Subtypes Sst Research Articles

Metataxomics reveals Blastocystis subtypes mixed infections in Colombian children.

Blastocystis sp., is an intestinal protist with a broad host range and a high prevalence in human populations worldwide, even in developed Western countries. The publication of conflicting evidence has divided the scientific community about the pathogenic role of this parasite. Even though, genetic studies on Blastocystis sp. revealed associations between genotypes and different pathogenic profiles. Conventionally, the detection of this parasite is based on microscopic or PCR methods, which offer meager or null performance in detecting mixed infections. In this work, we applied a metataxonomic NGS approach targeting the V4 region of the eukaryotic SSU-rRNA gene and classical phylogenetic methods. This approach allowed us to detect Blastocystis sp. in stool samples from infected children living in an urban setting in the city of Medellin attending the same daycare center. Phylogenetic analysis identified the subtypes present in the children as ST1, ST2, and ST3. Besides, mixed infections of subtypes ST1+ST3 were spotted in 16% of the analyzed stool samples.

A cell-type-specific error-correction signal in the posterior parietal cortex

Neurons in the posterior parietal cortex contribute to the execution of goal-directed navigation1 and other decision-making tasks2–4. Although molecular studies have catalogued more than 50 cortical cell types5, it remains unclear what distinct functions they have in this area. Here we identified a molecularly defined subset of somatostatin (Sst) inhibitory neurons that, in the mouse posterior parietal cortex, carry a cell-type-specific error-correction signal for navigation. We obtained repeatable experimental access to these cells using an adeno-associated virus in which gene expression is driven by an enhancer that functions specifically in a subset of Sst cells6. We found that during goal-directed navigation in a virtual environment, this subset of Sst neurons activates in a synchronous pattern that is distinct from the activity of surrounding neurons, including other Sst neurons. Using in vivo two-photon photostimulation and ex vivo paired patch-clamp recordings, we show that nearby cells of this Sst subtype excite each other through gap junctions, revealing a self-excitation circuit motif that contributes to the synchronous activity of this cell type. These cells selectively activate as mice execute course corrections for deviations in their virtual heading during navigation towards a reward location, for both self-induced and experimentally induced deviations. We propose that this subtype of Sst neurons provides a self-reinforcing and cell-type-specific error-correction signal in the posterior parietal cortex that may help with the execution and learning of accurate goal-directed navigation trajectories.

Pilot Study on the Prevalence of Entamoeba gingivalis in Austria-Detection of a New Genetic Variant.

Entamoeba gingivalis is a parasitic protist that resides in the oral cavity. Although E. gingivalis has been frequently detected in individuals with periodontitis, its precise role in this context remains to be established, since E. gingivalis is also regularly found in healthy individuals. Sequence data on E. gingivalis are still scarce, with only a limited number of sequences available in public databases. In this study, a diagnostic PCR protocol was established in order to obtain a first impression on the prevalence of E. gingivalis in Austria and enable a differentiation of isolates by targeting the variable internal transcribed spacer regions. In total, 59 voluntary participants were screened for E. gingivalis and almost 50% of the participants were positive, with a significantly higher prevalence of participants with self-reported gingivitis. Moreover, in addition to the established subtypes ST1 and ST2, a potentially new subtype was found, designated ST3. 18S DNA sequencing and phylogenetic analyses clearly supported a separate position of ST3. Interestingly, subtype-specific PCRs revealed that, in contrast to ST2, ST3 only occurred in association with ST1. ST2 and ST1/ST3 were more often associated with gingivitis; however, more data will be necessary to corroborate this observation.

Enterocytozoon bieneusi and Encephalitozoon intestinalis (microsporidia) in HIV-positive patients in central Spain.

Microsporidia are fungi-related eukaryotic intracellular parasites that opportunistically infect immunocompromised individuals such as those infected by the human immunodeficiency virus (HIV). Among them, Enterocytozoon bieneusi and Encephalitozoon spp. are the most clinically relevant species. We investigated the occurrence and genetic diversity of microsporidial and protist infections in mostly immunocompetent HIV-positive patients in Madrid, Spain. A structured questionnaire was used to retrieve data on factors potentially associated with an increased risk of infection, including sexual attitudes and sex-risk behaviour. Faecal samples (n = 96) from 81 HIV-positive patients were collected and analysed by molecular (PCR and Sanger sequencing) methods. Two microsporidial pathogens were detected: Ent. bieneusi (2.5%, 95% CI: 0.3-8.6) and Enc.intestinalis (4.9%, 95% CI: 1.4-12.2). The two Ent. bieneusi isolates were identified as zoonotic genotype A. Among protists, Entamoeba dispar was the species most prevalently found (33.3%, 95% CI: 23.2-44.7), followed by Blastocystis spp. (19.8%, 95% CI: 11.7-30.1), Giardia duodenalis (13.6%, 95% CI: 7.0-23.0), and Cryptosporidium spp. and Entamoeba histolytica (2.5%, 95% CI: 0.3-8.6 each). Cyclospora cayetanensis and Cystoisospora belli were not detected. Subtypes ST1 (70.6%, 12/17) and ST3 (29.4%, 5/17) were identified within Blastocystis sp., sub-assemblages AII and BIII (50%, 1/2 each) within G. duodenalis, and Cry. parvum and canine-adapted Cry. canis (50%, 1/2 each) within Cryptosporidium spp. Microsporidial and protist parasites were frequent in well-controlled, mostly immunocompetent HIV-positive patients and should be included in diagnostic algorithms when diarrhoea is present.

First identification of Blastocystis sp. subtypes in Rex rabbits in China

Blastocystis sp. is a common protist that colonizes the intestinal tract in both humans and animals worldwide. A total of 666 fecal samples of Rex rabbits were collected from 12 farms in three administrative regions in Henan, China. Blastocystis sp. was screened and subtyped by PCR amplification of the small subunit ribosomal DNA. The results indicated that 31 (4.7%, 31/666) rabbits were positive for Blastocystis sp. across three farms (25.0%, 3/12). The infection rate of Blastocystis sp. in Rex rabbits was highest in Jiyuan at 9.1% (30/331), followed by Luoyang (0.5%, 1/191), with no positive infections found in Zhengzhou. The Blastocystis sp. infection rate in adults (10.2%, 14/287) was higher than that in young rabbits (4.5%, 17/379) (χ2=0.0027, P>0.50). Four Blastocystis sp. subtypes (ST1, ST3, ST4, and ST17) were identified in rabbits in the present study. Among them, the subtypes ST1 (n=15) and ST3 (n=14) were dominant, followed by ST4 (n=1) and ST17 (n=1). Blastocystis sp. ST1 was the dominant subtype in adult rabbits, and ST3 was the dominant subtype in young rabbits. This study enriches the data on the prevalence and subtype distributions of Blastocystis sp. in rabbits. More studies are needed among humans, domestic animals, and wild animals to obtain a better understanding of their role in the spread of Blastocystis sp.

Draft genomes of Blastocystis subtypes from human samples of Colombia

BackgroundBlastocystis is one of the most common eukaryotic microorganisms colonizing the intestines of both humans and animals, but the conditions under which it may be a pathogen are unclear.MethodsTo study the genomic characteristics of circulating subtypes (ST) in Colombia, we established nine xenic cultures from Blastocystis isolated from human fecal samples, we identified 10 different subtypes, since one sample had a mixed infection. Thus, the genomes of the subtypes ST1 (n = 3), ST2 (n = 1), ST3 (n = 2), ST6 (n = 1), ST7 (n = 1), and ST8 (n = 2) were sequenced using Illumina and Oxford Nanopore Technologies (ONT).ResultsAnalyses of these draft nuclear genomes indicated remarkable diversity in terms of genome size and guanine-cytosine (GC) content among the compared STs. Illumina sequencing-only draft genomes contained 824 to 2077 scaffolds, with total genome size ranging from 12 to 13.2 Mb and N50 values ranging from 10,585 to 29,404 base pairs (bp). The genome of one ST1 isolate was sequenced using ONT. This assembly was more contiguous, with a size of 20 million base pairs (Mb) spread over 116 scaffolds, and an N50 of 248,997 bp.ConclusionThis work represents one of the few large-scale comparative genomic analyses of Blastocystis isolates, providing an additional glimpse into its genomic diversity.Graphical

Molecular Epidemiology of Blastocystis in Confined Slow Lorises, Macaques, and Gibbons.

Blastocystis sp. is a common intestinal anaerobic parasite infecting non-human primates and many other animals. This taxon threatens the health of NHPs due to its high genetic diversity, impeding efforts to improve confined management and subsequent conservation practices. This study collected 100 and 154 fecal samples from captive macaques, gibbons, and slow lorises in the summer and winter, respectively. The Blastocystis infection, its gene subtypes, and its zoonotic potential based on small subunit ribosomal RNA (SSU rRNA) were analyzed. The prevalence of Blastocystis in the three primate genera was 57.79% (89/154) in the summer (2021) and 29.00% (29/100) in the winter (2020). Four zoonotic subtypes-ST1, ST2, ST3, and ST4-were identified. ST2 was the most prevalent subtype, suggesting that these animals may serve as reservoirs for pathogens of human Blastocystis infections. The macaques showed a more significant variation in Blastocystis infection between seasons than gibbons and slow lorises. The slow lorises in small cages and enclosure areas were potentially more infected by Blastocystis in the summer, indicating that inappropriate captive management may have detrimental effects on their health.

Molecular subtyping of Blastocystis sp. detected in patients at a large tertiary referral hospital in Lusaka, Zambia

BackgroundBlastocystis sp. is a common enteric eukaryote of humans whose pathogenicity is still debatable. However, a number of reported Blastocystis colonization associated with enteric disease exist. In Zambia, presence of the pathogen has previously been reported in children. However, the molecular epidemiology of Blastocystis colonization remains unclarified in Zambia.Methods and resultsArchived stool samples submitted for routine parasitological diagnosis at Zambia’s largest tertiary referral hospital positive for Blastocystis sp. by microscopic examination were selected for the study. Subtyping of the Blastocystis was done based on polymerase chain reactions (PCR) amplification, sequencing and subsequent phylogenetic analysis of the 18S small subunit (SSU) rDNA gene. Four subtypes, ST1 (allele 4), ST2 (allele 12), ST3 (allele 34, 36, 37, 38, 39) and ST6 (allele 122), were identified by molecular procedures in the study, with some Zambian sequences showing close relationships with those detected in non-human primates and common rats.ConclusionsThe study revealed the circulation of multiple Blastocystis subtypes ST1, 20% (9/45), ST2, 15% (7/45), ST3 24.4% (11/45), and ST6, 2.2% (1/45) in the study population. The close clustering of some Zambian sequences with those detected from animals suggests the possibility of the presence of both anthroponotic and zoonotic transmission cycles in the country. Further studies in animal populations are recommended for a better understanding of the epidemiology of Blastocystis and for implementation of effective evidence-based control strategies.

Reassessment of somatostatin receptor SST4 expression in bronchopulmonary and gastroenteropancreatic neuroendocrine neoplasms using the novel rabbit monoclonal anti-human SST4 antibody 7H49L61

Somatostatin receptors SST1, SST2, and SST5 are overexpressed in neuroendocrine neoplasms (NENs), but little is known about SST4 expression in NENs because of a lack of specific monoclonal antibodies. We recently developed and thoroughly characterised a rabbit monoclonal anti-human SST4 antibody, 7H49L61, and showed that it is well suited for identifying SST4 expression in routine pathology samples. The present study aimed to re-evaluate SST4 expression in a large set of NEN samples using this antibody. For this purpose, we assessed SST4 expression in 722 formalin-fixed, paraffin-embedded NEN samples from 274 patients by immunohistochemistry using the novel antibody 7H49L61. The immunostaining was semiquantitatively evaluated using the 12-point immunoreactivity score (IRS), and the results were correlated with clinicopathological data. SST4 was detected in 39.3% of all NENs, but with a median IRS of 2.0, its expression intensity was negligible overall. In all cases, both cytoplasmic and membraneous staining was observed. SST4 expression was somewhat higher in bronchopulmonary NEN (BP-NEN) than in gastroenteropancreatic NEN (GEP-NEN) but still very low. SST4 expression positively correlated with favourable patient outcomes in BP-NEN but had a positive association with Ki-67 index or tumour grading and a negative interrelationship with overall survival in GEP-NEN. In conclusion, unlike that of other SST subtypes, SST4 expression in both BP-NEN and GEP-NEN is negligible and of no diagnostic or therapeutic relevance.

Identification of α-L-fucosidase (ALFuc) of Blastocystis sp. subtypes ST1, ST2 and ST3.

ABSTRACT Blastocystis sp. is a common intestinal microorganism. The α-L-fucosidase (ALFuc) is an enzyme long associated with the colonization of the gut microbiota. However, this enzyme has not been experimentally identified in Blastocystis cultures. The objective of the present study was to identify ALFuc in supernatants of axenic cultures of Blastocystis subtype (ST)1 ATCC-50177 and ATCC-50610 and to compare predicted ALFuc proteins of alfuc genes in sequenced STs1–3 isolates in human Blastocystis carriers. Excretion/secretion (Es/p) and cell lysate proteins were obtained by processing Blastocystis ATCC cultures and submitting them to SDS–PAGE and immunoblotting. In addition, 18 fecal samples from symptomatic Blastocystis human carriers were analyzed by sequencing of amplification products for subtyping. A complete identification of the alfuc gene and phylogenetic analysis were performed. Immunoblotting showed that the amplified band corresponding to ALFuc (~51 kDa) was recognized only in the ES/p. Furthermore, prediction analysis of ALFuc 3D structures revealed that the domain α-L-fucosidase and the GH29 family's catalytic sites were conserved; interestingly, the galactose-binding domain was recognized only in ST1 and ST2. The phylogenetic inferences of ALFuc showed that STs1–3 were clearly identifiable and grouped into specific clusters. Our results show, for the first time through experimental data that ALFuc is a secretion product of Blastocystis sp., which could have a relevant role during intestinal colonization; however, further studies are required to clarify this condition. Furthermore, the alfuc gene is a promising candidate for a phylogenetic marker, as it shows a conserved classification with the SSU-rDNA gene.

Octreotide Lar in Patients with Autosomal Dominant Polycystic Kidney Disease: From Bench to A Novel Perspective of Therapy

Polycystins 1 or 2 congenital defects result in impaired Ca2+ inflow through the tubular cell membrane with reduced intracellular Ca2+ concentration and secondary adenylcyclase over-activation with increased intracellular cAMP. This activates chloride-driven fluid secretion and tubular cell proliferation and de-differentiation with cyst formation and growth. Thus, medications, such as the somatostatin analogue Octreotide LAR or the vasopressin antagonist Tolvaptan, that reduce intracellular cAMP, have been tested to inhibit cAMP-mediated chloride secretion and cell proliferation in experimental and human polycystic kidney disease. Seminal studies conducted in the early ‘80s by Franklin Epstein showed that in the shark rectal gland chloride secretion is markedly inhibited by somatostatin in a way suggesting inhibition of adenylcyclase. Evidence that specific receptors for somatostatin, in particular the sst2 subtype, are present in tubular cell membranes suggested that Octreotide LAR binding to its specific renal receptors could exert similar effects in ADPKD cells. In a pilot, cross-over safety study we found that 6-month Octreotide LAR therapy was safe and well tolerated in 12 patients with ADPKD and significantly decreased total kidney volume growth as compared to placebo. Then, the ALADIN and ALADIN II academic, prospective, randomized, placebo-controlled clinical trials found that 3-year Octreotide LAR treatment significantly slowed total kidney and cyst volume growth. In ALADIN treatment slowed chronic decline of directly measured GFR in 79 patients with estimated GFR ≥ 40 ml/min/1.73 m2. In ALADIN II treatment slowed progression to doubling of serum creatinine or ESKD in 100 patients with stage 3b-4 CKD. Treatment was equally safe and well tolerated in both studies. Sub-studies also showed that 3-year Octreotide LAR therapy reduced total liver volume in 27 ADPKD patients with associated polycystic livers and improved left ventricular twisting and untwisting function in 34 ADPKD patients assessed by speckled-tracked echocardiography. Future trials should confirm the long-term benefits of Octreotide LAR in larger populations of ADPKD patients. Moreover, recent studies found that somatostatin analogues and Tolvaptan have additional beneficial effects in experimental polycystic kidney disease. Thus, clinical trials should also explore whether Octreotide LAR and Tolvaptan in combined therapy may have an additional beneficial effect even in human disease.

Robust cell-type-specific associations with Alzheimer's disease within excitatory and inhibitory neuronal subclasses.

Cortical neuron loss is a pathological hallmark of late-onset Alzheimer's disease (AD). However, despite recent single-cell transcriptomic analyses of human AD brain tissue, it remains unclear which neuronal subtypes are most vulnerable to degeneration. Here we applied cell-type proportion estimation methods to multiple RNA sequencing (RNAseq) datasets derived from human postmortem brain to identify the neuronal subtypes most strongly associated with AD. Postmortem bulk-brain RNAseq data collected from three AD studies were analyzed (n=953), comprising six datasets of different neocortical regions. Relative cell type proportions were estimated for each brain sample using the markerGeneProfile package and marker genes for specific neuronal subtypes derived from ultra-high depth single-nucleus RNAseq (Hodge et al., 2019). Linear models were used to test associations of cell-type proportion estimations (CTPs) with AD diagnosis in each dataset separately, followed by random-effects meta-analysis. Pseudo-bulk investigation of the Mathys et al. (2019) single-nucleus RNAseq dataset was used to further validate findings. Follow-up in the Religious Orders Study and Memory and Aging Project (ROS/MAP) cohorts (n=593) permitted the identification of specific neuropathologies associated with each neuronal subtype. Meta-analysis revealed several neuronal subtypes significantly associated with AD, with the strongest signals from intra-telencephalic (IT) excitatory neurons (meta p=4.82x10-13 ) and somatostatin (SST) inhibitory neurons (meta p= 1.55x10-10 ). Pseudo-bulk single-nucleus analysis suggested that these signals are most likely due to differences in cell proportions rather than differences in expression of any predominant marker gene for each class. In-depth analysis of CTPs in ROS/MAP consistently implicated a decrease in IT and SST CTPs with increasing amyloid and tau neuropathology, as well as more severe cognitive decline prior to death. Decreases in SST and IT neuron subtypes, estimated from bulk RNAseq data, were significantly associated with AD diagnosis across multiple cortical regions. The SST signal in particular is robust to the exclusion of the SST mRNA from CTP calculations, suggesting the pathological relevance of a loss of SST-expressing neurons rather than a downregulation of the SST gene specifically. These findings support seminal work implicating somatostatin in the pathogenesis of AD and helps to re-focus our current understanding of neuronal vulnerability in AD.

Prevalence of intestinal parasites and molecular characterization of Giardia intestinalis, Blastocystis spp. and Entamoeba histolytica in the village of Fort\xedn Mboror\xe9 (Puerto Iguaz\xfa, Misiones, Argentina)

BackgroundIntestinal parasites (IPs) are widely distributed worldwide and are one of the major contributors to gastrointestinal disease. Their prevalence is associated with poor access to water, sanitation and hygiene (WASH). The objective of this study was to identify the prevalence of IPs, including soil-transmitted helminths (STH), and their relation to socioeconomic characteristics, as well as a first approach to molecularly characterize the types of Giardia intestinalis, Blastocystis spp. and Entamoeba histolytica present in an indigenous community from Puerto Iguazú, Misiones, Argentina.MethodsA cross-sectional study was conducted in the rural settlement of Fortin Mbororé between January and March 2018. Socioeconomic variables, household characteristics, and stool and blood samples were collected. Standard coprological techniques were used to analyze stool samples, and a complete hemogram was performed on the blood samples. Giardia intestinalis microscopy-positive samples were genetically typed by the β-giardin (bg) gene. Molecular identification of Blastocystis spp. subtypes and E. histolytica were carried out by amplification and sequencing of a partial fragment of the small subunit ribosomal RNA gene (SSU rDNA).ResultsThe overall prevalence of IPs was 92.7%, with 72.0% specifically for hookworm. IPs were significantly more prevalent in preschool- and school-age children (P < 0.05). No formal education (P = 0.035), the presence of unimproved floors (P = 0.001) and overcrowding (P = 0.005) were significantly associated with IP infection. Hookworm was associated with anemia (P = 0.019). Molecular characterization revealed the presence of E. histolytica sub-assemblages AII (12.5%), AIII (87.5%) and BIV (100%); one case of sub-assemblage D for G. intestinalis; and the presence of subtypes ST1 (14.8%), ST2 (14.8%) and ST3 (70.4%) of Blastocystis spp.ConclusionsProtozoans detected in this study are transmitted mainly through water contaminated with fecal matter, evidencing the need to improve the quality of water and sanitation for the inhabitants of Fortín Mbororé. Molecular characterization showed that domestic animals can be implicated in the zoonotic transmission of G. intestinalis and Blastocystis spp. to humans. A hyperendemic area for STH was found, with hookworm prevalence greater than 50%. Therefore, improvements in WASH as well as mass deworming programs need to be implemented in this area to control and decrease the prevalence of IPs in general and STH in particular.Graphical

Molecular epidemiology and genotype/subtype distribution of Blastocystis sp., Enterocytozoon bieneusi, and Encephalitozoon spp. in livestock: concern for emerging zoonotic infections

Intestinal parasitic infections have high prevalence rate in many regions especially in developing countries. The aim of this study was to determine the presence and genotype/subtype of some intestinal protozoa in livestock in Iran. Stool samples were collected from cattle, sheep, chickens, and horses. The presence of targeted parasites was evaluated using real-time PCR. Genotyping/subtyping of positive samples was characterized using sequencing of the ITS and barcoding region, respectively. Blastocystis sp., 27.7% (48/173) and Enterocytozoon bieneusi 26.0% (45/173) were the most frequent protozoa followed by Encephalitozoon spp., 0.57% (1/173). Cryptosporidium spp. were not detected among samples. Encephalitozoon spp., was detected only in chickens 2.2% (1/45). A statistically correlation was seen between animals and the prevalence of targeted protozoa. E. bieneusi genotypes I (9/38; 23.68%), BEB6 (22/38; 57.89%), D (6/38; 15.79%), and horse1 (1/38; 2.63%) were detected among samples. A statistically significant correlation was seen between the genotypes and animals (P ≤ 0.05). Blastocystis sp., ST1 (1/45; 2.22%), ST5 3/45; 6.66%), ST7 (1/45; 2.22%), ST10 (24/45; 53.33%), and ST14 (16/45; 35.55%) were characterized among samples. There was no significant correlation between certain subtypes and animals (P = 0.173). The presence of zoonotic potential genotypes of E. bieneusi in animals and zoonotic potential subtypes ST1 and ST7 among our samples provide a clue about the transmission dynamic of E. bieneusi and Blastocystis sp. between animals–animals and humans–animals.

Wide Genetic Diversity of Blastocystis in White-Tailed Deer (Odocoileus virginianus) from Maryland, USA.

Blastocystis is a gastrointestinal protist frequently reported in humans and animals worldwide. Wildlife populations, including deer, may serve as reservoirs of parasitic diseases for both humans and domestic animals, either through direct contact or through contamination of food or water resources. However, no studies of the occurrence and subtype distribution of Blastocystis in wildlife populations have been conducted in the United States. PCR and next generation amplicon sequencing were used to determine the occurrence and subtypes of Blastocystis in white-tailed deer (Odocoileus virginianus). Blastocystis was common, with 88.8% (71/80) of samples found to be positive. Twelve subtypes were identified, ten previously reported (ST1, ST3, ST4, ST10, ST14, ST21, and ST23–ST26) and two novel subtypes (ST30 and ST31). To confirm the validity of ST30 and ST31, MinION sequencing was used to obtain full-length SSU rRNA gene sequences, and phylogenetic and pairwise distance analyses were performed. ST10, ST14, and ST24 were the most commonly observed subtypes. Potentially zoonotic subtypes ST1, ST3, or ST4 were present in 8.5% of Blastocystis-positives. Mixed subtype infections were common (90.1% of Blastocystis-positives). This study is the first to subtype Blastocystis in white-tailed deer. White-tailed deer were found to be commonly infected/colonized with a wide diversity of subtypes, including two novel subtypes, zoonotic subtypes, and subtypes frequently reported in domestic animals. More studies in wildlife are needed to better understand their role in the transmission of Blastocystis.

Epidemiology, species composition and genetic diversity of tetra- and octonucleated Entamoeba spp. in different Brazilian biomes

BackgroundEntamoeba species harbored by humans have different degrees of pathogenicity. The present study explores the intra- and interspecific diversity, phylogenetic relationships, prevalence and distribution of tetra- and octonucleated cyst-producing Entamoeba in different Brazilian regions.MethodsCross-sectional studies were performed to collect fecal samples (n = 1728) and sociodemographic data in communities located in four Brazilian biomes: Atlantic Forest, Caatinga, Cerrado, and Amazon. Fecal samples were subjected to molecular analysis by partial small subunit ribosomal DNA sequencing (SSU rDNA) and phylogenetic analysis.ResultsLight microscopy analysis revealed that tetranucleated cysts were found in all the studied biomes. The highest positivity rates were observed in the age group 6–10 years (23.21%). For octonucleated cysts, positivity rates ranged from 1 to 55.1%. Sixty SSU rDNA Entamoeba sequences were obtained, and four different species were identified: the octonucleated E. coli, and the tetranucleated E. histolytica, E. dispar, and E. hartmanni. Novel haplotypes (n = 32) were characterized; however, new ribosomal lineages were not identified. The Entamoeba coli ST1 subtype predominated in Atlantic Forest and Caatinga, and the ST2 subtype was predominant in the Amazon biome. E. histolytica was detected only in the Amazon biome. In phylogenetic trees, sequences were grouped in two groups, the first containing uni- and tetranucleated and the second containing uni- and octonucleated cyst-producing Entamoeba species. Molecular diversity indexes revealed a high interspecific diversity for tetra- and octonucleated Entamoeba spp. (H ± SD = 0.9625 ± 0.0126). The intraspecific diversity varied according to species or subtype: E. dispar and E. histolytica showed lower diversity than E. coli subtypes ST1 and ST2 and E. hartmanni.ConclusionsTetra- and octonucleated cyst-producing Entamoeba are endemic in the studied communities; E. histolytica was found in a low proportion and only in the Amazon biome. With regard to E. coli, subtype ST2 was predominant in the Amazon biome. The molecular epidemiology of Entamoeba spp. is a field to be further explored and provides information with important implications for public health.Graphical

Gut microbiota profiles in diarrheic patients with co-occurrence of Clostridioides difficile and Blastocystis.

Blastocystis and Clostridioides difficile co-occurrence is considered a rare event since the colonization by Blastocystis is prevented under a decrease in beneficial bacteria in the microbiota when there is C. difficile infection (CDI). This scenario has been reported once, but no information on the gut microbiota profiling is available. The present study is motivated by knowing which members of the microbiota can be found in this rare scenario and how this co-occurrence may impact the abundance of other bacteria, eukaryotes or archaea present in the gut microbiota. This study aimed to describe the bacterial and eukaryotic communities using amplicon-based sequencing of the 16S- and 18S-rRNA regions of three patient groups: (1) Blastocystis and C. difficile infection (B+/C+, n = 31), (2) C. difficile infection only (B˗/C+, n = 44), and (3) without Blastocystis or C. difficile (B˗/C˗, n = 40). Blastocystis was subtyped using amplicon-based sequencing of the 18S-rRNA gene, revealing circulation of subtypes ST1 (43.4%), ST3 (35.85%) and ST5 (20.75%) among the study population. We found that B+/C+ patients had a higher abundance of some beneficial bacteria (such as butyrate producers or bacteria with anti-inflammatory properties) compared with non-Blastocystis-colonized patients, which may suggest a shift towards an increase in beneficial bacteria when Blastocystis colonizes patients with CDI. Regarding eukaryotic communities, statistical differences in the abundance of some eukaryotic genera between the study groups were not observed. Thus, this study provides preliminary descriptive information of a potential microbiota profiling of differential presence by Blastocystis and C. difficile.

Molecular detection and subtype distribution of Blastocystis in farmed pigs in southern China

Blastocystis is one of the most common causative agents of intestinal diseases, which can cause enteric diseases in animals and humans. However, limited data is available on the prevalence or subtypes of Blastocystis infections in farmed pigs in southern China. In this study, a total of 396 fecal samples were collected from farmed pigs in three provinces in southern China in 2016, and screened for Blastocystis by PCR amplification of the small subunit rRNA (SSU rRNA) gene fragment. One hundred and seventy (42.93%) of the examined fecal samples were detected Blastocystis-positive, and two known zoonotic subtypes ST1 and ST5 were identified, with ST5 being the predominate subtype. Moreover, gender, age and region were considered as risk factors that associated with Blastocystis infection in farmed pigs. The present study revealed the prevalence and subtypes of Blastocystis infections in farmed pigs in southern China, which provided essential data for the control of Blastocystis infections in pigs, other animals and humans in China.

Genetic characterization and zoonotic potential of Blastocystis from wild animals in Sichuan Wolong National Natural Reserve, Southwest China.

Blastocystis is a prevalent eukaryotic parasite that has been identified in a wide range of hosts. Several species are considered potential sources of Blastocystis infection in humans, but little is known about the prevalence of Blastocystis in wild animals. In this study, the prevalence and subtypes of Blastocystis were investigated to assess the zoonotic potential of wild animals in Sichuan Wolong National Natural Reserve. A total of 300 fecal samples were collected from 27 wildlife species in three areas of the Reserve. The subtype (ST), genetic characteristics, and prevalence of Blastocystis were determined by PCR amplification of part (~600 bp) of the SSU rRNA gene. Thirty fecal samples (10.0%) were Blastocystis-positive. The highest prevalence of Blastocystis was found in Yinchanggou (18.3%), with significantly less found in Niutoushan (7.5%) and Genda (5.5%) (p < 0.05). No significant differences were associated with different orders of animals in prevalence, which may be because of the small number of positive samples obtained. Sequence analysis showed five subtypes (ST1, ST3, ST5, ST13, and ST14), with ST13 and ST14 being predominant (33% each), followed by ST1 (20%). This is the first molecular investigation of Blastocystis infection in the wild animals of southwestern China. Subtypes ST1, ST3, ST5, and ST14 have previously been identified in humans, suggesting that wild animals may be potential reservoirs of Blastocystis for humans.

Detection of enteric parasites and molecular characterization of Giardia duodenalis and Blastocystis sp. in patients admitted to hospital in Ankara, Turkey.

This epidemiological study assesses the occurrence of enteric parasites in 4303 patients attended at two public hospitals in Ankara (Turkey) during 2018-2019. Microscopy was used as a screening test. Giardia duodenalis was also identified using a commercial ELISA for the detection of parasite-specific coproantigens. Giardia-positive samples by microscopy/ELISA were confirmed by real-time PCR and characterized using a multilocus genotyping scheme. Blastocystis sp. was genotyped in a sample subset. Blastocystis sp. (11.1%, 95% CI 11.4‒14.8%) and G. duodenalis (1.56%, 95% CI 1.22‒1.96) were the most prevalent pathogens found. Cryptosporidium spp., Entamoeba histolytica and intestinal helminths were only sporadically (<0.5%) found. For G. duodenalis, sequence (n = 30) analyses revealed the presence of sub-assemblages AII (23.3%), discordant AII/AIII (23.3%) and mixed AII + AIII (6.7%) within assemblage A, and BIII (10.0%), BIV (3.3%) and discordant BIII/BIV (23.3%) within assemblage B. Two additional sequences (6.7%) were assigned to the latter assemblage but sub-assemblage information was unknown. No associations between G. duodenalis assemblages/sub-assemblages and sociodemographic and clinical variables could be demonstrated. For Blastocystis sp., sequence (n = 6) analyses identified subtypes ST1, ST2 and ST3 at equal proportions. This is the first molecular characterization of G. duodenalis based on MLG conducted in Turkey to date.