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Epidemiologia e frequência de Aspergillus em doentes com suspeita de patologia respiratória fúngica
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Advisor(s)
Abstract(s)
O género Aspergillus é composto por centenas de espécies de fungos ubíquos que são agentes
patogénicos oportunistas do ser humano, particularmente dos indivíduos imunocomprometidos e/ou
com danos estruturais nos pulmões resultado de patologias prévias como tuberculose pulmonar.
Aspergilose é o nome dado ao espetro de patologias causadas por Aspergillus e que se estendem
desde a reação hipersensível do hospedeiro aos fungos até à infeção invasiva cuja taxa de letalidade
é elevada. O tratamento destas patologias depende essencialmente dos antifúngicos da classe dos
azóis, contudo a emergência de resistência por A. fumigatus sensu stricto assim como a resistência
intrínseca das espécies crípticas pode comprometer esta terapêutica. O diagnóstico de aspergilose
está dependente da deteção laboratorial direta e/ou indireta de Aspergillus nas amostras biológicas
dos pacientes através das técnicas culturais e imunoenzimáticas, mas a sensibilidade e a
especificidade destas técnicas varia amplamente. Posto isto, este trabalho foi realizado com os
seguintes objetivos: (i) a vigilância de Aspergillus em pacientes com sintomas de patologia
respiratória fúngica, nomeadamente em grupos de risco de aspergilose pulmonar crónica (VIH
positivo e tuberculose pulmonar ativa ou prévia) e (ii) a otimização da deteção molecular de
Aspergillus e/ou de outros fungos potencialmente patogénicos através de PCR multiplex em tempo
real com o kit AsperGenius® e de técnicas de next generation sequencing, respetivamente.
No âmbito do estudo da epidemiologia de aspergilose em doentes com suspeita de infeção
fúngica do trato respiratório foram analisadas retrospetivamente as amostras respiratórias de 146
pacientes que se encontravam conservadas no Laboratório Nacional de Referência de Infeções
Parasitárias e Fúngicas. Cinquenta e sete destes pacientes (39,0%) foram positivos para Aspergillus
através das técnicas cultural, imunoenzimática e molecular. Cinquenta e oito colónias de Aspergillus
foram isoladas e identificadas através de sequenciação dos genes para a calmodulina ou a β-tubulina
e por PCR em tempo real. Detetaram-se 6 secções de Aspergillus distintas, sendo a mais frequente a
Nigri (42,1%) seguida de Fumigati (33,3%) e de Flavi (8,6%). Quanto às espécies, as mais
frequentemente identificadas foram A. niger sensu stricto (33,9%) seguida de A. fumigatus sensu
stricto (32,1%). Identificaram-se também 9 espécies crípticas cuja frequência foi 21,4%.
Paralelamente ao estudo epidemiológico indicado, analisou-se o perfil de suscetibilidade aos azóis
de Aspergillus da secção Fumigati que haviam sido isolados de produtos respiratórios e conservados
em coleção de culturas no âmbito do programa de Vigilância em Aspergillus. Os isolados
identificados como Aspergillus fumigatus sensu stricto (n=45), A. lentulus (n=4), A. udagawae (n=2)
e A. pseudofelis (n=1) foram inoculados em meios de screening com voriconazol, itraconazol e
posaconazol e o seu crescimento foi caracterizado. Os resultados foram corroborados pelas técnicas
de E-test e através de PCR multiplex em tempo real para deteção de mutações no gene Cyp51A.
Nenhum isolado de A. fumigatus sensu stricto revelou resistência aos azóis, mas foi detetada menor
suscetibilidade ao voriconazol num isolado da espécie A. udagawae e ao voriconazol (CMI = 1
μg/ml) e ao itraconazol (CMI = 2 μg/ml) em A. pseudofelis.
Com o objetivo de comparar os resultados obtidos por técnicas convencionais com os das
técnicas moleculares, o DNA extraído do sobrenadante de 44 amostras respiratórias que haviam sido
previamente analisadas através de ELISA para deteção de galactomanano e/ou pela técnica cultural
foi sujeito ao PCR multiplex em tempo real com o kit AsperGenius® e compararam-se os resultados
obtidos pelas várias técnicas. Verificou-se que o PCR multiplex em tempo real permitiu obter uma
taxa de positividade superior à das técnicas convencionais de diagnóstico de aspergilose,
particularmente nos pacientes em risco de desenvolver aspergilose pulmonar crónica (VIH positivo
e tuberculose pulmonar ativa ou prévia). Procedeu-se também à otimização da amplificação e
vii
sequenciação por técnicas de next generation sequencing da região ITS e do gene calmodulina do
DNA extraído de amostras respiratórias. Este estudo piloto de análise metagenómica permitiu detetar
os fungos Pneumocystis e Cryptococcus mo micobioma dos pacientes VIH positivo que
frequentemente desenvolvem pneumocistose e criptococose. Detetaram-se também outros fungos
potencialmente patogénicos nomeadamente dos géneros Aspergillus, Trichosporon, Saccharomyces
e Schizophyllum.
Conclui-se assim que a distribuição de Aspergillus é complexa e diversa e que a resistência aos
azóis não aparenta ser um problema nas estirpes analisadas, contudo a monitorização contínua é
fundamental. Essa monitorização deve incluir não só as técnicas de determinação do perfil de
suscetibilidade in vitro aos antifúngicos como a identificação molecular das espécies,
particularmente devido à elevada frequência com que as espécies crípticas foram detetadas neste
trabalho assim como a resistência intrínseca aos antifúngicos que algumas delas apresentaram.
Conclui-se também que as técnicas moleculares têm uma grande capacidade de detetar rápida e
eficazmente fungos potencialmente patogénicos nas amostras biológicas humanas pelo que esforços
para a sua otimização, padronização e validação clínica devem ser realizados no futuro.
The genus Aspergillus comprises hundreds of fungal species which are ubiquitous and opportunistic pathogens of immunocompromised patients and/or individuals whose lungs are structurally damaged due to previous pathologies such as pulmonary tuberculosis. Aspergillosis is the name given to the spectrum of diseases caused by Aspergillus which ranges from a hypersensitive reaction to the fungi by the hosts’ organism to a highly deadly invasive infection. The treatment of aspergillosis depends on azoles, however the emergence of acquired resistance in Aspergillus fumigatus sensu stricto as well as the intrinsic resistance of the cryptic species may compromise the use of this therapy. The diagnosis of aspergillosis depends on the direct and/or indirect detection of Aspergillus in biological samples trough culture or immunoenzimatic techniques, but the sensitivity and the specificity of these methodologies varies widely. Thus, this work had the following objectives: (i) the surveillance of Aspergillus in patients with symptoms of a potential fungal infection of the respiratory tract, namely those in higher risk of developing chronic pulmonary aspergillosis (HIV positive and previous or active pulmonary tuberculosis) and (ii) the optimization of the detection of Aspergillus and/or other potentially pathogenic fungi trough real-time multiplex PCR with the AsperGenius® commercial kit and with next generation sequencing techniques, respectively. In order to determine the epidemiology of Aspergillus in patients with suspicion of fungal infection of the respiratory tract, the respiratory samples of 146 patients which were conserved in the National Reference Laboratory of Parasitic and Fungal Infections were retrospectively analysed. Fifty-seven of these patients (39.0%) were positive for Aspergillus by culture, immunoenzimatic and molecular techniques. Fifty-eight colonies of Aspergillus were isolated and identified by sequencing of the calmodulin and β-tubulin genes. Six Aspergillus sections were detected and the most frequent were the Nigri section (42.1%) followed by the Fumigati (33.3%) and the Flavi sections (8.6%). Regarding the species, the most identified were A. niger sensu stricto (33.9%) followed by A. fumigatus sensu stricto (32.1%). Nine cryptic species were also identified which frequency was 21.4%. In parallel to the referred epidemiological study, we described the susceptibility profile of Aspergillus of the Fumigati section which had been isolated from respiratory samples and conserved in a culture collection in the scope of the program on Aspergillus surveillance. The isolates identified as Aspergillus fumigatus sensu stricto (n=45), A. lentulus (n=4), A. udagawae (n=2) and A. pseudofelis (n=1) were inoculated into screening media supplemented with voriconazole, itraconazole and posaconazole and their growth was characterized. The results were corroborated by E-test and by real-time multiplex PCR for the detection of mutations in the Cyp51A gene. None of the A. fumigatus sensu stricto isolates tested were resistant to azoles, but a decrease in susceptibility to voriconazole was shown in an A. udagawae strain and to voriconazole (MIC =1 μg/ml) and itraconazole (MIC = 2 μg/ml) in an A. pseudofelis strain. In order to compare the results obtained by the conventional and by the molecular techniques, the DNA extracted from the supernatant of 44 respiratory samples that had been previously analysed by ELISA for the detection of galactomannan and/or by cultural techniques was amplified by realtime multiplex PCR with the AsperGenius® kit and the results obtained by these different methodologies were compared. The real-time PCR was found to have a higher positivity rate than the conventional techniques used for the diagnosis of aspergillosis, particularly in the group of patients who have a higher risk of developing chronic pulmonary aspergillosis (HIV positive and active or previous pulmonary tuberculosis). We also optimized the amplification and sequencing by next generation techniques of the ITS region and the calmodulin gene of the DNA extracted from respiratory samples. This pilot study of metagenomic analysis allowed the detection of Pneumocystis and Cryptococcus in the mycobiome of HIV positive patients who frequently develop pneumocystosis and cryptococcosis. Other potentially pathogenic fungi such as Aspergillus, Trichosporon, Saccharomyces and Schizophyllum were also detected. In conclusion, the distribution of Aspergillus is complex and diverse and resistance to azoles is not a problem found in the tested isolates, but a continuous monitorization is essential. Monitoring should include not only the determination of the in vitro susceptibility profile but also the molecular identification of the species, particularly due to the high frequency in which cryptic species were detected in this work as well as the intrinsic resistance to antifungal drugs that characterizes some of them. We also conclude that molecular techniques have a great ability to detect potentially pathogenic fungi in human biological samples rapidly and efficiently. Efforts towards the optimization, standardization and clinical validation should be carried out in the future.
The genus Aspergillus comprises hundreds of fungal species which are ubiquitous and opportunistic pathogens of immunocompromised patients and/or individuals whose lungs are structurally damaged due to previous pathologies such as pulmonary tuberculosis. Aspergillosis is the name given to the spectrum of diseases caused by Aspergillus which ranges from a hypersensitive reaction to the fungi by the hosts’ organism to a highly deadly invasive infection. The treatment of aspergillosis depends on azoles, however the emergence of acquired resistance in Aspergillus fumigatus sensu stricto as well as the intrinsic resistance of the cryptic species may compromise the use of this therapy. The diagnosis of aspergillosis depends on the direct and/or indirect detection of Aspergillus in biological samples trough culture or immunoenzimatic techniques, but the sensitivity and the specificity of these methodologies varies widely. Thus, this work had the following objectives: (i) the surveillance of Aspergillus in patients with symptoms of a potential fungal infection of the respiratory tract, namely those in higher risk of developing chronic pulmonary aspergillosis (HIV positive and previous or active pulmonary tuberculosis) and (ii) the optimization of the detection of Aspergillus and/or other potentially pathogenic fungi trough real-time multiplex PCR with the AsperGenius® commercial kit and with next generation sequencing techniques, respectively. In order to determine the epidemiology of Aspergillus in patients with suspicion of fungal infection of the respiratory tract, the respiratory samples of 146 patients which were conserved in the National Reference Laboratory of Parasitic and Fungal Infections were retrospectively analysed. Fifty-seven of these patients (39.0%) were positive for Aspergillus by culture, immunoenzimatic and molecular techniques. Fifty-eight colonies of Aspergillus were isolated and identified by sequencing of the calmodulin and β-tubulin genes. Six Aspergillus sections were detected and the most frequent were the Nigri section (42.1%) followed by the Fumigati (33.3%) and the Flavi sections (8.6%). Regarding the species, the most identified were A. niger sensu stricto (33.9%) followed by A. fumigatus sensu stricto (32.1%). Nine cryptic species were also identified which frequency was 21.4%. In parallel to the referred epidemiological study, we described the susceptibility profile of Aspergillus of the Fumigati section which had been isolated from respiratory samples and conserved in a culture collection in the scope of the program on Aspergillus surveillance. The isolates identified as Aspergillus fumigatus sensu stricto (n=45), A. lentulus (n=4), A. udagawae (n=2) and A. pseudofelis (n=1) were inoculated into screening media supplemented with voriconazole, itraconazole and posaconazole and their growth was characterized. The results were corroborated by E-test and by real-time multiplex PCR for the detection of mutations in the Cyp51A gene. None of the A. fumigatus sensu stricto isolates tested were resistant to azoles, but a decrease in susceptibility to voriconazole was shown in an A. udagawae strain and to voriconazole (MIC =1 μg/ml) and itraconazole (MIC = 2 μg/ml) in an A. pseudofelis strain. In order to compare the results obtained by the conventional and by the molecular techniques, the DNA extracted from the supernatant of 44 respiratory samples that had been previously analysed by ELISA for the detection of galactomannan and/or by cultural techniques was amplified by realtime multiplex PCR with the AsperGenius® kit and the results obtained by these different methodologies were compared. The real-time PCR was found to have a higher positivity rate than the conventional techniques used for the diagnosis of aspergillosis, particularly in the group of patients who have a higher risk of developing chronic pulmonary aspergillosis (HIV positive and active or previous pulmonary tuberculosis). We also optimized the amplification and sequencing by next generation techniques of the ITS region and the calmodulin gene of the DNA extracted from respiratory samples. This pilot study of metagenomic analysis allowed the detection of Pneumocystis and Cryptococcus in the mycobiome of HIV positive patients who frequently develop pneumocystosis and cryptococcosis. Other potentially pathogenic fungi such as Aspergillus, Trichosporon, Saccharomyces and Schizophyllum were also detected. In conclusion, the distribution of Aspergillus is complex and diverse and resistance to azoles is not a problem found in the tested isolates, but a continuous monitorization is essential. Monitoring should include not only the determination of the in vitro susceptibility profile but also the molecular identification of the species, particularly due to the high frequency in which cryptic species were detected in this work as well as the intrinsic resistance to antifungal drugs that characterizes some of them. We also conclude that molecular techniques have a great ability to detect potentially pathogenic fungi in human biological samples rapidly and efficiently. Efforts towards the optimization, standardization and clinical validation should be carried out in the future.
Description
Trabalho desenvolvido no laboratório de Micologia do Instituto Nacional de Saúde Doutor Ricardo Jorge.
Dissertação de mestrado em Biologia Humana e Ambiente, apresentado à Faculdade de Ciências da Universidade de Lisboa, 2019.
Dissertação de mestrado em Biologia Humana e Ambiente, apresentado à Faculdade de Ciências da Universidade de Lisboa, 2019.
Keywords
Aspergillus Resistência em Aspergillus Aspergilose Tuberculose Fungos PCR Infeções Sistémicas e Zoonoses Infecções Respiratórias