Molecular Identification and Subtype Analysis of Blastocystis sp. Isolates from Wild Mussels (Mytilus edulis) in Northern France.

Blastocystis sp. is the most common single-celled eukaryote colonizing the human gastrointestinal tract worldwide. Because of the proven zoonotic potential of this protozoan, sustained research is therefore focused on identifying various reservoirs of transmission to humans, and in particular animal sources. Numerous groups of animals are considered to be such reservoirs due to their handling or consumption. However, some of them, including mollusks, remain underexplored. Therefore, a molecular epidemiological survey conducted in wild mussels was carried out in Northern France (Hauts-de-France region) to evaluate the frequency and subtypes (STs) distribution of Blastocystis sp. in these bivalve mollusks. For this purpose, 100 mussels (Mytilus edulis) were randomly collected in two sampling sites (Wimereux and Dannes) located in the vicinity of Boulogne-sur-Mer. The gills and gastrointestinal tract of each mussel were screened for the presence of Blastocystis sp. by real-time polymerase chain reaction (qPCR) assay followed by direct sequencing of positive PCR products and subtyping through phylogenetic analysis. In parallel, sequences of potential representative Blastocystis sp. isolates that were previously obtained from temporal surveys of seawater samples at marine stations offshore of Wimereux were integrated in the present analysis. By taking into account the qPCR results from all mussels, the overall prevalence of the parasite was shown to reach 62.0%. In total, more than 55% of the positive samples presented mixed infections. In the remaining mussel samples with a single sequence, various STs including ST3, ST7, ST14, ST23, ST26 and ST44 were reported with varying frequencies. Such distribution of STs coupled with the absence of a predominant ST specific to these bivalves strongly suggested that mussels might not be natural hosts of Blastocystis sp. and might rather be carriers of parasite isolates from both human and animal (bovid and birds) waste. These data from mussels together with the molecular identification of isolates from marine stations were subsequently discussed along with the local geographical context in order to clarify the circulation of this protozoan in this area. The identification of human and animal STs of Blastocystis sp. in mussels emphasized the active circulation of this protozoan in mollusks and suggested a significant environmental contamination of fecal origin. This study has provided new insights into the host/carrier range and transmission of Blastocystis sp. and emphasized its potential as an effective sentinel species for water quality and environmental contamination.

Frequency and Molecular Identification of Cryptosporidium in Adult Prim'Holstein Dairy Cattle Farms in the North of France.

Cryptosporidium apicomplexan protozoa are ubiquitous intracellular agents affecting humans and animals. In particular, bovine cryptosporidiosis is recognized as endemic worldwide. However, epidemiological investigations remain limited in France regarding the burden of these parasites in cattle. To improve our understanding of the epidemiology of cryptosporidiosis, the main aim of this study was to determine the frequency and the genetic diversity of Cryptosporidium in adult Prim'Holstein dairy cattle farms in the north of France. Fecal specimens were collected from 1454 non-diarrheic and non-pregnant animals (nulli-, primi-, or multiparous) throughout 20 farms in an area of 110 km around Lille. For Cryptosporidium species identification, nested PCR followed by sequence and phylogenetic analyses were used. The overall frequency of Cryptosporidium spp. in-fection was 30.00% (C.I. 95%: 12.83-54.33) in farms and 0.89% (C.I. 95%: 0.498-1.57) at the individual level. In primi- or multiparous cows, only C. andersoni was found. C. ryanae, C. bovis/xiaoi and C. andersoni were detected in heifers. The phylogenetic tree confirmed that analyzed sequences were grouped with known reference sequences reported in dairy cattle. Further studies on the cumulative prevalence, risks factors and pathogenicity are needed to give a more accurate assessment of the impact of Cryptosporidium infection in dairy cattle in France.

Bact-to-Batch: A Microbiota-Based Tool to Determine Optimal Animal Allocation in Experimental Designs.

The basis of any animal experimentation begins with the housing of animals that should take into account the need for splitting animals into similar groups. Even if it is generally recommended to use the minimum number of animals necessary to obtain reliable and statistically significant results (3Rs rule), the allocation of animals is currently mostly based on randomness. Since variability in gut microbiota is an important confounding factor in animal experiments, the main objective of this study was to develop a new approach based on 16S rRNA gene sequencing analysis of the gut microbiota of animals participating in an experiment, in order to correctly assign the animals across batches. For this purpose, a pilot study was performed on 20 mouse faecal samples with the aim of establishing two groups of 10 mice as similar as possible in terms of their faecal microbiota fingerprinting assuming that this approach limits future analytical bias and ensures reproducibility. The suggested approach was challenged with previously published data from a third-party study. This new method allows to embrace the unavoidable microbiota variability between animals in order to limit artefacts and to provide an additional assurance for the reproducibility of animal experiments.

A multiplex serological assay for the characterization of IgG immune response to SARS-CoV-2.

In the fight against SARS-COV-2, the development of serological assays based on different antigenic domains represent a versatile tool to get a comprehensive picture of the immune response or differentiate infection from vaccination beyond simple diagnosis. Here we use a combination of the Nucleoprotein (NP), the Spike 1 (S1) and Spike 2 (S2) subunits, and the receptor binding domain (RBD) and N-terminal domain (NTD) of the Spike antigens from the CoViDiag® multiplex IgG assay, to follow the immune response to SARS-CoV-2 infection over a long time period and depending on disease severity. Using a panel of 209 sera collected from 61 patients up to eight months after infection, we observed that most patients develop an immune response against multiple viral epitope, but anti-S2 antibodies seemed to last longer. For all the tested IgGs, we have found higher responses for hospitalized patients than for non-hospitalized ones. Moreover the combination of the five different IgG responses increased the correlation to the neutralizing antibody titers than if considered individually. Multiplex immunoassays have the potential to improve diagnostic performances, especially for ancient infection or mild form of the disease presenting weaker antibody responses. Also the combined detection of anti-NP and anti-Spike-derived domains can be useful to differentiate vaccination from viral infection and accurately assess the antibody potential to neutralize the virus.

Changes in the Human Gut Microbiota Associated With Colonization by Blastocystis sp. and Entamoeba spp. in Non-Industrialized Populations.

Human gut microbial communities are mainly composed of bacteria, but also include fungi, viruses, archaea, and protozoa, whose role in the gut ecosystem has only recently begun to be recognized. For example, humans colonized by Blastocystis (a gut protozoan with controversial pathogenicity) host a more diverse bacterial microbiota than individuals not carrying it, suggesting that its presence may be beneficial for the host. In parallel, the presence of non-pathogenic Entamoeba spp. has been associated with an increased diversity and compositional shifts in the bacterial microbiota of healthy rural individuals in Cameroon. However, Entamoeba and Blastocystis, the two most prevalent human gut protozoa, have never been studied in the same individuals, preventing the study of their interaction. As Blastocystis is one of the few gut protozoa commonly found in industrialized populations, which are otherwise mostly devoid of gut eukaryotes, we need to focus on rural "traditional" populations, who harbor a higher diversity of gut eukaryotes (whether pathogenic or commensal) in order to study protozoa interactions in the gut ecosystem. To this end, we profiled the gut bacterial microbiota of 134 healthy Cameroonian adults using 16S rRNA gene amplicon sequencing data. Entamoeba and Blastocystis presence and co-occurrence pattern in the same individuals were determined using metagenomic shotgun data. We found that, when taking into account both protozoa jointly, Blastocystis was associated with both a higher richness and a higher evenness of the gut bacterial microbiota, while Entamoeba was associated only with a higher richness. We demonstrated a cumulative influence of these protozoa on bacterial microbiome diversity. Furthermore, while the abundance of several common taxa (for example, Ruminococcaceae, Coprococcus and Butyrivibrio) varied according to Blastocystis colonization, only a single Bacteroides amplicon sequence variant was found to be differentially abundant between Entamoeba-negative and Entamoeba-positive samples. Given the specific signature of each protozoan on the gut microbiota and the seemingly stronger association for Blastocystis, our results suggest that Blastocystis and Entamoeba interact with gut bacteria each in its own way, but experimental studies are needed to explore the precise mechanisms of these interactions.

Agrodiag PorCoV: A multiplex immunoassay for the differential diagnosis of porcine enteric coronaviruses.

Three different porcine enteric coronaviruses (PECs), i.e., porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine Deltacoronavirus (PDCoV) are currently circulating in U.S. commercial swine herds. Differential diagnosis of PECs relies on laboratory methods. This study describes the development of an ELISA-like multiplex planar immunoassay based on virus-specific recombinant S1 proteins printed in an array of spots at the bottom of a 96-well microplate for simultaneous detection differential serodiagnosis of PEDV, TGEV, PDCoV in a single sample. The technology overall format and working principle is similar to the solid-phase standard ELISA. After the three typical incubation steps, the reaction was visualized as blue spots which intensity correlated with antibody levels to specific viral antigen target in the array. The diagnostic performance of the assay was evaluated on known status serum samples (n = 480) collected over time (day post-inoculation -7, 0, 7, 14, 21, 28, 35, and 42) from pigs inoculated with PEDV, TGEV Purdue, TGEV Miller, PDCoV (USA/IL/2014), or mock inoculated with culture media under experimental conditions. Antigen-specific cut-offs were selected to ensure 100% diagnostic and analytical specificity for each given antigen target. The overall diagnostic sensitivity was 92% (44/48 positives, 95% confidence interval (CI) 98,100) for PEDV S1, 100% (95/95 positives, 95% CI 98, 100) for TGEV S1, and 98% (47/48 positives, 95% CI 97, 100) for PDCoV S1. The results of this study demonstrate that the AgroDiag PEC multiplex immunoassay is an efficient and reliable test for differential detection and serodiagnosis of PEDV, TGEV and PDCoV.

Genetic basis for virulence differences of various Cryptosporidium parvum carcinogenic isolates.

Cryptosporidium parvum is known to cause life-threatening diarrhea in immunocompromised hosts and was also reported to be capable of inducing digestive adenocarcinoma in a rodent model. Interestingly, three carcinogenic isolates of C. parvum, called DID, TUM1 and CHR, obtained from fecal samples of naturally infected animals or humans, showed higher virulence than the commercially available C. parvum IOWA isolate in our animal model in terms of clinical manifestations, mortality rate and time of onset of neoplastic lesions. In order to discover the potential genetic basis of the differential virulence observed between C. parvum isolates and to contribute to the understanding of Cryptosporidium virulence, entire genomes of the isolates DID, TUM1 and CHR were sequenced then compared to the C. parvum IOWA reference genome. 125 common SNVs corresponding to 90 CDSs were found in the C. parvum genome that could explain this differential virulence. In particular variants in several membrane and secreted proteins were identified. Besides the genes already known to be involved in parasite virulence, this study identified potential new virulence factors whose functional characterization can be achieved through CRISPR/Cas9 technology applied to this parasite.

The Impact of Bioinformatics Pipelines on Microbiota Studies: Does the Analytical "Microscope" Affect the Biological Interpretation?

Targeted metagenomics is the solution of choice to reveal differential microbial profiles (defined by richness, diversity and composition) as part of case-control studies. It is well documented that each data processing step may have the potential to introduce bias in the results. However, selecting a bioinformatics pipeline to analyze high-throughput sequencing data from A to Z remains one of the critical considerations in a case-control microbiota study design. Consequently, the aim of this study was to assess whether the same biological conclusions regarding human gut microbiota composition and diversity could be reached using different bioinformatics pipelines. In this work, we considered four pipelines (mothur, QIIME, kraken and CLARK) with different versions and databases, and examined their impact on the outcome of metagenetic analysis of Ion Torrent 16S sequencing data. We re-analyzed a case-control study evaluating the impact of the colonization of the intestinal protozoa Blastocystis sp. on the human gut microbial profile. Although most pipelines reported the same trends in this case-control study, we demonstrated how the use of different pipelines affects the biological conclusions that can be drawn. Targeted metagenomics must therefore rather be considered as a profiling tool to obtain a broad sense of the variations of the microbiota, rather than an accurate identification tool.

The Formation of Glycan-Specific Natural Antibodies Repertoire in GalT-KO Mice Is Determined by Gut Microbiota.

Gut commensal bacteria are known to have a significant role in regulating the innate and adaptive immune homeostasis. Alterations in the intestinal microbial composition have been associated with several disease states, including autoimmune and inflammatory conditions. However, it is not entirely clear how commensal gut microbiota modulate and contribute to the systemic immunity, and whether circulating elements of the host immune system could regulate the microbiome. Thus, we have studied the diversity and abundance of specific taxons in the gut microbiota of inbred GalT-KO mice during 7 months of animal life by metagenetic high-throughput sequencing (16S rRNA gene, variable regions V3-V5). The repertoire of glycan-specific natural antibodies, obtained by printed glycan array technology, was then associated with the microbial diversity for each animal by metagenome-wide association studies (MWAS). Our data show that the orders clostridiales (most abundant), bacteriodales, lactobacillales, and deferribacterales may be associated with the development of the final repertoire of natural anti-glycan antibodies in GalT-KO mice. The main changes in microbiota diversity (month-2 and month-3) were related to important changes in levels and repertoire of natural anti-glycan antibodies in these mice. Additionally, significant positive and negative associations were found between the gut microbiota and the pattern of specific anti-glycan antibodies. Regarding individual features, the gut microbiota and the corresponding repertoire of natural anti-glycan antibodies showed differences among the examined animals. We also found redundancy in different taxa associated with the development of specific anti-glycan antibodies. Differences in microbial diversity did not, therefore, necessarily influence the overall functional output of the gut microbiome of GalT-KO mice. In summary, the repertoire of natural anti-carbohydrate antibodies may be partially determined by the continuous antigenic stimulation produced by the gut bacterial population of each GalT-KO mouse. Small differences in gut microbiota diversity could determine different repertoire and levels of natural anti-glycan antibodies and consequently might induce different immune responses to pathogens or other potential threats.

[Prospects for applications in human health of nanopore-based sequencing]. / Séquençage par nanopores - Perspectives d'applications en santé humaine.

High throughput sequencing has opened up new clinical opportunities moving towards a medicine of precision. Oncology, infectious diseases or human genomics, many applications have been developed in recent years. The introduction of a third generation of nanopore-based sequencing technology, addressing some of the weaknesses of the previous generation, heralds a new revolution. Portability, real time, long reads and marginal investment costs, these promising new technologies point to a new shift of paradigm. What are the perspectives opened up by nanopores for clinical applications?

MICRA: an automatic pipeline for fast characterization of microbial genomes from high-throughput sequencing data.

The increase in available sequence data has advanced the field of microbiology; however, making sense of these data without bioinformatics skills is still problematic. We describe MICRA, an automatic pipeline, available as a web interface, for microbial identification and characterization through reads analysis. MICRA uses iterative mapping against reference genomes to identify genes and variations. Additional modules allow prediction of antibiotic susceptibility and resistance and comparing the results of several samples. MICRA is fast, producing few false-positive annotations and variant calls compared to current methods, making it a tool of great interest for fully exploiting sequencing data.

Targeted metagenomic sequencing data of human gut microbiota associated with Blastocystis colonization.

In the past decade, metagenomics studies have become widespread due to the arrival of second-generation sequencing platforms characterized by low costs, high throughput and short read lengths. Today, although benchtop sequencers are considered to be accurate platforms to deliver data for targeted metagenomics studies, the limiting factor has become the analysis of these data. In a previous paper, we performed an Ion Torrent PGM 16S rDNA gene sequencing of faecal DNAs from 48 Blastocystis-colonized patients and 48 Blastocystis-negative subjects, in order to decipher the impact of this widespread protist on gut microbiota composition and diversity. We report here on the Ion Torrent targeted metagenomic sequencing and analysis of these 96 human faecal samples, and the complete datasets from raw to analysed data. We also provide the key steps of the bioinformatic analyses, from library preparation to data filtering and OTUs tables generation. This data represents a valuable resource for the scientific community, enabling re-processing of these targeted metagenomic datasets through various pipelines and a comparative evaluation of microbiota analysis methods.

Assessment of Common and Emerging Bioinformatics Pipelines for Targeted Metagenomics.

Targeted metagenomics, also known as metagenetics, is a high-throughput sequencing application focusing on a nucleotide target in a microbiome to describe its taxonomic content. A wide range of bioinformatics pipelines are available to analyze sequencing outputs, and the choice of an appropriate tool is crucial and not trivial. No standard evaluation method exists for estimating the accuracy of a pipeline for targeted metagenomics analyses. This article proposes an evaluation protocol containing real and simulated targeted metagenomics datasets, and adequate metrics allowing us to study the impact of different variables on the biological interpretation of results. This protocol was used to compare six different bioinformatics pipelines in the basic user context: Three common ones (mothur, QIIME and BMP) based on a clustering-first approach and three emerging ones (Kraken, CLARK and One Codex) using an assignment-first approach. This study surprisingly reveals that the effect of sequencing errors has a bigger impact on the results that choosing different amplified regions. Moreover, increasing sequencing throughput increases richness overestimation, even more so for microbiota of high complexity. Finally, the choice of the reference database has a bigger impact on richness estimation for clustering-first pipelines, and on correct taxa identification for assignment-first pipelines. Using emerging assignment-first pipelines is a valid approach for targeted metagenomics analyses, with a quality of results comparable to popular clustering-first pipelines, even with an error-prone sequencing technology like Ion Torrent. However, those pipelines are highly sensitive to the quality of databases and their annotations, which makes clustering-first pipelines still the only reliable approach for studying microbiomes that are not well described.

Effects of Propidium Monoazide (PMA) Treatment on Mycobiome and Bacteriome Analysis of Cystic Fibrosis Airways during Exacerbation.

INTRODUCTION AND PURPOSE: Propidium monoazide (PMA)-pretreatment has increasingly been applied to remove the bias from dead or damaged cell artefacts, which could impact the microbiota analysis by high-throughput sequencing. Our study aimed to determine whether a PMA-pretreatment coupled with high-throughput sequencing analysis provides a different picture of the airway mycobiome and bacteriome. RESULTS AND DISCUSSION: We compared deep-sequencing data of mycobiota and microbiota of 15 sputum samples from 5 cystic fibrosis (CF) patients with and without prior PMA-treatment of the DNA-extracts. PMA-pretreatment had no significant effect on the entire and abundant bacterial community (genera expressed as operational taxonomic units (OTUs) with a relative abundance greater than or equal to 1%), but caused a significant difference in the intermediate community (less than 1%) when analyzing the alpha biodiversity Simpson index (p = 0.03). Regarding PMA impact on the airway mycobiota evaluated for the first time here; no significant differences in alpha diversity indexes between PMA-treated and untreated samples were observed. Regarding beta diversity analysis, the intermediate communities also differed more dramatically than the total and abundant ones when studying both mycobiome and bacteriome. Our results showed that only the intermediate (or low abundance) population diversity is impacted by PMA-treatment, and therefore that abundant taxa are mostly viable during acute exacerbation in CF. Given such a cumbersome protocol (PMA-pretreatment coupled with high-throughput sequencing), we discuss its potential interest within the follow-up of CF patients. Further studies using PMA-pretreatment are warranted to improve our "omic" knowledge of the CF airways.

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.

Relationship Between Pneumocystis carinii Burden and the Degree of Host Immunosuppression in an Airborne Transmission Experimental Model.

To quantitatively assess the risk of contamination by Pneumocystis depending on the degree of immunosuppression (ID) of the exposed rat hosts, we developed an animal model, where rats went through different doses of dexamethasone. Then, natural and aerial transmission of Pneumocystis carinii occurred during cohousing of the rats undergoing gradual ID levels (receivers) with nude rats developing pneumocystosis (seeders). Following contact between receiver and seeder rats, the P. carinii burden of receiver rats was determined by toluidine blue ortho staining and by qPCR targeting the dhfr monocopy gene of this fungus. In this rat model, the level of circulating CD4(+) and CD8(+) T lymphocytes remained significantly stable and different for each dose of dexamethasone tested, thus reaching the goal of a new stable and gradual ID rat model. In addition, an inverse relationship between the P. carinii burden and the level of circulating CD4(+) or CD8(+) T lymphocytes was evidenced. This rat model may be used to study other opportunistic pathogens or even co-infections in a context of gradual ID.

Photodynamic therapy relieves colitis and prevents colitis-associated carcinogenesis in mice.

BACKGROUND: Inflammatory bowel diseases are incurable illnesses of the gastrointestinal tract, which substantially enhance the risk of developing colorectal carcinogenesis. Conventional photodynamic therapy is a clinically approved therapeutic modality used in the treatment of neoplastic diseases. Recent preclinical and clinical studies have shown that photodynamic therapy with low doses of photosensitizer and/or light improves inflammatory conditions, including colitis. This study aims therefore at investigating the therapeutic potential of low-dose photodynamic therapy (LDPDT) with a liposomal formulation of meta-tetra(hydroxyphenyl)chlorin (namely Foslip) in the prevention of colitis-associated cancer in mice. METHODS: LDPDT efficacy was evaluated by endoscopic, macroscopic, and histological analysis. Myeloperoxidase levels were quantified by enzyme linked immunosorbent assay and cytokines expression by quantitative RT-PCR analysis. The integrity of the intestinal barrier was evaluated by immunostaining, and bacterial composition of the fecal microbiota was determined by 454 pyrosequencing of V3-V4 region of bacterial 16S rRNA genes. RESULTS: LDPDT reduced intestinal tumor growth by decreasing the expression of a wide range of inflammatory mediators and by lowering neutrophil influx. LDPDT treatment prevents onset of a dysbiotic microbiota in the colitis-associated cancer model. CONCLUSIONS: LDPDT with Foslip could be considered as a novel treatment modality to prevent colorectal carcinogenesis in patients with inflammatory bowel disease.

High-Throughput Sequencing, a VersatileWeapon to Support Genome-Based Diagnosis in Infectious Diseases: Applications to Clinical Bacteriology.

The recent progresses of high-throughput sequencing (HTS) technologies enable easy and cost-reduced access to whole genome sequencing (WGS) or re-sequencing. HTS associated with adapted, automatic and fast bioinformatics solutions for sequencing applications promises an accurate and timely identification and characterization of pathogenic agents. Many studies have demonstrated that data obtained from HTS analysis have allowed genome-based diagnosis, which has been consistent with phenotypic observations. These proofs of concept are probably the first steps toward the future of clinical microbiology. From concept to routine use, many parameters need to be considered to promote HTS as a powerful tool to help physicians and clinicians in microbiological investigations. This review highlights the milestones to be completed toward this purpose.