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.

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.

Genetic diversity of Blastocystis hominis sensu lato isolated from humans in Poland

INTRODUCTION AND OBJECTIVES: Blastocystis hominis s. l. is one of the most commonly detected protozoa in the human large intestine. The aim of the study was to determine the genetic subtypes of Blastocystis hominis s. l. occurring in humans in Poland. MATERIALS AND METHODS: Stool samples from patients diagnosed in the Laboratory of the Department of Parasitology, National Institute of Public Health ­ National Institute of Hygiene (NIZP-PZH) and in the Parasitology Laboratory of the Hospital for Infectious Diseases in Warsaw were examined. Blastocystis subtypes were assayed based on the fragment of small-subunit ribosomal RNA gene sequences (SSU rDNA). RESULTS: The examined isolates were classified into five Blastocystis subtypes (STs), fifteen of which belonged to ST3, three to ST1, two to ST2, two to ST6, and one isolate belonged to ST7. In three cases the subtype of isolate was not identified. DISCUSSION AND CONCLUSIONS: In Poland, the subtypes ST1, ST2, ST3, ST4, ST6 and ST7 have been reported in humans so far. The ST6 and ST7 subtypes are rarely detected in humans in Europe. In Poland, the ST6 subtype was previously described in chickens. On the basis of the studies, it was found that Blastocystis isolated from humans in Warsaw show high genetic diversity. In order to determine the possible pathogenic potential of individual Blastocystis subtypes, special epidemiological studies are required.

[Blastocystis hominis- parasites or commensals? ]. / Blastocystis hominis - komensal czy patogen?

Blastocystis hominis (B. hominis) is a cosmopolitan pro- tozoa which parasitizes the human large intestine. This parasite had been considered to be commensal of the large intestine for a long time, because even an intense invasion may be asymptomatic. However, this species is now being regarded as a parasitic organism. In this paper the latest data concerning the epidemiology, diagnostics and treatment of B. hominis invasion have been cited and discussed.

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.

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.

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.

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.

Phenotypic and genotypic characterisation of Blastocystis hominis isolates implicates subtype 3 as a subtype with pathogenic potential.

Despite frequent reports on the presence of Blastocystis hominis in human intestinal tract, its pathogenicity remains a matter of intense debate. These discrepancies may be due to the varying pathogenic potential or virulence of the isolates studied. The present study represents the first to investigate both phenotypic and genotypic characteristics of B. hominis obtained from symptomatic and asymptomatic individuals. Symptomatic isolates had a significantly greater size range and lower growth rate in Jones' medium than asymptomatic isolates. The parasite cells of symptomatic isolates exhibited rougher surface topography and greater binding affinity to Canavalia ensiformis (ConA) and Helix pomatia (HPA). The present study also identifies further phenotypic characteristics, which aided in differentiating the pathogenic forms from the non-pathogenic forms of B. hominis. Blastocystis subtype 3 was found to be correlated well with the disease.

A possible link between subtype 2 and asymptomatic infections of Blastocystis hominis.

Blastocystis hominis is one of the most common eukaryotic organisms in the intestinal tract of humans, while its pathogenic potential is still controversial. A total of 286 stool samples obtained from adult and pediatric patients with or without gastrointestinal symptoms in two hospitals in Manisa, Turkey, were cultured to detect B. hominis infection. Forty-one and 51 isolates were obtained from the adults and children, respectively, and these isolates were subjected to subtyping by polymerase chain reaction (PCR) with the known sequence-tagged site primers. The correlation between the genotype and the symptoms was evaluated. PCR subtyping indicated that subtype 3 was the most common genotype in both symptomatic and asymptomatic groups, and the second common genotype was subtypes 1 and 2 in symptomatic and asymptomatic groups, respectively. A significant correlation between subtype 2 and the asymptomatic groups was found among both in pediatric and adult patients (chi(2) (cal) = 4.38, df = 1, p = 0.044). However, there were no significant differences between the other genotypes and the symptomatic or asymptomatic groups, as well as both the age and sex of the patients. The present study suggests that subtype 2 is a non-pathogenic genotype of B. hominis.

Pathophysiological variability of different genotypes of human Blastocystis hominis Egyptian isolates in experimentally infected rats.

The genotyping of Blastocystis hominis clinical isolates obtained from 28 gastrointestinal symptomatic patients and 16 asymptomatic individuals were identified by polymerase chain reaction using sequenced-tagged site (STS) primers. Then, pathophysiological variability between different B. hominis genotypes was evaluated in experimentally infected rats. Only four B. hominis subtypes (1, 2, 3, and 4) were detected (18.2%, 9.1%, 54.5%, and 18.2%, respectively) in human isolates. In symptomatic isolates, subtypes 1, 3, and 4 were detected in 8 (28.6%), 16 (57.1%), and 4 (14.3%) patients, respectively. In asymptomatic isolates, subtypes 2, 3, and 4 were identified in 4 (25%), 8 (50%), and 4 (25%), respectively. Subtype 3 was the commonest in humans. Different degrees of pathological changes were found among infected rats by symptomatic subtypes compared with asymptomatic subtypes. The moderate and severe degrees of pathological changes were found only in symptomatic subtypes infected rats while mild degree was found only in asymptomatic subtypes infected rats. Only subtype 1 induced mortality rate with 25% among infected rats. On evaluation of the intestinal cell permeability in the Ussing chamber, a prominent increase in short circuit current (DeltaIsc) was found in symptomatic subtype 1 compared to symptomatic subtypes 3 and 4 infected rats. Minimal effects were found in the asymptomatic and control groups. The results proved that subtype 1 was clinically and statistically highly relevant to the pathogenicity of B. hominis while subtype 2 was irrelevant. Also, the results suggest the presence of pathogenic and nonpathogenic strains among subtypes 3 and 4.

[Is Blastocystis hominis an opportunist agent?]. / Blastocystis hominis firsatçi bir patojen mi?

Despite its high prevalence throughout the world, major issues about Blastocystis hominis remain unresolved, including fundamental areas such as taxonomy and pathogenicity. Sequences of the SSUrRNA gene place Blastocystis in the stramenophiles. Analysis of the elongation factor 1-alpha gene, however, indicates similarity to Entamoeba histolytica. There is considerable morphological variability and karyotype diversity, and it appears that more than one species is present in humans and animals. In culture, three major forms predominate: vacuolar, granular, and ameboid. The vacuolated form (usually 10 to 30 mum) was most frequently detected in fecal specimens. The prevalence of Blastocystosis in humans appears to be higher in developing countries (30% to 50%) than in developed countries (1.5% to 10%), and has been associated with travel. B. hominis is the most common parasite isolated from stool specimens in symptomatic and asymptomatic persons in a variety of settings. Isolates resembling B. hominis have been described in a variety of mammals, birds, reptiles, and even insects. The significance of this human infection is uncertain.

Infectivity of different genotypes of human Blastocystis hominis isolates in chickens and rats.

Most Blastocystis hominis isolates from humans are believed to be potentially zoonotic. This is because B. hominis isolates found in a variety of other host species have been found to have identical or relatively similar genotypes to those found in human isolates. However, the transmission of human B. hominis isolates to other animals has not been confirmed experimentally. In this study, the infectivity associated with several unique human Blastocystis genotypes (subtypes 2, 3, 4 and 7) was therefore investigated by infecting chickens and rats with two isolates of each subtype experimentally. The results showed that one isolate of subtype 4 and one isolate of subtype 7 was capable of infecting both chickens and rats, while two isolates of subtype 2, another isolate of subtype 4, and another isolate of subtype 7 could only infect chickens. Conversely, two isolates of subtype 3 failed to infect either of the animals. These results confirmed that several genotypes from human isolates could infect chickens and/or rats, indicating that chickens and rats are suitable experimental animal models for studying the zoonotic potential of human Blastocystis isolates.

PCR fingerprinting of Blastocystis isolated from symptomatic and asymptomatic human hosts.

Genomic DNA from 16 Blastocystis hominis isolates comprising of eight asymptomatic isolates (A1-A8) and eight symptomatic isolates (S1-S8) was amplified by arbitrarily primed polymerase chain reaction (AP-PCR) using 38 arbitrary 10-mer primers. Six primers (A10, B5, C20, D1, F6, and F10) generated reproducible DNA fingerprints. AP-PCR amplification revealed similar DNA fingerprints among all symptomatic isolates (S1-S8) with common bands at 850 bp using primer A10, 920 bp using primer B5, and 1.3 kbp using primer D1. Isolates A1, A3, A4, A5, A6, and A7 showed similar DNA banding patterns and all asymptomatic isolates (A1-A8) shared a major band at 1 kbp using primer B5. Isolates A2 and A8 showed distinct DNA banding patterns that differed from the remainder of the isolates. The results of the phylogenetic analyses showed that all symptomatic isolates (S1-S8) formed a clade with >70% similarity among the isolates and which were clearly separate from asymptomatic isolates A1, A3, A4, A5, A6, and A7. Asymptomatic isolates A2 and A8 formed two distinct and separate clades. AP-PCR revealed higher genetic variability within the asymptomatic isolates than within the symptomatic isolates. The present study suggests that AP-PCR can be a valuable method for differentiating between isolates of B. hominis and our results support the hypothesis that our asymptomatic and symptomatic B. hominis isolates may represent two different strains/species with varying pathogenic potential.

Molecular characterization of Blastocystis isolates in the Philippines by riboprinting.

Extensive genomic polymorphism has been demonstrated among morphologically identical Blastocystis isolates. To this end, 32 Blastocystis isolates from the Philippines (12 from humans, 12 from pigs and 8 from chickens) were analyzed genetically by riboprinting or restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) amplified small subunit rDNA. Three distinct riboprint patterns were observed from the HinfI digestion, while four patterns resulted from the RsaI digestion of Blastocystis SSU rDNA. Restriction fragment profiles between Blastocystis isolates from different hosts were generally different from each other. However, Blastocystis isolates within each host group were practically the same. Cluster analysis of the riboprint patterns revealed seven distinct groups of the Blastocystis isolates, including a zoonotic strain. These results demonstrate the genetic heterogeneity of Blastocystis in the Philippines and a support to the idea of the organism's zoonotic potential.

Fecal-oral transmission of the cyst form of Blastocystis hominis in rats.

The infectivity of two Blastocystis hominis strains, RN94-9 and NIH:1295:1, was examined in 3-week-old SPF Wistar rats. The NIH:1295:1 strain, originally isolated from a guinea pig, was only able to infect rats via intracecal inoculation of the cultured organisms, while the RN94-9 strain, originally isolated from a laboratory rat, was able to infect rats by oral inoculation of the cultures due to the presence of a cystic form in the in vitro culture. Since many cysts were discharged in the feces of the infected rats, the infectivity of the concentrated cysts was compared between the two strains. Successful oral infection was observed in rats inoculated with 1 x 10(2)-1 x 10(6) cysts of the RN94-9 and NIH:1295:1 strains. The infectivity of the ten cysts varied in the three experiments of ten rats, being 20-100% and 30-100% in the RN94-9 and NIH:1295:1 strains, respectively. When an uninfected normal rat was housed with five experimentally inoculated rats, the normal rat became infected, demonstrating the fecal-oral transmission of the cyst form of this parasite. These results show that the Wistar rat is an ideal host for the propagation of strains RN94-9 and NIH:1295:1 of B. hominis, and demonstrate that the cyst form is the only transmissible form of this parasite.

Molecular and phylogenetic analysis of Blastocystis isolates from various hosts.

Blastocystis hominis is a protozoan parasite found in humans. B. hominis-like organisms have been found in a variety of animals, but have been called Blastocystis sp. because the isolates from animals were indistinguishable from B. hominis morphologically. Recent molecular studies show that some isolates from animals have genetic similarity with B. hominis. However, it has been unclear whether the isolates from animals have zoonotic potential or not. In the present study, the SSUrDNA of 19 Blastocystis isolates from these animals was sequenced in its entirety, and the phylogenetic relationship among isolates from humans and animals was clarified using available nucleotide sequences of the same locus. Phylogenetic analysis showed that the 19 isolates analyzed in the present study could be classified into seven groups (I-VII): Group I consisted of the isolates from humans, primates, cattle, pigs and birds; Group II of the isolates from humans and primates; Group III of the isolates from humans, cattle and pigs; Group IV of the isolates from primates, birds and rodents; Group V of the isolates from cattle and pigs; Groups VI and VII of the isolates from humans and birds. These results indicate that many of the isolates harboring in animals have zoonotic potential, or have cross-transmissibility among heterogeneous hosts.