Resistance of Blastocystis to chlorine and hydrogen peroxide.

Blastocystis is a ubiquitous, widely distributed protist inhabiting the gastrointestinal tract of humans and other animals. The organism is genetically diverse, and so far, at least 28 subtypes (STs) have been identified with ST1-ST9 being the most common in humans. The pathogenicity of Blastocystis is controversial. Several routes of transmission have been proposed including fecal-oral (e.g., zoonotic, anthroponotic) and waterborne. Research on the latter has gained traction in the last few years with the organism having been identified in various bodies of water, tap water, and rainwater collection containers including water that has been previously filtered and/or chlorinated. Herein, we assessed the resistance of 11 strains maintained in culture, spanning ST1-ST9 to various chlorine and hydrogen peroxide concentrations for 24 h, and performed recovery assays along with re-exposure. Following the treatment with both compounds, all subtypes showed increased resistance, and viability could be visualized at the cellular level. These results are hinting at the presence of mechanism of resistance to both chlorine and hydrogen peroxide. As such, this pilot study can be the platform for developing guidelines for water treatment processes.

Molecular testing for clinical diagnosis and epidemiological investigations of intestinal parasitic infections.

Over the past few decades, nucleic acid-based methods have been developed for the diagnosis of intestinal parasitic infections. Advantages of nucleic acid-based methods are numerous; typically, these include increased sensitivity and specificity and simpler standardization of diagnostic procedures. DNA samples can also be stored and used for genetic characterization and molecular typing, providing a valuable tool for surveys and surveillance studies. A variety of technologies have been applied, and some specific and general pitfalls and limitations have been identified. This review provides an overview of the multitude of methods that have been reported for the detection of intestinal parasites and offers some guidance in applying these methods in the clinical laboratory and in epidemiological studies.

Last of the human protists: the phylogeny and genetic diversity of Iodamoeba.

Iodamoeba is the last genus of obligately parasitic human protist whose phylogenetic position is unknown. Iodamoeba small subunit ribosomal DNA sequences were obtained using samples from three host species, and phylogenetic analyses convincingly placed Iodamoeba as a sister taxon to Endolimax. This clade in turn branches among free-living amoeboflagellates of the genus Mastigamoeba. Two Iodamoeba ribosomal lineages (RL1 and RL2) were detected whose sequences differ by 31%, each of which is found in both human and nonhuman hosts.

Diversity and distribution of Blastocystis sp. subtypes in non-human primates.

Blastocystis SSU-rDNA sequence data from 317 captive and free-living non-human primates (NHPs) representing 30 genera of apes, Old and New World (OW and NW) monkeys and prosimians were analysed to investigate subtype (ST) and allele distribution among hosts. Excluding 20 mixed ST infections, 27% of the sequences belonged to ST1, 22% to ST2, 34% to ST3, 1% to ST4, 4% to ST5, 11% to ST8, <1% to ST13 and 1% to ST15. The study confirmed cryptic host specificity of ST1 and ST3; conversely, considerable overlap in ST2 alleles exists among humans and NHPs. Subtype distribution in humans and NHPs differs mainly in that ST4 is rarely reported in NHPs while ST5 and ST8 are both unusual in humans. This may be due to host specificity and/or the apparent geographically restricted range of some subtypes. While the distribution of ST1, ST2 and ST3 was independent of NHP group or geographical association, ST5 was seen only in apes and OW monkeys and ST8 primarily in arboreal NHPs and only in species native to Asia or South America.

Comparison of molecular diagnostic approaches for the detection and differentiation of the intestinal protist Blastocystis sp. in humans.

Blastocystis is the most commonly found intestinal protist in the world. Accurate detection and differentiation of Blastocystis including its subtypes (arguably species) are essential to understand its epidemiology and role in human health. We compared (i) the sensitivity of conventional PCR (cPCR) and qPCR in a set of 288 DNA samples obtained from stool samples of gut-healthy individuals, and (ii) subtype diversity as detected by next-generation sequencing (NGS) versus Sanger sequencing. Real-time PCR resulted in more positive samples than cPCR, revealing high fecal load of Blastocystis based on the quantification curve in most samples. In subtype detection, NGS was largely in agreement with Sanger sequencing but showed higher sensitivity for mixed subtype colonization within one host. This fact together with use of the combination of qPCR and NGS and obtaining information on the fecal protist load will be beneficial for epidemiological and surveillance studies.

Title: Comparaison des approches de diagnostic moléculaire pour la détection et la différenciation du protiste intestinal Blastocystis sp. chez l'homme. Abstract: Blastocystis est le protiste intestinal le plus répandu dans le monde. La détection et la différenciation précises de Blastocystis, y compris ses sous-types (sans doute des espèces), sont essentielles pour comprendre son épidémiologie et son rôle dans la santé humaine. Nous avons comparé (i) la sensibilité de la PCR conventionnelle (cPCR) et de la qPCR dans un ensemble de 288 échantillons d'ADN obtenus à partir d'échantillons de selles d'individus en bonne santé intestinale et (ii) la diversité des sous-types détectée par le séquençage de nouvelle génération (NGS) par rapport au séquençage Sanger. La PCR en temps réel a donné plus d'échantillons positifs que la cPCR, révélant une charge fécale élevée de Blastocystis sur la base de la courbe de quantification dans la plupart des échantillons. Dans la détection des sous-types, le NGS était largement en accord avec le séquençage de Sanger mais a montré une sensibilité plus élevée pour la colonisation de sous-types mixtes au sein d'un hôte. Ce fait, associé à l'utilisation de la combinaison de qPCR et de NGS et à l'obtention d'informations sur la charge fécale de protistes, sera bénéfique pour les études épidémiologiques et de surveillance.

Zoonotic pathogens in wild muskoxen (Ovibos moschatus) and domestic sheep (Ovis aries) from Greenland.

The present study aimed to estimate the prevalence of zoonotic pathogens Giardia duodenalis, Cryptosporidium spp., Toxoplasma gondii and Erysipelothrix in muskoxen (Ovibos moschatus) and sheep (Ovis aries) from Greenland. In 2017 and 2018, faecal samples were collected from wild muskoxen from three distinct populations (Zackenberg, Kangerlussuaq, and Ivittuut) and from domestic sheep from southwest Greenland. Blood samples were collected from muskoxen from Kangerlussuaq and Ivittuut and from sheep. Faecal samples were tested for specific DNA of G. duodenalis and Cryptosporidium spp., and blood samples were tested for antibodies against T. gondii and Erysipelothrix. The estimated prevalence of G. duodenalis was 0% (0/58), 17% (7/41) and 0% (0/55) in muskoxen from Zackenberg, Kangerlussuaq and Ivittuut, respectively, and 37% (16/43) in sheep. The estimated prevalence of Cryptosporidium was 0% (0/58), 2% (1/41), 7% (4/55) in muskoxen from Zackenberg, Kangerlussuaq, Ivittuut, respectively, and 2% (1/43) in sheep. Neither Giardia nor Cryptosporidium were detected in winter samples (0/78). Of the positive samples, Giardia from one muskox sample only was successfully typed as G. duodenalis assemblage A, and Cryptosporidium from two muskoxen was successfully typed as C. parvum, subtype IIdA20G1e. The estimated T. gondii seroprevalence was 2% (1/44) and 0% (0/8) in muskoxen from Kangerlussuaq and Ivittuut, respectively, and 1% (1/155) in sheep. The estimated Erysipelothrix seroprevalence was 2% (1/45) and 13% (1/8) in muskoxen from Kangerlussuaq and Ivittuut, respectively, and 7% (10/150) in sheep. The results of this study add to the scarce knowledge on zoonotic pathogens in the Arctic.

Parasites modulate the gut-microbiome in insects: A proof-of-concept study.

Host-parasite interactions may be modulated by host- or parasite-associated microbes, but the role of these are often overlooked. Particularly for parasites with intestinal stages (either larval or adult), the host gut microbiome may play a key role for parasite establishment; moreover, the microbiome may change in response to invading parasites. Hypothesis testing at the organismal level may be hampered, particularly in mammalian definitive hosts, by ethical, logistical, and economical restrictions. Thus, invertebrates naturally serving as intermediate hosts to parasites with complex life cycles may inform the development of mammalian models as an early-stage host-parasite model. In addition, several important pathogens are vectored by insects, and insect gut microbiome-pathogen interactions may provide essential base-line knowledge, which may be used to control vectorborne pathogens. Here, we used the grain beetle, Tenebrio molitor, a host of the tapeworm Hymenolepis diminuta, to explore interactions between infection status and resident gut microbiota at two pre-determined time points (day two and seven) post infection. Using 16S/18S microbial profiling, we measured key parameters of the composition, relative abundance, and diversity of the host gut bacteriome and mycobiome. In addition, we quantified the systemic beetle immune response to infection by Phenoloxidase activity and hemocyte abundance. We found significant changes in the gut bacteriome and mycobiome in relation to infection status and beetle age. Thus, the relative abundance of Proteobacteria was significantly higher in the gut of infected beetles and driven mostly by an increased abundance of Acinetobacter. In addition, the mycobiome was less abundant in infected beetles but maintained higher Shannon diversity in infected compared with non-infected beetles. Beetles treated with a broad-spectrum antibiotic (Tetracycline) exhibited significantly reduced parasite establishment compared with the untreated control group, indicating that the host microbiome may greatly influence hatching of eggs and subsequent establishment of H. diminuta larvae. Our results suggest that experimental work using invertebrates may provide a platform for explorative studies of host-parasite-microbe interactions and their underlying mechanisms.

Dietary Inulin and Trichuris suis Infection Promote Beneficial Bacteria Throughout the Porcine Gut.

The gut microbiota (GM) displays a profound ability to adapt to extrinsic factors, such as gastrointestinal pathogens and/or dietary alterations. Parasitic worms (helminths) and host-associated GM share a long co-evolutionary relationship, exerting mutually modulatory effects which may impact the health of the host. Moreover, dietary components such as prebiotic fibers (e.g. inulin) are capable of modulating microbiota toward a composition often associated with a healthier gut function. The effect of helminth infection on the host microbiota is still equivocal, and it is also unclear how parasites and prebiotic dietary components interact to influence the microbiota and host health status. Some helminths, such as Trichuris suis (porcine whipworm), also exhibit strong immunomodulatory and anti-inflammatory effects. We therefore explored the effects of T. suis, alone and in interaction with inulin, both in fecal microbiota during the infection period and luminal microbiota across four intestinal segments at the end of a 4-week infection period. We observed that T. suis generally had minimal, but mainly positive, effects on the microbiota. T. suis increased the relative abundance of bacterial genera putatively associated with gut health such as Prevotella, and decreased bacteria such as Proteobacteria that have been associated with dysbiosis. Interestingly, dietary inulin interacted with T. suis to enhance these effects, thereby modulating the microbiota toward a composition associated with reduced inflammation. Our results show that administration of T. suis together with the consumption of prebiotic inulin may have the potential to positively affect gut health.

Pursuing the clinical significance of Blastocystis--diagnostic limitations.

The clinical significance of one of the most prevalent single-celled intestinal parasites worldwide, Blastocystis, remains unsettled. A plethora of clinical and epidemiological studies have been undertaken to generate data on its prevalence in different populations and investigate the role of the parasite as a cause of gastro- and extra-intestinal disease. In this article, we pinpoint limitations of studies that seek to determine the clinical significance of Blastocystis, based on shortcomings in our understanding of Blastocystis diagnosis and biology, and identify methodologies for further studies aimed at determining the molecular epidemiology and clinical impact of this parasite.

Mucosal Barrier and Th2 Immune Responses Are Enhanced by Dietary Inulin in Pigs Infected With Trichuris suis.

Diet composition may play a crucial role in shaping host immune responses and commensal gut microbiota populations. Bioactive dietary components, such as inulin, have been extensively studied for their bioactive properties, particularly in modulating gut immune function and reducing inflammation. It has been shown that colonization with gastrointestinal parasitic worms (helminths) may alleviate chronic inflammation through promotion of T-helper cell type (Th) 2 and T-regulatory immune responses and alterations in the gut microbiome. In this study, we investigated if dietary inulin could modulate mucosal immune function in pigs during colonization with the porcine whipworm Trichuris suis. T. suis infection induced a typical Th2-biased immune response characterized by transcriptional changes in Th2- and barrier function-related genes, accompanied by intestinal remodeling through increased epithelial goblet and tuft cell proliferation. We observed that inulin also up-regulated Th2-related immune genes (IL13, IL5), and suppressed Th1-related pro-inflammatory genes (IFNG, IL1A, IL8) in the colon. Notably, inulin augmented the T. suis-induced responses with increased transcription of key Th2 and mucosal barrier genes (e.g., IL13, TFF3), and synergistically suppressed pro-inflammatory genes, such as IFNG and CXCL9. 16S rRNA sequencing of proximal colon digesta samples revealed that inulin supplementation reduced the abundance of bacterial phyla linked to inflammation, such as Proteobacteria and Firmicutes, and simultaneously increased Actinobacteria and Bacteroidetes. Interestingly, pigs treated with both inulin and T. suis displayed the highest Bacteroidetes: Firmicutes ratio and the lowest gut pH, suggesting an interaction of diet and helminth infection that stimulates the growth of beneficial bacterial species. Overall, our data demonstrate that T. suis infection and inulin co-operatively enhance anti-inflammatory immune responses, which is potentially mediated by changes in microbiota composition. Our results highlight the intricate interactions between diet, immune function and microbiota composition in a porcine helminth infection model. This porcine model should facilitate further investigations into the use of bioactive diets as immunomodulatory mediators against inflammatory conditions, and how diet and parasites may influence gut health.

Terminology for Blastocystis subtypes--a consensus.

Blastocystis is a ubiquitous enteric protistan parasite that has extensive genetic diversity and infects humans and many other animals. Distinct molecular methodologies developed to detect variation and obtain information about transmission patterns and clinical importance have resulted in a confusing array of terminologies for the identification and designation of Blastocystis subtypes. In this article, we propose a standardization of Blastocystis terminology to improve communication and correlate research results. Based primarily on published small-subunit ribosomal RNA gene analyses, we propose that all mammalian and avian isolates should be designated Blastocystis sp. and assigned to one of nine subtypes.

Detecting Blastocystis using parasitologic and DNA-based methods: a comparative study.

Few studies have targeted the relative performance of diagnostic methods used for the detection of Blastocystis, a unicellular organism often present in fecal specimens from individuals with and without gastrointestinal symptoms. Aims of this study included a comparison of the formol ethyl acetate concentration technique (FECT), permanent trichrome staining of feces fixed in sodium acetate-acetic acid-formalin (SAF-PST), xenic in vitro culture (XIVC), and polymerase chain reaction (PCR) regarding Blastocystis screening of 107 samples from 93 patients with suspected enteroparasitic disease. Compared with PCR, the sensitivity/specificity of XIVC, SAF-PST, and FECT was 89%/100%, 82%/100%, and 50%/100%, respectively. False-negative results generated by the FECT and SAF-PST appeared to be associated with Blastocystis sp. subtype 3. A comparison of results obtained by dideoxy sequencing of positive PCR products amplified from DNA extracted directly from feces and DNA extracted from 5- and 28-day-old XIVC of 10 randomly chosen Blastocystis isolates showed no disparities, indicating that XIVC has very little or no impact on subtype distribution or variation within a given specimen. It is recommended that short-term XIVC be used for cost-effective screening of fresh fecal specimens for Blastocystis infection to generate valid prevalence estimates and to identify isolates for molecular characterization in studies aiming to illuminate the molecular epidemiology of Blastocystis.

Prevalence and subtype identification of Blastocystis isolated from humans in Ahvaz, Southwestern Iran.

AIM: The aim of the present study was to determine the prevalence and subtype distribution of Blastocystis and its relation with demographic data and symptoms in humans referred to medical centers in Ahvaz 2014-2015. BACKGROUND: Infections with intestinal parasites are one of the most important threats to human health worldwide, especially in tropical and subtropical areas. Blastocystis sp. is a common parasite of humans with a vast variety of non-human hosts. We aimed to study the prevalence and subtypes of Blastocystis sp. in individuals referred to medical laboratories in Ahvaz city, southwest Iran. METHODS: From September 2014 to September 2015, 618 stool samples were collected from 16 medical laboratories in Ahvaz, and examined using direct wet mount, formalin-ether concentration, a modified version of the Ziehl-Neelsen staining technique, and cultivation in xenic HSr + S medium. Subtypes of positive Blastocysts sp. were obtained using the "barcoding" method. The results were analyzed using SPSS software, version 16, with Chi-square and Fisher's exact test. RESULTS: Totally, 325 (52.6%) of the referred individuals were men and 293 (47.4%) were women. Blastocystis sp. was observed in 146 (23.6%) samples. Co-infections with other intestinal parasites were found in 32 (5.17%) cases. Out of the 146 positive isolates, 20.83%, 20.83% and 58.34% belonged to ST1, ST2, ST3 respectively. CONCLUSION: Blastocystis sp. was quite common in the study population, with a carrier rate corresponding to nearly one in every four individuals. The subtype distribution identified in the present study was largely identical to that reported from other studies in Iran, with ST3 being the most common.

Prevalence and genetic diversity of Trichomonas vaginalis in the general population of Granada and co-infections with Gardnerella vaginalis and Candida species.

PURPOSE: Purulent or exudative genitourinary infections are a frequent cause of consultation in primary and specialized healthcare. The objectives of this study were: to determine the prevalence of Trichomonas vaginalis and co-infections with Candida spp. and Gardnerella vaginalis in vaginal secretion; and to use multilocus sequence typing (MLST) to analyse the genetic diversity of T. vaginalis strains. METHODOLOGY: The samples were submitted for analysis (n=5230) to a third-level hospital in Granada (Southern Spain) between 2011 and 2014; eight T. vaginalis strains isolated during 2015 were randomly selected for MLST analysis. Culture and nucleic acid hybridization techniques were used to detect microorganisms in the samples. RESULTS: The prevalence of T. vaginalis was 2.4 % between 2011 and 2014, being higher during the first few months of both 2011 and 2012. Among samples positive for T. vaginalis, co-infection with G. vaginalis was detected in 29 samples and co-infection with Candida spp. in 6, while co-infection with all three pathogens was observed in 3 samples. The only statistically significant between-year difference in co-infection rates was observed for T. vaginalis with G. vaginalis due to an elevated rate in 2011. MLST analysis results demonstrated a high genetic variability among strains circulating in our setting. CONCLUSION: These findings emphasize the need for the routine application of diagnostic procedures to avoid the spread of this sexually transmitted infection.

Immune responses and parasitological observations induced during probiotic treatment with medicinal Trichuris suis ova in a healthy volunteer.

Ingestion of eggs (ova) of the porcine nematode parasite Trichuris suis (TSO) may reduce the severity of autoimmune disorders, however the development of TSO treatment as a useful therapy for autoimmune diseases is hampered by a lack of knowledge on the development of the parasite and the nature of the local immune responses in humans. Here, we used colonoscopy to investigate the development of T. suis and related mucosal and systemic immune responses during TSO treatment in an intestinally healthy male volunteer. TSO treatment induced T. suis-specific serum antibodies, a transient blood eosinophilia, and increases in IFNγ+ and IL4+ cells within the circulating CD4+ T-cell population. Increased expression of genes encoding cytokines (IL4, IL10, IL17 and TGF-ß), and transcription factors (FOXP3, GATA3 and RORC) were apparent in the ascending and transverse colon (the predilection site of the worms), whereas only limited changes in gene expression were observed proximally (ileum) and distally (descending colon) to the infected tissue. We further show that T. suis is able to colonise the human colon, with a number of worms developing to a similar size and morphology observed in the natural pig host, and a small number of unembryonated eggs were passed in the faeces, indicating patent infection. Notably, the volunteer experienced a substantial improvement in psoriasis during the course of TSO treatment. Thus, TSO treatment induced a mixed Th1/Th2/T regulatory response at the local site of infection, which was also reflected to some extent in the peripheral circulation. These results, together with the first definitive observations that T. suis can mature to adult size and reproduce in humans, shed new light on the interaction between the human immune system and probiotic helminth treatment, which should facilitate further development of this novel therapeutic option.

Blastocystis: Isolation, Xenic Cultivation, and Cryopreservation.

Blastocystis is an intestinal parasite that is very easily isolated in culture from fresh stool samples. In fact, the parasite grows so readily in culture that short-term in vitro culture is sometimes used as a diagnostic tool in the absence of DNA-based methods. While axenizing Blastocystis cultures remains a significant challenge, the parasite can be propagated for several months in the presence of metabolically active bacteria (xenic culture). Hence, culture can be used for maintaining live Blastocystis strain libraries. This enables the production of a stable resource of reference material, which for instance can be used for DNA-based assays and research. Blastocystis isolates can also be cryopreserved with a view to reestablishing them in culture. Here, we provide protocols for xenic in vitro culture and cryopreservation of Blastocystis. © 2016 by John Wiley & Sons, Inc.

Molecular Identification and Subtype Analysis of Blastocystis.

Several typing methods have been used in studies aiming to unravel the molecular epidemiology of Blastocystis, which is one of the most common intestinal parasites in human and many non-human hosts. Such studies have the potential to add to knowledge on Blastocystis transmission, host specificity, phylogeography, and clinical and public health significance, but rely on robust, standardized methods by which data can be generated and compared directly between studies. One of the most used methods is "barcoding,", which involves single-round PCR amplification and sequencing of partial small subunit ribosomal RNA genes of the parasites. Recently, a publicly available online facility was developed for quick and standardized identification of subtypes (ribosomal lineages) and subtype alleles (variation within subtypes) based on sequence data obtained by barcoding PCR. Moreover, a modified barcoding approach is now available using nested PCR, which enables detection of mixed subtype infections. © 2016 by John Wiley & Sons, Inc.

Current status of Blastocystis: A personal view.

Despite Blastocystis being one of the most widespread and prevalent intestinal eukaryotes, its role in health and disease remains elusive. DNA-based detection methods have led to a recognition that the organism is much more common than previously thought, at least in some geographic regions and some groups of individuals. Molecular methods have also enabled us to start categorizing the vast genetic heterogeneity that exists among Blastocystis isolates, wherein the key to potential differences in the clinical outcome of Blastocystis carriage may lie. In this review we summarize some of the recent developments and advances in Blastocystis research, including updates on diagnostic methods, molecular epidemiology, genetic diversity, host specificity, clinical significance, taxonomy, and genomics. As we are now in the microbiome era, we also review some of the steps taken towards understanding the place of Blastocystis in the intestinal microbiota.

Blastocystis Mitochondrial Genomes Appear to Show Multiple Independent Gains and Losses of Start and Stop Codons.

Complete mitochondrion-related organelle (MRO) genomes of several subtypes (STs) of the unicellular stramenopile Blastocystis are presented. Complete conservation of gene content and synteny in gene order is observed across all MRO genomes, comprising 27 protein coding genes, 2 ribosomal RNA genes, and 16 transfer RNA (tRNA) genes. Despite the synteny, differences in the degree of overlap between genes were observed between subtypes and also between isolates within the same subtype. Other notable features include unusual base-pairing mismatches in the predicted secondary structures of some tRNAs. Intriguingly, the rps4 gene in some MRO genomes is missing a start codon and, based on phylogenetic relationships among STs, this loss has happened twice independently. One unidentified open reading frame (orf160) is present in all MRO genomes. However, with the exception of ST4 where the feature has been lost secondarily, orf160 contains variously one or two in-frame stop codons. The overall evidence suggests that both the orf160 and rps4 genes are functional in all STs, but how they are expressed remains unclear.

Transmission of Dientamoeba fragilis: pinworm or cysts?

Recently, conflicting evidence has been published on the mode of transmission of the trichomonad Dientamoeba fragilis. Detection of D. fragilis DNA inside Enterobius vermicularis eggs agrees with the prediction of Dobell in 1940 that the eggs of a nematode act as a vector for transmission. However, the identification of a cyst stage of D. fragilis in the stool of rodents infected with a human isolate has also been reported, and this implies a life cycle similar to those of most other intestinal protistan parasites. Herein we discuss the recent data, identify gaps in the experimental evidence, and propose a method for determining which view of the life cycle of this organism is correct.