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.

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.

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.

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.

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.

[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.

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.

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.

Association of Blastocystis hominis genetic subtypes with urticaria.

Although intestinal parasites are a possible cause of skin disorders, there are few case reports concerning the role of Blastocystis hominis in urticaria. To clarify this association, we determined the frequency of B. hominis genetic subtype in urticarial patients by stool culture and polymerase chain reaction (PCR) and evaluated the clinical and parasitological recovery of urticarial patients after treatment with metronidazole. Of 54 urticarial patients (group I), 18 (33.3%) were diagnosed as acute urticaria (group IA) and 36 (66.7%) were diagnosed as chronic (group IB). Thirty-three (61.1%) out of 54 urticarial (group I) patients were Blastocystis positive by stool culture and PCR. Out of these 33 patients, 21 were symptomatic and 12 were asymptomatic. The amoeboid form was found in 20 (95.2%) out of 21 symptomatic Blastocystis urticarial patients assuring their pathogenic potential. Of 50 normal control group (group II), four (8%) Blastocystis isolates were found with no amoeboid form. B. hominis subtype 3 was the only detected genotype in both groups. Of 20 symptomatic Blastocystis urticarial patients, 12 (60%) patients recovered symptomatically and parasitologically after one course of metronidazole. Recovery reached 100% on repeating the treatment for a second course with disappearance of the amoeboid form. It was concluded that acute urticaria of unknown etiology and chronic idiopathic urticaria patients who are resistant to the ordinary regimen of urticaria treatment might be examined for infection with B. hominis, in order to prescribe the proper specific anti-protozoan treatment.

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.

Evaluation of the transmission mode of B. hominis by using PCR method.

Blastocystis hominis is a common intestinal parasite observed in fecal examination. On the other hand, the transmission of this parasite is certainly unknown. The transmission of B. hominis can be realized by animal contact and the contamination by water and food with excreted cysts from the reservoir hosts. B. hominis isolated from 25 humans, their pets, and tap water was identified by polymerase chain reaction using sequenced tag site primers in this study. B. hominis isolates obtained from humans and pets were identified as subtype1, subtype2, and subtype3 while B. hominis isolates obtained from tap water were also identified as subtype1. The B. hominis isolates obtained from humans in this study were defined as the same as the subtypes of the B. hominis isolates obtained from the pets, of which these people keep at their homes, and the tap water. These findings reveal that the source of B. hominis infection could be pets and tap water.

Irritable bowel syndrome: is it associated with genotypes of Blastocystis hominis.

Blastocystis hominis is the most common intestinal parasite in humans. An extensive genetic variability has been described recently in B. hominis isolates. The aim of this study was to analyze genotypes of B. hominis isolates obtained from the healthy individuals and patients with irritable bowel syndrome-diarrhea (IBS-D). The patients with IBS-D were enrolled from gastroenterology outpatient department at the Aga Khan University Hospital. History and physical examination was done. Stool microscopy, culture, and polymerase chain reaction for B. hominis genotyping were carried out. The study included 158 patients with IBS-D, mean age 41 +/- 15, age range 16-83 years, and male/female ratio of 109:49. One hundred fifty-seven (49.8%) were taken as healthy control. The dominant B. hominis genotypes were genotype 1 in 87 (65%) and type 3 in 49 (37%). In IBS-D, genotype 1 was present in 75 (86%; P < 0.001) compared to 12 (14%) in controls while type 3 was present in 23 (47%) compared to 26 (53%) in controls (P < 0.001), respectively. Infection with single genotype of B. hominis was present in 70 (73%) with IBS-D and in 26 (27%) in control group while with multiple genotypes in 25 (64%) in IBS-D and 14 (36%) in control group (P = 0.30), respectively. Majority of our patients had typeable B. hominis infection. The genotype of B. hominis in IBS-D was type 1 while in control genotype 3 was predominant.

Blastocystis hominis and Dientamoeba fragilis in patients fulfilling irritable bowel syndrome criteria.

Studies have suggested a possible role for Blastocystis hominis and Dientamoeba fragilis in the etiology of irritable bowel syndrome (IBS). We studied the prevalence of B. hominis and D. fragilis in patients with IBS-diarrhea (IBS-D). Three hundred and thirty patients were enrolled, 171 (52%) with IBS-D and 159 (48%) were controls, respectively. Stool microscopy, culture, and polymerase chain reaction (PCR) for B. hominis and D. fragilis were done. B. hominis was positive by stool microscopy in 49% (83/171) of IBS compared to 24% (27/159) in control (p < 0.001). B. hominis culture was positive in 53% (90/171) in IBS compared to 16% (25/159) in control (p < 0.001). B. hominis PCR was positive in 44% (75/171) in IBS compared to 21% (33/159) in control (p < 0.001). D. fragilis microscopy was positive in 3.5% (6/171) in IBS-D compared to 0.6% (1/159) in control (p = 0.123). D. fragilis culture was positive in 4% (7/171) in IBS compared to 1.3% (2/159) in control (p = 0.176). D. fragilis PCR was positive in 4% (6/171) in IBS-D compared to 0% (0/159) in control (p = 0.030). B. hominis is common, while D. fragilis was less prevalent in our patients with IBS-D. B. hominis and D. fragilis culture had a better yield compared to stool microscopy and PCR.

High prevalence of subtype 4 among isolates of Blastocystis hominis from symptomatic patients of a health district of Valencia (Spain).

In order to know the genetic diversity of Blastocystis hominis from a health district of Valencia (Spain) 51 clinical isolates from symptomatic patients, 31 axenic and 20 monoxenic, were ribotyped by analysing the restriction fragment length polymorphism (RFLP) of amplicons obtained by polymerase chain reaction (PCR) of small-subunit of ribosomal DNA genes (SSU-rDNA). For this purpose, DNA was subjected to two independent PCR (RD3-RD5, F1-R1) and to three independent treatments with restrictases (AluI, HinfI and RsaI). The digested DNA was separated electrophoretically, the isolates were clustered into ribotypes (ribodemes, RD3-RD5; subgroups, F1-R1) according to their profiles and the results were translated into genetic subtypes (ST) proposed by a consensus terminology. The results show that the isolates studied are an heterogeneous population and that both PCR-RFLP SSU-rDNA protocols have a similar discriminative power, since it allowed the ribotyping of all isolates and their clustering into four demes: ribodemes 1, 3 and 3-r and 6, which include isolates belonging to subgroup III, IV, V and V-r, respectively; which were assigned to ST1 (2%), ST2 (3.9%) and ST4 (94.1%). The most common of which is a zoonotic subtype (Blastocystis ratti) which includes, according to recent studies, non-pathogenic and pathogenic variants.

Identification of Blastocystis hominis isolates from asymptomatic and symptomatic patients by PCR.

Despite years of study, the pathogenic role of Blastocytis hominis is still controversial. Genotypic differences between the asymptomatic and symptomatic isolates should assist in determining the pathogenicity of Blastocystis. In this study, we genotyped 32 Blastocystis isolates obtained from 12 asymptomatic healthy individuals and 20 symptomatic patients pain by polymerase chain reaction using known seven kinds of sequence tagged site primers in this study. When we compared genotype of Blastocystis isolates between the symptomatic and asymptomatic patient group, we found that subtype3 is the most dominant genotype in asymptomatic individual (9/12) and subtype1 determined all of symptomatic patients (20/20).

[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.