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Développement D'outils Moléculaires De Détection Des Moisissures

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Développement D'outils Moléculaires De Détection Des Moisissures ACADEMIE UNIVERSITAIRE WALLONIE-EUROPE UNIVERSITE DE LIEGE FACULTE DE MEDECINE VETERINAIRE DEPARTEMENT GIGA-INFLAMMATION, INFECTION AND IMMUNITY LABORATOIRE D'IMMUNOLOGIE CELLULAIRE ET MOLÉCULAIRE Développement d’outils moléculaires de détection des moisissures présentes dans l’air intérieur afin de déterminer leur impact sur la santé publique Development of molecular tools for rapid detection and quantification of indoor airborne molds to assess their impact on public health Libert Xavier THESE PRESENTEE EN VUE DE L’OBTENTION DU GRADE DE DOCTEUR EN SCIENCES VETERINAIRE ANNEE ACADEMIQUE 2016-2017 ACADEMIE UNIVERSITAIRE WALLONIE-EUROPE UNIVERSITE DE LIEGE FACULTE DE MEDECINE VETERINAIRE DEPARTEMENT GIGA-INFLAMMATION, INFECTION AND IMMUNITY LABORATOIRE D'IMMUNOLOGIE CELLULAIRE ET MOLÉCULAIRE Développement d’outils moléculaires de détection des moisissures présentes dans l’air intérieur afin de déterminer leur impact sur la santé publique Development of molecular tools for rapid detection and quantification of indoor airborne molds to assess their impact on public health Libert Xavier THESE PRESENTEE EN VUE DE L’OBTENTION DU GRADE DE DOCTEUR EN SCIENCES VETERINAIRE ANNEE ACADEMIQUE 2016-2017 Supervisors Prof. F. Bureau, ULg Dr. Ir. S. De Keersmaecker, WIV-ISP Dr. Ir. A. Packeu, WIV-ISP Dr. Ir N. Roosens, WIV-ISP Members of the Examination Committee Prof. B. Dewals, ULg Prof. P. Le Cann, EHESP, France Prof. J.-B. Watelet, UGent Prof. C. Charlier, ULg Prof. D. Cataldo, ULg Prof. B. Mignon, ULg Dissertation presented for the degree of Prof. D. Peeters, ULg Doctor in Veterinary Sciences (ULg) January 2017 Résumé Résumé Aujourd’hui, la contamination de l'environnement intérieur par des moisissures aéroportées est considérée comme un problème de santé publique. Les méthodes analytiques classiques de surveillances, basées sur la culture et l'identification microscopique, présentent des limitations liées à la dépendance vis-à-vis de la culture et du temps requis pour les analyses. Par conséquent des biais peuvent être introduits, notamment concernant la fraction morte (champignons non-cultivables ou morts), pouvant avoir potentiellement un impact sur la santé humaine. Dans ce contexte, les outils moléculaires semblent être d’excellentes alternatives pour la surveillance des contaminations fongiques aéroportées d’intérieurs. Ainsi, différents outils moléculaires ont été développés lors de cette thèse pour détecter et identifier les champignons dans l'air intérieur (qPCR SYBR ®green, High Resolution Melting (HRM), Luminex xMAP ® et NGS). L'objectif étant d'améliorer la détection des contaminants fongiques, la fraction morte inclue, par rapport aux méthodes de surveillance classiquement utilisées, mais également d’améliorer les connaissances actuelles sur la contamination fongique aéroportée d’intérieur. Ainsi, un test PCR en temps réel (qPCR) a été développé pour la détection d’ Aspergillus versicolor, une moisissure pathogène de l’air intérieur ,et d’ Exophiala jeanselmei , une moisissure pathogène suspectée de faire partie de la "fraction morte". Bien que validée selon des critères stricts prouvant la qualité des outils développés, des limites, notamment concernant la discrimination des espèces génétiquement proches et le multiplexage ont été observées. Dans cette thèse, la première question a été résolue par l'utilisation d’une analyse post-PCR High Resolution Melting (HRM). Utilisé tel un proof of concept , le HRM a été testé sur 3 Aspergillus génétiquement proches ( A. versicolor , Aspergillus creber et Aspergillus sydowii ) démontrant que son utilisation peut améliorer le suivi de ce type de contaminants. La problématique du multiplexage a été résolue grâce à la technologie Luminex xMAP ®. Développé pour la détection simultanée de 10 moisissures fréquemment observées dans l'air intérieur. Ainsi, il a été démontré que l’introduction de cette technique dans un protocole de monitoring permettrait de réduire le temps d’analyse et, in fine , le temps de transmission des résultats à l’équipe médicale en charge du patient. Cependant, puisqu’une sélection des espèces à identifier est nécessaire avant l’analyse, cette technologie ne convient pas pour l'étude de la diversité fongique. Dans ce contexte, la technologie next generation sequencing (NGS) semble offrir une alternative valable en tant qu’outil universel d’identification des moisissures intérieures. Dans cette étude, la fraction « morte » a été investiguée grâce à une analyse NGS métagénomique, permettant par la i Résumé même occasion de détecter pour la première fois E. jeanselmei dans l'air intérieur. De plus, une analyse de la diversité fongique de l’air intérieur provenant de résidences contaminées a été réalisée démontrant qu’une analyse NGS métagénomique peut contribuer à l'amélioration des données sur la diversité fongique aéroportée d’intérieur, nécessaire au développement de méthodes de détections, ainsi que de tests immunologiques représentatifs de cette diversité. Les méthodes développées ainsi que les résultats obtenus lors de ce doctorat sont une première étape pour une meilleure compréhension du lien existant entre les champignons aéroportés présents dans l’intérieur de l’habitat et la santé publique. ii Summary Summary Currently, contamination of the indoor environment by fungi is suggested to be a public health problem, although scientific evidence on the causal link is still limited. The monitoring of indoor airborne fungal contamination is a common tool to help understanding the link between fungi in houses and respiratory problems. Classical monitoring methods, based on cultivation and microscopic identification, have some limitations. For example, uncultivable or dead fungi (“unknown” fraction) cannot be identified, although they could have an impact on human health. In this context, molecular tools seem to be a valuable alternative. In this PhD work, different molecular tools were developed, from simplex to multiplex, to detect and identify indoor airborne fungi. The goal was to improve the detection of fungal contaminants, including the “unknown” fraction, as compared to the currently used classical monitoring methods. The necessary air sampling and DNA extraction protocols, adapted to the downstream molecular monitoring methods have also been developed. Through the application of the developed tools to specific case studies, we aimed to improve the current knowledge on fungal contamination. At first, we developed a specific ITS-based SYBR ®green real-time PCR (qPCR) assay for Aspergillus versicolor , a species frequently observed in indoor air and known to be allergenic. Additionally, an ITS-based qPCR assay was developed for the specific detection of Exophiala jeanselmei, a pathogenic yeast suspected to be a part of the “unknown fraction”. The performance of these qPCR methods was assessed. This comparison demonstrated that SYBR ®green qPCR assays can be used as a molecular alternative for monitoring of contaminated samples while eliminating the need for culturing and thereby considerably decreasing the required analysis time. However, qPCR has some limitations especially concerning the discrimination of genetically close species and multiplexing. The first issue was addressed through the use of post-qPCR high resolution melting (HRM) analysis, providing a proof-of-concept for this approach, using 3 closely related Aspergillus, i.e., A. versicolor , Aspergillus creber and Aspergillus sydowii . This HRM tool will allow a more accurate monitoring of these closely related indoor air contaminants, thereby contributing to an improved insight in the causal link between the specific presence of these species and health issues . The multiplexing issue was overcome through a Luminex xMAP® assay, developed for the simultaneous detection of the 10 most frequently in indoor air found fungi. All the species identified with the classical method were also detected with the xMAP ® assay, however in a shorter time frame, and using less sample material. This assay will improve the communication with the involved medical team and the patient. To provide scientific evidence for the causal link between indoor airborne fungi and health iii Summary problems, the full diversity needs however to be identified. This cannot be achieved by using a targeted assay. Therefore, next generation sequencing (NGS) could offer a valuable alternative as an open approach multiplex monitoring method. An NGS-based metagenomics approach was used to investigate the “unknown” agents in air samples of offices in contact with air-conditioning reservoirs and showed the first detection of E. jeanselmei in indoor air. Finally, a metagenomics analysis was performed to investigate the indoor airborne fungal diversity in contaminated residences in Brussels where people with health problems were living. This demonstrated that NGS could contribute to improved data concerning the indoor airborne fungal diversity, as compared to the currently used classical methods. The methods developed in this PhD work and the insights obtained are a first step for a better understanding of the causal link between indoor airborne fungi and public health. iv Abbreviations Abbreviations µl microliter ∆ Cq quantitative cycle difference ∆G Gibb’s energy Ac Avogadro’s constant Aversi_ITS assay qPCR SYBR ®green assay developed for the detection of Aspergillus versicolor BCCM/IHEM Biomedical Fungi and Yeasts Collection BLAST basic local alignment search tool BLAST standard nucleotide BLAST bp base pair CFU colony forming unit CFU/m³
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