Aspergillus species are responsible for a variety of human diseases, ranging from allergic bronchopulmonary aspergillosis to invasive aspergillosis. Identification of Aspergillus species could facilitate the selection of antifungal regimens and epidemiological studies. Most of the clinical microbiology laboratories identify Aspergillus species by traditional phenotypic and/or antigen detection methods, which are laborious, time-consuming and inaccurate. In recent years, sequence analysis of β-tubulin and calmodulin genes becomes widely used for the identification of fungal species due to their relatively high resolving power, universality of usage, and high availability in the public databases. It can also be used for the taxonomic classification and the identification of rare and even novel fungal species. In this study, we aim to evaluate the effectiveness of analyzing the β-tubulin and calmodulin gene sequences for the identification of Aspergillus species and subsequently to determine the clinical spectrum of Aspergillus infections in Hong Kong during 2012-2014. In this study, 48 Aspergillus strains isolated from patients over a 3-year period were characterised to the species level using sequencing of β-tubulin and calmodulin genes and the clinical spectrum of the 48 patients was described. Sequencing of β-tubulin and calmodulin genes showed that all 48 strains were known Aspergillus species. Ten different Aspergillus species were identified, including A. fumigatus (n=16), Aspergillus species of the A. flavus clade (n=7), A. awamori (n=7), A. terreus (n=6), A. tubingensis (n=4), A. sydowii (n=3), A. pseudocaelatus (n=2), A. uniguis (n=1), Aspergillus species of the A. tamarii clade (n=1), and A. austroafricanus (n=1). These Aspergillus species were shown to be associated with respiratory infections, infections of nail, ear canal infections, invasive infections, and eye infection. This study also described the first reported cases of ear infection associated with A. pseudocaelatus. To conclude, sequence analysis of β-tubulin and calmodulin genes enables accurate and rapid Aspergillus species differentiation. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

No description available.

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.

published_or_final_version / Microbiology / Master / Master of Philosophy

Chronic pulmonary aspergillosis (CPA) is a serious disease that occurs secondary to tuberculosis and is estimated to affect 1.2 million persons globally. Pulmonary aspergillosis is found in 2-3% of all AIDS autopsies, but 90% of cases go undiagnosed ante-mortem. Here the sensitivity and specificity of optimal diagnostic thresholds for CPA have been defined in relation to six Aspergillus-specific IgG assays. The prevalence of CPA in an area of high tuberculosis prevalence has been measured. Receiver operating characteristic (ROC) curves were used to compare results of testing with six Aspergillus-specific IgG assays in 241 patients with CPA and 100 healthy controls. ThermoFisher Scientific ImmunoCAP and Siemens Immulite had ROC area under curve (AUC) results of 0.995 and 0.991 respectively. Both were statistically significantly superior to all other assays. Both had a sensitivity of 96% and specificity of 98% using diagnostic cut offs of 20 mg/L and 10 mg/L respectively. Eighty patients with allergic bronchopulmonary aspergillosis (ABPA) were also assessed. ROC AUC results were 0.959 for ImmunoCAP and 0.932 for Immulite. The new thresholds produced specificities of 98% for both assays and sensitivities of 78% and 81% respectively. Levels in ABPA patients were also compared to asthmatic controls.398 patients with treated tuberculosis in Gulu, Uganda were assessed in a cross-sectional survey. CCPA diagnostic criteria were; 1 – Cough or haemoptysis for one month, 2 – Progressive cavitation on serial chest X-ray or either paracavitary fibrosis or aspergilloma on CT scan and 3 – Raised Siemens Immulite Aspergillus-specific IgG. All three were required for diagnosis. CCPA was present in 5.7% of patients and simple aspergilloma in 0.7% of patients. There was a non-significant trend to less frequent CCPA in HIV positive patients (p=0.18). Aspergillus-specific IgG levels were measured in stored sera from two adult in patient groups at Mulago Hospital, Kampala, Uganda. 26% of 39 patients with HIV infection and subacute respiratory illness and no diagnosis after extensive investigation had raised levels. 47% of 57 patients with proven active pulmonary tuberculosis had raised levels. The Immulite and ImmunoCAP assays both have good sensitivity and specificity for the diagnosis of CPA. New diagnostic thresholds improve the performance of all assays. CCPA has been shown to complicate pulmonary tuberculosis in Gulu, Uganda. Subacute invasive pulmonary aspergillosis is likely to affect many patients with AIDS and subacute respiratory illness. CPA may begin during active pulmonary tuberculosis infection. CPA associated with tuberculosis constitutes a significant unrecognized public health problem, which is probably being incorrectly identified as ‘smear-negative tuberculosis’ clinically and in public health data. Prospective studies are now needed to confirm the prevalence of CPA secondary to tuberculosis and define the optimal strategy for routine CPA screening, followed by studies to define optimal treatment regimes for use in research poor-settings, where most cases of CPA are likely to occur.

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.

A pneumonia fúngica é uma infecção pulmonar profunda causada principalmente por fungos do gênero Aspergillus, incluindo A. fumigatus, A. niger, A. flavus e A. terreus. Se encontram mais comumente em matéria orgânica em decomposição. Seus propágulos estão presentes na poeira e no ar, o que favorece a inalação, porta principal de entrada do agente no organismo. Produzem grande quantidade de conídios com menos de 8 μ, fazendo com que, quando inalados, alcancem o leito pulmonar. É o gênero considerado o mais comum em nosso planeta e o A. fumigatus é a espécie mais frequentemente descrita em invasão tecidual. Em animais e humanos, os sistemas respiratório e imunológico saudáveis são suficientes para eliminar os propágulos das vias aéreas, evitando sua colonização. Doenças e fármacos imunossupressores têm sido descritos como fatores predisponentes da infecção pulmonar fúngica, considerada oportunística. É considerada rara em cães. Os sinais clínicos são variados, mas a tosse persistente e a não cessação dos sinais com o uso de antimicrobianos são dados para suspeita de pneumonia fúngica. No exame radiológico de pulmões, são descritas várias imagens, das quais a mais relatada é a de padrão intersticial nodular miliar generalizado, embora não seja imagem patognomônica. Este trabalho teve como objetivo verificar a ocorrência de aspergilose pulmonar em cães com sintomatologia respiratória atendidos no Hospital de Clínicas Veterinárias da Universidade Federal do Rio Grande do Sul (HCV-UFRGS), localizado na cidade de Porto Alegre. A amostragem foi composta por 46 cães nos quais foi realizado Biópsia por Aspiração com Agulha Fina (BAAF) nos dois pulmões, coleta de sangue e exame radiológico torácico. O material obtido de uma punção foi homogeneizado com caldo Sabouraud Dextrose líquido. Parte deste homogeneizado foi semeado em Agar Malte para Aspergillus spp. e feito exame direto em lâmina e com colotração de Grocott. A outra parte foi semeada em vários meios de cultura para exame bacteriológico. Do material obtido na outra punção foi realizado exame citopatológico corado pelo método Panótico Rápido. Do soro obtido do sangue coletado foi realizado exame soromicológico para A. fumigatus, A. flavus e A. niger. Os resultados dos exames micológico, citopatológico, soromicológico e bacteriológico foram negativos para todas as amostras testadas. Não havendo positividade dos exames, não foi possível correlacionar a pneumonia fúngica com as imagens radiológicas obtidas dos pulmões dos 46 cães radiografados. A maioria dos cães viviam em áreas abertas, em contato com matéria orgânica onde o fungo frequentemente se encontra. Os exames para detecção de fungos devem ser analisados com cautela, pois o resultado positivo em um único exame não confirma a presença da infecção, pois o agente pode estar presente frequentemente no trato respiratório superior de cães. / Fungal pneumonia is a deeply lung infection caused by fungal agents of the genus Aspergillus, including A. fumigatus, A. niger, A. flavus and A. terreus. They are found most commonly in decaying organic matter. Their fungal propagules are present in dust and air, which favors the inhalation, the main entrance of the agent in the body. They produce large amounts of conidia with less than 8 μ, so that, when inhaled, reaching the pulmonary bed. It is the considered the most common kind genus on our planet and A. fumigatus is the most often reported species in tissue invasion. In animals and humans, a healthy immune and respiratory systems is sufficient to remove the seedlings of the airways, preventing colonization. Diseases and immunosuppressive drugs have been described as predisposing factors of pulmonary fungal infection, considered opportunistic. It is considered rare in dogs. Clinical symptoms are varied, but a persistent cough and no cessation of symptoms with the use of antimicrobials are data for suspected fungal pneumonia. In the radiological examination of the lungs, several images have been described, and the most described is a generalized miliary nodular interstitial pattern, but it is not a pathognomonic image. This work aimed to verify the occurrence of pulmonary aspergillosis in dogs with respiratory symptoms examined at the Veterinary Clinical Hospital of Federal University of Rio Grande do Sul (HCVUFRGS), located in Porto Alegre city, in Brasil. The sampling was composed of 46 dogs in which biopsy was performed by fine needle aspiration (FNAC) in both lungs, blood sampling and radiological examination of the chest. The material obtained from a puncture was homogenized with Sabouraud dextrose liquid. Part of this homogenate was seeded on malt agar for Aspergillus spp. and taken directly on slides and stained with “Grocott”. The other party was seeded in various culture media to bacteriological examination. The material obtained in another puncture was performed cytopathological examination with “Panótico” coloring. Serum obtained from blood collected was performed for serology to A. fumigatus, A. flavus and A. niger. The results of mycological examination, cytology, bacteriology and serology examinations were negative for all samples tested. In the absence of positive tests, it was not possible to correlate fungal pneumonia with radiological images obtained from the lungs of the 46 dogs radiographed. Most dogs live in open areas in contact with organic matter where the fungus is usually found. Tests to detect fungi should be analyzed with caution, because a positive result in a single test does not confirm the presence of infection, since the agent can be present normally in the upper respiratory tract of dogs.

O objetivo deste estudo foi avaliar os achados na tomografia computadorizada de alta resolução (TCAR) de pacientes transplantados de pulmão diagnosticados com infecção pulmonar por Aspergillus. Foram revisados retrospectivamente os exames de TCAR de 23 pacientes diagnosticados com aspergilose. Os exames de imagem foram realizados entre 2-5 dias após o início dos sintomas. A amostra de pacientes incluiu 12 homens e 11 mulheres com idades entre 22-59 anos (idade média: 43,6 anos). Todos os pacientes apresentaram taquipnéia, dispnéia e tosse. O diagnóstico foi estabelecido com o ensaio imunoenzimático (Platelia Aspergillus) para a detecção do antígeno galactomanana no lavado broncoalveolar e recuperação dos sintomas e dos achados de TCAR após tratamento com voriconazol. As TCAR foram analisadas independentemente por dois observadores que chegaram a uma decisão em consenso. O principal padrão na TCAR encontrado foi o de nódulos centrolobulares com padrão de árvore-em-brotamento associados com espessamento de paredes brônquicas que foi visualizado em 65% (n=15) dos pacientes. Este padrão foi descrito em associação com áreas de consolidação e opacidades em vidro-fosco em 13% (n=3) dos pacientes. Consolidação e opacidades em vidro-fosco foi o padrão principal em 22% (n=5) dos pacientes. O padrão de nódulos grandes com e sem o sinal do halo foi observado em 13% (n=3) dos pacientes e, em um caso, esteve associado com consolidação e opacidades em vidro-fosco. Conclui-se que os achados predominantes na TCAR em pacientes transplantados de pulmão com aspergilose foram espessamento de paredes brônquicas e opacidades centrolobulares com padrão de árvore-em-brotamento bilateralmente. Além disso, opacidades em vidro-fosco e / ou áreas de consolidação bilaterais foram achados comuns. Os nódulos com o sinal do halo foram encontrados em apenas 13% dos pacientes. / The aim of this study was to assess high-resolution computed tomographic (HRCT) findings at presentation in lung transplant patients diagnosed with pulmonary Aspergillus infection. We retrospectively reviewed HRCT findings from 23 patients diagnosed with pulmonary aspergillosis. Imaging studies were performed 2–5 days after the onset of symptoms. The patient sample comprised 12 men and 11 women aged 22–59 years (mean age, 43.6 years). All patients had dyspnea, tachypnea, and cough. Diagnoses were established with Platelia Aspergillus enzyme immunoassays for galactomannan antigen detection in bronchoalveolar lavage and recovery of symptoms, and HRCT findings after voriconazole treatment. The HRCT scans were reviewed independently by two observers who reached a consensus decision. The main HRCT pattern, found in 65% (n = 15) of patients, was centrilobular tree-in-bud nodules associated with bronchial thickening. This pattern was described in association with areas of consolidation and ground-glass opacities in 13% (n = 3) of patients. Consolidation and ground-glass opacities were the main pattern in 22% (n = 5) of patients. The pattern of large nodules with and without the halo sign was observed in 13% (n = 3) of patients, and were associated with consolidation and ground-glass opacities in one case. In conclusion, the predominant HRCT findings in lung transplant patients with pulmonary aspergillosis were bilateral bronchial wall thickening and centrilobular opacities with the tree-in-bud pattern. Ground-glass opacities and/or bilateral areas of consolidation were also common findings. Pulmonary nodules with the halo sign were found in only 13% of patients.

O objetivo deste estudo foi avaliar os achados na tomografia computadorizada de alta resolução (TCAR) de pacientes transplantados de pulmão diagnosticados com infecção pulmonar por Aspergillus. Foram revisados retrospectivamente os exames de TCAR de 23 pacientes diagnosticados com aspergilose. Os exames de imagem foram realizados entre 2-5 dias após o início dos sintomas. A amostra de pacientes incluiu 12 homens e 11 mulheres com idades entre 22-59 anos (idade média: 43,6 anos). Todos os pacientes apresentaram taquipnéia, dispnéia e tosse. O diagnóstico foi estabelecido com o ensaio imunoenzimático (Platelia Aspergillus) para a detecção do antígeno galactomanana no lavado broncoalveolar e recuperação dos sintomas e dos achados de TCAR após tratamento com voriconazol. As TCAR foram analisadas independentemente por dois observadores que chegaram a uma decisão em consenso. O principal padrão na TCAR encontrado foi o de nódulos centrolobulares com padrão de árvore-em-brotamento associados com espessamento de paredes brônquicas que foi visualizado em 65% (n=15) dos pacientes. Este padrão foi descrito em associação com áreas de consolidação e opacidades em vidro-fosco em 13% (n=3) dos pacientes. Consolidação e opacidades em vidro-fosco foi o padrão principal em 22% (n=5) dos pacientes. O padrão de nódulos grandes com e sem o sinal do halo foi observado em 13% (n=3) dos pacientes e, em um caso, esteve associado com consolidação e opacidades em vidro-fosco. Conclui-se que os achados predominantes na TCAR em pacientes transplantados de pulmão com aspergilose foram espessamento de paredes brônquicas e opacidades centrolobulares com padrão de árvore-em-brotamento bilateralmente. Além disso, opacidades em vidro-fosco e / ou áreas de consolidação bilaterais foram achados comuns. Os nódulos com o sinal do halo foram encontrados em apenas 13% dos pacientes. / The aim of this study was to assess high-resolution computed tomographic (HRCT) findings at presentation in lung transplant patients diagnosed with pulmonary Aspergillus infection. We retrospectively reviewed HRCT findings from 23 patients diagnosed with pulmonary aspergillosis. Imaging studies were performed 2–5 days after the onset of symptoms. The patient sample comprised 12 men and 11 women aged 22–59 years (mean age, 43.6 years). All patients had dyspnea, tachypnea, and cough. Diagnoses were established with Platelia Aspergillus enzyme immunoassays for galactomannan antigen detection in bronchoalveolar lavage and recovery of symptoms, and HRCT findings after voriconazole treatment. The HRCT scans were reviewed independently by two observers who reached a consensus decision. The main HRCT pattern, found in 65% (n = 15) of patients, was centrilobular tree-in-bud nodules associated with bronchial thickening. This pattern was described in association with areas of consolidation and ground-glass opacities in 13% (n = 3) of patients. Consolidation and ground-glass opacities were the main pattern in 22% (n = 5) of patients. The pattern of large nodules with and without the halo sign was observed in 13% (n = 3) of patients, and were associated with consolidation and ground-glass opacities in one case. In conclusion, the predominant HRCT findings in lung transplant patients with pulmonary aspergillosis were bilateral bronchial wall thickening and centrilobular opacities with the tree-in-bud pattern. Ground-glass opacities and/or bilateral areas of consolidation were also common findings. Pulmonary nodules with the halo sign were found in only 13% of patients.

A pneumonia fúngica é uma infecção pulmonar profunda causada principalmente por fungos do gênero Aspergillus, incluindo A. fumigatus, A. niger, A. flavus e A. terreus. Se encontram mais comumente em matéria orgânica em decomposição. Seus propágulos estão presentes na poeira e no ar, o que favorece a inalação, porta principal de entrada do agente no organismo. Produzem grande quantidade de conídios com menos de 8 μ, fazendo com que, quando inalados, alcancem o leito pulmonar. É o gênero considerado o mais comum em nosso planeta e o A. fumigatus é a espécie mais frequentemente descrita em invasão tecidual. Em animais e humanos, os sistemas respiratório e imunológico saudáveis são suficientes para eliminar os propágulos das vias aéreas, evitando sua colonização. Doenças e fármacos imunossupressores têm sido descritos como fatores predisponentes da infecção pulmonar fúngica, considerada oportunística. É considerada rara em cães. Os sinais clínicos são variados, mas a tosse persistente e a não cessação dos sinais com o uso de antimicrobianos são dados para suspeita de pneumonia fúngica. No exame radiológico de pulmões, são descritas várias imagens, das quais a mais relatada é a de padrão intersticial nodular miliar generalizado, embora não seja imagem patognomônica. Este trabalho teve como objetivo verificar a ocorrência de aspergilose pulmonar em cães com sintomatologia respiratória atendidos no Hospital de Clínicas Veterinárias da Universidade Federal do Rio Grande do Sul (HCV-UFRGS), localizado na cidade de Porto Alegre. A amostragem foi composta por 46 cães nos quais foi realizado Biópsia por Aspiração com Agulha Fina (BAAF) nos dois pulmões, coleta de sangue e exame radiológico torácico. O material obtido de uma punção foi homogeneizado com caldo Sabouraud Dextrose líquido. Parte deste homogeneizado foi semeado em Agar Malte para Aspergillus spp. e feito exame direto em lâmina e com colotração de Grocott. A outra parte foi semeada em vários meios de cultura para exame bacteriológico. Do material obtido na outra punção foi realizado exame citopatológico corado pelo método Panótico Rápido. Do soro obtido do sangue coletado foi realizado exame soromicológico para A. fumigatus, A. flavus e A. niger. Os resultados dos exames micológico, citopatológico, soromicológico e bacteriológico foram negativos para todas as amostras testadas. Não havendo positividade dos exames, não foi possível correlacionar a pneumonia fúngica com as imagens radiológicas obtidas dos pulmões dos 46 cães radiografados. A maioria dos cães viviam em áreas abertas, em contato com matéria orgânica onde o fungo frequentemente se encontra. Os exames para detecção de fungos devem ser analisados com cautela, pois o resultado positivo em um único exame não confirma a presença da infecção, pois o agente pode estar presente frequentemente no trato respiratório superior de cães. / Fungal pneumonia is a deeply lung infection caused by fungal agents of the genus Aspergillus, including A. fumigatus, A. niger, A. flavus and A. terreus. They are found most commonly in decaying organic matter. Their fungal propagules are present in dust and air, which favors the inhalation, the main entrance of the agent in the body. They produce large amounts of conidia with less than 8 μ, so that, when inhaled, reaching the pulmonary bed. It is the considered the most common kind genus on our planet and A. fumigatus is the most often reported species in tissue invasion. In animals and humans, a healthy immune and respiratory systems is sufficient to remove the seedlings of the airways, preventing colonization. Diseases and immunosuppressive drugs have been described as predisposing factors of pulmonary fungal infection, considered opportunistic. It is considered rare in dogs. Clinical symptoms are varied, but a persistent cough and no cessation of symptoms with the use of antimicrobials are data for suspected fungal pneumonia. In the radiological examination of the lungs, several images have been described, and the most described is a generalized miliary nodular interstitial pattern, but it is not a pathognomonic image. This work aimed to verify the occurrence of pulmonary aspergillosis in dogs with respiratory symptoms examined at the Veterinary Clinical Hospital of Federal University of Rio Grande do Sul (HCVUFRGS), located in Porto Alegre city, in Brasil. The sampling was composed of 46 dogs in which biopsy was performed by fine needle aspiration (FNAC) in both lungs, blood sampling and radiological examination of the chest. The material obtained from a puncture was homogenized with Sabouraud dextrose liquid. Part of this homogenate was seeded on malt agar for Aspergillus spp. and taken directly on slides and stained with “Grocott”. The other party was seeded in various culture media to bacteriological examination. The material obtained in another puncture was performed cytopathological examination with “Panótico” coloring. Serum obtained from blood collected was performed for serology to A. fumigatus, A. flavus and A. niger. The results of mycological examination, cytology, bacteriology and serology examinations were negative for all samples tested. In the absence of positive tests, it was not possible to correlate fungal pneumonia with radiological images obtained from the lungs of the 46 dogs radiographed. Most dogs live in open areas in contact with organic matter where the fungus is usually found. Tests to detect fungi should be analyzed with caution, because a positive result in a single test does not confirm the presence of infection, since the agent can be present normally in the upper respiratory tract of dogs.