Novel clinical and laboratorial challenges in Aspergillosis

© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).In recent years, research in the areas of Aspergillus and aspergillosis has continued to advance rapidly, including advancements in genomics, immunological studies, clinical areas, and diagnostic areas. Recently, new risk groups for the development of aspergillosis have emerged—patients with influenza- or COVID-19-ssociated pulmonary aspergillosis. The rise and spread of antifungal resistances have also become a clinical concern in some geographic areas and have drawn the attention of clinicians due to difficulties in treating these infections. In this paper, a snapshot of these issues is presented, emphasizing these novel clinical and laboratorial challenges in the aspergillosis field and focusing on their actual relevance.info:eu-repo/semantics/publishedVersio

Culture media and sampling collection method for Aspergillus spp. assessment : tackling the gap between recommendations and the scientific evidence

Copyright: © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license.Culturing is still the most widely used method for determining fungal growth. Thus, is important to identify the most suitable culture media to assess Aspergillus spp. The aim of this study was to analyze data obtained from previous studies, aiming at identifying the most suitable culture media (malt extract agar (MEA) or dichloran-glycerol agar (DG18) to assess Aspergillus spp. isolation and growth. This study was conducted by using environmental samples (n = 1153). Most of the active sampling methods (air samples) were impacted directly onto both culture media. As for passive sampling methods, fungi were extracted from environmental matrices inoculated onto both media. Overall, total Aspergillus counts were higher in MEA (n = 617, 53.5%) than in DG18 (n = 536, 46.5%). Regarding Aspergillus sections, significant associations were detected with the media (χ2 (7) = 241.118, p < 0.001), the sampling approach (p < 0.001, 95% CI = (0.3 × 10−4), and the indoor environment (p < 0.001, 95% CI = (0.3 × 10−4)). As such, sampling approach and the culture media should be accurately selected when dealing with Aspergillus spp. exposure assessment.This work was supported by FCT—Fundação para Ciência e Tecnologia for funding the project EXPOsE—Establishing protocols to assess occupational exposure to microbiota in clinical settings (02/SAICT/2016—Project n° 23222) and Instituto Politécnico de Lisboa, Lisbon, Portugal for funding the Project “Waste Workers’ Exposure to Bio-burden through Filtering Respiratory Protective Devices” (IPL/2018/WasteFRPD_ESTeSL) and the Project “Occupational exposure of ambulance drivers to bioburden” (IPL/2020/BIO-AmbuDrivers_ESTeSL). H&TRC authors gratefully acknowledge the FCT/MCTES national support through the UIDB/05608/2020 and UIDP/05608/2020.info:eu-repo/semantics/publishedVersio

Candida auris, an agent of hospital-associated outbreaks : which challenging issues do we need to have in mind?

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).The emergence of Candida auris is considered as one of the most serious problems associated with nosocomial transmission and with infection control practices in hospital environment. This multidrug resistant species is rapidly spreading worldwide, with several described outbreaks. Until now, this species has been isolated from different hospital surfaces, where it can survive for long periods. There are multiple unanswered questions regarding C. auris, such as prevalence in population, environmental contamination, effectiveness of infection prevention and control, and impact on patient mortality. In order to understand how it spreads and discover possible reservoirs, it is essential to know the ecology, natural environment, and distribution of this species. It is also important to explore possible reasons to this recent emergence, namely the environmental presence of azoles or the possible effect of climate change on this sudden emergence. This review aims to discuss some of the most challenging issues that we need to have in mind in the management of C. auris and to raise the awareness to its presence in specific indoor environments as hospital settings.info:eu-repo/semantics/publishedVersio

Candida auris : the recent emergence of a multiresistant pathogenic fungi

Copyright © Ordem dos Médicos 2020Candida auris was first described as a new species back in 2009. Although it differs markedly from other Candida species, this species can be misidentified as other yeasts in the routine microbiology laboratories. Therefore, its identification to species level should be confirmed by reference laboratories. Candida auris exhibits potential to cause invasive infections, and frequently shows a multidrugresistance pattern, and it is associated with high mortality rates. Outbreaks caused by Candida auris and associated with health care institutions have been reported in several countries around the world, including some European countries, such as the United Kingdom and Spain. In Portugal, to our knowledge, there are no known infections or colonization cases caused by Candida auris. This species can survive in the environment for several weeks and once introduced into the hospital environment, the risk of transmission is high, requiring strict infection control measures in order to prevent transmission. This paper intends to raise the awareness of the emergence of this fungal species, as well as to discuss the consequences of this situation.info:eu-repo/semantics/publishedVersio

Aspergillosis, avian species and the one health perspective : the possible importance of birds in azole resistance

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).The One Health context considers health based on three pillars: humans, animals, and environment. This approach is a strong ally in the surveillance of infectious diseases and in the development of prevention strategies. Aspergillus spp. are fungi that fit substantially in this context, in view of their ubiquity, as well as their importance as plant pathogens, and potentially fatal pathogens for, particularly, humans and avian species. In addition, the emergence of azole resistance, mainly in Aspergillus fumigatus sensu stricto, and the proven role of fungicides widely used on crops, reinforces the need for a multidisciplinary approach to this problem. Avian species are involved in short and long distance travel between different types of landscapes, such as agricultural fields, natural environments and urban environments. Thus, birds can play an important role in the dispersion of Aspergillus, and of special concern, azole-resistant strains. In addition, some bird species are particularly susceptible to aspergillosis. Therefore, avian aspergillosis could be considered as an environmental health indicator. In this review, aspergillosis in humans and birds will be discussed, with focus on the presence of Aspergillus in the environment. We will relate these issues with the emergence of azole resistance on Aspergillus. These topics will be therefore considered and reviewed from the “One Health” perspective.This study was supported by the International Cooperation Program financed by Brazilian Federal Agency for Support and Evaluation of Graduate Education within the Ministry of Education of Brazil (CAPES), under the Capes-Print Program and PDSE Program—Finance Code 001info:eu-repo/semantics/publishedVersio

Azole-resistant Aspergillus fumigatus harboring the TR34/L98H mutation : first report in Portugal in environmental samples

Copyright: © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).The frequency in detection of azole-resistant Aspergillus fumigatus isolates has increased since 2010. In Portugal, the section Fumigati is one of the most frequent, and resistant strains to have been found in clinical and environmental contexts. Although several cryptic species within the Fumigati section show intrinsic resistance to azoles, one factor driving (acquired) resistance is selective pressure deriving from the extensive use of azoles. This is particularly problematic in occupational environments where high fungal loads are expected, and where there is an increased risk of human exposure and infection, with impact on treatment success and disease outcome. The mechanisms of resistance are diverse, but mainly associated with mutations in the cyp51A gene. Despite TR34/L98H being the most frequent mutation described, it has only been detected in clinical specimens in Portugal. Methods: We analyzed 99 A. fumigatus isolates from indoor environments (healthcare facilities, spas, one dairy and one waste sorting unit) collected from January 2018 to February 2019 in different regions of Portugal. Isolates were screened for resistance to itraconazole, voriconazole and posaconazole by culture, and resistance was confirmed by broth microdilution. Sequencing of the cyp51A gene and its promoter was performed to detect mutations associated with resistance. Results: Overall, 8.1% of isolates were able to grow in the presence of at least one azole, and 3% (isolated from the air in a dairy and from filtering respiratory protective devices in a waste sorting industry) were pan-azole-resistant, bearing the TR34/L98H mutation. Conclusion: For the first time in Portugal, we report environmental isolates bearing the TR34/L98H mutation, isolated from occupational environments. Environmental surveillance of the emergence of azole-resistant A. fumigatus sensu stricto strains is needed, to ensure proper and timely implementation of control policies that may have a positive impact on public and occupational health.H&TRC authors gratefully acknowledge the FCT/MCTES national supportthrough the UIDB/05608/2020 and UIDP/05608/2020info:eu-repo/semantics/publishedVersio

Molecular epidemiology studies of candidiasis in oncological patients and development of new polymorphic microsatellite markers to distinguish Candida parapsilosis strains and investigate genetic diversity

Tese de doutoramento em Ciências (área de conhecimento de Biologia)As infecções fúngicas constituem um problema relevante em hospitais de todo o mundo. A fácil transmissão dos fungos através do ar ou pelo contacto directo ou indirecto com pessoas, superfícies ou objectos contaminados conduz a uma crescente preocupação principalmente em pacientes imunodeprimidos. Os doentes oncológicos são um dos grupos mais sujeitos a infecções fúngicas, nomeadamente causadas por leveduras do género Candida. Os dados epidemiológicos disponíveis em Portugal sobre candidemia são escassos e, como tal, o objectivo geral deste trabalho foi estudar o status epidemiológico do nosso país no que respeita às colonizações, infecções por Candida e candidemia em pacientes oncológicos. Além disso, considerando a grande relevância de C. parapsilosis como agente etiológico de infecções sistémicas e de surtos hospitalares, o objectivo específico deste trabalho foi descrever e caracterizar novos marcadores moleculares baseados em regiões de DNA microssatélite que permitissem distinguir estirpes de C. parapsilosis e investigar a sua diversidade genética. Na primeira parte deste trabalho, efectuou-se um estudo sobre a incidência de candidemia num hospital oncológico Português. Verificou-se ser C. albicans a levedura mais frequentemente isolada, seguida de C. parapsilosis, C. tropicalis, C. krusei e C. glabrata. Estirpes de Saccharomyces cerevisiae e Rhodotorula mucilaginosa foram também isoladas. Candida albicans foi isolada mais frequentemente em pacientes com tumores sólidos, nomeadamente da mama e dos tractos gastrointestinal e genitourinário (p=0.005), enquanto outras espécies de Candida são mais frequentemente isoladas de pacientes hematológicos (p=0.007). A taxa de mortalidade associada a candidemia foi de 31.9% (p=0.016). Todos os isolados de C. albicans e C. parapsilosis foram susceptíveis ao fluconazol, voriconazol e itraconazol. Resistência à caspofungina foi apenas observada em isolados de C. albicans e R. mucilaginosa. Todas as estirpes de C. parapsilosis mostraram-se susceptíveis ao posaconazol mas foram detectadas estirpes de C. albicans, C. tropicalis, C. krusei e C. glabrata resistentes a este antifúngico. Este estudo facultou informações úteis e importantes sobre a epidemiologia local de candidemia em pacientes oncológicos. Com o intuito de avaliar o risco de infecção endógena nestes pacientes, os resultados obtidos em culturas de rotina de 345 pacientes deste mesmo hospital foram analisados. A expectoração e as secreções brônquicas foram os produtos mais contaminados, com 28.9% do total de leveduras isoladas. A Gastrenterologia foi a unidade hospitalar donde provieram maior número de isolados, seguida da Hematologia. Também neste estudo C. albicans foi a espécie mais frequentemente isolada, sendo a incidência de C. parapsilosis bastante menor do que nos casos de candidemia. Não foi encontrada associação positiva entre as espécies de leveduras e o produto biológico analisado ou entre as espécies de leveduras e a unidade hospitalar. Como o ambiente hospitalar tem um papel crucial na epidemiologia das infecções fúngicas nosocomiais, a carga fúngica ambiental da Unidade de Hematologia foi avaliada com o intuito de determinar a diversidade fúngica e o seu potencial risco de infecção. No total, e durante quatro diferentes períodos de colheita, foram analisadas 304 superfícies, bem como as mãos do pessoal hospitalar dessa mesma unidade. As espécies mais frequentemente isoladas das superfícies foram Aspergillus spp., Fusarium spp., Penicillium spp., Chrysonilia sitophila, Candida spp e Rhodotorula spp., enquanto Aspergillus spp. e Rhizopus arrhizus foram isoladas com mais frequência do ar. No que respeita aos resultados obtidos nas colheitas das mãos do pessoal hospitalar, foram isoladas espécies relacionadas com infecções da pele e unhas como Trichophyton spp., Fusarium spp., Scytalidium dimidiatum e Chrysosporium spp., tendo C. parapsilosis sido a levedura mais frequentemente encontrada. A amostragem efectuada antes e depois da lavagem das mãos mostrou que uma desinfecção adequada era 100% eficaz na remoção de microrganismos. Quatro novos loci polimórficos de DNA microssatélite úteis na distinção de isolados de C. parapsilosis foram identificados e usados na distinção de 233 isolados independentes de C. parapsilosis sensu stricto. Em análise multiplex, obtiveram-se 192 genótipos multilocus diferentes, tendo-se determinado um poder discriminatório combinado de 0.99, o mais alto descrito até hoje. Realizou-se a comparação dos genótipos obtidos por genotipagem microssatélite com os obtidos por RAPD, RFLP e ITS e verificou-se que a primeira conseguia distinguir todos isolados, enquanto os outros métodos não. Utilizando o método de genotipagem desenvolvido, observou-se que em casos de isolados múltiplos do mesmo paciente o genótipo multilocus de estirpes colonizadoras e causadoras de infecção é, de forma geral, mantido. O mesmo genótipo multilocus ou muito similar foi também encontrado em estirpes isoladas de superfícies hospitalares, mãos do pessoal hospitalar e pacientes internados. O alto grau de similitude genética entre estes isolados indica uma possível via de transmissão da estirpe através das mãos do pessoal hospitalar e de superfícies contaminadas, como maçanetas e carros de tratamento. Demonstrou-se assim que este método pode ser uma ferramenta valiosa em investigações epidemiológicas. Isolados clínicos e ambientais e de diferentes genótipos multilocus foram seleccionados e testados no que respeita à sua capacidade de produzir pseudo-hifas, resistir à fagocitose e morte intracelular, e capacidade de causar dano em macrófagos, medida pela libertação de lactato desidrogenase. A produção de TNF-α pelos macrófagos após incubação com as estirpes seleccionadas foi também quantificada. Observaram-se diferenças significativas entre isolados ambientais e clínicos, causando os primeiros, maior dano nos macrófagos e uma menor produção de TNF-α. Isolados das espécies geneticamente relacionadas C. orthopsilosis e C. metapsilosis mostraram ser menos citotóxicas para os macrófagos. Contrariamente ao descrito para C. albicans, não se demonstrou associação entre formação de pseudo-hifas e dano celular. O controlo das infecções nosocomiais por Candida requer um conhecimento aprofundado quer da estirpe infectante quer do estado imunológico do paciente. Este conhecimento, para o qual este trabalho visa contribuir, abre a possibilidade de pesquisas futuras neste campo.Fungal infections constitute a relevant problem in hospitals from all over the world. Their easy transmission through the air or by direct or indirect contact with contaminated persons, surfaces or objects lead to an increasing preoccupation with those infections, particularly in immunocompromised patients. Amongst them, cancer patients are one of the risk groups in acquiring fungal infections, namely caused by yeasts belonging to genus Candida. Epidemiological data on candidemia in our country is scarce therefore the overall objectives of this work were to study the epidemiological status of Candida infection such as colonization, cross-transmission, and candidemia in Portuguese cancer patients. Furthermore, and regarding the enormous importance of C. parapsilosis as agent of bloodstream infections and hospital outbreaks a specific objective was to describe and characterize new polymorphic microsatellite markers able to distinguish among C. parapsilosis strains and to investigate genetic diversity. In the first part of this work incidence of candidemia in a Portuguese oncology hospital during a 6-year period was evaluated. The most frequent species found was C. albicans, followed by C. parapsilosis, C. tropicalis, C. krusei and C. glabrata, but Saccharomyces cerevisiae and Rhodotorula mucilaginosa were also isolated. Candida albicans was more frequently associated with solid tumors of the gastrointestinal and genitourinary tracts and breast (p=0.005), while other species were most frequently recovered from hematological patients (p=0.007). The mortality rate associated with candidemia was 31.9% (p=0.016). All C. albicans and C. parapsilosis isolates were susceptible to fluconazole, voriconazole and itraconazole. Resistance to caspofungin was only observed in C. albicans and in the R. mucilaginosa isolates. Posaconazole was active against all C. parapsilosis isolates tested but resistant strains were found among C. albicans, C. tropicalis, C. krusei, and C. glabrata. This study provided useful information about the local epidemiology of candidemia in cancer patients, and to evaluate the risk of endogenous infections the results of surveillance cultures from 345 patients of the same hospital were analyzed. Sputum and bronchial secretions were the most contaminated, with 28.9% of the total yeast isolates. Gastroenterology was the hospital ward with more isolates obtained, followed by the Hematology Unit. Again, the most frequently isolated species was C. albicans, but the incidence of C. parapsilosis was much lower than it was found for the candidemia episodes. No positive association was seen between yeast species and biological products or between yeast species and hospital ward. As hospital environment plays a crucial role in the epidemiology of nosocomial fungal infections the environmental fungal burden of the Hematology unit of the Hospital was evaluated in order to determine fungal diversity and potential risk for infection. In total, 304 surfaces were analyzed, in four different collection periods, as well as hands of healthcare workers of the same unit. The most frequent species isolated from surfaces were Aspergillus spp., Fusarium spp., Penicillium spp., Chrysonilia sitophila, Candida spp and Rhodotorula spp while from the air, Aspergillus spp. and Rhizopus arrhizus were the most frequently found. In what respects the hands of heath care workers, species related with skin and nail infections like Trichophyton spp., Fusarium spp., Scytalidium dimidiatum and Chrysosporium spp were isolated, among others, and C. parapsilosis was also frequently isolated. Sampling before and after hand washing showed that hand hygiene compliance was 100% effective. Four new polymorphic microsatellite loci able to distinguish among C. parapsilosis isolates were identified and used to genotype 233 independent C. parapsilosis sensu stricto isolates. In a multiplex analysis, 192 genotypes were obtained and the combined discriminatory power of the four microsatellites was 0.99, the highest reported. Comparison of the genotypes obtained by microsatellite analysis and those obtained by RAPD, RFLP and ITS grouping was performed and showed that the microsatellite method could distinguish individual isolates while none of the other methods could. Using the developed microsatellite genotyping methodology it was observed that in cases of multiple isolates from the same patient the multilocus genotype of colonizing and bloodstream isolates was mainly maintained. The same or very similar multilocus genotypes were also found among isolates from hospital surfaces, from the hands of health care workers and from patients. High degree of genetic similarity was found among isolates collected from the hospital environment, hands of healthcare workers and from the patients, indicating that the possible route of transmission of C. parapsilosis is through the hands of healthcare workers and the contaminated medical trolleys and doors knobs. We demonstrate that this typing method is able to distinguish clonal clusters from genetically unrelated genotypes and can be a valuable tool to support epidemiological investigations. Selected C. parapsilosis isolates from clinical and environmental origins and different microsatellite multilocus genotypes were studied regarding their ability to produce pseudohyphae, to resist to phagocytosis and intracellular killing and to cause macrophage damage, measured by lactate dehydrogenase release. Quantification of TNFα production by macrophages upon incubation with the isolates was also performed. Significant differences amongst environmental and clinical isolates were observed, the first being able to cause higher macrophage damage and less induction of TNFα production. Isolates of the genetically related species C. orthopsilosis and C. metapsilosis were found to be less cytotoxic to host cells. Contrarily to what is described for C. albicans, association between pseudohyphae production and host cellular damage was not observed. Control of nosocomial infections due to Candida species requires both fundamental knowledge about the infecting yeast and of the characteristics and immunological status of the patient. This knowledge, to which the present work aimed to contribute, opens up the possibility for future research in this field.Fundação para a Ciência e a Tecnologia (FCT) - Bolsa de Doutoramento SFRH/BD/22100/2005

Environmental and bioclimatic factors influencing yeasts and molds distribution along European shores

© 2022 Elsevier B.V. All rights reserved.The present study employed data collected during the Mycosands survey to investigate the environmental factors influencing yeasts and molds distribution along European shores applying a species distribution modelling approach. Occurrence data were compared to climatic datasets (temperature, precipitation, and solar radiation), soil datasets (chemical and physical properties), and water datasets (temperature, salinity, and chlorophyll-a concentration) downloaded from web databases. Analyses were performed by MaxEnt software. Results suggested a different probability of distribution of yeasts and molds along European shores. Yeasts seem to tolerate low temperatures better during winter than molds and this reflects a higher suitability for the Northern European coasts. This difference is more evident considering suitability in waters. Both distributions of molds and yeasts are influenced by basic soil pH, probably because acidic soils are more favorable to bacterial growth. Soils with high nitrogen concentrations are not suitable for fungal growth, which, in contrast, are optimal for plant growth, favored by this environment. Finally, molds show affinity with soil rich in nickel and yeasts with soils rich in cadmium resulting in a distribution mainly at the mouths of European rivers or lagoons, where these metals accumulate in river sediments.Ana Sampaio thanks to the Foundation for Science and Technology and FEDER under Programe PT2020 for financial support to CITAB (UID/AGR/04033/2020)info:eu-repo/semantics/publishedVersio