Blastocystis Species Research Articles

Subtypes identification and frequency of Blastocystis isolated from patients in Kashan, Central Iran

Aims: Blastocystis species are one of the most common enteric protist infections in humans and some animals worldwide. Molecular studies have shown that there is a high level of genetic variation among Blastocystis isolates. The aim of this study was to identify the subtypes and frequency of Blastocystis isolates in patients who referred to the medical diagnostic laboratories in Kashan, Central Iran. Materials and Methods: This cross-sectional study was conducted on 1118 patients, from December 2017 to June 2018. Fecal specimens were evaluated by the microscopic examination. Positive samples were cultivated in Robinson media. After massive growth and DNA extraction, a 550 bp from the small subunit ribosomal RNA gene was amplified by the polymerase chain reaction (PCR) for subtype identification. The PCR products have been sequenced, identified, and compared at the NCBI site. The results were analyzed using the SPSS software version 16. Results: The frequency of Blastocystis sp. was 8.58%, (confidence interval = 6.94%–10.22%) from which 76% were men and 24% were women. Of the 51 PCR positive samples, ST3 (41.2%), ST1 (39.2%), ST2 (11.8%), and 7.8% isolates were identified as mixed. ST3 and ST1 have been more common. The highest levels of infection were observed in the food-handlers, the age group of 31–40, and people with high school education. Conclusion: The results showed that the frequency of Blastocystis was lower than other studies and the most common Blastocystis subtype was subtype 3, followed by subtype 1, and subtype 2.

Molecular characterization of Blastocystis subtypes isolated in the city of Uberaba, Minas Gerais State, Brazil

INTRODUCTIONBlastocystis is an intestinal protozoan that may play a role in the pathogenicity of humans. This study aimed to (i) genetically characterize Blastocystis isolates obtained from human fecal samples and the water supply of the city of Uberaba, Minas Gerais, Brazil, and (ii) to verify the phylogenetic relationship between these isolates. METHODSBlastocystis species present in 26 fecal samples obtained from humans and animals from Uberaba were genetically characterized by polymerase chain reaction-restriction fragment length polymorphism and polymerase chain reaction-sequence-tagged sites. All amplicons were partially sequenced and/or defined according to the GenBank classification. RESULTS Polymerase chain reaction amplicons were generated from 21 human isolates and 18 water samples. The subtypes defined were ST1 (53.3%), ST3 (40.0%), and ST2 (6.7%) for human isolates; ST10 (100%) for bovine isolates; and ST5 (50.0%), ST1 (25%), and ST3 (25%) for pigs. Sequencing of polymerase chain reaction products showed a 98%-99% identity for the Blastocystis sequences deposited in GenBank, except for sequences from water samples that showed the identity of algae sequences. Phylogenetic analysis of Blastocystis sequences showed two distinct groups, one of which was principally formed by ST1, ST5, and ST10, and the other by isolates characterized as ST3 and ST7. Both clades showed human and animal sequences, reinforcing the notion that Blastocystis subtypes are not host-specific.CONCLUSIONS The data showed that Blastocystis subtypes circulating in Uberaba are ST1-ST3, ST5, and ST10, present in both humans and animals, demonstrating that the Blastocystis subtypes are not host-specific; that is, zoonotic transmission is possible.

Current view on Blastocystis species: clinical forms, diagnosis and treatment

The symptomatic form of Blastocystis spp. infection not only with mild diarrhea or dysentery-like syndrome, but also with the development of severe ulcerative necrotic lesions of the intestine. Meanwhile, the pathogenicity of these microorganisms should not be exaggerated, due to majority asymptomatic cases or infection transmission with minor impaired bowel function.

"Polymerase chain reaction-based subtype diversity of Blastocystis species isolates obtained from hospitalized patients with blastocystosis"

"Polymerase chain reaction-based subtype diversity of Blastocystis species isolates obtained from hospitalized patients with blastocystosis"

Distribution and relevance of Dientamoeba fragilis and Blastocystis species in gastroenteritis: results from a case-control study.

The actual role of Dientamoeba fragilis and Blastocystis in patients with gastrointestinal symptoms is still under debate. A multicenter case-control study was performed in The Netherlands to elucidate the clinical relevance of molecular diagnostics results in gastroenteritis (GE). Samples from this case-control study were used to perform a detailed analysis on the presence of D. fragilis and Blastocystis in relation to gastrointestinal symptoms. In the present study, a real-time PCR for Blastocystis was performed on 1374 case samples and 1026 control samples from the multicenter gastroenteritis case-control study previously tested for D. fragilis. Prevalence of both micro-organisms was highest in children under 20 years of age and lowest in the oldest age group. A significantly lower overall detection of D. fragilis and Blastocystis was found in cases (both 25.8%) as compared to controls (37.6% and 40.0%, respectively). The difference for D. fragilis was statistically significant for subjects above 20 years of age. For Blastocystis, the difference was statistically significant in all age groups, except in children less than 5 years of age. A negative relation between D. fragilis-positive cases and diarrhea was found in this study population. More GE symptoms were reported in cases without D. fragilis or Blastocystis. In the present study, prevalence of both D. fragilis and Blastocystis is lower in cases with gastroenteritic symptoms than in controls. Besides, in cases with D. fragilis or Blastocystis, no association is shown between any of the GE symptoms. Interestingly, this suggests that the presence of these protozoans may be considered characteristic of a healthy intestinal microbiome.

Substantial prevalence of enteroparasites Cryptosporidium spp., Giardia duodenalis and Blastocystis sp. in asymptomatic schoolchildren in Madrid, Spain, November 2017 to June 2018.

BackgroundProtozoan enteroparasites Cryptosporidium species and Giardia duodenalis are major contributors to the burden of gastrointestinal illness in children globally, whereas the stramenopile Blastocystis species has been associated with irritable bowel syndrome and skin disorders.AimTo investigate the carriage of these parasites in voluntary asymptomatic schoolchildren (4‒14 years) in 12 different primary and secondary schools in Leganés (Madrid, Spain).MethodsIn a prospective cross-sectional study, stool samples and epidemiological questionnaires on demographics and potential risk factors were collected from participating schoolchildren. Detection of enteric parasites was conducted by PCR-based methods and confirmed by sequence analysis. We calculated prevalence and odds ratios (OR) with logistic regression.ResultsStool samples and questionnaires were provided by 1,359 schoolchildren from 12 schools. The individual prevalence for any parasite was 28%; Blastocystis sp.: 13%; G. duodenalis: 18%; Cryptosporidium spp.: 1%. Two schoolchildren were infected with all three species and 53 with two species. Multivariable risk factor analysis using logistic regression models indicated that an existing infection with one parasite increased the odds for an additional infection with another parasite. The odds of Blastocystis sp. carriage increased up to the age of 10 years and being female increased the odds of Cryptosporidium spp. infection. Washing vegetables before preparing a meal was protective for Blastocystis sp. infection.ConclusionWe detected a larger than expected proportion of asymptomatic cases in the participanting schoolchildren. Further investigation of asymptomatic children should be considered. Good hygiene measures should be encouraged for individuals of all ages to protect from protozoal infections.

Surface ultrastructure, protein profile and zymography of Blastocystis species isolated from patients with colorectal carcinoma.

Oncogenic potential of Blastocystis species have been predicted on reporting the enhanced proliferation of human colorectal cancer cells by the parasite solubilized antigen in vitro, and the enhanced drug-induced carcinogenesis by infection in vivo. The present study is the first to investigate some phenotypic characters, namely the surface ultrastructure, protein profiles and protease activity of Blastocystis sp. isolated from three different clinical groups: colorectal carcinoma (CRC) patients, symptomatic and asymptomatic infected persons. Under SEM, all CRC Blastocystis sp. isolates had a very rough intensely folded surface in comparison to the less rough and completely smooth surface of all symptomatic and asymptomatic Blastocystis sp. Non-CRC isolates, respectively. Under reducing conditions, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis had shown a significant presence of 2 protein bands of 230 and 32KDa in 42.9% of Blastocystis sp. CRC isolates with their complete absence from Non-CRC isolates. While using non-reducing condition with the incorporation of gelatin in the gel to study the protease activity of the parasite, no significant difference existed between isolates of the three groups. In conclusion, the significant difference in surface ultrastructure and in protein profiles exists between Blastocystis sp. of CRC and Non-CRC isolates. These differences might be either secondary to the altered gut environment in the presence of CRC or are indicators of a different pathogenic potential of the parasite isolates inducing malignancy.

Subtype analysis of Blastocystis spp. isolated from domestic mammals and poultry and its relation to transmission to their in-contact humans in Ismailia governorate, Egypt.

Background: Blastocystis is a common protist detected in fecal samples of humans and a wide range of animals. The parasite exhibits extensive genetic diversity with seventeen distinct subtypes (STs) identified collectively from humans, other mammals and birds. Shared STs between animal and human hosts were considered to be potentially zoonotic. However, Blastocystis infection among non-human hosts, in Egypt has not been investigated so far.Objective: To determine the occurrence and ST distribution of Blastocystis species isolated from domestic mammals, poultry and their in-contact humans.Material and Methods: A total of 416 fecal samples from domestic animals (mammals and poultry) as well as their in-contact humans were screened by inoculation into Jones' media. Positive samples were subtyped using seven pairs of ST-specific sequence-tagged-site (STS) primers.Results: The occurrence of Blastocystis spp. infection was 69.8% in poultry, 17.7% in domestic mammals and 35.7% in humans. Among the studied animal species ST1-ST7 were identified with varying percentages; however, only four STs (ST1-ST4) were identified in humans. A minority of human subjects examined (16/56, 28.5%) were carrying the same ST detected in their domestic animals.Conclusion: The detection of all the tested STs among the infected animal species examined highlights the broad genetic diversity observed among Blastocystis spp. isolated from animals. However, the detection of only four STs among humans suggests that these STs can easily infect humans and the animals carrying the same STs could be possible reservoirs. Surprisingly, direct handling of animals was not found to be a major contributor to human blastocystosis in Egypt, denoting the role of anthroponotic transmission and the possibility of fecal cross-contamination from other potential reservoir animals in the surrounding environment.

Molecular phylogeny of Blastocystis isolates from wild rodents captured in Indonesia and Japan.

Blastocystis sp. is a common intestinal protist found worldwide in a variety of animals, including humans. Currently, 17 subtypes (STs) of Blastocystis isolates from mammalian and avian host species have been reported based on the small subunit ribosomal RNA gene (SSU rDNA). Among these, human Blastocystis were only identified among STs 1-9. Except ST9, all other STs comprised isolates from humans and other animal species. Entire sequence data of the SSU rDNA of nine Blastocystis isolates from laboratory rats or guinea pigs previously showed ST4, whereas Blastocystis isolates from wild rodents have not been addressed genetically. In this study, Blastocystis infection in wild rodents was surveyed in Indonesia and Japan, and 11 and 12 rodent Blastocystis parasites were obtained from Rattus exulans and R. novercious, respectively. All new Blastocystis isolates from wild rodents were identified as ST4 based on the SSU rDNA sequences. The best tree inferred with the entire sequences of the SSU rDNA of all ST4 isolates including 17 data registered in GenBank clearly showed monophyletic ST4A and ST4B clades. Although ST4 isolates from laboratory rats were separated into these two clades, all Blastocystis isolates from wild rodents in the present study were positioned into the clade ST4A and further separated into two sub-clusters within the clade ST4A according to the location of the host species. Considering the fact that laboratory rats were susceptible to both ST4A and ST4B, separation of the monophyletic sub-clusters of Blastocystis isolates from Indonesian Polynesian rats and Japanese brown rats may indicate the presence of geographical variations rather than a host-specific separation. In either way, the robust host preference to rodent species of ST4 Blastocystis was also confirmed.

Prevalence and molecular characterization of Strongyloides stercoralis, Giardia duodenalis, Cryptosporidium spp., and Blastocystis spp. isolates in school children in Cubal, Western Angola

BackgroundHuman infections by the gastrointestinal helminth Strongyloides stercoralis and the enteric protozoans Giardia duodenalis, Cryptosporidium spp. and Blastocystis spp. are not formally included in the list of 20 neglected tropical diseases prioritised by the World Health Organization. Although largely underdiagnosed and considered of lower public health relevance, these infections have been increasingly demonstrated to cause significant morbidity and even mortality globally, particularly among children living in resource-poor settings.MethodsIn this cross-sectional survey the prevalence, frequency and molecular diversity of S. stercoralis, G. duodenalis, Cryptosporidium spp. and Blastocystis spp. were investigated in a school children population in the province of Benguela (Angola). A total of 351 stool samples were collected during January to June 2015. The presence of S. stercoralis and G. duodenalis was confirmed by qPCR methods. Giardia duodenalis assemblages and sub-assemblages were determined by multilocus sequence-based genotyping of the glutamate dehydrogenase and β-giardin genes of the parasite. Detection and identification of Cryptosporidium and Blastocystis species and subtypes was carried out by amplification and sequencing of a partial fragment of the small-subunit ribosomal RNA gene of both protozoan. Analyses of risk factors potentially associated with the transmission of these pathogens were also conducted.ResultsPrevalences of S. stercoralis, G. duodenalis, Cryptosporidium spp., and Blastocystis spp. were estimated at 21.4% (95% CI: 17.1–25.7%), 37.9% (95% CI: 32.8–43.0%), 2.9% (95% CI: 1.1–4.5%) and 25.6% (95% CI: 21.18–30.2%), respectively. Overall, 64.1% (225/351) of the children were infected by at least one of the pathogens investigated. Sequence analyses of the 28 G. duodenalis isolates that were successfully genotyped allowed the identification of sub-assemblages AI (14.3%), AII (14.3%), BIII (7.1%) and BIV (25.0%). Discordant typing results AII/AIII and BIII/BIV were identified in 7.1% and 14.3% of the isolates, respectively. A total of five additional isolates (17.9%) were identified as assemblage B. Three Cryptosporidium species including C. hominis (70%), C. parvum (20%) and C. canis (10%) were found circulating in the children population under study. A total of 75 Blastocystis isolates were assigned to the subtypes ST1 (30.7%), ST2 (30.7%), ST3 (36.0%), ST5 (1.3%) and ST7 (1.3%), respectively. Children younger than seven years of age had significantly higher risk of infections by protozoan enteropathogens (PRR: 1.35, P < 0.01), whereas being underweight seemed to have a protective effect against these infections (PRR: 0.74, P = 0.005).ConclusionsThe burden of disease attributable to human strongyloidiasis, giardiosis, cryptosporidiosis and blastocystosis in Angola is considerably higher than initially estimated in previous surveys. Surveillance and control of these infections should be jointly tackled with formally considered neglected tropical diseases in order to maximize effort and available resources. Our data also demonstrate the added value of using molecular diagnostic methods in high transmission areas.

BLASTOCYSTIS HOMINIS: A MYSTERIOUS AND COMMONLY DISREGARDED PARASITE

Blastocystis hominis (B. hominis) is an anaerobic, single-cell protozoan, commonly present in human and animal stool samples. It can be found in healthy people as well and it still has not been elucidated whether it is a commensal organism or a pathogen. Blastocystosis is a disease caused by the protozoan in humans. The prevalence of the parasitosis varies both between the countries, and between certain population groups within individual countries. Due to poor hygienic conditions, common exposure to animals and intake of contaminated water and food, people in the developing countries have got a higher prevalence of blastocystosis, but economically developed countries have not been spared either. The taxonomy of B. hominis is still a matter of debates. For the reasons of genetic diversity, it has been suggested that the name B. hominis should be replaced with „Blastocystis species‟. Seventeen subtypes of the species have been so far identified, and a definitive characterization of Blastocystis spp. is possible at the molecular level only. The parasite is transferred by the fecal-oral route. A variety of hosts have been identified, and animal-to-human and vice versa transfers have been documented. The most common manifestations of the infection with the organism are diarrhea, abdominal pain, nausea, and bloating. This infection has also been associated with the irritable bowel syndrome (IBS), non-specific colitis, chronic inflammatory bowel disease (CIBD), and urticaria. The diagnosis can be made using the methods of conventional microscopy (CVM), phase-contrast and electron microscopy, cultivation, serodiagnosis, and by using molecular methods. The infection caused by the parasite does not always require treatment. In symptomatic patients, the first line medical treatment is metronidazole. Further studies are required to resolve all dilemmas regarding the parasite.

Mysterious chronic urticaria caused by Blastocystis spp.?

Species of the genus Blastocystis, which are single-cell, intestinal protozoan parasites of humans and animals, remain mysterious, with unclear clinical and epidemiologic significance. In recent years, many researchers have suggested a possible connection between Blastocystis spp. infection and chronic urticaria. In the present article, we review the literature and discuss the possible associations between the clinical symptomatology and pathogenicity of this organism in terms of its subtypes, morphologic forms, genetic diversity, and interactions with other intestinal microbiota.

Blastocystis Species: Guilty or Innocent?

Blastocystis spp. was previously considered to be a member of normal intestinal flora; it is now receiving increased attention as a potential causative agent of human intestinal disease. The present study attempts to investigate the pathogenicity of Blastocystis spp. in clinically symptomatic patients. Eighty-six immunocompetent patients with intestinal Blastocystis spp. were evaluated clinically. Two consecutive stool samples were examined by wet mount, modified formol ethyl acetate concentration method and trichrome and Kinyoun acid-fast staining. Presence of more than 10 various morphological forms of Blastocystis spp. under 40× magnification was taken as a criterion for the presence of protozoan. Patients with other pathogens were excluded from the study. Metronidazole was administered for five to seven days. After treatment, stool sample of patients were re-examined for Blastocystis spp. There were 39 males and 47 females with a mean age of 39.8 years ± 18.8 (range 19–62 years). Abdominal pain was the most frequent symptom (77.9%) followed by diarrhea (65.1%). A second stool specimen was obtained from 61 (70.9%) patients after metronidazole therapy. Consecutive investigation revealed no intestinal protozoa in 56 (91.8%) patients. Symptoms persisted in five patients. Out of 56 patients who complained of diarrhea, 51 showed improvement in symptomology with no protozoan found. If all other pathogens and factors causing intestinal pathologies are ruled out and only Blastocystis spp. is present in symptomatic patients, it is logical to consider them as pathogenic rather than commensals and treatment can be initiated in such cases. However, further studies for genetic association and prevalence are needed to strengthen this view.

Leucocytic Variations Associated With Blastocystis Species: Are They Symptom Specific?

Leucocytic Variations Associated With Blastocystis Species: Are They Symptom Specific?

Blastocystis specific serum immunoglobulin in patients with irritable bowel syndrome (IBS) versus healthy controls

BackgroundBlastocystis species are common enteric human parasites and carriage has been linked to Irritable Bowel Syndrome (IBS), particularly diarrhoea-predominant IBS. The spectrum of immune reactivity to Blastocystis proteins has been reported previously in symptomatic patients. We investigated differences in serum immunoglobulin profiles between patients with IBS, both positive and negative for Blastocystis carriage, and healthy controls (HC).MethodsForty diarrhoea-predominant IBS patients (26 patients positive for Blastocystis sp., 14 negative patients) and forty HC (24 positive, 16 Blastocystis-negative) were enrolled. Age, gender, ethnicity and serum immunoglobulin A (IgA) levels were recorded and faecal specimens were analysed using smear, culture and polymerase chain reaction amplification of ribosomal DNA. Sera were tested in Western blots and the reactivities compared to known targets using monoclonal antibodies Blastofluor® (Blastocystis specific antibody), MAb1D5 (cytopathicto Blastocystis cells), anti-promatrix metalloprotease-9 (anti-MMP-9) and SDS-PAGE zymograms.ResultsLevels of serum IgA were significantly lower in Blastocystis carriers (p < 0.001) but had no relationship to symptoms. Western blots demonstrated serum IgG antibodies specific for Blastocystis proteins of 17,27,37,50,60-65, 75–90, 95–105 and 150 kDa MW. Reactivity to the 27, 50 and 75-95 kDa proteins were found more frequently in the IBS group compared to the HC’s (p < 0.001) and correlation was greater for Blastocystis-positive IBS patients (p < 0.001) than for negative IBS patients (p < 0.05). MAb1D5 reacted with proteins of 27 and 100 kDa, and anti-MMP-9 with 27, 50 and 75-100 kDa proteins. Bands were seen in zymograms around 100 kDa.ConclusionsLow serum IgA levels are associated with Blastocystis carriage. All IBS patients were more likely to demonstrate reactivity with Blastocystis proteins of 27 kDa (likely a cysteine protease), 50 and 75-95 kDa MW compared to HC. The presence of antibodies to these Blastocystis proteins in some Blastocystis-negative subjects suggests either prior exposure to Blastocystis organisms or antibody cross reactivities. The anti-proMMP-9 reaction at 50 and 75–100 kDa and the zymogram result suggest that metalloproteases may be important Blastocystis antigens.Trial registrationAustralian and New Zealand Clinical Trials registry ACTRN: 12611000918921Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1069-x) contains supplementary material, which is available to authorized users.

Characterization of Blastocystis species infection in humans and mantled howler monkeys, Alouatta palliata aequatorialis, living in close proximity to one another.

This study characterizes Blastocystis species infections in humans and mantled howler monkeys, Alouatta palliata aequatorialis, living in close proximity to one another in northwestern Ecuador. Blastocystis species were identified from 58 of 96 (60.4%) mantled howler monkey fecal samples, and 44 of 55 human fecal samples (81.5%) by polymerase chain reaction. Using single-stranded conformation polymorphism, we were able to efficiently separate and sequence subtypes (STs) within mixed samples without the need for cloning. Blastocystis ST1, ST2, and ST3 were found in people, and two individuals were infected with more than one subtype. All monkey samples were ST8. The lack of shared subtypes between humans and monkeys suggests that no Blastocystis transmission occurs between these species in spite of close proximity in some instances. Based on analysis of demographic data from a questionnaire given to human participants, individuals who boiled their water before consumption were significantly less likely to be infected with Blastocystis (44.4%) compared to those who did not (93.8%) (p = 0.002). No other risk factors were significant, although hunters, females, individuals living in large families, and those living closer to forested habitat tended to have a higher proportion of Blastocystis infections.

Enteric pathogens of food sellers in rural Gambia with incidental finding of Myxobolus species (Protozoa: Myxozoa)

Ongoing surveillance of enteric pathogens of public health significance among casual food sellers is undertaken in many resource-limited countries. We report the results of a survey in Kiang West province, The Gambia, and provide an exemplar methodology for such surveys in resource-limited laboratories. Unpreserved, unrefrigerated stool samples were subjected to Salmonella, Shigella and agar plate culture for rhabditoid nematodes. Direct microscopy, formalin-ethyl acetate concentration and iron-hematoxylin staining was performed later, following preservation. Of 128 specimens received, no Shigella spp. was recovered, while four serovars of non-typhoidal Salmonella enterica, including Chandans, were isolated. Pathogenic parasitic infections were Necator americanus 10/128 (7.8%), Strongyloides stercoralis 3/128 (2.8%), Blastocystis species 45/128 (35.1%), Entamoeba histolytica complex 19/128 (14.8%) and Giardia intestinalis 4/128 (3.1%). A single case each of Hymenolepis diminuta and S. mansoni infection were detected. In one participant, myxozoan spores identical to those of Myxobolus species were found. Rare parasitoses and serovars of Salmonella enterica may occur relatively commonly in rural Africa. This paper describes intestinal pathogens found in a cohort of food sellers in such a setting. Furthermore, it describes two parasites rarely recovered from humans and demonstrates the need for methods other than microscopy to detect S. stercoralis infections.

Recent insights into the genetic diversity, epidemiology and clinical relevance of Blastocystis species.

Blastocystis is a widely prevalent intestinal protozoan of man and other animals. It is associated with gastro intestinal disorders and symptoms. Emerging literature suggests that several subtypes of this parasite exist and are responsible for virulence, pathogenicity and symptoms. A variety of new diagnostic methods have been developed which help to recognize genotypic variations, subtype relationship and determination of carrier state. In symptomatic patients, when the other etiological agents have been excluded, it is prudent to treat Blastocystis infection. Metronidazole remains the drug of choice for treatment. Further research is required to shed light on taxonomy, biology and virulence of this organism.

Investigation of the presence of Blastocystis spp. in stool samples with microscopic, culture and molecular methods

Blastocystis species are enteric protozoa frequently detected in human and animals. Seventeen subtypes (STs) have now been identified, nine of them isolating from humans. The pleomorphic structure and genetic diversity of Blastocystis spp. and the absence of standardized diagnostic methods complicate the evaluation of current data. Microscopic methods such as native-lugol and trichrome staining are most frequently used methods in routine diagnosis, while culture and molecular methods are preferred for research purposes. The aims of this study were to investigate the presence of Blastocystis spp. in the stool samples of patients with gastrointestinal complaints by microscopic and culture methods, and to detect the subtypes of isolates by polymerase chain reaction (PCR). A total of 350 stool samples collected from patients with diarrhea (n= 157) and without diarrhea (n= 193) were included in the study. Presence of Blastocystis spp. in the samples were investigated by native-lugol examination, trichrome staining and direct fluorescent antibody (DFA) methods. Ringer's solution containing 10% horse serum and 0.05% asparagine was used for cultivation. The cultures were evaluated after 3-4 days of incubation at 37°C by microscopic examination. The subtypes of Blastocystis spp. strains isolated from the cultures have been identified by PCR using sequence-tagged site primers. A total of 66 (19%) stool samples, of them 26 (16.6%) were from diarrheal and 40 (21%) from non-diarrheal cases, yielded Blastocystis sp. growth in culture. Among the evaluated samples, 12% (42/350) were found positive with native-lugol examination, 17% (58/350) with trichrome staining, and 19% (66/350) with DFA method. The agreement of culture and native-lugol method was estimated as strong (κ= 0.752), while it was very strong between culture with trichrome staining and DFA methods (κ= 0.922 and κ= 1.00, respectively). When the culture was accepted as reference method, the sensitivity and specificity rates of native-lugol method were 65% and 100%, trichrome staining method were 88% and 100%, and DFA method were 100% and 100%, respectively. Forty-three (65%) of Blastocystis spp. positive samples were subtyped by PCR, while 23 isolates could not be subtyped. The most frequent detected subtype was ST3 (12/43; 28%), followed by ST1 (6/43; 13.9%), ST4 (5/43; 11.6%) and ST7 (5/43; 11.6%), ST2 (3/43; 7%) and ST6 (1/43; 2.3%). ST5 was not detected in this study and 11 (25.6%) samples have been identified to have mixed subtypes. The differences of Blastocystis spp. positivity rates and the distribution of the subtypes between the patients with or without diarrhea were not found statistically significant (p> 0.05). In our study, ST3 was the most frequently identified Blastocystis spp. subtype, similar to the previous national studies, however ST6 and ST7 have been identified for the first time. In conclusion, as the sensitivity of native-lugol examination is low, culture is time-consuming and laborious and PCR methods are costly and non-standardized, rapid, practical and high sensitive DFA is considered as the favourable method in the diagnosis of Blastocystis spp. in routine laboratories.

Clinical pilot study: efficacy of triple antibiotic therapy in

Blastocystis species are common human enteric parasites. Carriage has been linked to Irritable Bowel Syndrome (IBS). Treatment of Blastocystis spp. with antimicrobials is problematic and insensitive diagnostic methods and re-infection complicate assessment of eradication. We investigated whether triple antibiotic therapy comprising diloxanide furoate, trimethoprim/sulfamethoxazole and secnidazole (TAB) given to diarrhoea-predominant IBS (D-IBS) patients positive for Blastocystis would achieve eradication. In a longitudinal, prospective case study 10 D-IBS Blastocystis-positive patients took 14 days of diloxanide furoate 500 mg thrice daily, trimethoprim/sulfamethoxazole 160/80 mg twice daily and secnidazole 400 mg thrice daily. Faecal specimens were collected at baseline, day 15 and 4 weeks after completion of TAB. Specimens were analysed using faecal smear, culture and polymerase chain reaction (PCR) of the 16 SSU rRNA. Patients kept a concurrent clinical diary. Six (60%) patients cleared Blastocystis spp. after TAB, including three who had failed previous therapy. Subtypes detected were ST3 (60%), ST4 (40%), ST1 (20%) and ST7, 8 (10%); four patients had mixed ST infections. Serum immunoglobulin A (IgA) levels were low in 40% of patients. Higher rates of Blastocystis clearance were observed in patients symptomatic for less than a year (Mann-Whitney, p?=?0.032, 95% confidence) with no associations found with age, previous antibiotic therapy, faecal parasite load, ST, IgA level or clinical improvement. Clearance of Blastocystis spp. was achieved with TAB in 60% of D-IBS patients, an improvement over conventional monotherapy. Higher clearance rates are needed to facilitate investigation of the relevance of this parasite in clinically heterogenous IBS.