Estudos recentes têm sugerido que doença pulmonar obstrutiva crônica (DPOC) possa ser um fator de risco para aspergilose invasiva (AI), particularmente no contexto de ventilação mecânica e uso de esteróides. Neste trabalho, realizamos estudo de coorte prospectivo multicêntrico (2009-2010) em três unidades de terapia intensiva no Sul do Brasil. Foram incluídos no estudo pacientes com DPOC que apresentassem novo infiltrado pulmonar enquanto em ventilação mecânica e sob uso de corticosteróides. Para estes pacientes, foram realizados os seguintes testes, em amostras respiratórias (maioria aspirado traqueal): exame micológico direto, cultura quantitativa para fungos, pesquisa de antígeno galactomanana (GM) (Platelia Aspergillus) e PCR em tempo real para Aspergillus. O DNA das amostras respiratórias foi extraído utilizando-se o kit de extração MycXtra (Myconostica, UK), sendo a amplificação feita com dois kits comerciais de q-PCR: Aspergillus spp q-PCR Alert kit (Nanogen, Itália) e MycAssayTM Aspergillus kit (Myconostica, UK). Foi também obtido soro destes pacientes, onde foi testada GM, precipitinas para Aspergillus e IgE total. O estudo foi aprovado no comitê de ética dos dois hospitais. Foram incluídos no estudo 47 pacientes (40,4% do sexo masculino), sendo a idade média de 68,6 anos (±9,9). A maioria (72,8%) dos pacientes possuía DPOC grave (GOLD III/IV). A dosagem de esteróides (equivalentes de prednisona) variou de 100-4125 mg (mediana: 900 mg). Exame micológico (direto e cultivo) foi positivo para Aspergillus seção Fumigatti em apenas dois pacientes (4,2%). Outros fungos identificados foram Scedosporium apiospermum (n=1) e Histoplasma capsulatum (n=1). Precipitinas para Aspergillus foram positivas em três pacientes, com títulos baixos (<1:2). Os níveis de IgE variaram de 2 a >3000 UI/ml (mediana de 74 UI/ml). Em sua grande maioria, os índices de GM no soro foram <0,5, enquanto que nas amostras respiratórias, os índices de GM foram >0,5, >1,0 e >1,5 em 74,5%, 40,5% e 21,3%, respectivamente. PCR da Myconostica foi positivo em 10 pacientes, enquanto PCR Nanogen detectou apenas um paciente. A mortalidade geral foi de 53,2%. Este estudo prospectivo multicêntrico mostrou uma baixa incidência (4,2%) de AI em pacientes com DPOC. A determinação de GM mostrou altos índices nas amostras analisadas (50% com índices ópticos >1,3), possivelmente necessitando um maior ponto de corte para excluir resultados falso-positivos. A combinação de PCR e GM para o diagnóstico de AI em amostras respiratórias merece investigação adicional, devido à baixa sensibilidade dos métodos de cultivo observados nos estudos clínicos realizados. / Recent data have suggested that chronic obstructive pulmonary disease (COPD) may be an important risk factor for invasive aspergillosis (IA), particularly in the context of mechanical ventilation (MV) and therapy with corticosteroids. Here we present the results of a prospective multicentric study (2009-2010) conducted in three intensive care units (ICUs) in Southern Brazil. COPD patients on steroids showing a new lung infiltrate while on mechanical ventilation were included and the following tests were performed in respiratory samples (mostly tracheal aspirates): microscopy, quantitative fungal culture, galactomannan (GM) (Platelia Aspergillus EIA) and real-time PCR to detect Aspergillus DNA. DNA was extracted using MycXtra kit (Myconostica, UK) and amplification was performed using two q-PCR commercial kits: Aspergillus spp q-PCR Alert kit (Nanogen, Italy) and MycAssayTM Aspergillus kit (Myconostica, UK). Serum was also obtained and tested for Aspergillus precipitins, GM and total IgE levels. Ethical approval was obtained in each of the participant hospitals. A total of 47 patients were enrolled in the study (male 59.6%). Mean age was 68.6 years-old (± 9.9). Most patients had severe COPD (GOLD stages III/IV in 72.8%). Steroid dosage (prednisone equivalent) ranged from 100-4125 mg (median 900 mg). Microscopy and culture were positive for Aspergillus section Fumigatti in only 2 patients (4.2%). Other fungi included H. capsulatum (n=1) and S. apiospermum (n=1). Aspergillus precipitins were positive for three patients, at low titers (<1:2). IgE levels ranged from 2 to >3,000 IU/ml (median 74 IU/ml). All serum GM indexes were <0.5 and respiratory samples, GM indexes of >0.5, >1.0 and >1.5 were observed in 74.5%, 40.5%, and 21.3%, respectively. Myconostica PCR was positive in 10 patients, while Nanogen PCR detected only one patient. Overall mortality was 53.2%. This prospective multicenter study showed a low incidence (4.2%) of IA in critically ill patients with COPD. High optical indices were observed when GM was tested in respiratory samples (50% of the results showed indices of >1.3). Therefore, the test did not discriminate IA and a a higher cutoff would be needed to exclude false-positive results. The combination of PCR and GM for the diagnosis of IA in respiratory samples deserves further investigation due to the low diagnostic sensitivity of the classical mycology methods.

Descrição: Bola fúngica é definida como uma macrocolônia composta por emaranhado de hifas, células inflamatórias, fibrina, muco e fragmentos de tecidos. Aspergillus fumigatus é o agente etiológico mais frequente, responsável por cerca de 90% dos casos, seguido de A niger e A flavus, respectivamente. O antecedente mais comum para o desenvolvimento da bola fúngica é cavidade secundária à tuberculose e a manifestação clínica mais presente e causadora de óbitos nesses pacientes é a hemoptise. Objetivos: Investigar as espécies de Aspergillus causadoras de bola fúngica pulmonar, determinar as condições predisponentes e/ou associadas e a comprovação laboratorial para o diagnóstico etiológico e observar a resposta as diferentes medidas terapêuticas dos pacientes com bola fúngica pulmonar. Delineamento: Foram analisados retrospectivamente, prontuários de pacientes para a caracterização da bola fúngica pulmonar por Aspergillus. Local do estudo: Laboratório de Micologia da Santa Casa Complexo Hospitalar, no período de 1980 a 2009. Pacientes e métodos: Foram incluídos neste estudo todos os pacientes com diagnóstico de bola fúngica pulmonar aspergilar de uma população de 750 casos de aspergilose, de 1980 a 2009. Os critérios para o diagnóstico foram os seguintes: isolamento da espécie de Aspergillus proveniente do material de cavidade pulmonar associado à imagem radiográfica compatível; isolamento da espécie de Aspergillus em outros materiais do trato respiratório, excluindo material da cavidade, associado ou não ao exame direto positivo; imunodifusão radial dupla positiva para Aspergillus associada ao exame de imagem compatível. Resultados: Foram incluídos 391 pacientes com bola fúngica pulmonar aspergilar, a idade variou de 18-78 anos, sendo 67,3% do gênero masculino. O diagnóstico foi baseado nos achados clínicos, radiológicos e laboratoriais. Em todos os pacientes foram detectados achados característicos de bola fúngica tanto no radiograma quanto na tomografia de tórax e bola fúngica complexa foi detectado em 97,4% da casuística. Tuberculose curada foi a principal condição predisponente (89%). Hemoptise foi manifestação clínica mais frequente (89%). A espécie A. fumigatus foi o agente etiológico mais isolado, 89,3% dos casos, seguido de A niger 7,1% e menos frequente A flavus 3,3%. A positividade no cultivo foi de 84,7% nos espécimes clínicos e a imunodifusão radial dupla de 81,6% dos pacientes. A principal medida terapêutica foi ressecção cirúrgica apresentando desfecho favorável em 88,3%. A eliminação da bola fúngica por lise espontânea ocorreu em 2,3% dos casos. Mortalidade foi atribuída à cirurgia e a hemoptise em 32,3 e 13,8%, respectivamente. Conclusões: Tuberculose curada e hemoptise é a primeira hipótese diagnóstica de bola fúngica pulmonar. O sinal radiológico indicativo de bola fúngica é cavidade contendo produto patológico com densidade de partes moles, espessamento da parede da cavidade e da pleura circunjacente. A detecção de anticorpos séricos por imunodifusão radial dupla, e o cultivo de espécimes do trato respiratório inferior determinaram A. fumigatus como o principal agente etiológico da bola fúngica pulmonar. A medida terapêutica mais utilizada nos pacientes do presente estudo foi ressecção cirúrgica, e a metade da ocorrência de óbito esteve presente nestes casos. / Background: Pulmonary fungus ball is defined as a conglomeration, within a cavity of intertwined Aspergillus hyphae, inflammatory cells, fibrin, mucus and cellular debris. Aspergillus fumigatus is the most frequent etiologic agent, about 90% of cases, followed by A niger and A flavus, respectively. The most common condiction to develop fungus ball is residual cavities of healed tuberculosis, and the most prevalent clinical manifestation and cause of death is hemoptysis in these patients. Objectives: To investigate the species of Aspergillus causing pulmonary fungus ball, we compared underlying conditions, laboratory evidence to the etiological diagnosis, and response of the different therapy, and outcome of patients with pulmonary fungus ball. Design: We analyzed retrospectively the medical records of patients for the characterization of pulmonary Aspergillus fungus ball. Settings: A university-based tertiary care hospital in Porto Alegre, Rio Grande do Sul, Brazil. Patients and methods: The study included patients diagnosed with pulmonary Aspergillus fungus ball in a population of 750 cases of aspergillosis, from 1980 to 2009. The criteria for the diagnosis were: isolation of Aspergillus species from the material of the pulmonary cavity associated with the compatible radiographic image; isolation of Aspergillus species from other materials of the respiratory tract, excluding cavity material, with or without direct examination positive; double immunodiffusion positive for Aspergillus associated with compatible image. Results: We included 391 patients with pulmonary Aspergillus fungus ball, age ranged from 18 to 78 years, 67.3% were male. The diagnosis was based on clinical, radiological, and laboratory findings. In all patients we detected the characteristic findings of fungal ball, on X-ray and tomography; and complex fungal ball, on their radiological appearance, was detected in 97.4% of cases. Healed tuberculosis was the commonest pre-existing disease (89%). Hemoptysis was the major symptoms (89%). The species A. fumigatus was the most common etiologic agent, 89.3% of cases, followed by 7.1% A niger and A flavus less frequent in 3.3%. Culture was positive in 84.7% specimes, and immunodiffusion in 81.6% patients. The main treatment was surgical resection in 88.3% that had a favorable outcome. Spontaneous lysis was obtained in 2.3% of cases. Mortality was attributed to the surgery and hemoptysis in 32.3 and 13.8%, respectively. Conclusions: Patient with healed tuberculosis and hemoptysis is the first hypothesis diagnostic fungus ball. The most frequent radiological signs were rounded dense opacity surrounded with a halo of air in a thick cavity wall and thickening of the pleura over cavity. The detection of serum antibodies by double immunodiffusion, and the cultive of the lower respiratory tract specimens determined A. fumigatus as the main agent of pulmonary fungal ball. The detection of serum antibodies by double immunodiffusion, and the cultivation of the lower respiratory tract specimens determined A. fumigatus as the main agent of pulmonary fungal ball. The measure most commonly used therapy in patients of this study was to surgical resection, and half of the patients who died were in these cases.

Lors de travaux, la mise en suspension des spores d’Aspergillus constitue un facteur de risque reconnu dans le développement d’une aspergillose invasive. Durant l’année 2015, un pavillon de 6000 m2 (60 lits) de notre établissement a été entièrement déconstruit. L’objectif principal de cette étude a été d’évaluer l’association entre la concentration des spores d’Aspergillus fumigatus (AF) dans l’environnement extérieur et intérieur des pavillons, mais également avec la coexistence de cas cliniques, afin de proposer des recommandations d’amélioration (pratiques & techniques). Pour cela, durant 11 mois, une surveillance prospective de la contamination à Aspergillus fumigatus (AF) de l’air extérieur et intérieur par impaction sur gélose, mais aussi une investigation épidémiologique des patients à risque ont été mis en place. Au total, 3885 prélèvements d’air ont été réalisés (1744 extérieurs et 2141 intérieurs) permettant, par calcul des ratios de contamination (extérieurs vs intérieurs), de confirmer une efficacité des mesures de précautions pour réduire l’aérocontamination. Des prélèvements extérieurs continus des spores d’Aspergillacées (spore/m3/jour) ont également été réalisés par un capteur Hirst. Ce capteur, mais aussi le suivi des conditions météorologiques se sont révélés être des systèmes d’alerte utiles pour prévenir les pics de contamination. Enfin, 394 (383 environnementaux, 11 cliniques) isolats d’AF sensibles aux antifongiques ont été génotypés (MLVA). L’analyse des génotypes a montré 7 génotypes similaires entre des isolats d’AF cliniques et environnementaux confirmant un rôle de l’environnement hospitalier dans l’infection ou la colonisation des patients / Invasive aspergillosis (IA) due to Aspergillus has been associated with building construction, which may increase spores emission nearby immunocompromised patients. In 2015, one blocks of 6,000 m2 (60 beds) form our hospital has been entirely demolished. The aim of this study was to evaluate possible association between concentration of A. fumigatus (AF) spores in the outdoor and indoor environment and also with the clinical cases in order to propose some improvements in actuals methods and practices. A daily surveillance of fungal contamination was implemented during 11-months. Environmental survey was realized by air samplings, outdoor and indoor, with an automatic agar sampler. In parallel, surveillance of IA infection cases was conducted by epidemiological investigation. A total of 3885 air samples (1744 outdoor samples and 2141 indoor samples) were collected, allowing calculation of ratios (outdoor vs indoor) to confirm efficacy of preventives measures applied to reduce indoor aerocontamination. Outdoor continuous sampling of Aspergillaceae spores (spore/m3/day) was also realized by a Hirst collector. This collector was useful as alarm system to detect contamination peaks. Similarly, monitoring of meteorological parameters seems to be an interesting tool, to prevent Aspergillus peaks. Finally, 394 isolates of AF, susceptible to antifungals (383 environmental and 11 clinical isolates) were genotyped using MLVA. Analysis of genotypes showed 7 similar genotypes shared by environmental and clinical isolates, suggesting that clinical colonization and/or infection may originate from the hospital environment

Depuis la disponibilité des combinaisons antirétrovirales (cART) en 1996, l’incidence des infections opportunistes classantes SIDA (IO), dont la pneumocystose (PCP) a très fortement diminué. Malgré tout, chez les patients infectés par le VIH, la PCP était la 2ème IO la + fréquente en France en 2001-2003 et les infections fongiques, avec 1 million de nouveaux cas/an de cryptococcose, restent un problème de santé publique majeur au niveau mondial. Cependant, depuis l’ère des cART, très peu de recherches épidémiologiques sur les infections fongiques dans les pays industrialisés ont été entreprises. C’est dans ce contexte que nous avons mené une étude épidémiologique de 2 infections fongiques chez les patients infectés par le VIH en France sur la French Hospital Database on HIV ANRS CO4 (FHDH) : la pneumocystose et l’aspergillose invasive. Concernant la pneumocystose, sur la période 2004-2011, dans la base FHDH, la moitié des 1259 cas de PCP étaient survenus chez des patients qui avaient interrompus leur suivi, et, pour ceux qui avaient déjà eu une IO avant la PCP, leur mortalité était de 25% à 3 ans. Pour l’aspergillose invasive (AI), après un retour national aux dossiers des cas déclarés sur 20 ans sur la base FHDH, un comité d’experts a validé 242 cas d’AI. Les données montrent que, chez les patients infectés par le VIH, seulement la moitié des AI validées répondaient aux critères EORTC. La mortalité à 3 mois après une AI s’est améliorée après l’ère des cART et un rôle protecteur du voriconazole sur la survie à 3 mois a également été démontré pour la 1ère fois chez les patients infectés par le VIH. / The advent of combined antiretroviral therapy (cART) in 1996 resulted in a dramatic fall in the incidence of AIDS-defining illness (ADI), including Pneumocystis jirovecii pneumonia (PCP). Nevertheless, PCP was the second most frequent ADI in France in 2001-2003 and fungal infections remain a major threat for HIV-infected individuals worldwide. Epidemiological data on fungal infections in the late cART period in resource-rich settings are scarce. The purpose of our work was to study changes in the epidemiology of fungal infections among HIV-infected individuals in France in the late cART period, focusing on PCP and invasive aspergillosis (IA) in the French Hospital Database on HIV ANRS CO4 (FHDH). In the FHDH, during the 2004-2011 period, half of the 1259 PCP cases occurred among HIV-infected individuals who had waning adherence to care, and for those who had a prior ADI before PCP the 3-year mortality rate was 25%. For the second study on IA, a review committee validated IA cases among all the cases that included a diagnostic code for aspergillosis (ICD-9 or ICD-10) in the FHDH over a 20-year period. Our study demonstrated that only half of validated IA cases among HIV-infected individuals met EORTC criteria. The 3-months survival rate after IA diagnosis improved after the advent of cART and a protective role of voriconazole was observed in the period after 2001.

The opportunistic fungus, Aspergillus fumigatus, is a leading cause of morbidity and mortality among the immunocompromised population. Experimental and clinical findings have established that phagocytic defenses are critical in the recognition and clearance of A. fumigatus. Previous studies found that Toll-like receptors (TLRs), specifically TLR2 and TLR4, were essential in the detection of the mold. Furthermore, one study found that mice deficient in TLR9 lived longer than their wild-type counterparts following challenge with A. fumigatus. We sought to determine the role of TLR9 during A. fumigatus infection. Our results show that A. fumigatus contains unmethylated CpG DNA, the natural ligand of TLR9. Furthermore, A. fumigatus DNA stimulates a potent pro-inflammatory response in mouse bone marrow derived dendritic cells (BMDCs) and human plasmacytoid dendritic cells (pDCs). A genome wide analysis showed that A. fumigatus DNA contains 87 human and 23 mouse putative immunostimulatory motifs. The response to A. fumigatus DNA is TLR9-dependent, as BMDCs from TLR9-/- mice were unresponsive to the fungal DNA. In addition, HEK293 cells cotransfected with human TLR9 and NFκB driven Luciferase conferred responsiveness to A. fumigatus CpG-rich sequences found in the fungal DNA. Our results show that TLR9 detects A. fumigatus DNA, resulting in the secretion of proinflammatory cytokines. While pDCs secrete IFNα in response to A. fumigatus DNA, these cells have been mainly described to play critical roles in the antiviral responses. The role of pDCs during fungal infections remains to be elucidated. Our data show that CD304+ peripheral blood pDCs challenged with A. fumigatus hyphae secrete large concentrations of IFNα and TNFα in response to infection. Furthermore, the response appears to be TLR9- independent. However, pDCs spread over the hyphae and inhibit fungal growth. Furthermore, pDCs undergo cell lysis upon incubation with A. fumigatus. The antifungal activity of the pDCs was retained in the cell lysates, suggesting that this response was mediated by an intracellular factor. Addition of exogenous Zn2+, but not Fe3+, partially restores hyphal growth. In addition, western blot of pDC lysates show that these cells have the Zn2+-binding protein calprotectin. Over 60% cell death is observed in the pDC population following a 2 hour incubation with A. fumigatus. The observed pDC cell death can be partially attributed to gliotoxin, as pDCs challenged with A. fumigatus stains deficient in production of the mycotoxin result in decreased pDC cytotoxicity. Furthermore, pDC cell death occurs independent of contact with the mold, confirming that pDC cell death is mediated by a secreted fungal factor. In addition, our results show that pDCs are required for the host response against A. fumigatus. Mice depleted of their pDCs are more susceptible to A. fumigatus infection than the control counterparts, suggesting that pDCs play a role in the antifungal response. Also, we observe a 5-fold increase in the pDC population in the lungs of infected mice. Therefore, the possibility of these cells playing a role in recruiting and communicating with other immune cells cannot be eliminated. Upon maturation, pDCs acquire characteristics of conventional DCs (cDCs) such as upregulation of major histocompatability complex (MHC) and becoming more phagocytic. Whether mature pDCs are involved in the detection of and responses against fungal pathogens remains to be determined. Here we show that mature pDC secrete IFNα and TNFα in response to A. fumigatus conidia as early as 6 hours post-challenge. While cytokine secretion of mature pDCs against A. fumigatus does not require opsonization, it requires for A. fumigatus being alive and growing. Furthermore, supernatants from conidial growth induced cytokine secretion by the mature pDCs. The work presented in this thesis establishes that the nucleic acids in A. fumigatus serve as a pathogen associated molecular pattern (PAMP) that can induce a TLR9- dependent response. Furthermore, I show that pDCs secrete cytokines and induce an antifungal response against A. fumigatus conidia and hyphae. While the pDC population in the blood appears to be small, our work shows that these cells could be intimately involved in the antifungal responses against A. fumigatus.

Among the different pulmonary fungal infections, aspergillosis, and in particular invasive pulmonary aspergillosis (IPA), are becoming the most worrying diseases in immunocompromised patients. This is due to their high incidence and mortality. Indeed, invasive aspergillosis manifests as invasive pulmonary disease accounting for 50/60% of all cases, with a mortality of 50-90% in severely immunocompromised patients. Triazoles act by inhibiting 14-α demethylase, a fungal cytochrome P450 enzyme implicated in the synthesis of ergosterol, an essential constituent of fungal cell walls. Moreover, they interact with the same cytochrome present in large quantities in the human liver, inducing possible drug-drug interactions in IPA patients. Consequently, interactions resulting from inhibitors, inductors, or substrates of cytochromes can modify the plasmatic concentrations of triazoles or other drugs administered concomitantly. To overcome these important issues, pulmonary delivery of triazoles could be an interesting alternative to conventional routes.The aim of this work was to develop triazole-based dry powders for inhalation able to be deposited adequately in the lungs, with a release of drug and a lung retention that can optimize its pharmacological action. This work focused on two active pharmaceutical ingredients (API): itraconazole (ITZ), for which improved solubility was needed, and voriconazole (VCZ), for which slow release was required.Concerning ITZ, solid dispersions for inhalation (SDIs) comprising ITZ and mannitol were previously developed in our laboratory. The selected SDI showed interesting results in terms of improved dissolution and lung retention in vivo in mice during a pharmacokinetic study. Therefore, this SDI was tested in a murine preclinical model of IPA and showed promising results in terms of prophylaxis efficacy. One aim of this work was to continue the pharmaceutical development of this promising SDI by making a scaling-up study. These methods were intended to improve the SDI’s ecological footprint and productivity by increasing the production yield and decreasing the amount of solvents and time used in its manufacture. During the first step of this study, the obtained SDI showed interesting results obtaining similar powder characteristics (i.e. amorphous content, aerodynamic performance, and dissolution profiles) from concentrated solutions using a laboratory-scale spray-dryer B-290 (Büchi, Switzerland) before using a pilot-scale spray-dryer (GEA Niro, Denmark). Then, the upscaling was performed on the pilot spray-dryer allowing the production of SDIs with increased productivity (yield and process duration). These SDIs had similar powder characteristics than the optimized lab-scale SDIs. During the second part of this work we developed VCZ based dry powder for inhalation. The aim was to slow down the release of this highly permeable and very slightly soluble API and to prolong its lung residence. To this end, various lipidic excipients were chosen. The selection took into account the potential good pulmonary tolerance of the lipids and their hydrophobicity to evaluate their ability to slow down the VCZ release (FPFs 20-25%, slowed release up to 24h, burst effect of ± 58% of VCZ dissolved within 30min). Immediate-release SDIs were also developed to have a comparator reference for the pharmacokinetic and efficacy studies (FPFs of 40%).Then, a pharmacokinetic study in mice was performed following the pulmonary administration of one immediate-release and two sustained-release SDIs (with or without PEG excipient). With an 80-fold higher pulmonary exposure over 24 hours, the slow-release SDIs presented a real interest compared to the immediate-release SDI. Moreover, in accordance with these results, VCZ plasma exposure following the administration of the SDI with PL90-H was more than 1.5-fold higher than its pulmonary exposure (AUC0-24 of 8.70 µg.h/g in the lungs and 14.70 µg.h/mL in the plasma). The slow-release formulations presented plasma exposures at least 15 times lower than their pulmonary exposures (AUC0-24 in lung of 741.40 and 686.85 µg.h/g vs plasmatic AUC0-24 of 37.44 and 42.81 µg.h.mL, respectively with and without PEG excipient). Moreover, the presence of PEG excipient did not influence the residence time and the exposure of the VCZ within the lungs. Finally, the sustained-release SDIs administration by inhalation led to VCZ lung and plasma concentrations higher than the minimal inhibitory concentration (MIC) of VCZ against Aspergillus fumigatus (1 μg/mL) over 24 h. Finally, a murine model of IPA was developed in our lab. The immunosuppression model was fixed and performed by the intraperitoneal (IP) injection of corticosteroids to induce a neutropenia state. Then, different doses of spores (from 1.10^4 to 5.10^6 spores) were inoculated to the neutropenic mice via an endotracheal instillation and the survival rate of each group was observed. Unfortunately, the survival rate resulting from the different infections were not reproducible. Therefore, these models were not suitable to conduct the efficacy study. This underlined the link between the immunosuppressive model and the infection. Indeed, the IPA murine model should be developed according to the immune state of the animal, the Aspergillus conidia species and its concentration to be used. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Die vorliegende Arbeit beschäftigte sich mit zwei Methoden zur Diagnostik der Aspergillose bei Greifvögeln, um neue Erkenntnisse über die Aussagekraft dieser nicht invasiven Diagnostika zu gewinnen. In der ersten Studie wurden bei ausschließlich Aspergillose-positiven Falken (Falco spp.) (n = 110) spezifische Röntgenzeichen an digital erstellten Röntgenbildern systematisch ermittelt und mit den typischen Röntgenzeichen von Papageien mit Erkrankungen des unteren Respirationstrakts verglichen. In der zweiten Studie wurden gesunde (n = 73) und an Aspergillose erkrankte (n = 32) Jagdfalken (Falco spp.) mittels Serumelektrophorese untersucht, Referenzwerte für die gesunden Falken erstellt und mit den Werten der erkrankten Falken verglichen. In beiden Studien stammten die Tiere aus dem Patientengut derselben Klinik. Bei der Auswertung von Röntgenbildern Aspergillose-positiver Falken wurden hauptsächlich subtile Röntgenzeichen beschrieben. Von den 110 Tieren waren 29 (26,4 %) radiologisch vollkommen unauffällig. Die am häufigsten beschriebenen Befunde waren inhomogene Verschattungen des Lungenfeldes (38,2 % laterolateral [ll]) und strichförmige Verschattungen der kaudalen Lungengrenze (30,0 % ll) sowie inhomogene (34,5 % ll; 29,1 % ventrodorsal [vd]) und streifige (26,4 % ll) Verschattungen der Luftsäcke, aber auch eine schlechte Abgrenzbarkeit des Herzschattens in der laterolateralen Projektion (42,7 %). Im Vergleich zu an Papageien mittels konventioneller Projektionsradiographie durchgeführten Studien war der Anteil an subtilen Röntgenzeichen geringer und der Anteil an massiven Röntgenzeichen größer. Verglichen mit Referenzwerten diverser Greifvogelspezies aus der Literatur zeigten die Referenzwerte der gesunden Falken dieser Studie unter Verwendung des hochauflösenden Elektrophoresesystems SAS 1 unit (Helena, Saint Leu La Forest, Frankreich) relativ niedrige Gesamtproteinwerte und relativ hohe Präalbuminwerte auf. Bei den 32 Serumproben der an Aspergillose erkrankten Falken ließ sich im Gegensatz zu den 73 Serumproben der gesunden Falken ein signifikant erniedrigter Totalalbuminwert (Albumin + Präalbumin) sowie ein hoch signifikant erniedrigter Präalbuminwert mittels Serumelektrophorese feststellen. Obwohl die Falken meist schon in frühen Krankheitsstadien vorgestellt wurden und die Diagnostik in diesen Stadien besonders schwierig ist, konnten mit beiden Untersuchungsmethoden von gesunden Tieren differierende Befunde erhoben werden. Diese in Zusammenhang mit Aspergillose erhobenen Befunde wichen jedoch teilweise deutlich von den in der Literatur beschriebenen „typischen“ Befunden bei an Aspergillose erkrankten Vögeln ab. Dies kann damit erklärt werden, dass die meisten vergleichbaren Studien an als Heimtiere gehaltenen Papageien oder gefangen gehaltenen Zoovögeln (von Falken abweichende Haltungsform, Anatomie und Physiologie sowie Leistungsniveau) und mit unterschiedlicher Technik (digitale versus konventionelle Projektionsradiographie, unterschiedliche Elektrophoresesysteme und Verwendung von Serum anstelle von Plasma) durchgeführt wurden. Die digitale Projektionsradiographie kann aufgrund ihrer schonenden, einfachen und schnellen Durchführbarkeit sowohl den Vogelmedizin spezialisierten Institutionen als auch den Kleintierpraktikern uneingeschränkt empfohlen werden. Die Proteinelektrophorese kann bis zum heutigen Zeitpunkt nur bedingt für den Praktiker, wohl aber für spezialisierte Institutionen bei Beachtung aller Besonderheiten als zusätzliches Diagnostikum empfohlen werden. / The present study concentrates on two methods for diagnosing birds of prey with aspergillosis with the intent to increase the knowledge of the validity of these non-invasive diagnostic methods. In the first study, specific radiographic signs of digitally created radiographs of falcons (Falco spp.) which were exclusively positive for aspergillosis (n = 110) were systematically analyzed and compared to the typical radiographic signs of parrots with diseases of the lower respiratory tract. In the second study, healthy falcons (n = 73) and falcons affected with aspergillosis (n = 32) (Falco spp.) were examined by using serum protein electrophoresis in order to create reference values for healthy falcons and compare them with the values of the affected falcons. In both studies, the animals were patients of the same clinic. While evaluating the radiographs of the falcons with aspergillosis, mainly subtle radiographic signs were described. Radiographically within normal limits were 29 (26.4%) of the 110 animals. The most commonly reported findings were inhomogeneous increased radiodensity of the lung area (38.2% laterolateral [ll]), line-shaped shadowings of the caudal lung border (II 30.0%) as well as an inhomogeneous (34.5% ll, 29.1% ventrodorsal [vd]) and streaky (26.4% II) radiodensity of the air sacs, but also a poor delineation of the cardiac silhouette in the laterolateral projection (42.7%). Compared to studies performed on parrots by conventional radiography, the portion of subtle radiographic signs was lower and the portion of severe signs was higher. Compared to reference values of various raptor species from the literature, this study, which made use of the high-resolution electrophoresis SAS 1 unit (Helena, Saint Leu La Forest, France), revealed relatively low values for total proteins and relatively high values for prealbumin in the reference values of the healthy falcons. The 32 serum samples of the falcons suffering from aspergillosis showed a significantly reduced total albumin (albumin + prealbumin) level and a highly significantly reduced prealbumin level compared to the 73 serum samples of healthy falcons. Although the falcons were for the most part already brought to the clinic in one of the early stages of the disease, when diagnosing aspergillosis is particularly difficult, both examination methods revealed different results for the healthy and diseased animals. However, the findings related to aspergillosis were in some cases significantly different from those described in the literature as the \"typical\" findings in birds suffering from aspergillosis. This can be explained by the fact that most of the comparable studies were conducted with parrots held as pets or with captive zoo birds (when husbandry, anatomy and physiology, as well as performance level are different from falcons) and with a different technique (digital versus conventional radiography, different electrophoresis systems and the use of serum instead of plasma). The digital radiography can be fully recommended for specialized medical institutions for avian medicine as well as for small animal practitioners because of its easy, rapid and gentle feasibility. To date, the protein electrophoresis can only be recommended with restrictions for practitioners, however for specialized institutions, it can be useful as additional diagnostic tool if all its specific features are taken into account.

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No description available.

A Aspergilose Broncopulmonar alérgica (ABPA) é uma doença complexa,desencadeada por uma reação de hipersensibilidade ao Aspergillus fumigatus, que apresenta vários estágios, sendo que no estágio mais grave, os pacientes apresentam bronquiectasias. O diagnóstico da doença é difícil e o maior problema é a falta de antígenos padronizados necessários para a determinação de anticorpos específicos. O objetivo do presente estudo é avaliar se os testes cutâneos com os análogos de Asp f 1 podem auxiliar no diagnóstico e no estadiamento da ABPA. Três grupos de pacientes classificados por testes sorológicos foram obtidos a saber 20 ABPA (16BQ+; 4BQ-), 25 possíveis -ABPA (14BQ+;11BQ-) e 24 asmáticos sem ABPA (11BQ+;13BQ-). Fizeram parte do estudo 10 pessoas sem asma . Todos foram submetidos a testes intradérmicos com três antígenos a-sarcina, mitogilina e estrictocina.Houve uma intensa reação a todos os antígenos e as reações produzidas foram semelhantes para os três antígenos. As reações de leitura tardia positivas à mitogilina foram biopsiadas. As biopsias de 2 (12,5%) dos pacientes BQ+ do grupo ABPA e 5 do grupo ABPA possível com BQ+ (35,6%) mostraram vasculite por depósito de imunocomplexos. 11 pacientes do terceiro grupo não apresentaram vasculite. O quarto grupo não apresentou reação tardia. Todos os pacientes com reação positiva apresentaram BQ+. alfa-sarcina, a mitogilina e a restrictocina diferenciaram pacientes com ABPA por testes intradérmicos e podem ser aplicados no diagnóstico da doença. A maior incidência de bronquiectasias foi encontrada no primeiro grupo (80%) e no segundo (56%). No terceiro grupo nenhum caso foi encontrado em 23 pacientes com asma e teste ID positivo ao aspergillus fumigatus todos os pacientes com vasculite tinham bronquiectasia. Há possibilidade de que as lesões produzidas nos pulmões sejam produzidas por vasculite. / Allergic Bronchopulmonary Aspergillosis (ABPA) is a complex disease, triggered by a hypersensitivity reaction to Aspergillus fumigatus. The disease diagnosis is difficult, and a major problem is the lack of standardized allergens for the determination of specific antibodies. The aim of the present study is to evaluate if intradermal (ID) tests with analogs of Asp f 1 can aid in the diagnosis and stage assessment of abpa. Three groups of patients classified by serological tests were obtained. 20 ABPA (16BQ+; 4BQ-), 25 possible-ABPA (14BQ+; 11BQ-), 24 asthmatic-ABPAfree (11BQ+; 13BQ-) and 10 asthma-free people were submitted to id tests with three antigens: mitogillin, a-sarcin and restrictocin. There was intense reaction to all three antigens and the response was similar. The positive reactions to mitogillin were biopsied. The skin biopsies of two (12,5%) bq+ patients of the first group and 5 BQ+ (35,6%) patients of the second one showed vasculitis by immune complexes (IC) deposition. 11 patients of the third group had negative biopsies. The fourth group didn\'t have late-reaction. All patients with positive reaction were BQ+. By ID test, alfa-sarcin, mitogillin and restrictocin could differentiate patients with abpa and can be applicable in disease diagnosis. The higher incidence of bronchiectasis was found in the first (80%) and second (56%) groups. In the third group, IC wasn\'t found in 23 asthma patients and id test was positive to A. fumigatus. All patients with vasculitis by IC had bronchiectasis. Therefore, the results indicate that this kind of pulmonary lesion is caused by vasculitis.