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Background: Invasive pulmonary aspergillosis (IPA) is a common cause of mortality in haemato-oncology patients and early diagnosis is vital for improving outcomes. Since lung biopsy in this acute setting is rarely performed due to the associated risks, an empirical strategy remains the standard of care in many haematology units, but leads to overtreatment with antifungal drugs, which have significant side-effects. This project has developed novel approaches for detecting IPA, allowing early and specific treatment of genuine fungal infection. Methods: A combination marker approach involving a new Aspergillus qPCR assay, an EORTC/MSG-endorsed GM ELISA and an Aspergillus LFD, was used to establish a robust diagnosis of IPA from clinical broncho-alveolar lavage (BAL) fluid samples. The inflammatory cytokine profile associated with IPA biomarker positive BAL fluid was also evaluated. Finally, antigen and qPCR detection were combined in a proximity ligation assay (PLA), to demonstrate proof-of-principle for a diagnostic assay for the earliest possible detection of fungal infections. Results: A dual testing approach involving a novel MIQE-compliant Aspergillus qPCR assay and an Aspergillus LFD showed a sensitivity and specificity of 100% and 94%, respectively in BAL fluid, unlike in blood where this approach was not sensitive. Results confirmed previously published concerns over the repeatability of GM in serum, whereas BAL GM results appear stable. Biomarker detection results in exhaled breath condensate did not correlate well with results in BAL fluid samples. Respiratory samples did not identify a distinct inflammatory marker profile in IPA. Finally, antibodies raised against JF5 mannoprotein were used to develop a PLA test to detect active growth of Aspergillus. Conclusions: The optimised qPCR is a very sensitive and highly specific aid in IPA diagnosis. A combination biomarker approach could be incorporated into a diagnostic-driven approach to patient management to direct antifungal treatment to patients with evidence of invasive fungal disease.

Due au champignon Aspergillus fumigatus, l’aspergillose pulmonaire invasive représente une grave menace pour les individus souffrant d’immunodépression sévère. En parallèle d’un diagnostic manquant de spécificité, les traitements actuels présentent une forte toxicité. Ces travaux se sont dans un premier temps intéressés au développement d’anticorps thérapeutiques dirigés contre les protéines pariétales Chitin ring formation du champignon. Le ciblage de ces protéines impliquées dans la croissance fongique a permis la mise en évidence d’effets modérés in vitro, et ont induit, in vivo, un recrutement significatif de cellules immunitaires impliquées dans la défense anti-aspergillaire. Dans un second temps, ces travaux se sont intéressés au ciblage du microenvironnement et de la réponse de l’hôte au cours de l’aspergillose, afin de mieux comprendre les processus physiopathologiques induits au cours de la maladie, et de permettre l’identification de nouveaux biomarqueurs et cibles thérapeutiques. L’utilisation de la spectrométrie de masse iTRAQ®, chez des rats et des manchots, a permis la mise en évidence de plusieurs voies de signalisation surreprésentées. Ces travaux se sont également intéressés à la caractérisation immunologique d’un modèle rat d’API. En plus de la mise en évidence des effets du champignon sur le recrutement de certaines populations de cellules immunitaires, l’utilisation de l’iTRAQ® a permis la mise en évidence de la surexpression de l’interleukine-33 et de son récepteur ST2 au cours de la maladie. Ces travaux ouvrent d’intéressantes perspectives dans la mise en place de nouveaux traitements contre l’API. / Caused by the fungus Aspergillus fumigatus, invasive pulmonary aspergillosis is a serious threat for individuals suffering from severe immunosuppression. In parallel of a diagnosis lacking specificity, current treatments present a high toxicity. This work first focused on the development of therapeutic antibodies directed against cell wall proteins Chitin ring formation of the fungus. Targeting of these proteins involved in fungal growth highlighted moderate effects in vitro, and induced, in vivo, a significant recruitment of immune cells involved in anti-aspergillary defense. In a second time, this work focused on targeting the microenvironment and the host response during aspergillosis, in order to better understand pathophysiological processes induced during the disease, and allow the identification of new biomarkers and therapeutic targets. Use of iTRAQ® mass spectrometry in rat and penguins allowed the identification of several overrepresented signaling pathways. This work also focused on the immune characterization of a rat model of IPA. In addition of highlighting the effects of the fungus in the recruitment of some immune cell populations, use of iTRAQ® exhibited an overexpression of interleukin-33 and its receptor ST2 during the disease. Overall, this work is bringing interesting insights in the establishment of new treatments against IPA.

Ziel: Die Studie zielte auf die Beurteilung von klinischer Information und Röntgenthorax jeweils alleine und in Kombination für Unterscheidung der invasiven pulmonalen Aspergillose von seinen pulmonalen Differentialdiagnosen. Design: Zur Evaluierung der diagnostischen Leistungen wurde eine Receiver Operating Characteristic Studie angewandt. Methodik: Die diagnostischen Leistungen der alleinigen Klinik, des alleinigen Röntgenthorax und der Kombination von Klinik und Röntgenthorax wurden ermittelt. An der Studie nahmen jeweils acht mit Aspergillose erfahrene Internisten und Radiologen teil. Dazu wurde eine aus 25 gesicherten Fällen mit Aspergillose und 25 differentialdiagnostischen Fällen bestehende Sammlung zusammengestellt. Die Fälle der multizentrischen Studie wurden verblindet, randomisiert und auf einer Fall-CD-ROM präsentiert. Ergebnisse: Die Internisten erreichten mittels alleiniger Klinik die höchste diagnostische Leistung und sie zeigten die größte Variabilität in ihrer Gruppe. Durch die zusätzliche Verfügbarkeit von Röntgenbildern, veränderte sich ihre diagnostische Leistung nicht. Die Radiologen konnten durch zusätzlich zum Röntgenthorax verfügbare klinische Informationen ihre diagnostische Leistung signifikant erhöhen. Schlussfolgerungen: Die klinischen Angaben hatten für die Differentialdiagnose der Aspergillose für Internisten und Radiologne eine hohen diagnostischen Wert. Radiologen brauchen deshalb einen kompletten Zugang zu den spezifischen klinischen Informationen. Der Röntgenthorax zeigte für die radiologisch charakteristischen Aspergillosen und für die Differentialdiagnosen eine hohe Aussagekraft. Als Basis in der diagnostischen Bildgebung sollte sich der Diagnostiker der Stärken und Limitationen dieser konventionellen Bildgebung bewußt sein. / Objective: The role of clinical information and chest film alone and combined for the discrimination between invasive pulmonary aspergillosis (IPA) and its differential diagnoses in human immunodeficiency virus (HIV) infection was studied. Design: Receiver operating characteristics (ROC) methodology was employed to determine separate diagnostic performances. Methods: The diagnostic performance of clinical information and chest film alone and in combination was studied separately for 8 internists and 8 radiologists with regular exposure to HIV and IPA patients. The multicenter ROC case sample consisted of 25 patients with proven IPA and 25 with other proven pulmonary disease entities typical for HIV. The cases were blinded, randomized and presented on a CD-ROM using HTML. Results: With clinical information alone internists achieved the highest diagnostic performance in the discrimination between IPA and its differential diagnoses. Viewing the chest films did not contribute to their performance. The radiologist's performance on the basis of viewing the chest film alone increased significantly when clinical information was supplied. Conclusion: For internists with regular exposure to HIV patients chest films do not provide information essential for the verification or differentiation of potential IPA. Radiologists with regular exposure to HIV patients need full access to the clinical data to reach a comparable performance. Chest films hold relevant information and contribute to the determination of cases with characteristic radiological appearance. Close cooperation between internists and radiologists is mandatory in challenging cases.

Infection fongique opportuniste, l’aspergillose pulmonaire invasive reste redoutée au sein des services hospitaliers d’onco-hématologie, de réanimation ou de transplantations d’organes. Le diagnostic, tant clinique que biologique, souffre globalement d’un manque de sensiblité et de spécificité. De ce fait, le développement de nouveaux marqueurs d’infection semble nécessaire pour améliorer la prise en charge des patients à risque. Dans ce sens, nous avons d’abord mis au point un modèle murin nous permettant d’explorer la maladie aspergillaire avec reproductibilité. Nous avons ensuite étudié le compartiment pulmonaire des rats grâce à deux techniques permettant l’exploration protéomique de leurs liquides de lavages broncho-alvéolaires (LBA). La spectrométrie de masse MALDI-TOF a premièrement dessiné des profils protéiques reproductibles, spécifiques de l’état infecté. L’électrophorèse bidimensionnelle, suivie d’une étude comparative statistique, a ensuite sélectionné 20 spots protéiques surrepreséntés au cours de l’aspergillose expérimentale. Leur caractérisation en spectrométrie de masse a abouti à l’identification de 16 protéines, dont une présentait un intérêt tout particulier car jamais décrite jusqu’ici : ITIH4. Une analyse par western blotting a confirmé la surabondance de cette protéine dans tous les LBA de rats malades, ainsi que son augmentation relative dans le sérum après initiation de l’aspergillose expérimentale. De même, une tendance similaire a été observée dans des LBA d’origine humaine. / Opportunistic fungal infection, invasive aspergillosis is still much feared in the hematologyoncology departments, intensive care units and organ transplant centres. Diagnosis globally suffers from a lack of sensibility and specificity. Therefore, the development of new markers of infection seems necessary to improve the management of patients at risk. In this sense, we first developed a rat model which closely mimics the human disease. We were then able to study the pulmonary compartment of infected rats by the means of two proteomic techniques carried out within their bronchial-oalveolar lavage fluids (BALF). MALDI-TOF mass spectrometry first designed reproducible protein profiles of infected BALF, like a finger print. Two-dimensional electrophoresis, followed by a comparative statistical study, then selected 20 spots overrepresented in experimental aspergillosis. Their characterization by mass spectrometry led to the successful identification of 16 proteins. One of them was of a particular interest because never described so far: ITIH4. Analysis by western blotting confirmed the overabundance of this protein in all infected rat BALF, as well as a relative increase in the serum after initiation of the experimental aspergillosis. Likewise, a similar trend was observed in BALF of human origin.

Aspergilose Pulmonar Invasiva é uma doença fúngica oportunista, causada principalmente por Aspergillus fumigatus, que acomete pacientes imunodeprimidos. Para melhor compreensão dessa micose inicialmente estabelecemos em camundongos C57BL/6 um modelo experimental de depleção de neutrófilos por inoculação intraperitoneal de anticorpos anti-GR-1/Ly-6G, confirmado por contagem total e diferencial de leucócitos sanguíneos. A seguir, avaliamos a evolução da infecção pulmonar experimental utilizando duas amostras de A. fumigatus, caracterizadas previamente em fraca (amostra 699) e forte (amostra 1753) produtoras de elastase. Nenhum dos animais imunocompetentes e infectados evoluiu para o óbito, no período de 7 dias de observação. Os animais neutropênicos, infectados por ambas as amostras, apresentaram 100% de mortalidade após 5 dias, com curvas de sobrevivência praticamente sobrepostas, sugerindo que a maior contribuição para a virulência foi a condição imunológica e não a atividade de elastase da amostra fúngica. Para análise do comprometimento pulmonar, os animais foram sacrificados nos tempos 24, 48 e 72 horas pós-infecção. Durante a evolução da infecção experimental foi observada uma redução da carga fúngica nos pulmões dos animais, para ambas as amostras de A. fumigatus, mas não foi observada uma redução da carga fúngica, diferenciada e estatisticamente significativa, entre os grupos de animais neutropênicos e imunocompetentes. O padrão celular do infiltrado inflamatório observado nos pulmões dos animais neutropênicos, infectados por qualquer uma das amostras de A. fumigatus, mostrou predominância de células mononucleares, em infiltrados difusos, indícios de angioinvasão e invasão brônquica com ruptura de fibras elásticas em ambas as estruturas, além de exuberância de filamentação dos conídios para ambas as amostras fúngicas, desde os tempos iniciais da infecção experimental. O processo inflamatório observado nos pulmões dos animais imunocompetentes, infectados por ambas as amostras de fungos, foi constituído nos tempos iniciais por neutrófilos e se tornou exuberante após 72 horas, com predomínio de macrófagos. Foi observada integridade de vasos sanguíneos e discreta ruptura de parede brônquica no parênquima pulmonar. Para estes animais, salienta-se a ausência de transformação dos conídios de A. fumigatus em hifas para a amostra 699, em todos os períodos de observação. A contagem total de leucócitos no lavado broncoalveolar (LBA) foi significativamente maior, 72 horas pós-infecção, para os animais neutropênicos e imunocompetentes, infectados por ambas as amostras do fungo. A contagem diferencial revelou a presença de macrófagos e neutrófilos, com a primeira célula sempre em maior quantidade no LBA dos animais neutropênicos em comparação com os animais imunocompetentes, independentemente do período da infecção e da amostra fúngica infectante. Ao contrário, o número de neutrófilos foi sempre mais relevante nos animais imunocompetentes. Por microscopia eletrônica de transmissão foi observado que a interação do fungo (conídios ou hifas) com as células de defesa do LBA envolveu íntima adesão e fusão entre os componentes de superfície de ambas as células. A presença de hemoglobina no LBA foi oriunda de lesão alvéolo-capilar causada pelo crescimento e invasão provocados pelas amostras fúngicas ou por lesão determinada pela própria reação inflamatória. Concluímos que os neutrófilos são essenciais na defesa contra A. fumigatus, pois na ausência dessa população celular os fungos rapidamente invadem e lesam o parênquima pulmonar. No entanto, deve-se considerar que a simples presença do fungo em animais imunocompetentes induz a migração de neutrófilos para o sítio da infecção, os quais também causam dano tecidual. As amostras de A. fumigatus com perfis distintos de produção de elastase não refletiram em diferenças significantes para a mortalidade ou gênese das lesões pulmonares observadas em camundongos neutropênicos, sugerindo que embora a elastase contribua para a ruptura das fibras elásticas observadas no tecido pulmonar, outros fatores de virulência, como a morfogênese, podem assumir um papel mais relevante para a patogênese da API experimental. / Invasive Pulmonary Aspergillosis (IPA) is an opportunistic fungal disease, caused mainly by Aspergillus fumigatus, that affects immunocompromised patients. To better understand this mycoses, we originally established in C57BL/6 mice an experimental model of neutrophils depletion by intraperitoneal inoculation of antibodies anti GR-1/Ly-6G, confirmed by total and differential leukocyte counts from blood. Next, we evaluated the evolution of experimental pulmonary infection using two strains of A. fumigatus, previously characterized as weak (strain 699) and strong (strain 1753) elastase producers. None of immunocompetent infected mice died with 7 days of observation, while neutropenic mice, infected with both strains, showed 100% mortality after 5 days, with survival curves nearly overlap, suggesting that the major contribution to the virulence was the immune status instead of elastase activity of each fungal strain. For analysis of lung parenchyma, mice were sacrificed 24, 48 and 72 hours post-infection. During the course of experimental infection it was observed a reduction of fungal burden in the lungs, for both strains of A. fumigatus, but this reduction was not statistically significant between the infected groups (neutropenic and immunocompetent). The cellular pattern of the inflammatory infiltrate observed in lungs from neutropenic mice, infected with both strains of A. fumigatus, revealed a predominance of mononuclear cells, a diffuse pattern and clear evidences of angioinvasion, bronchial disruption with break of elastic fibers in both structures, besides exuberance of conidia filamentation for both fungal strains, since the early period of experimental infection. The inflammatory process observed in lungs from immunocompetent mice, infected with both fungal strains, was composed on early times by neutrophils and became exuberant after 72 hours, with predominance of macrophages. It was observed integrity of blood vessels and moderate bronchial wall disruption in lung parenchyma. A relevant observation was the lack of transformation of conidia in hyphae for 699 A. fumigatus strain, in all periods of observation. Total leukocytes count in bronchoalveolar lavage (BAL) was significantly higher at 72 hours post-infection for both groups infected with both strains. The differential count revealed the presence of macrophages and neutrophils, with the former always in greater percentage in BAL from neutropenic mice and the latter always more elevated in immunocompetent group. Analysis by transmission electron microscopy demonstrated that the interaction of fungal structures (conidia or hyphae) with the defense cells (neutrophlis or macrophages) of BAL involved an intimate adhesion and fusion between the surface components from both cells. The presence of hemoglobin in BAL was a result of alveolar injury caused by the fungal development and invasion, but also by injuries determined by the inflammatory process itself. We concluded that neutrophils have a critical role against A. fumigatus since the pathogen quickly invades and damages the lung parenchyma in its absence. However, we must consider that the mere presence of A. fumigatus in immunocompetent mice induces the neutrophils migration to the infection site, which can also cause a tissue injury. Strains of A. fumigatus with distinct patterns of elastase production did not reflect in significant differences in mortality or origin of pulmonary lesions observed in neutropenic mice, suggesting that although elastase contributes to elastic disruptions observed in pulmonary tissue, another virulence factors, such as morphogenesis, can assume a more relevant role for pathogenesis of experimental IPA.

Aspergilose Pulmonar Invasiva é uma doença fúngica oportunista, causada principalmente por Aspergillus fumigatus, que acomete pacientes imunodeprimidos. Para melhor compreensão dessa micose inicialmente estabelecemos em camundongos C57BL/6 um modelo experimental de depleção de neutrófilos por inoculação intraperitoneal de anticorpos anti-GR-1/Ly-6G, confirmado por contagem total e diferencial de leucócitos sanguíneos. A seguir, avaliamos a evolução da infecção pulmonar experimental utilizando duas amostras de A. fumigatus, caracterizadas previamente em fraca (amostra 699) e forte (amostra 1753) produtoras de elastase. Nenhum dos animais imunocompetentes e infectados evoluiu para o óbito, no período de 7 dias de observação. Os animais neutropênicos, infectados por ambas as amostras, apresentaram 100% de mortalidade após 5 dias, com curvas de sobrevivência praticamente sobrepostas, sugerindo que a maior contribuição para a virulência foi a condição imunológica e não a atividade de elastase da amostra fúngica. Para análise do comprometimento pulmonar, os animais foram sacrificados nos tempos 24, 48 e 72 horas pós-infecção. Durante a evolução da infecção experimental foi observada uma redução da carga fúngica nos pulmões dos animais, para ambas as amostras de A. fumigatus, mas não foi observada uma redução da carga fúngica, diferenciada e estatisticamente significativa, entre os grupos de animais neutropênicos e imunocompetentes. O padrão celular do infiltrado inflamatório observado nos pulmões dos animais neutropênicos, infectados por qualquer uma das amostras de A. fumigatus, mostrou predominância de células mononucleares, em infiltrados difusos, indícios de angioinvasão e invasão brônquica com ruptura de fibras elásticas em ambas as estruturas, além de exuberância de filamentação dos conídios para ambas as amostras fúngicas, desde os tempos iniciais da infecção experimental. O processo inflamatório observado nos pulmões dos animais imunocompetentes, infectados por ambas as amostras de fungos, foi constituído nos tempos iniciais por neutrófilos e se tornou exuberante após 72 horas, com predomínio de macrófagos. Foi observada integridade de vasos sanguíneos e discreta ruptura de parede brônquica no parênquima pulmonar. Para estes animais, salienta-se a ausência de transformação dos conídios de A. fumigatus em hifas para a amostra 699, em todos os períodos de observação. A contagem total de leucócitos no lavado broncoalveolar (LBA) foi significativamente maior, 72 horas pós-infecção, para os animais neutropênicos e imunocompetentes, infectados por ambas as amostras do fungo. A contagem diferencial revelou a presença de macrófagos e neutrófilos, com a primeira célula sempre em maior quantidade no LBA dos animais neutropênicos em comparação com os animais imunocompetentes, independentemente do período da infecção e da amostra fúngica infectante. Ao contrário, o número de neutrófilos foi sempre mais relevante nos animais imunocompetentes. Por microscopia eletrônica de transmissão foi observado que a interação do fungo (conídios ou hifas) com as células de defesa do LBA envolveu íntima adesão e fusão entre os componentes de superfície de ambas as células. A presença de hemoglobina no LBA foi oriunda de lesão alvéolo-capilar causada pelo crescimento e invasão provocados pelas amostras fúngicas ou por lesão determinada pela própria reação inflamatória. Concluímos que os neutrófilos são essenciais na defesa contra A. fumigatus, pois na ausência dessa população celular os fungos rapidamente invadem e lesam o parênquima pulmonar. No entanto, deve-se considerar que a simples presença do fungo em animais imunocompetentes induz a migração de neutrófilos para o sítio da infecção, os quais também causam dano tecidual. As amostras de A. fumigatus com perfis distintos de produção de elastase não refletiram em diferenças significantes para a mortalidade ou gênese das lesões pulmonares observadas em camundongos neutropênicos, sugerindo que embora a elastase contribua para a ruptura das fibras elásticas observadas no tecido pulmonar, outros fatores de virulência, como a morfogênese, podem assumir um papel mais relevante para a patogênese da API experimental. / Invasive Pulmonary Aspergillosis (IPA) is an opportunistic fungal disease, caused mainly by Aspergillus fumigatus, that affects immunocompromised patients. To better understand this mycoses, we originally established in C57BL/6 mice an experimental model of neutrophils depletion by intraperitoneal inoculation of antibodies anti GR-1/Ly-6G, confirmed by total and differential leukocyte counts from blood. Next, we evaluated the evolution of experimental pulmonary infection using two strains of A. fumigatus, previously characterized as weak (strain 699) and strong (strain 1753) elastase producers. None of immunocompetent infected mice died with 7 days of observation, while neutropenic mice, infected with both strains, showed 100% mortality after 5 days, with survival curves nearly overlap, suggesting that the major contribution to the virulence was the immune status instead of elastase activity of each fungal strain. For analysis of lung parenchyma, mice were sacrificed 24, 48 and 72 hours post-infection. During the course of experimental infection it was observed a reduction of fungal burden in the lungs, for both strains of A. fumigatus, but this reduction was not statistically significant between the infected groups (neutropenic and immunocompetent). The cellular pattern of the inflammatory infiltrate observed in lungs from neutropenic mice, infected with both strains of A. fumigatus, revealed a predominance of mononuclear cells, a diffuse pattern and clear evidences of angioinvasion, bronchial disruption with break of elastic fibers in both structures, besides exuberance of conidia filamentation for both fungal strains, since the early period of experimental infection. The inflammatory process observed in lungs from immunocompetent mice, infected with both fungal strains, was composed on early times by neutrophils and became exuberant after 72 hours, with predominance of macrophages. It was observed integrity of blood vessels and moderate bronchial wall disruption in lung parenchyma. A relevant observation was the lack of transformation of conidia in hyphae for 699 A. fumigatus strain, in all periods of observation. Total leukocytes count in bronchoalveolar lavage (BAL) was significantly higher at 72 hours post-infection for both groups infected with both strains. The differential count revealed the presence of macrophages and neutrophils, with the former always in greater percentage in BAL from neutropenic mice and the latter always more elevated in immunocompetent group. Analysis by transmission electron microscopy demonstrated that the interaction of fungal structures (conidia or hyphae) with the defense cells (neutrophlis or macrophages) of BAL involved an intimate adhesion and fusion between the surface components from both cells. The presence of hemoglobin in BAL was a result of alveolar injury caused by the fungal development and invasion, but also by injuries determined by the inflammatory process itself. We concluded that neutrophils have a critical role against A. fumigatus since the pathogen quickly invades and damages the lung parenchyma in its absence. However, we must consider that the mere presence of A. fumigatus in immunocompetent mice induces the neutrophils migration to the infection site, which can also cause a tissue injury. Strains of A. fumigatus with distinct patterns of elastase production did not reflect in significant differences in mortality or origin of pulmonary lesions observed in neutropenic mice, suggesting that although elastase contributes to elastic disruptions observed in pulmonary tissue, another virulence factors, such as morphogenesis, can assume a more relevant role for pathogenesis of experimental IPA.

A aspergilose invasiva (AI) é a infecção por fungos filamentosos mais comum em pacientes imunodeprimidos, especialmente em transplantes de células tronco hematopoiético e neoplasias hematológicas. Objetivo: Geral: Estabelecer a comparação entre a dosagem de Galactomanana (GM), 1,3betaD-glucana (BDG) e dados tomográficos no diagnóstico da AI bem como seu papel no desfecho clínico. Específicos: 1. Verificar a sensibilidade e especificidade dos ensaios de Galactomanana e de 1,3betaD-glucana no soro e lavado broncoalveolar. 2. Comparar os resultados da galatomanana e 1,3betaD-glucana com os dados de imagem em pacientes com suspeita de AI. 3. Verificar a relação entre a evolução dos níveis de GM e desfecho clínico (óbito e sobrevida). Casuística, Materiais e Métodos: Realizou-se um estudo tipo coorte prospectiva, incluindo 398 sujeitos das diversas enfermarias de pacientes imunodeprimidos do HCFMUSP, sendo incluídos dois grupos de pacientes: 202(51%) AI e 198(49%) controles. Resultados: Dos casos, 18 (8,8%) tinham aspergilose provada, 28 (13,7%) provável e 158 possível (77,5%), de acordo a classificação de 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer / Mycoses Study Group). Os sujeitos submetidos ao TCTH eram 42,7%, com neoplasias hematológicas 37%, TOS 9% e outras doenças 11,3%. Os fatores de risco associados ao desenvolvimento da AI foram neutropenia, monocitopenia, uso de corticóide, presença de doença pelo citomegalovírus e rejeição ou doença do enxerto contra o hospedeiro. O fator de risco associado à evolução para o óbito foi a presença de AI. Foram observados bons desempenhos para a GM tanto no soro como no LBA com LR menores que os registrados na literatura. O melhor desempenho da GM no soro para aspergilose+provável ocorreu com LR de 0,35 com sensibilidade-S, especificidade-E, valor preditivo positivo- VPP), valor preditivo negativo-VPN) e área sob a curva-ASC de 54,4%, 73,4%, 50,8%, 76,2% e 0,64, sendo os valores superiores para aspergilose provada tanto na S, como E, VPN. No LBA os valores de S-E-VPP-VPN-ASC para GM para LR de 0,65 para aspergilose provável + provada foram 58,3%, 92,6%, 87,5%,71,4% e 0,75, sendo na aspergilose provada os valores de S, e VPN superiores. Nesta casuística, o melhor desempenho para BDG no soro apontou para uma LR de 100 pg/mL na aspergilose provável+provada, com 54,5%, 73,4%, 50,8% e 76,2%, 0,64 respectivamente para S-E-VPP-VPN-ASC. Para BDG no LBA, a LR na aspergilose provável + provada foi de 140 pg/mL, com os mesmos valores de 46,7%, 76,7%, 70%, 55,6% e 0.62, respectivamente. Conclusão: A GM no LBA e no soro foram úteis no diagnóstico da aspergilose mediante emprego de LR menores, sendo mais sensível na LBA, principalmente em estágios iniciais da forma angioinvasiva. A persistência de GM sérica foi relacionada ao óbito em relação à negativação da mesma. A proporção de concordância entre a TC e os biomarcadores no soro e no LBA variou de 0,5 a 0,6, com pequena concordância na estatística kappa. Excelente concordância foi observada entre dois radiologistas independentes, que analisaram de maneira cega as TC de sujeitos com aspergilose provada. Nesta casuística com inclusão de doenças sistêmicas e endêmicas, a BDG teve baixo desempenho diagnóstico / Invasive aspergillosis (IA) has become the leading infectious cause of death in immunocompromised hosts, particularly in subjects under SCTH and hematologic neoplasias. Objectives: General: To compare the performance of GM and BG tests in serum and bronchoalveolar lavage fluid (BAL) and computer tomography (CT) scans in the diagnosis of IA in immunocompromised hosts as well as their role in the patient outcome. Specific: 1. To analyse the sensitivity and specificity of Galactomannan and 1,3 betaD-glucan assays in the serum and bronchoalveolar lavage. 2. To compare the results of Galactomannan and 1,3betaD-glucan assays with CT scans in patients with invasive aspergilosis. 3. To analyse the relationship between the evolution of galactomannan levels and clinical outcome (death or survival). Patients, Materials and Methods: From December 2008 to March 2013, a prospective cohort of 398 patients from several wards of immunocompromised patients of Hospital das Clínicas, Faculdade de Medicina, University of São Paulo was included classified in two groups of patients: 202 (51%) with invasive aspergillosis (IA) and 198 (49%) control patients. Results: Considering 202 cases, 18(8.8%) were subjects with proven, 28(13.7%) with probable aspergillosis and 156(77.5%), with possible aspergillosis, according to 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) criteria. The most common underlying disease were: HSCT (42.7%), hematologic malignancy (37%), SOT (9%), or other diseases (11.3%). The main risk factors associated with IA were neutropenia, monocytopenia, patients under corticosterois, presence of CMV disease, and rejection or graft versus host disease. The risk factor associated with death was the presence of invasive aspergillosis. Good performances for serum and BAL GM were registered with lower cutoffs in the present workin relationship to those found in the literature. The best cutoff for proven + probable aspergillosis for serum GM was observed at 0.35 vallue with Sensitivity-S, Specificity-Sp, Positive Predictive value-PPV), Negative Predictive Value-NPV) and AUC of 54.4%, 73.4%, 50.8%, 76.2% and 0.64; the values for proven aspergillosis alone were higher for S, Sp and NPV. On BAL tests for GM (cutoff value of 0.65) in proven+probable aspergillosis we observed 58.3%, 92.6%, 87.5%,71.4%, 0.75, respectively as S-Sp-PPV-NPVAUC; the sensitivity and VPN were higher in proven aspergillosis alone. In this work, the best performance in proven+probable aspergillosis for serum BDG showed 100 pg/ML as cutoff value, with 54.5%, 73.4%, 50.8%,76.2%, 0.64 for S-Sp-PPVNPV- AUC, respectively. For BAL- BDG, the cut off for proven+probable aspergillosis was 140 pg/mL, and we observed 46.7%, 76.7%, 70.0%, 55.,6%, 0.62, respectively for for S-Sp-PPV-NPV-AUC. Conclusion: The serum and BAL GM are useful tests for diagnosis in early stages of angioinvasive form at lower cutoffs; BAL GM is more sensitive. Agreement proportion between CT scan and each biomarker in the serum or BAL ranged from 0.5-0.6, with low ? index. Perfect ? statistic was observed for analysis of CT scan of subjects in proven aspergillosis by two independent radiologists, blinded for diagnosis. Persistence of serum GM was associated to death in relationship with its negativation. BDG test showed low performance in this work, where systemic and endemic diseases were included

A aspergilose invasiva (AI) é a infecção por fungos filamentosos mais comum em pacientes imunodeprimidos, especialmente em transplantes de células tronco hematopoiético e neoplasias hematológicas. Objetivo: Geral: Estabelecer a comparação entre a dosagem de Galactomanana (GM), 1,3betaD-glucana (BDG) e dados tomográficos no diagnóstico da AI bem como seu papel no desfecho clínico. Específicos: 1. Verificar a sensibilidade e especificidade dos ensaios de Galactomanana e de 1,3betaD-glucana no soro e lavado broncoalveolar. 2. Comparar os resultados da galatomanana e 1,3betaD-glucana com os dados de imagem em pacientes com suspeita de AI. 3. Verificar a relação entre a evolução dos níveis de GM e desfecho clínico (óbito e sobrevida). Casuística, Materiais e Métodos: Realizou-se um estudo tipo coorte prospectiva, incluindo 398 sujeitos das diversas enfermarias de pacientes imunodeprimidos do HCFMUSP, sendo incluídos dois grupos de pacientes: 202(51%) AI e 198(49%) controles. Resultados: Dos casos, 18 (8,8%) tinham aspergilose provada, 28 (13,7%) provável e 158 possível (77,5%), de acordo a classificação de 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer / Mycoses Study Group). Os sujeitos submetidos ao TCTH eram 42,7%, com neoplasias hematológicas 37%, TOS 9% e outras doenças 11,3%. Os fatores de risco associados ao desenvolvimento da AI foram neutropenia, monocitopenia, uso de corticóide, presença de doença pelo citomegalovírus e rejeição ou doença do enxerto contra o hospedeiro. O fator de risco associado à evolução para o óbito foi a presença de AI. Foram observados bons desempenhos para a GM tanto no soro como no LBA com LR menores que os registrados na literatura. O melhor desempenho da GM no soro para aspergilose+provável ocorreu com LR de 0,35 com sensibilidade-S, especificidade-E, valor preditivo positivo- VPP), valor preditivo negativo-VPN) e área sob a curva-ASC de 54,4%, 73,4%, 50,8%, 76,2% e 0,64, sendo os valores superiores para aspergilose provada tanto na S, como E, VPN. No LBA os valores de S-E-VPP-VPN-ASC para GM para LR de 0,65 para aspergilose provável + provada foram 58,3%, 92,6%, 87,5%,71,4% e 0,75, sendo na aspergilose provada os valores de S, e VPN superiores. Nesta casuística, o melhor desempenho para BDG no soro apontou para uma LR de 100 pg/mL na aspergilose provável+provada, com 54,5%, 73,4%, 50,8% e 76,2%, 0,64 respectivamente para S-E-VPP-VPN-ASC. Para BDG no LBA, a LR na aspergilose provável + provada foi de 140 pg/mL, com os mesmos valores de 46,7%, 76,7%, 70%, 55,6% e 0.62, respectivamente. Conclusão: A GM no LBA e no soro foram úteis no diagnóstico da aspergilose mediante emprego de LR menores, sendo mais sensível na LBA, principalmente em estágios iniciais da forma angioinvasiva. A persistência de GM sérica foi relacionada ao óbito em relação à negativação da mesma. A proporção de concordância entre a TC e os biomarcadores no soro e no LBA variou de 0,5 a 0,6, com pequena concordância na estatística kappa. Excelente concordância foi observada entre dois radiologistas independentes, que analisaram de maneira cega as TC de sujeitos com aspergilose provada. Nesta casuística com inclusão de doenças sistêmicas e endêmicas, a BDG teve baixo desempenho diagnóstico / Invasive aspergillosis (IA) has become the leading infectious cause of death in immunocompromised hosts, particularly in subjects under SCTH and hematologic neoplasias. Objectives: General: To compare the performance of GM and BG tests in serum and bronchoalveolar lavage fluid (BAL) and computer tomography (CT) scans in the diagnosis of IA in immunocompromised hosts as well as their role in the patient outcome. Specific: 1. To analyse the sensitivity and specificity of Galactomannan and 1,3 betaD-glucan assays in the serum and bronchoalveolar lavage. 2. To compare the results of Galactomannan and 1,3betaD-glucan assays with CT scans in patients with invasive aspergilosis. 3. To analyse the relationship between the evolution of galactomannan levels and clinical outcome (death or survival). Patients, Materials and Methods: From December 2008 to March 2013, a prospective cohort of 398 patients from several wards of immunocompromised patients of Hospital das Clínicas, Faculdade de Medicina, University of São Paulo was included classified in two groups of patients: 202 (51%) with invasive aspergillosis (IA) and 198 (49%) control patients. Results: Considering 202 cases, 18(8.8%) were subjects with proven, 28(13.7%) with probable aspergillosis and 156(77.5%), with possible aspergillosis, according to 2002 EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) criteria. The most common underlying disease were: HSCT (42.7%), hematologic malignancy (37%), SOT (9%), or other diseases (11.3%). The main risk factors associated with IA were neutropenia, monocytopenia, patients under corticosterois, presence of CMV disease, and rejection or graft versus host disease. The risk factor associated with death was the presence of invasive aspergillosis. Good performances for serum and BAL GM were registered with lower cutoffs in the present workin relationship to those found in the literature. The best cutoff for proven + probable aspergillosis for serum GM was observed at 0.35 vallue with Sensitivity-S, Specificity-Sp, Positive Predictive value-PPV), Negative Predictive Value-NPV) and AUC of 54.4%, 73.4%, 50.8%, 76.2% and 0.64; the values for proven aspergillosis alone were higher for S, Sp and NPV. On BAL tests for GM (cutoff value of 0.65) in proven+probable aspergillosis we observed 58.3%, 92.6%, 87.5%,71.4%, 0.75, respectively as S-Sp-PPV-NPVAUC; the sensitivity and VPN were higher in proven aspergillosis alone. In this work, the best performance in proven+probable aspergillosis for serum BDG showed 100 pg/ML as cutoff value, with 54.5%, 73.4%, 50.8%,76.2%, 0.64 for S-Sp-PPVNPV- AUC, respectively. For BAL- BDG, the cut off for proven+probable aspergillosis was 140 pg/mL, and we observed 46.7%, 76.7%, 70.0%, 55.,6%, 0.62, respectively for for S-Sp-PPV-NPV-AUC. Conclusion: The serum and BAL GM are useful tests for diagnosis in early stages of angioinvasive form at lower cutoffs; BAL GM is more sensitive. Agreement proportion between CT scan and each biomarker in the serum or BAL ranged from 0.5-0.6, with low ? index. Perfect ? statistic was observed for analysis of CT scan of subjects in proven aspergillosis by two independent radiologists, blinded for diagnosis. Persistence of serum GM was associated to death in relationship with its negativation. BDG test showed low performance in this work, where systemic and endemic diseases were included