Use of shotgun metagenomics for the identification of protozoa in the gut microbiota of healthy individuals from worldwide populations with various industrialization levels.

Protozoa have long been considered undesirable residents of the human gut, but recent findings suggest that some of them may positively affect the gut ecosystem. To better understand the role and ecological dynamics of these commensal and potentially beneficial protozoan symbionts, we need efficient methods to detect them, as well as accurate estimates of their prevalence across human populations. Metagenomics provides such an opportunity, allowing simultaneous detection of multiple symbionts in a single analytical procedure. In this study, we collected fecal samples of 68 individuals from three Cameroonian populations with different subsistence modes and compared metagenomics-based and targeted methods of detection for two common protozoan genera: Blastocystis and Entamoeba. In addition, we analyzed our data along with publicly available fecal metagenomes from various worldwide populations to explore the prevalence and association patterns of ten protozoan genera. Regarding the detection method, microscopy was much less sensitive than metagenomics for Entamoeba, whereas qPCR was at least as sensitive as metagenomics for Blastocystis sp. However, metagenomics was more likely to detect co-colonizations by multiple subtypes. Out of the ten examined genera in 127 individuals from Cameroon, Tanzania, Peru, Italy or USA, only three (Blastocystis, Entamoeba and Enteromonas) had an overall prevalence exceeding 10%. All three genera were more common in less industrialized populations and their prevalence differed between continents and subsistence modes, albeit not in a straightforward manner. The majority (72.5%) of colonized individuals carried at least two protozoan species, indicating that mixed-species colonizations are common. In addition, we detected only positive and no negative association patterns between different protozoa. Despite the pitfalls of the metagenomic approach, ranging from the availability of good-quality sequencing data to the lack of standard analytical procedures, we demonstrated its utility in simultaneous detection of multiple protozoan genera, and especially its ability to efficiently detect mixed-species colonizations. Our study corroborates and expands prevalence results previously obtained for Blastocystis sp. and provides novel data for Entamoeba spp. and several other protozoan genera. Furthermore, it indicates that multiple protozoa are common residents of the healthy human gut worldwide.

Prevalence, transmission, and host specificity of Cryptosporidium spp. in various animal groups from two French zoos.

Cryptosporidium represents a major cause of gastrointestinal illness in humans and animals including domestic, wild, and in captivity animals, and more than 30 validated species of Cryptosporidium are recognized as infectious to different hosts such as mammals, birds, reptiles, amphibians, and fish. Therefore, numerous investigations have been conducted worldwide in order to shed light on the epidemiology of this parasite and to explore its potential reservoirs. Few surveys, targeting humans and animals have been carried out regarding the epidemiology of Cryptosporidium spp. in France and no data are available about the circulation of this parasite in French zoological gardens. Herein, we determined the prevalence of Cryptosporidium in animals housed in two French zoos. A total of 307 fecal samples belonging to 161 species were screened by nested PCR. Overall, Cryptosporidium DNA was detected in 1.9% of the 161 species and 1% of the total number of fecal samples tested. Additionally, three Cryptosporidium species were identified: C. galli, C. andersoni, and C. tyzzeri. To our knowledge, this is the first molecular study focused on Cryptosporidium infection in captivity animals in France. This study is of interest considering the exposure of a large number of humans and animals to this waterborne protozoan, found ubiquitously in the environment.

Proteogenomic Insights into the Intestinal Parasite Blastocystis sp. Subtype 4 Isolate WR1.

Blastocystis sp. is known for years as a highly prevalent anaerobic eukaryotic parasite of humans and animals. Several monophyletic clades have been delineated based on molecular data, and the occurrence of each subtype in humans and/or animal hosts has been documented. The genome of several representatives has been sequenced revealing specific traits such as an intriguing 3'-end processing of primary transcripts. Here, a first high-throughput proteomics dataset acquired on this difficult-to-cultivate parasite is presented for the zoonotic subtype T4 isolate WR1. Amongst the 2766 detected proteins, we highlighted the role of a small ADP ribosylation factor GTP-binding protein involved in intracellular traffic as major regulator of vesicle biogenesis and a voltage-dependent anion-selective channel protein because both were unexpectedly highly abundant. We show how these data may be used for gaining proteogenomics insights into Blastocystis sp. specific molecular mechanisms. We evidenced for the first time by proteogenomics a functional termination codon derived from transcript polyadenylation for seven different key cellular components.

Molecular Epidemiology of Blastocystis sp. in Various Animal Groups from Two French Zoos and Evaluation of Potential Zoonotic Risk.

Blastocystis sp. is a common intestinal parasite infecting humans and a wide range of animals worldwide. It exhibits an extensive genetic diversity and 17 subtypes (STs) have thus far been identified in mammalian and avian hosts. Since several STs are common to humans and animals, it was proposed that a proportion of human infections may result from zoonotic transmission. However, the contribution of each animal source to human infection remains to be clarified. Therefore, the aim of this study was to expand our knowledge of the epidemiology and host specificity of this parasite by performing the largest epidemiological survey ever conducted in animal groups in terms of numbers of species screened. A total of 307 stool samples from 161 mammalian and non-mammalian species in two French zoos were screened by real-time PCR for the presence of Blastocystis sp. Overall, 32.2% of the animal samples and 37.9% of the species tested were shown to be infected with the parasite. A total of 111 animal Blastocystis sp. isolates were subtyped, and 11 of the 17 mammalian and avian STs as well as additional STs previously identified in reptiles and insects were found with a varying prevalence according to animal groups. These data were combined with those obtained from previous surveys to evaluate the potential risk of zoonotic transmission of Blastocystis sp. through the comparison of ST distribution between human and animal hosts. This suggests that non-human primates, artiodactyls and birds may serve as reservoirs for human infection, especially in animal handlers. In contrast, other mammals such as carnivores, and non-mammalian groups including reptiles and insects, do not seem to represent significant sources of Blastocystis sp. infection in humans. In further studies, more intensive sampling and screening of potential new animal hosts will reinforce these statements and expand our understanding of the circulation of Blastocystis sp. in animal and human populations.

Prevalence, risk factors for infection and subtype distribution of the intestinal parasite Blastocystis sp. from a large-scale multi-center study in France.

BACKGROUND: Blastocystis sp. is the most common intestinal parasite of humans. Despite its potential public health impact, epidemiological data regarding the prevalence and molecular subtype distribution of Blastocystis sp. in Europe are rarely reported. Therefore, the first multi-center epidemiological survey performed in Europe was conducted in France to diagnose and subtype Blastocystis sp. and to identify risk factors for infection. METHODS: Stool samples from 788 patients were collected either in summer or winter in 11 hospitals throughout France together with patient data. All stool samples were tested for the presence of Blastocystis sp. by quantitative PCR targeting the SSU rDNA gene. Positive samples were sequenced to determine the distribution of the subtypes in our cohort. Statistical analyses were performed to identify potential risk factors for infection. RESULTS: Using quantitative PCR, the overall prevalence of Blastocystis sp. was shown to reach 18.1 %. The prevalence was significantly higher in summer (23.2 %) than in winter (13.7 %). Travellers or subjects infected with other enteric parasites were significantly more infected by Blastocystis sp. than non-travellers or subjects free of other enteric parasites, respectively. Different age-related epidemiological patterns were also highlighted from our data. The prevalence of Blastocystis sp. was not significantly higher in patients with digestive symptoms or diagnosed with chronic bowel diseases. Among symptomatic patients, Blastocystis sp. infection was significantly associated with abdominal pain. Gender, socioeconomic status, and immune status were not identified as potential risk factors associated with infection. Among a total of 141 subtyped isolates, subtype 3 was predominant (43.3 %), followed by subtype 1 and subtype 4 (20 %), subtype 2 (12.8 %), subtype 6 and subtype 7 (2.1 %). No association between ST and clinical symptoms was statistically evidenced. CONCLUSIONS: A high prevalence of Blastocystis sp. infection was found in our French patient population. Seasonal impact on the prevalence of Blastocystis sp. was highlighted and recent travels and age were identified as main risk factors for infection. Most cases were caused by subtypes 1 to 4, with a predominance of subtype 3. Large variations in both prevalence and ST distribution between hospitals were also observed, suggesting distinct reservoirs and transmission sources of the parasite.

Colonization with the enteric protozoa Blastocystis is associated with increased diversity of human gut bacterial microbiota.

Alterations in the composition of commensal bacterial populations, a phenomenon known as dysbiosis, are linked to multiple gastrointestinal disorders, such as inflammatory bowel disease and irritable bowel syndrome, or to infections by diverse enteric pathogens. Blastocystis is one of the most common single-celled eukaryotes detected in human faecal samples. However, the clinical significance of this widespread colonization remains unclear, and its pathogenic potential is controversial. To address the issue of Blastocystis pathogenicity, we investigated the impact of colonization by this protist on the composition of the human gut microbiota. For that purpose, we conducted a cross-sectional study including 48 Blastocystis-colonized patients and 48 Blastocystis-free subjects and performed an Ion Torrent 16S rDNA gene sequencing to decipher the Blastocystis-associated gut microbiota. Here, we report a higher bacterial diversity in faecal microbiota of Blastocystis colonized patients, a higher abundance of Clostridia as well as a lower abundance of Enterobacteriaceae. Our results contribute to suggesting that Blastocystis colonization is usually associated with a healthy gut microbiota, rather than with gut dysbiosis generally observed in metabolic or infectious inflammatory diseases of the lower gastrointestinal tract.

Prevalence and Risk Factors for Intestinal Protozoan Infections with Cryptosporidium, Giardia, Blastocystis and Dientamoeba among Schoolchildren in Tripoli, Lebanon.

BACKGROUND: Intestinal protozoan infections are confirmed as major causes of diarrhea, particularly in children, and represent a significant, but often neglected, threat to public health. No recent data were available in Lebanon concerning the molecular epidemiology of protozoan infections in children, a vulnerable population at high risk of infection. METHODOLOGY AND PRINCIPAL FINDINGS: In order to improve our understanding of the epidemiology of intestinal pathogenic protozoa, a cross-sectional study was conducted in a general pediatric population including both symptomatic and asymptomatic subjects. After obtaining informed consent from the parents or legal guardians, stool samples were collected in January 2013 from 249 children in 2 schools in Tripoli, Lebanon. Information obtained from a standard questionnaire included demographic characteristics, current symptoms, socioeconomic status, source of drinking water, and personal hygiene habits. After fecal examination by both microscopy and molecular tools, the overall prevalence of parasitic infections was recorded as 85%. Blastocystis spp. presented the highest infection rate (63%), followed by Dientamoeba fragilis (60.6%), Giardia duodenalis (28.5%) and Cryptosporidium spp. (10.4%). PCR was also performed to identify species and genotypes of Cryptosporidium, subtypes of Blastocystis, and assemblages of Giardia. Statistical analysis using a logistic regression model showed that contact with family members presenting gastrointestinal disorders was the primary risk factor for transmission of these protozoa. CONCLUSIONS: This is the first study performed in Lebanon reporting the prevalence and the clinical and molecular epidemiological data associated with intestinal protozoan infections among schoolchildren in Tripoli. A high prevalence of protozoan parasites was found, with Blastocystis spp. being the most predominant protozoans. Although only 50% of children reported digestive symptoms, asymptomatic infection was observed, and these children may act as unidentified carriers. This survey provides necessary information for designing prevention and control strategies to reduce the burden of these protozoan infections, especially in children.

Draft genome sequence of the intestinal parasite Blastocystis subtype 4-isolate WR1.

The intestinal protistan parasite Blastocystis is characterized by an extensive genetic variability with 17 subtypes (ST1-ST17) described to date. Only the whole genome of a human ST7 isolate was previously sequenced. Here we report the draft genome sequence of Blastocystis ST4-WR1 isolated from a laboratory rodent at Singapore.

Acute blastocystis-associated appendicular peritonitis in a child, Casablanca, Morocco.

Despite increasing reports that Blastocystis infection is associated with digestive symptoms, its pathogenicity remains controversial. We report appendicular peritonitis in a 9-year-old girl returning to France from Morocco. Only Blastocystis parasites were detected in stools, appendix, peritoneal liquid, and recto-uterine pouch. Simultaneous gastroenteritis in 26 members of the child's family suggested an outbreak.

Blastocystis is associated with decrease of fecal microbiota protective bacteria: comparative analysis between patients with irritable bowel syndrome and control subjects.

Blastocystis is a protistan parasite living in the digestive tract of many animals, including humans. This highly prevalent intestinal parasite is suspected to be linked to Irritable Bowel Syndrome (IBS), a chronic functional bowel disorder. Here, we first compared the prevalence of Blastocystis among 56 IBS patients (40 IBS with constipation (IBS-C), 9 IBS with diarrhea (IBS-D), 4 mixed IBS (IBS-M) and 3 unsubtyped IBS (IBS-U) according to the Rome III criteria) and 56 control (i.e. without any diagnosed chronic or acute gastrointestinal disorder) subjects. The highest prevalence of Blastocystis spp. was observed in the IBS group, but was only statistically significant in men (36.8% in the IBS group versus 4.8% in the control group). We then conducted a meta-analysis including epidemiological studies attempting to determine whether Blastocystis carriage could be linked to IBS, and highlighted that IBS patients had a relative risk of 2.34 to be infected by Blastocystis when compared to non-IBS subjects. We also looked for Dientamoeba fragilis, which is often associated with IBS, and identified this parasite only in some IBS patients (n = 6/56). Several studies provided evidence for a major role of the gut microbiota in the pathophysiology of IBS. Thus, we investigated the possible impact of Blastocystis carriage on the enteric bacterial community through quantification of 8 major bacterial groups from the enteric flora. Our data indicated that men with IBS-C had a significant decrease in Bifidobacterium sp. when infected by Blastocystis. Interestingly, in control subjects (i.e. without any gastrointestinal disorder) positive for Blastocystis, Faecalibacterium prausnitzii, which is known for its anti-inflammatory properties, was significantly decreased in men. Our results support the hypothesis that Blastocystis might be linked to the pathophysiology of IBS-C and intestinal flora imbalance.

Children of Senegal River Basin show the highest prevalence of Blastocystis sp. ever observed worldwide.

BACKGROUND: Blastocystis sp. is currently the most common intestinal protist found in human feces and considered an emerging parasite with a worldwide distribution. Because of its potential impact in public health, we reinforced the picture of Blastocystis sp. prevalence and molecular subtype distribution in Africa by performing the first survey of this parasite in Senegal. METHODS: Stool samples from 93 symptomatic presenting with various gastrointestinal disorders or asymptomatic children living in three villages of the Senegal River Basin were tested for the presence of Blastocystis sp. by non-quantitative and quantitative PCR using primer pairs targeting the SSU rDNA gene. Positive samples were subtyped to investigate the frequency of Blastocystis sp. subtypes in our cohort and the distribution of subtypes in the symptomatic and asymptomatic groups of children. RESULTS: By the use of molecular tools, all 93 samples were found to be positive for Blastocystis sp. indicating a striking parasite prevalence of 100%. Mixed infections by two or three subtypes were identified in eight individuals. Among a total of 103 subtyped isolates, subtype 3 was most abundant (49.5%) followed by subtype 1 (28.2%), subtype 2 (20.4%) and subtype 4 (1.9%). Subtype 3 was dominant in the symptomatic group while subtypes 1 and 2 were detected with equal frequency in both symptomatic and asymptomatic groups. The distribution of subtypes was compared with those available in other African countries and worldwide. Comparison confirmed that subtype 4 is much less frequently detected or absent in Africa while it is commonly found in Europe. Potential sources of Blastocystis sp. infection including human-to-human, zoonotic, and waterborne transmissions were also discussed. CONCLUSIONS: The prevalence of Blastocystis sp. in our Senegalese population was the highest prevalence ever recovered worldwide for this parasite by reaching 100%. All cases were caused by subtypes 1, 2, 3 and 4 with a predominance of subtype 3. More than half of the children infected by Blastocystis sp. presented various gastrointestinal disorders. Such high prevalence of blastocystosis in developing countries makes its control a real challenge for public health authorities.

Prevalence, risk factors of infection and molecular characterization of trichomonads in puppies from French breeding kennels.

The trichomonad species Tritrichomonas fetus and Pentatrichomonas hominis were recently identified in the feces of dogs with diarrhea. However the prevalence and pathogenicity of these parasites in the canine population still remained poorly resolved. Therefore the aim of the present study was (1) to determine the prevalence of trichomonads infecting puppies living in French breeding kennels, (2) to confirm the predominance of P. hominis in dogs, (3) to investigate the genetic diversity of P. hominis isolates identified in the French canine population and (4) to evaluate the risk factors for infection by P. hominis and the influence of the parasite on feces consistency. A total of 215 both diarrheic and non-diarrheic puppies from 25 French breeding kennels were included in this epidemiological survey. Fecal samples from each puppy were examined for 6 gastrointestinal pathogens: parvovirus type 2 (CPV2), coronavirus, Toxocara canis, Cystoisospora ohioensis-complex, Cystoisospora canis, and Giardia intestinalis. A part of each collected stool was also tested for the presence of motile trichomonads by microscopy after culturing. The prevalence of trichomonad infection was 15.8% (34/215) among puppies and 20% (5/25) among breeding kennels. DNA from 26 of the 34 positive samples was successfully amplified using a trichomonad-specific primer pair. Analysis of the sequences of PCR products indicated that P. hominis was the only trichomonad infecting the canine population. All the puppies infected with P. hominis belonged to large breed dogs. Moreover, puppies from large breeding kennels, excreting a high level of G. intestinalis and/or excreting a high level of C. canis oocysts showed a higher probability of being positive for P. hominis infection. Univariate analysis also revealed an increased risk for P. hominis infection in puppies with abnormal feces. However, in a multivariate analysis, CPV2 was the only gastrointestinal pathogen associated with abnormal feces. Since enteropathogens were commonly found in dogs infected by P. hominis, the pathogenic potential of this trichomonad species remained uncertain and has to be further evaluated by experimental infection studies.

Molecular epidemiology of Blastocystis in Lebanon and correlation between subtype 1 and gastrointestinal symptoms.

Blastocystis is the most common eukaryotic parasite in the intestinal tract of humans. Because of its potential impact in public health, we acquired the first data concerning the prevalence of this parasite and the frequency of the Blastocystis subtypes (STs) in the Lebanese population. In this study, fecal samples from 220 Lebanese symptomatic and asymptomatic patients were collected and a total of 42 patients (19%) were identified as positive for this parasite by direct-light microscopy of smears. Among these, 36 Blastocystis isolates were genotyped using partial small subunit ribosomal RNA gene sequencing. The ST distribution in the present Lebanese population was as follows: ST3 (33.3%), ST2 (33.3%), ST1 (30.6%), and ST4 (2.8%). These data were compared with those available in other Middle Eastern and neighboring countries. Finally, ST1 was significantly more prevalent among symptomatic patients of this Lebanese population.

Prevalence of Tritrichomonas foetus infections in French catteries.

Recently, Tritrichomonas foetus, the known etiologic agent of bovine trichomonosis was identified in domestic cats in many countries around the world. In felids, this parasite would be a significant cause of large-bowel diarrhoea. Therefore the aim of the present study was to determine for the first time the prevalence of T. foetus infection in French catteries. In this epidemiological survey, rectal swabs from 140 cats participating in three international shows were tested for the presence of motile parasites by microscopy after culturing. The prevalence of T. foetus infection was 14.3% among cats (20/140) and 15.9% among catteries (18/117). These values were similar to those previously obtained in other European countries. Except for the age, no significant associations were found between the presence of T. foetus and various risk factors of infection such as the size of the cattery, the type of food, or the vicinity of a dog. Internal transcribed region of the ribosomal DNA unit was sequenced from the 20 T. foetus isolates identified in this study. They exhibited 100% identity and are homologous with other sequences of strains isolated from domestic cats in other countries.

Exploring and quantifying fungal diversity in freshwater lake ecosystems using rDNA cloning/sequencing and SSU tag pyrosequencing.

Water samples were collected along transects from the shore to the centre of two French lakes: the deep, volcanic, oligomesotrophic and low allochthonic-impacted Lake Pavin, and the productive and higher allochthonic-impacted Lake Aydat. The biodiversity was analysed using two approaches: the classical approach consisting of cloning/sequencing of the 18S, ITS1, 5.8S, ITS2 and partial 28S region using primers designed for fungus sequences, and the pyrosequencing of 18S rRNA hypervariable V2, V3 and V5 regions using two primer sets (one universal for eukaryotes and one for fungi). The classical approach yielded 146 (Lake Pavin) and 143 (Lake Aydat) sequences, corresponding to 46 and 63 operational taxonomic units (OTUs) respectively. Fungi represented half of the OTUs identified in Lake Pavin and 30% in Lake Aydat, and were dominated by sequences from Chytridiomycota found throughout Lake Pavin but mostly in the central pelagic zone of Lake Aydat. The pyrosequencing approach yielded 42,064 (Pavin) and 61,371 (Aydat) reads, of which 12-15% and 9-19% reads were assigned to fungi in Lakes Pavin and Aydat respectively. Chytridiomycota members were also dominant among these reads, with OTUs displaying up to > 33-fold overrepresentation in the centre compared with the riparian areas of Lake Aydat. Besides fungi, both approaches revealed other major eukaryote groups, with the highest diversity in the central areas of lakes. One of the major findings of our study was that the two lakes displayed contrasting spatial distributions, homogenous for Lake Pavin and heterogeneous for Lake Aydat, which may be related to their peculiarities. This study represents the first unveiling of microbial eukaryote and fungus diversity assessed with two complementary molecular methods, and is considered a major milestone towards understanding the dynamics and ecology of fungi in freshwater lake ecosystems, which are directly link to the abundance and distribution of taxa.

Subtype analysis of Blastocystis isolates from symptomatic patients in Egypt.

Blastocystis sp. has been described as the most common intestinal parasite in humans and has an increased impact in public health. To improve our understanding of the molecular epidemiology of this human-emerging parasite, we determined the Blastocystis subtypes (STs) and their relative frequency in Egyptian patients living in or in the vicinity of Cairo and presenting gastrointestinal symptoms. We obtained a total of 20 stool samples identified as positive for Blastocystis by microscopic examination of smears. Genotyping using partial small subunit ribosomal RNA gene analysis identified a total of 21 Blastocystis isolates corresponding to 19 single infections and one mixed infection (ST1 and ST3). Three STs were identified: ST3 was the most common ST in the present Egyptian population (61.90%) followed by ST1 (19.05%) and ST2 (19.05%). Together with previous studies carried out in different areas in Egypt, a total of five STs (ST1, ST2, ST3, ST4, and ST6) have been found in symptomatic patients. These data were compared to those available in the literature, and we underlined variations observed in the number and relative proportions of STs between and within countries. On the whole, it seemed that Blastocystis infection is likely not associated with specific STs even if some STs are predominant in the epidemiologic studies, but rather with a conjunction of factors in the course of infection including environmental risk and parasite and host factors.

Molecular epidemiology of human Blastocystis isolates in France.

Blastocystis sp. is the most common eukaryotic parasite in the intestinal tract of humans. Due to its strong impact in public health, in this study, we determined the frequency of different Blastocystis subtypes in patients in France. We hypothesized on the mode of transmission and tested a possible relationship between the subtype and symptomatic status. We obtained a total of 40 stool samples identified as positive for Blastocystis by microscopic examination of smears. Participants consisted of 25 symptomatic and 15 asymptomatic patients, for whom clinical and parasitological data were collected. For nested-polymerase chain reaction and genotyping, DNA was extracted directly from fecal samples or from fecal cultures. Morphological forms observed in fecal cultures were uncorrelated with symptomatic status. Genotyping using partial small subunit rRNA gene analysis identified a total of 43 Blastocystis isolates corresponding to 37 single infections and three mixed infections by two different subtypes. These 43 isolates belonged to five subtypes (1, 2, 3, 4, and 7) with predominance of subtype 3 (53.5%). Patient symptomatic status was uncorrelated with Blastocystis subtype.