Improving the diagnostic management of invasive fungal diseases

Les infections fongiques systémiques (IFS) sont un problème de santé publique car elles concernent des patients à risque en constante augmentation et leur incidence a augmenté ces dernières années. La performance du diagnostic biologique des IFS reste insuffisante et leur létalité à 3 mois est d’environ 40% en France. Le diagnostic précoce est un axe majeur de progrès, dans le but d’améliorer la prise en charge des patients et réduire la létalité.Dans la première partie, nous avons réalisé un catalogue des champignons filamenteux isolés chez l’Homme, répertoriant 565 espèces de champignons filamenteux d’intérêt clinique. Les sites anatomiques et le cadre nosologique de ces infections ont été décrits.La seconde partie de notre travail visait à optimiser le diagnostic des levuroses invasives. Nous avons d’abord sélectionné les techniques les plus performantes pour extraire l’ADN des cinq principales espèces de Candida d’intérêt clinque. Nous en avons retenu trois techniques d’extraction d’ADN, parmi 11 techniques évaluées, dont la performance était adéquate. Ensuite, nous avons cherché à améliorer la détection des Candida à partir d’échantillon liquide par une méthode de capture en solution au moyen de billes magnétiques couplées à des anticorps spécifiques. Enfin, nous nous sommes intéressés au diagnostic des IFS à levures rares, les trichosporonoses, pour lesquelles les outils diagnostiques spécifiques sont inexistants.La dernière partie s’intéresse à l’identification des sources potentielles des infections fongiques invasives à travers l’investigation d’une épidémie d’infection à Saprochaete clavata dans un centre de cancérologie.Invasive fungal infections (IFI) are a public-health issue because they concern patients at risk, which is constantly increasing and their incidence has risen in recent years. The performance of biological diagnosis of IFI remains insufficient and the overall 3-month IFIs fatality rate is about 40% in France. Early diagnosis is a major improvement target that would allow enhance the patients’ management and curb this fatality risk. In the first part, we made a catalog of filamentous fungi isolated in humans, listing 565 species of filamentous fungi of clinical interest. The anatomical sites and the nosological entities associated with these fungi were described.The second part of our work aimed at optimizing the diagnosis of invasive fungal yeast infections. We first selected the most efficient techniques to extract DNA from the five main Candida species of clinical interest. We selected three DNA extraction techniques, among 11 techniques evaluated, which exhibited adequate performance. Next, we aimed to improve the detection of Candida from liquid suspensions by a solution capture using magnetic beads coupled to specific antibodies. Finally, we are interested in the diagnosis of rare invasive yeast infections, trichosporonosis, for which specific diagnostic tools are inexistent.The last part focuses on identifying potential sources of invasive fungal infections through the investigation of an outbreak of Saprochaete clavata infection in a cancer center

Étude épidémiologique portant sur la détection moléculaire d’un panel de dix parasites gastro-intestinaux, à Marseille

Il existe peu de données épidémiologiques récentes sur les infections parasitaires intestinales chez l’Homme en France. Nous avons mené une étude prospective visant à estimer la prévalence de dix protozoaires entériques au CHU de La Timone à Marseille, France, à l'aide d'un diagnostic basé sur la détection de l’ADN de ces parasites par la PCR en temps réel. Un total de 643 prélèvements de selles, provenant de 488 patients, adressées au laboratoire de parasitologie-mycologie du Centre Hospitalo-Universitaire de Marseille sur une période de 6 mois a été inclus. La moyenne d’âge des patients était de 44 ans (intervalle : 0 – 97 ans).En amont de cette étude nous avons comparé le kit manuel le plus représenté dans la littérature QIAmp® DNA Stool Mini Kit (Qiagen) avec une technique semi automatisée alliant prétraitement mécanique, chimique, enzymatique et extraction automatisée par EZ1® (Qiagen). A l’issu de cette étude préliminaire, nous avons choisi d’extraire L'ADN en utilisant la méthode semi-automatisée EZ1® (Qiagen). Les parasites d’intérêt ont été détectés à l’aide de PCR « singleplex » quantitatives (qPCR). Le choix des amorces pour chaque protozoaire a été réalisé à partir d’une recherche bibliographique dans pubmed. Pour les échantillons positifs, le sous-type Blastocystis a été déterminé par analyse de séquence. Au cours de l'étude, la prévalence globale des protozoaires entériques était de 17%. Blastocystis spp. a été l’espèce la plus fréquente (10,5%), suivie par Dientamoeba fragilis (2,3%) et Giardia intestinalis (2,3%). La prévalence des autres protozoaires était inférieure à 1% chacun. Pour Blastocystis spp. les sous types (ST) retrouvés ont été les suivants : le sous-type 3 (ST3) prédominant (43,6%) et les autres sous-types ST1, ST2, ST4 et ST6. A notre connaissance, c'est la première fois qu'un diagnostic basé sur la qPCR est utilisé pour étudier la prévalence de dix protozoaires entériques dans un hôpital universitaire français

Conception et validation d'un substitut vasculaire naturel, fonctionnalisé par un film multicouche de polyélectrolytes et cellularisé par un endothelium autologue orienté

Les taux élevés de mortalité et de morbidité associés aux maladies vasculaires en font des pathologies dont les conséquences physiopathologiques, chirurgicales et socio-économiques sont d une importance majeure pour le système de santé. Malgré leurs avantages, la disponibilité limitée des vaisseaux autologues a conduit au développement de prothèses synthétiques. Cependant, leur surface hautement thrombogène limite leur utilisation dans la substitution des vaisseaux de petit calibre (< 6 mm). De ce fait, à cause de leur obstruction précoce, la reconstitution d une surface luminale proche de l endothélium natif est incontournable. Pourtant, les revêtements de surface actuellement disponibles possèdent de médiocres qualités de rétention des néo-endothélium lorsqu ils sont soumis à des contraintes de cisaillement physiologiques. Dans ce travail, nous proposons un substitut vasculaire de petit calibre endothélialisé réalisé à partir de trois éléments : 1) une matrice préparée à partir d une artère ombilicale désendothélialisée, 2) un recouvrement de surface innovant constitué du film multicouche de polyélectrolytes (MPE) (PAH-PSS)3-PAH, et 3) un néo-endothélium constitué de cellules endothéliales matures ou progénitrices. Les études in vitro menées sur ces substituts ont montré que la formation, la rétention sous contraintes de cisaillement et la fonctionnalité du néoendothélium élaboré sur la surface luminale étaient améliorées par le film MPE. L implantation du substitut par pontage termino-latéral sur le lapin a montré que le cahier des charges imputé aux substituts de petit calibre était rempli, principalement en termes de perméabilité et de diamètre, mais aussi de résistance à la suture et aux infections. En conclusion, le film MPE favorise le développement d un substitut vasculaire de petit diamètre perméable à long terme et qui pourrait répondre aux exigences des chirurgiensVascular diseases with their high rate of mortality and morbidity belong to the pathologies involving important socio-economic factors for health system. Despite the advantages of autografts, the limited availability of autologous vessels has led to the development of synthetic prostheses. However, their high thrombogenic surface limits their use as small calibre vascular substitutes (< 6 mm). To prevent narrowing of small diameter vascular grafts, the reconstruction of a luminal surface close to the native endothelium is essential. However, the retention of the neo-endothelium subjected to shear stress is poor on the coatings currently available. In this work, we developed a small calibre endothelialized vascular substitute thanks to three elements: 1) a natural matrix prepared from umbilical artery, 2) an innovative coating based on the polyelectrolytes multilayer film (PEM) (PAH-PSS)3-PAH, and 3) used for cell culture of mature or progenitor endothelial cells. In vitro studies have shown that the formation, the retention under shear stress and the endothelial function of the neoendothelium on the luminal surface were improved by PEM film. The anastomosis of this substitute on rabbits has shown that the specifications essential to small calibre vascular grafts were reached, mainly in terms of permeability and diameter but also of resistance to suture and infections. In conclusion, PEM films helped us to develop a small diameter vascular substitute with long term patencyNANCY1-Bib. numérique (543959902) / SudocSudocFranceF

Eukaryotic and Prokaryotic Microbiota Interactions

The nature of the relationship between the communities of microorganisms making up the microbiota in and on a host body has been increasingly explored in recent years. Microorganisms, including bacteria, archaea, viruses, parasites and fungi, have often long co-evolved with their hosts. In human, the structure and diversity of microbiota vary according to the host's immunity, diet, environment, age, physiological and metabolic status, medical practices (e.g., antibiotic treatment), climate, season and host genetics. The recent advent of next generation sequencing (NGS) technologies enhanced observational capacities and allowed for a better understanding of the relationship between distinct microorganisms within microbiota. The interaction between the host and their microbiota has become a field of research into microorganisms with therapeutic and preventive interest for public health applications. This review aims at assessing the current knowledge on interactions between prokaryotic and eukaryotic communities. After a brief description of the metagenomic methods used in the studies were analysed, we summarise the findings of available publications describing the interaction between the bacterial communities and protozoa, helminths and fungi, either in vitro, in experimental models, or in humans. Overall, we observed the existence of a beneficial effect in situations where some microorganisms can improve the health status of the host, while the presence of other microorganisms has been associated with pathologies, resulting in an adverse effect on human health

Dual infection: What diptera has (have) bit her

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First case of B ALL with KMT2A-MAML2 rearrangement: a case report

Abstract Background A large number of chromosomal translocations of the human KMT2A gene, better known as the MLL gene, have so far been characterized. Genetic rearrangements involving KMT2A gene are frequently involved in lymphoid, myeloid and mixed lineage leukemia. One of its rare fusion partners, the mastermind like 2 (MAML2) gene has been reported in four cases of myeloid neoplasms after chemotherapy so far: two acute myeloid leukemias (AML) and two myelodysplasic syndrome (MDS), and two cases of secondary T-cell acute lymphoblastic leukemia (T-ALL). Case presentation Here we report the case of a KMT2A - MAML2 fusion discovered by Next-Generation Sequencing (NGS) analysis in front of an inv11 (q21q23) present in a 47-year-old female previously treated for a sarcoma in 2014, who had a B acute lymphoid leukemia (B ALL). Conclusion It is, to our knowledge, the first case of B acute lymphoblastic leukemia with this fusion gene. At the molecular level, two rearrangements were detected using RNA sequencing juxtaposing exon 7 to exon 2 and exon 9 to intron 1–2 of the KMT2A and MAML2 genes respectively, and one rearrangement using Sanger sequencing juxtaposing exon 8 and exon 2

Evaluation of 11 DNA Automated Extraction Protocols for the Detection of the 5 Mains Candida Species from Artificially Spiked Blood

International audienceThe molecular detection of Candida plays an important role in the diagnosis of candidaemia, a major cause of morbidity and mortality. The sensitivity of this diagnosis is partly related to the efficiency of yeast DNA extraction. In this monocentric study, we investigated the suitability of 11 recent automated procedures for the extraction of low and high amounts of Candida DNA from spiked blood. The efficacy of the DNA extraction procedures to detect Candida spp. in blood samples ranged from 31.4% to 80.6%. The NucliSENSTM easyMAGTM procedure was the most efficient, for each species and each inoculum. It significantly outperformed the other procedures at the lower Candida inocula mimicking the clinical setting. This study highlighted a heterogeneity in DNA extraction efficacy between the five main Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei). Up to five automated procedures were appropriate for C. krusei DNA extraction, whereas only one method yielded an appropriate detection of low amount of C. tropicalis. In the era of the syndromic approach to bloodstream infection diagnosis, this evaluation of 11 automated DNA extraction methods for the PCR diagnosis of candidaemia, puts the choice of an appropriate method in routine diagnosis within the reach of laboratories

Pericardial Effusion Due to Trichosporon japonicum: A Case Report and Review of the Literature

International audienceInvasive infections due to Trichosporon spp. are life-threatening opportunistic fungal infections that may affect a wide array of organs. Here, we described a case of pericardial effusion due to Trichosporon japonicum in a 42-year-old female after a heart transplantation. T. japonicum was isolated from the pericardial fluid, pericardial drain hole and the swab of the sternal surgery scar wound. The late mycological diagnosis due to blood culture negative, the ineffective control of pulmonary bacterial infection and the late start antifungal therapy were the contributing factors in the patient’s death

Measurement of the IgG Avidity Index in the Diagnosis of Clinical Toxocariasis Patients

International audienceToxocara spp. are parasitic nematodes responsible for human toxocariasis, a common zoonotic helminth infection. The five main features of human toxocariasis are the classical ocular toxocariasis and visceral larva migrans syndrome, followed by covert toxocariasis, common toxocariasis and neurotoxocariasis. The diagnosis of toxocariasis is feasible by considering clinical symptoms, anamnestic history and serology laboratory results; however, serological criteria cannot be used to distinguish active Toxocara infection from past exposure, which is an area of much discussion in clinical practice. In this context, we developed avidity tests (ELISA and immunoblotting) and evaluated their clinical usefulness in distinguishing past from active toxocariasis. Our study involved 46 patients divided into two groups: “active toxocariasis” (n = 14) and “chronic toxocariasis” (n = 32). According to the avidity indices obtained for both the chronic and active toxocariasis groups, we proposed two thresholds: first, an AI lower than 32% supports an active infection; secondly, a threshold above 42% can exclude an active infection. In order to use this assay in routine clinical practice, however, is still requires standardisation with regards to the method and threshold values, which can be established through studies involving larger populations

A Repertoire of Clinical Non-Dermatophytes Moulds

International audienceHumans are constantly exposed to micromycetes, especially filamentous fungi that are ubiquitous in the environment. In the presence of risk factors, mostly related to an alteration of immunity, the non-dermatophyte fungi can then become opportunistic pathogens, causing superficial, deep or disseminated infections. With new molecular tools applied to medical mycology and revisions in taxonomy, the number of fungi described in humans is rising. Some rare species are emerging, and others more frequent are increasing. The aim of this review is to (i) inventory the filamentous fungi found in humans and (ii) provide details on the anatomical sites where they have been identified and the semiology of infections. Among the 239,890 fungi taxa and corresponding synonyms, if any, retrieved from the Mycobank and NCBI Taxonomy databases, we were able to identify 565 moulds in humans. These filamentous fungi were identified in one or more anatomical sites. From a clinical point of view, this review allows us to realize that some uncommon fungi isolated in non-sterile sites may be involved in invasive infections. It may present a first step in the understanding of the pathogenicity of filamentous fungi and the interpretation of the results obtained with the new molecular diagnostic tools