose mothers were incomplete primary and primary education was reached 37.5% and 25%; respectively and the study showed the highest percentage was with those who kept animals at homes was 75%. CONCLUSION: Conclusions: According to the genetic analysis of the sequence of eight samples that were positive for Blastocystis hominis parasite using the conventional polymerase chain reaction and they were back to the subtypes 3.

Prevalence and genetic characteristics of Blastocystis hominis and Cystoisospora belli in HIV/AIDS patients in Guangxi Zhuang Autonomous Region, China.

Blastocystis hominis and Cystoisospora belli are considered to be common opportunistic intestinal protozoa in HIV/AIDS patients. In order to investigate the prevalence and genetic characteristics of B. hominis and C. belli in HIV/AIDS patients, a total of 285 faecal samples were individually collected from HIV/AIDS patients in Guangxi, China. B. hominis and C. belli were investigated by amplifying the barcode region of the SSU rRNA gene and the internal transcribed spacer 1 (ITS-1) region of the rRNA gene, respectively. Chi-square test or Fisher's exact test were conducted to assess the risk factors related to B. hominis and C. belli infection. The prevalence of B. hominis and C. belli was 6.0% (17/285) and 1.1% (3/285) respectively. Four genotypes of B. hominis were detected, with ST3 (n = 8) and ST1 (n = 6) being predominant, followed by ST6 (n = 2) and ST7 (n = 1). Females had a statistically higher prevalence of B. hominis (11.6%) than males (4.2%). The statistical analysis also showed that the prevalence of B. hominis was significantly associated with age group and educational level. Our study provides convincing evidence for the genetic diversity of B. hominis, which indicates its potential zoonotic transmission and is the first report on the molecular characteristics of C. belli in HIV/AIDS patients in China.

[Prevalence and risk factors of Blastocystis hominis infection in inpatients in Jiangjin District, Chongqing City].

OBJECTIVE: To understand the prevalence and risk factors of Blastocystis hominis infection in inpatients in Jiangjin District, Chongqing City. METHODS: A cross-sectional study was conducted in a community hospital in Jiangjin District, Chongqing City, and the inpatients were surveyed by questionnaires. After obtaining the informed consent from the inpatients or legal guardians, the stool and blood samples were collected and examined by microscopy and PCR from April 17 to May 1, 2018. The univariate analysis and logistic regression analysis were used to analyze the risk factors of the B. hominis infection. RESULTS: A total of 198 hospitalized patients were investigated, and the infection rate of B. hominis was 10.61% (21/198), and the infection rate of the females (12.10%) was higher than that of the males (8.11%), but the difference was not statistically significant. The highest rate of infection was 19.23% in the age group of 10 to 20 years, followed by 17.74% in the age group of 60 years and above, and the lowest rate was 2.38% in the age group of 20 to 40 years. The difference in infection rates of B. hominis among the different age groups was statistically significant (P < 0.05). The infection rate of B. hominis in the people who used dry pail latrines was 33.30%, which was higher than that of the people who used water flush toilets (9.10%) (P < 0.05). The genotypes of B. hominis were ST1, ST3, ST6 and ST7, and ST6 and ST3 being the most predominant genotypes which accounted for 47.62% (10/21) and 38.10% (8/21) respectively, and among the infected males, the genotypes were only ST3 and ST6. The multiple logistic regression analysis showed that among the factors affecting B. hominis infection, only keeping pets was a risk factor [OR = 3.798, 95% CI (1.245, 11.581), P < 0.05]. CONCLUSIONS: A high prevalence of B. hominis infection is found in the inpatients in Jiangjin District, Chongqing City, the predominant genotypes are ST6 and ST3, and keeping pets may be one of the main risk factors.

Higher prevalence of Blastocystis hominis in healthy individuals than patients with gastrointestinal symptoms from Ahvaz, southwestern Iran.

BACKGROUND: Blastocystis, a common intestinal protozoan of humans and animals, infected more than 1 billion people around the world. This enteric protozoan is frequently reported in both healthy individuals and patients with gastrointestinal (GI) symptoms. METHODS: Three hundred and forty-five fecal samples including 151 GI patients and 194 healthy individuals were examined by microscopy, culture and PCR-sequencing techniques to determine Blastocystis frequency and subtype (ST) variation. RESULTS: The occurrence of Blastocystis was detected 56 (16.2%) and 85 (24.6%) by microscopy, culture and PCR methods, respectively. Out of the 85 positive patients, 60 (70.6%) were asymptomatic and 25 (29.4%) were symptomatic. The results of 41 successfully sequenced isolates identified 8 (19.5%), 8 (19.5%), and 25 (61.0%) ST1, ST2, and ST3, respectively. CONCLUSION: This study has found that Blastocystis was more common in healthy individuals than GI patients. Another finding was that no correlation was found between clinical symptoms and Blastocystis STs.

Metagenomics for broad and improved parasite detection: a proof-of-concept study using swine faecal samples.

Efficient and reliable identification of emerging pathogens is crucial for the design and implementation of timely and proportionate control strategies. This is difficult if the pathogen is so far unknown or only distantly related with known pathogens. Diagnostic metagenomics - an undirected, broad and sensitive method for the efficient identification of pathogens - was frequently used for virus and bacteria detection, but seldom applied to parasite identification. Here, metagenomics datasets prepared from swine faeces using an unbiased sample processing approach with RNA serving as starting material were re-analysed with respect to parasite detection. The taxonomic identification tool RIEMS, used for initial detection, provided basic hints on potential pathogens contained in the datasets. The suspected parasites/intestinal protists (Blastocystis, Entamoeba, Iodamoeba, Neobalantidium, Tetratrichomonas) were verified using subsequently applied reference mapping analyses on the base of rRNA sequences. Nearly full-length gene sequences could be extracted from the RNA-derived datasets. In the case of Blastocystis, subtyping was possible with subtype (ST)15 discovered for the first known time in swine faeces. Using RIEMS, some of the suspected candidates turned out to be false-positives caused by the poor status of sequences in publicly available databases. Altogether, 11 different species/STs of parasites/intestinal protists were detected in 34 out of 41 datasets extracted from metagenomics data. The approach operates without any primer bias that typically hampers the analysis of amplicon-based approaches, and allows the detection and taxonomic classification including subtyping of protist and metazoan endobionts (parasites, commensals or mutualists) based on an abundant biomarker, the 18S rRNA. The generic nature of the approach also allows evaluation of interdependencies that induce mutualistic or pathogenic effects that are often not clear for many intestinal protists and perhaps other parasites. Thus, metagenomics has the potential for generic pathogen identification beyond the characterisation of viruses and bacteria when starting from RNA instead of DNA.

Evaluation of the Roche LightMix Gastro parasites multiplex PCR assay detecting Giardia duodenalis, Entamoeba histolytica, cryptosporidia, Dientamoeba fragilis, and Blastocystis hominis.

OBJECTIVES: Multiplex PCR assays offer highly sensitive and specific tools for the detection of enteric pathogens. This prospective study aimed at comparing the novel Roche LightMix Modular Assay Gastro Parasites (LMAGP) detecting Giardia duodenalis, Entamoeba histolytica, Cryptosporidium spp., Blastocystis hominis, and Dientamoeba fragilis with routine laboratory procedures. METHODS: Stool specimens (n = 1062 from 1009 patients) were consecutively examined by LMAGP, R-Biopharm Ridascreen enzyme immunoassays (EIAs) detecting G. duodenalis or E. histolytica/dispar, and microscopy of wet mounts. Discrepant results were analysed by in-house PCR. RESULTS: D. fragilis or B. hominis were detected by LMAGP in 131 (14.4%) and 179 (19.9%; 16 samples positive by microscopy; p < 0.0001) of 909 samples, respectively. Of 918 samples analysed for Cryptosporidium spp., six were positive by LMAGP (three could be confirmed by Kinyoun staining and one by in-house PCR). G. duodenalis was detected by LMAGP, EIA, or microscopy in 20, 16, or 9 of 1039 stool samples, respectively; all four samples missed by EIA were confirmed by in-house PCR. In total, 938 stool samples were analysed for E. histolytica/dispar. Nine of ten EIA-positive samples were negative by LMAGP but positive by in-house PCR for E. dispar. One E. histolytica infection (positive by both LMAGP and in-house PCR) was missed by EIA and microscopy. Parasites only detected by microscopy included Enterobius vermicularis eggs (n = 3) and apathogenic amoebae (n = 27). CONCLUSIONS: The data call for routine use of multiplex PCR assays for the detection of enteric protozoan parasites in laboratory diagnostics.

Diagnosis and Identification of Blastocystis Subtypes in Primary School Children in Jakarta.

Blastocystis hominis is an enteric protozoan with many subtypes. It is frequently found in children and may cause chronic diarrhea. This study revealed Blastocystis subtypes among primary school children and comparison of molecular technique and culture method in Blastocystis diagnosis. A total of 141 stools were collected, examined microscopically, selected into the Blastocystis and negative parasite groups, for diagnostic comparison between culture and 18S rRNA polymerase chain reaction (PCR) methods. Positive PCR amplicons were subsequently sequenced for subtyping. The PCR results revealed 89%, 78%, 80% and 88% sensitivity, specificity and positive and negative predictive values, respectively, in comparison with the culture method (McNemar, p > 0.05). Sixteen PCR samples were successfully sequenced and resulted in three Blastocystis subtypes 1, 3 and 4. In conclusion, PCR was sensitive enough and can be used to exclude Blastocystis infection up to 88% of the cases. Subtypes 3 and 1 were the main subtypes found in apparently healthy school children in Jakarta.

PCR-based molecular characterization of Blastocystis hominis subtypes in southwest of Iran.

Blastocystis hominis is the most common intestinal parasite found in humans and many other hosts. Pathogenicity of Blastocystis sp. remains controversial and it has been suggested that it may be associated with certain subtypes of organism. The aim of this study was to evaluate the molecular epidemiology of B. hominis and its subtype distribution in Ahvaz, southwest of Iran. During 2012-2014, a total of 481 samples were collected from patients referred to the medical laboratory centers in Ahvaz for stool examination. Samples were examined by wet mount, and genomic DNA was extracted from 50 positive samples. PCR was performed using seven primer pairs targeting the SSU rDNA gene and sequenced. 69 (14.35%) samples were found to be positive for B. hominis and the subtypes of 50 samples were identified. Five subtypes (STs) were identified, including: ST1 (22%), ST2 (6%), ST3 (40%), ST4 (2%), and ST5 (8%). 11 (22%) mixed infections were found, of which 5 were a mixture of ST3/ST4. Mixtures of ST1/ST3 and ST1/ST4 were 3, respectively. In this study people infected with ST3 showed the most gastrointestinal symptoms. This is the first study in the population of Ahvaz and indicates the high prevalence of ST3 in this area. The results suggest a possible association between this subtype and pathogenic potential of parasite.

Coproscopy and molecular screening for detection of intestinal protozoa.

BACKGROUND: Intestinal parasitosis is one of several health concerns about immigrants who travel from endemic to non-endemic regions. Reliable rapid sensitive diagnostic tools, for use in non-endemic regions, are urgently required to enable frequent assessment of immigrant workers in jobs where risk of local transmission is a particular concern (e.g. food-handlers). We assessed the burden of intestinal protozoa in newly arrived immigrants and those applying for renewal of work permits in Qatar (n = 735), by both microscopic examination of stool samples and by Real Time PCR methodology. RESULTS: Prevalence was considerably higher using RT-PCR compared with coproscopy (Blastocystis hominis: 65.2 vs 7.6%; Giardia duodenalis: 14.3 vs 2.9%; Entamoeba histolytica: 1.6 vs 1.2%). Dientamoeba fragilis was sought only by RT-PCR (prevalence of 25.4%). Prevalence of G. duodenalis was significantly higher in male subjects, associated with blue collar workers and declined over time. Prevalence of B. hominis varied significantly with region of origin of subjects with highest values recorded among African immigrants. Prevalence of D. fragilis also varied with region of origin of subjects, and was lower in young female subjects and in renewal applicants compared with first-time applicants for work permits. CONCLUSIONS: We strongly recommend that, henceforth, intestinal protozoa should be screened by RT-PCR, with a particular focus on frequent assessment of immigrant food-handlers.

Molecular Analysis of the Enteric Protozoa Associated with Acute Diarrhea in Hospitalized Children.

Pediatric diarrhea is a common cause of death among children under 5 years of age. In the current study, we investigated the frequency of intestinal parasites among 580 pediatric patients with chronic diarrhea. Parasitic protozoa (all species combined) were detected by molecular tools in 22.9% of the children and the most common parasite was Cryptosporidium spp. (15.1%). Blastocystis hominis was detected in 4.7%, Dientamoeba fragilis in 4%, Giardia duodenalis in 1.7%, and Entamoeba histolytica in 0.17%. Protozoan infections were observed among all regional groups, but prevalence was highest among Qatari subjects and during the winter season. Typing of Cryptosporidium spp. revealed a predominance of Cryptosporidium parvum in 92% of cases with mostly the IIdA20G1 subtype. Subtypes IIdA19G2, IIdA18G2, IIdA18G1, IIdA17G1, IIdA16G1, and IIdA14G1 were also detected. For Cryptosporidium hominis, IbA10G2 and IbA9G3 subtypes were identified. This study provides supplementary information for implementing prevention and control strategies to reduce the burden of these pediatric protozoan infections. Further analyses are required to better understand the local epidemiology and transmission of Cryptosporidium spp. in Qatar.

An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads.

Microbial eukaryotes (protists) are important components of terrestrial and aquatic environments, as well as animal and human microbiomes. Their relationships with metazoa range from mutualistic to parasitic and zoonotic (i.e., transmissible between humans and animals). Despite their ecological importance, our knowledge of protists in urban environments lags behind that of bacteria, largely due to a lack of experimentally validated high-throughput protocols that produce accurate estimates of protist diversity while minimizing non-protist DNA representation. We optimized protocols for detecting zoonotic protists in raw sewage samples, with a focus on trichomonad taxa. First, we investigated the utility of two commonly used variable regions of the 18S rRNA marker gene, V4 and V9, by amplifying and Sanger sequencing 23 different eukaryotic species, including 16 protist species such as Cryptosporidium parvum, Giardia intestinalis, Toxoplasma gondii, and species of trichomonad. Next, we optimized wet-lab methods for sample processing and Illumina sequencing of both regions from raw sewage collected from a private apartment building in New York City. Our results show that both regions are effective at identifying several zoonotic protists that may be present in sewage. A combination of small extractions (1 mL volumes) performed on the same day as sample collection, and the incorporation of a vertebrate blocking primer, is ideal to detect protist taxa of interest and combat the effects of metazoan DNA. We expect that the robust, standardized methods presented in our workflow will be applicable to investigations of protists in other environmental samples, and will help facilitate large-scale investigations of protistan diversity.

[PCR-based genotype classification of Blastocystis hominis isolates from college students of Guangxi].

Fifty-three Blastocystis hominis isolates were separated from the fecal specimens of carriers in college students from Guangxi and cultivated in vitro, and the genetic DNA was extracted. All the isolates were genotyped by PCR using seven pairs of known sequence-tagged site (STS) primers. The results showed there were five subtypes in the 53 isolates. Subtype 3 was the most popular one (32.1%, 17/53), followed by subtype 7 (9.4%, 5/53). Subtypes 1 (7.6%, 4/53), 4 (7.6%, 4/53), and 6 (1.9%, 1/53) were detected, while subtypes 2 and 5 were not detected. The genotypes of the other 22 isolates were unknown which were negative to all the STS primers.

[Genotype analysis and isoenzyme patterns of ten isolates of Blastocystis hominis from Guangxi].

OBJECTIVE: To analyze genotypes and lactate dehydrogenase (LDH) and esterase (EST) patterns in 10 isolates of Blastocystis hominis collected from Guangxi. METHODS: Ten B. hominis isolates (BhGX1-BhGX10) were obtained from the fecal specimens of patients and cultivated in vitro, and then the genomic DNA was extracted. The isolates were genotyped by PCR using seven pairs of known sequenced-tagged site (STS) primers. Isoenzyme patterns of LDH and EST were investigated by SDS-PAGE. RESULTS: Out of the 10 isolates, 8 were identified as genotype I and the genotypes of the other two (BhGX4 and BhGX7) were unknown which were negative to all the STS primers. Among the ten isolates, 10 LDH bands were found, more with Rm37, Rm49, Rm57, Rm68 and Rm92. 12 bands showed in EST patterns: Rm14, Rm18, Rm23, Rm27, Rm35, Rm41, Rm45, Rm50, Rm55, Rm68, Rm77 and Rm82. Difference existed with the LDH and EST patterns among the isolates. CONCLUSION: Genotype I is the major one in the 10 B. hominis isolates from Guangxi, and the isolates show different isoenzyme patterns for LDH and EST.

Blastocystis infection is associated with irritable bowel syndrome in a Mexican patient population.

In recent times, some common "non-pathogenic" parasites, such as Blastocystis and Dientamoeba fragilis, have been associated to the aetiology of irritable bowel syndrome (IBS), while host pro-inflammatory cytokine gene polymorphisms might have a role in the pathophysiology of the disease. Therefore, Blastocystis subtypes (ST), D. fragilis and gene promoter single nucleotide polymorphisms of interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) in IBS patients and controls were studied. After giving written consent, 45 patients with symptoms of IBS according to the Rome III criteria and 45 controls were enrolled. DNA was extracted from peripheral blood for SNP analysis at position -174 for IL-6 as well as -238 and -308 for TNF-α. Blastocystis was more common in the IBS group (p = 0.043). Interestingly, D. fragilis was found more frequently in the control group (p = 0.002); Blastocystis ST1 and 3 were most frequent in both groups. Haploview analysis revealed linkage disequilibrium in TNF-α (p < 0.0001); however, none of the SNPs for IL-6 and TNF-α were found to be significantly related with IBS. The clinical and molecular approaches undertaken for the first time in Latin American IBS patients demonstrated an association with Blastocystis that supports a pathogenic role of this parasite in IBS Furthermore, co-infections with ST1 and ST3 were frequent; thus, the genetic diversity proposed within ST polymorphisms does not rule out that particular strains might be associated with disease. In addition, our results do not support a major contribution of IL-6 and TNF-α gene polymorphisms in the susceptibility to IBS.

Study of Blastocystis hominis isolates in urticaria: a case-control study.

Blastocystis hominis is a common intestinal parasite, with a prevalence in developing countries of up to 50%. The aim of this study was to investigate the association of this parasite with urticaria by determining the genotypic isotypes in the Egyptian population. In total, 54 patients with urticaria and 50 controls were enrolled in the study. Stool samples were examined and assessed by PCR. The parasite was detected in a significantly higher number (P < 0.001) of the patient group than the control group. There was no significant difference between the patients with acute and those with chronic urticaria (P = 0.2). The amoeboid form was found in 60.6% of Blastocystis-positive patients with urticaria, but in none of the healthy controls. Subtype 3 was the only isolate found in both the patient and control groups. We recommend treatment for Blastocystis-positive patients with urticaria in developing countries. The prevalence is much lower (around 10%) in developed countries, where treatment should only be considered in the absence of other possible causes of urticaria.