Blastocystis spp. from epidemiology to pathophysiology. What is the link with irritable bowel syndrome?

Le protozoaire anaérobie Blastocystis spp. est le parasite le plus fréquemment identifié dans les selles humaines, quelle que soit la région du monde considérée, même si peu de données sont disponibles en France. Par ailleurs, ces chiffres varient grandement selon les techniques de diagnostic utilisées. Son caractère pathogène est controversé car, dans la majorité des cas, Blastocystis spp. est isolé chez des patients asymptomatiques. Néanmoins, (i) l’analyse des données génomiques de Blastocystis sp. montre que le parasite code pour un nombre important de protéases sécrétées, facteurs de virulence connus chez d’autres parasites ; (ii) plusieurs études de prévalence suggèrent une association avec le Syndrome de l’Intestin Irritable (SII), une colopathie chronique fonctionnelle caractérisée notamment par une dysbiose et une augmentation de la perméabilité paracellulaire. Au cours de nos travaux, nous avons complété les données épidémiologiques du parasite au travers d’une étude multicentrique en France et au Liban, en utilisant une technique de détection par biologie moléculaire. Ainsi, en France, la prévalence globale atteint 18,1% avec des chiffres significativement plus élevés en été, le sous-type 3 (ST3) est majoritaire (43,3% des cas). La notion de voyage dans l’année précédente apparaît comme un facteur de risque de contamination. La prévalence libanaise est de 63%, avec également une prédominance du ST3 (46,3%). Nous nous sommes ensuite intéressés au lien potentiel entre Blastocystis spp. et le SII. Nous avons montré que le risque d’être infecté par Blastocystis spp. était deux fois élevé chez les sujets atteints de SII comparativement à des sujets non-SII. Nous avons observé une dysbiose chez les individus porteurs de Blastocystis sp., qu’ils soient atteints de SII ou non, avec une diminution significative de bactéries considérées comme « bénéfiques », les bifidobactéries et Faecalibacterium prausnitzii, bactérie aux propriétés anti-inflammatoires. Compte tenu de ces modifications du microbiote intestinal et du manque de recul, Blastocystis spp. fait partie des microorganismes à rechercher chez un donneur avant transplantation de microbiote fécal. Dans cette optique, nous avons comparé 3 techniques de diagnotic par PCR en temps réel décrites dans la littérature ainsi que la trousse SeegeneTM (Eurobio), sur une cohorte de 140 individus et montré des différences de performances entre ces outils. Afin de mieux comprendre les impacts du parasite sur l’équilibre intestinal, nous avons développé des approches in vitro et in vivo. Nous avons mis en évidence in vitro une augmentation de la perméabilité para-cellulaire sous l’effet de 2 protéases à cystéine parasitaires sécrétées, fonctionnant en tandem, une légumaïne et une cathepsine B. Nous avons ensuite mis au point un modèle animal d’infection chronique par Blastocsytis sp. chez le rat. Nous avons procédé par gavage oral de kystes purifiés issus de selles humaines. Des immunomarquages réalisés sur des colons de rats infectés ont montré que Blastocystis sp. est capable de pénétrer dans la couche de mucus. Nous avons également pu observer par microscopie électronique que le parasite pouvait adhérer aux cellules épithéliales. Afin de standardiser ce modèle animal, nous avons travaillé sur la forme kystique du parasite, qui est la forme infectieuse. Nous avons caractérisé les kystes en mettant en évidence la présence de 1 à 4 noyaux et une paroi riche en chitine et D-mannose. Ces caractéristiques nous permettront de valider les formes obtenues par enkystement in vitro à partir de parasites en culture axénique. Nos résultats suggèrent donc un impact possible de Blastocystis spp. en santé humaine (patients colopathes, receveurs de transplantation de microbiote fécal, simple sujet porteur) et ouvrent de nouvelles pistes pour l’exploration de sa pathogénie et de son lien avec le SII.The anaerobic protozoan Blastocystis spp. is the most frequently identified parasite in human feces in all regions of the world, although few data are available in France. Moreover, these figures vary greatly according to the diagnostic techniques used. Its pathogenicity is controversial because, in the majority of cases, Blastocystis spp. is isolated from asymptomatic patients. Nevertheless, (i) analysis of genomic data of Blastocystis sp. shows that the parasite encodes a significant number of secreted proteases, known virulence factors in other parasites; (ii) several prevalence studies suggest an association with Irritable Bowel Syndrome (IBS), a chronic functional colopathy characterized by dysbiosis and increased paracellular permeability. In the course of our work, we completed the epidemiological data of the parasite through a multicenter study in France and Lebanon, using a molecular biology detection technique. Thus, in France, the overall prevalence reached 18.1% with significantly higher figures in summer, subtype 3 (ST3) is the majority (43.3% of cases). The notion of having travelled in the previous year appears to be a risk factor for contamination. The Lebanese prevalence is 63%, with ST3 also predominating (46.3%). We then looked at the potential link between Blastocystis spp. and IBS. We showed that the risk of being infected by Blastocystis spp. was twice as high in IBS subjects compared to non-IBS subjects. We observed a dysbiosis in individuals carrying Blastocystis sp., whether they had IBS or not, with a significant decrease in bacteria considered "beneficial", bifidobacteria and Faecalibacterium prausnitzii, a bacterium with anti-inflammatory properties. Given these changes in the intestinal microbiota and the lack of experience, Blastocystis spp. are among the microorganisms to look for in a donor before fecal microbiota transplantation. With this in mind, we compared 3 real-time PCR diagnostic techniques described in the literature and the SeegeneTM kit (Eurobio), on a cohort of 140 individuals and showed differences in performance between these tools. In order to better understand the impacts of the parasite on the intestinal balance, we developed in vitro and in vivo approaches. We have demonstrated in vitro an increase in paracellular permeability under the effect of two secreted parasite cysteine proteases, a legumain and a cathepsin B, working in tandem. We then developed an animal model of chronic infection by Blastocsytis sp. in rats. We proceeded by oral gavage of purified cysts from human feces. Immunolabeling of infected rat colons showed that Blastocystis sp. is able to penetrate the mucus layer. We were also able to observe by electron microscopy that the parasite could adhere to epithelial cells. In order to standardize this animal model, we worked on the cystic form of the parasite, which is the infectious form. We characterized the cysts by showing the presence of 1 to 4 nuclei and a wall rich in chitin and D-mannose. These characteristics will allow us to validate the forms obtained by encystation in vitro from parasites in axenic culture. Our results suggest a possible impact of Blastocystis spp. in human health (colopathic patients, fecal microbiota transplantation recipients, simple carrier subjects) and open new avenues for the exploration of its pathogenesis and its link with IBS

Evaluation of Two Commercial Kits on the Automated ELITe InGenius PCR Platform for Molecular Diagnosis of Toxoplasmosis

International audienceMolecular diagnosis of toxoplasmosis is essential for establishing the diagnosis of congenital contaminations and for primary infection or reactivation of immunocompromised patients. An integrated extraction and real-time PCR-based system is of particular interest in this context. Commercial kits for automated extraction and amplification steps are now available. Herein, we assessed two commercial PCR assays for this diagnosis, those of Bio-Evolution and Elitech, on the ELITe InGenius platform. The Bio-Evolution assay showed a specificity and a sensitivity of 100% on clinical samples, but a lower analytical detection threshold than the Elitech assay. The latter showed a specificity of 100% and a sensitivity of 96%. The SP1000 cartridges, which allow DNA extraction from 1 mL of template, showed interesting performances on amniotic fluid samples. Overall, the two kits had good performances on the InGenius platform, which offers a turn-key solution suitable for the molecular diagnosis of toxoplasmosis

[Pseudo-hypoglycemia and hyperleukocytosis: a case report].

International audienceMrs B., 39 years old, hospitalized in the department of respiratory medicine for lung cancer, has an undetectable and verified venous blood glucose concentration (measured in central laboratory) less than 0.1 mmol/L. The patient feels no symptom of hypoglycemia. A concomitant capillary determination realised by a bedside glucose reader gives a result of 4.7 mmol/L. This gap is due to glucose consumption in vitro by leukocytes between the time of sampling and laboratory analysis. Indeed the leukocyte count is 86.4 G/L (92% neutrophils) probably in a context of paraneoplastic syndrome. An efficient talk between biologist and clinician identified this phenomenon for this patient. This event allows us to recall the different causes of hypoglycemia (artifactual or real), and to describe the care of patients with true hypoglycemia

Encephalitis caused by an unusual human herpes virus type 6 and Toxoplasma gondii co-infection in a cord blood transplant recipient

International audienceBackground: The case of a central nervous system human herpes virus type 6 (HHV-6) and Toxoplasma gondii co-infection after an umbilical cord blood transplantation in a chronic myelomonocytic leukaemia patient is reported.Case report: A 65-year-old Caucasian man underwent an umbilical cord blood transplantation within the context of chronic myelomonocytic leukaemia. On day 37 post-graft, he presented with a severe headache; PCRs of cerebrospinal fluid and blood were positive for T. gondii and HHV-6. The patient was treated with pyrimethamine and sulfadiazine associated with ganciclovir.Conclusion: HHV-6 reactivation can trigger a reactivation of T. gondii. This case suggests that patients who are seropositive for T. gondii and who present with HHV-6 reactivation should be monitored closely for toxoplasmosis

Prokaryotic and Eukaryotic Fecal Microbiota in Irritable Bowel Syndrome Patients and Healthy Individuals Colonized With Blastocystis

Blastocystis is the most frequently isolated protozoan from human stool. Its role in human health is still debated, and a high prevalence was reported in irritable bowel syndrome (IBS) subjects, suggesting a potential link with microbiota. In the present study, we aimed to investigate prokaryotic and eukaryotic microbiota in both IBS-C (constipated) and healthy individuals. We recruited 35 IBS-C patients and 23 healthy subjects, from which 12 and 11 carried Blastocystis, respectively. We performed 16S and 18S rRNA high-throughput sequencing on feces. Whereas we did not observe differences between infected and non-infected controls, several phyla were significantly modified in IBS-C patients according to the presence of Blastocystis. Tenericutes phylum and Ruminococcaceae family were especially increased in Blastocystis carriers. Furthermore, colonization with Blastocystis was associated with discrete changes in the microbial eukaryome, particularly among the Fungi taxa. Depending on the group of patients considered, the mycobiota changes do not go in the same direction and seem more deleterious in the IBS-C group. These results encourage further in vivo and in vitro investigations concerning the role of Blastocystis in the gut environment

Comparison of DNA extraction methods and real-time PCR assays for the detection of blastocystis sp. in stool specimens

International audienceDiagnosis of Blastocystis in stool may be challenging, as microscopic examination and culture-based methods have demonstrated low sensitivity. Molecular detection assays are now available for this enteric parasite, based on “in-house” or commercial-developed techniques. The aim of this study was to assess and compare the performance of (i) two DNA extraction methods (manual versus automated), and (ii) four qPCR assays (three “in-house” and one commercialized), for detection of Blastocystis sp. in human stools. One hundred and forty stools were included, among which 76 were confirmed to be positive for Blastocystis. The manual DNA extraction method allowed for the identification of significantly more positive specimens than the automated method (p < 0.05). In particular, specimens with a low parasite load were negative when DNA was extracted with the automated process. The four qPCR assays also had variable performances, with the commercialized assay being the most sensitive (84%) but the least specific (82%). Overall, for all qPCR assays, the specificity decreased when the sensitivity increased. Blastocystis’ subtype, notably the subtype 4, influenced these performances. Our results indicate that the positivity rate for the detection of Blastocystis in stools could be variable according to the DNA extraction method and the qPCR assay used. These pitfalls need to be considered for the selection of method and interpretation of results, particularly considering the search of this intestinal parasite in a donor before fecal microbiota transplantation

Method validation of a set of 12 GEM® Premier™ 4000 blood gas analyzers for point-of-care testing in a university teaching hospital

International audienceBackground: Blood gas analyzers are o0.ften integrated into point-of-care testing provisions. International standards (ISO 22870 and 15189) as adapted to French COFRAC regulations make accreditation of point-ofta-care testintag obligatory. We installed and assessed 12 GEM PREMIER 4000 analyzers for pH, pCO 2 , pO 2 , Na + , K + , Cl-, Ca 2+ , lactate, hemoglobin and oxyhemoglobin (O 2 Hb) at Clermont-Ferrand Hospital. These instruments were distributed across 11 care sites in the hospital. Methods: Precision was studied at two control levels for each parameter. Comparisons between GEM analyzers were performed (on 30 samples) for pH, pCO 2 , pO 2 , Na + , K + , Cl-, Ca 2+ , lactate, hemoglobin and O 2 Hb; and between GEM analyzers and the central laboratory for Na + , K + , Cl-, Ca 2+ and hemoglobin (on 30-50 samples). Uncertainty in measurement (UM) was evaluated with an approach using reproducibility and accuracy data. Results: The coefficients of variation (CVs) were in line with recommendations, except for the repeatability CV for pO 2. All CVs were below 4%. All comparisons complied with recommendations. Uncertainties of measurement were also validated. Conclusion: Our results met standard requirements and the 12 analyzers were assessed as suitable for point-of-care testing in services of academic medical centers, as exemplified at Clermont-Ferrand hospital

OUP accepted manuscript

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Hormographiella aspergillata: an emerging basidiomycete in the clinical setting? A case report and literature review

International audienceBackground: Filamentous basidiomycetes are mainly considered to be respiratory tract colonizers but the clinical significance of their isolation in a specimen is debatable. Hormographiella aspergillata was first reported as a human pathogen in 1971. We discuss the role of this mold as a pathogen or colonizer and give an update on diagnostic tools and in vitro antifungal susceptibility. Case presentation: We identified three cases of H. aspergillata with respiratory symptoms in a short period of time. One invasive infection and two colonizations were diagnosed. Culture supernatants showed that H. aspergillata can produce galactomannan and β-D-glucan but not glucuronoxylomannan. For the first time, isavuconazole susceptibility was determined and high minimum inhibitory concentrations (MICs) were found. Liposomal amphotericin B and voriconazole have the lowest MICs. Conclusion: To date, 22 invasive infections involving H. aspergillata have been reported. On isolation of H. aspergillata, its pathogenic potential in clinical settings can be tricky. Molecular identification and antifungal susceptibility testing are essential considering high resistance against several antifungal therapies

Population Structure of Candida parapsilosis: No Genetic Difference Between French and Uruguayan Isolates Using Microsatellite Length Polymorphism

International audienceCandida parapsilosis is a human commensal yeast, frequently involved in infection worldwide and especially in neonates. It is the second species responsible for bloodstream infections in Uruguay and the third species in France. We were interested in knowing whether the population structure of isolates responsible for candidemia in France and in Uruguay was different. Genotyping methods based on microsatellite length polymorphism (MLP) have been described and are especially used for investigation of local outbreaks. We therefore determined the genotypes of 159 C. parapsilosis isolates recovered from 122 patients (84 French patients from 43 hospitals and 38 Uruguayan patients from 10 hospitals) using three microsatellites markers previously described. Our results confirmed that C. parapsilosis population has a high genetic diversity, clonal inheritance and that majority of patients were infected by a single isolate. But we described recurrent infections due to related or unrelated genotypes resulting from isolates harboring loss or gain of heterozygosity. We also described three cases of coinfections due to unrelated genotypes. We did not uncover geographic specificity but observed two linked genotypes that seem to be associated with voriconazole resistance. Finally, among eight isolates involved in grouped cases, the genotypes were similar in six cases supporting the hypothesis of inter-patient transmission. These results confirmed the usefulness of performing MLP genotyping analysis for grouped cases of C. parapsilosis isolates in order to reinforce preventive hygiene measures