Genetic diversity and evolution of Pneumocystis fungi infecting wild Southeast Asian murid rodents.

Pneumocystis organisms are airborne-transmitted fungal parasites that infect the lungs of numerous mammalian species with strong host specificity. In this study, we investigated the genetic diversity and host specificity of Pneumocystis organisms infecting Southeast Asian murid rodents through PCR amplification of two mitochondrial genes and tested the co-phylogeny hypothesis among these fungi and their rodent hosts. Pneumocystis DNA was detected in 215 of 445 wild rodents belonging to 18 Southeast Asian murid species. Three of the Pneumocystis lineages retrieved in our phylogenetic trees correspond to known Pneumocystis species, but some of the remaining lineages may correspond to new undescribed species. Most of these Pneumocystis species infect several rodent species or genera and some sequence types are shared among several host species and genera. These results indicated a weaker host specificity of Pneumocystis species infecting rodents than previously thought. Our co-phylogenetic analyses revealed a complex evolutionary history among Pneumocystis and their rodent hosts. Even if a significant global signal of co-speciation has been detected, co-speciation alone is not sufficient to explain the observed co-phylogenetic pattern and several host switches are inferred. These findings conflict with the traditional view of a prolonged process of co-evolution and co-speciation of Pneumocystis and their hosts.

Correction: What Do Pneumocystis Organisms Tell Us about the Phylogeography of Their Hosts? The Case of the Woodmouse Apodemus sylvaticus in Continental Europe and Western Mediterranean Islands.

[This corrects the article DOI: 10.1371/journal.pone.0120839.].

A charter for biomedical research ethics in a progressive, caring society.

BACKGROUND: Given that advances in research continuously raise new ethical issues, a multidisciplinary working group of investigators involved in biomedical research has gathered to discuss and compare ethical viewpoints in their daily practice. METHODS: The working group has drafted a Charter for Ethics in Biomedical Research that encompasses all the steps in the research process, i.e. from the initial idea to analysis and publication of the results. RESULTS: Based on key principles for ethically responsible research, the Charter may serve as a tool for performing research, discussing research issues and training researchers. CONCLUSIONS: The Charter should stimulate researchers to think about their responsibility for research in a progressive, caring society.

Identification of Cryptosporidium Species in Fish from Lake Geneva (Lac Léman) in France.

Cryptosporidium, a protozoan parasite that can cause severe diarrhea in a wide range of vertebrates including humans, is increasingly recognized as a parasite of a diverse range of wildlife species. However, little data are available regarding the identification of Cryptosporidium species and genotypes in wild aquatic environments, and more particularly in edible freshwater fish. To evaluate the prevalence of Cryptosporidiumspp. in fish from Lake Geneva (Lac Léman) in France, 41 entire fish and 100 fillets (cuts of fish flesh) were collected from fishery suppliers around the lake. Nested PCR using degenerate primers followed by sequence analysis was used. Five fish species were identified as potential hosts of Cryptosporidium: Salvelinus alpinus, Esox lucius, Coregonus lavaretus, Perca fluviatilis, and Rutilus rutilus. The presence of Cryptosporidium spp. was found in 15 out of 41 fish (37%), distributed as follows: 13 (87%) C. parvum, 1 (7%) C. molnari, and 1 (7%) mixed infection (C. parvum and C. molnari). C. molnari was identified in the stomach, while C. parvum was found in the stomach and intestine. C. molnari was also detected in 1 out of 100 analyzed fillets. In order to identify Cryptosporidium subtypes, sequencing of the highly polymorphic 60-kDa glycoprotein (gp60) was performed. Among the C. parvum positive samples, three gp60 subtypes were identified: IIaA15G2R1, IIaA16G2R1, and IIaA17G2R1. Histological examination confirmed the presence of potential developmental stages of C. parvum within digestive epithelial cells. These observations suggest that C. parvum is infecting fish, rather than being passively carried. Since C. parvum is a zoonotic species, fish potentially contaminated by the same subtypes found in terrestrial mammals would be an additional source of infection for humans and animals, and may also contribute to the contamination of the environment with this parasite. Moreover, the risk of human transmission is strengthened by the observation of edible fillet contamination.

SYTO-13, a Viability Marker as a New Tool to Monitor In Vitro Pharmacodynamic Parameters of Anti-Pneumocystis Drugs.

While Pneumocystis pneumonia (PcP) still impacts the AIDS patients, it has a growing importance in immunosuppressed HIV-negative patients. To determine the anti-Pneumocystis therapeutic efficacy of new compounds, animal and in vitro models have been developed. Indeed, well-designed mouse or rat experimental models of pneumocystosis can be used to describe the in vivo anti-Pneumocystis activity of new drugs. In vitro models, which enable the screening of a large panel of new molecules, have been developed using axenic cultures or co-culture with feeder cells; but no universally accepted standard method is currently available to evaluate anti-Pneumocystis molecules in vitro. Thus, we chose to explore the use of the SYTO-13 dye, as a new indicator of Pneumocystis viability. In the present work, we established the experimental conditions to define the in vitro pharmacodynamic parameters (EC50, Emax) of marketed compounds (trimethoprim/sulfamethoxazole, pentamidine, atovaquone) in order to specifically measure the intrinsic activity of these anti-P. carinii molecules using the SYTO-13 dye for the first time. Co-labelling the fungal organisms with anti-P. carinii specific antibodies enabled the measurement of viability of Pneumocystis organisms while excluding host debris from the analysis. Moreover, contrary to microscopic observation, large numbers of fungal cells can be analyzed by flow cytometry, thus increasing statistical significance and avoiding misreading during fastidious quantitation of stained organisms. In conclusion, the SYTO-13 dye allowed us to show a reproducible dose/effect relationship for the tested anti-Pneumocystis drugs.

Initial data on the molecular epidemiology of cryptosporidiosis in Lebanon.

Cryptosporidium spp. represent a major public health problem worldwide and infect the gastrointestinal tract of both immunocompetent and immunocompromised persons. The prevalence of these parasites varies by geographic region, and no data are currently available in Lebanon. To promote an understanding of the epidemiology of cryptosporidiosisin this country, the main aim of this study was to determine the prevalence Cryptosporidium in symptomatic hospitalized patients, and to analyze the genetic diversity of the corresponding isolates. Fecal specimens were collected in four hospitals in North Lebanon from 163 patients (77 males and 86 females, ranging in age from 1 to 88 years, with a mean age of 22 years) presenting gastrointestinal disorders during the period July to December 2013. The overall prevalence of Cryptosporidium spp. infection obtained by modified Ziehl-Neelsen staining and/or nested PCR was 11%, and children <5 years old showed a higher rate of Cryptosporidium spp. The PCR products of the 15 positive samples were successfully sequenced. Among them, 10 isolates (66.7%) were identified as C. hominis, while the remaining 5 (33.3%) were identified as C. parvum. After analysis of the gp60 locus, C. hominis IdA19, a rare subtype, was found to be predominant. Two C. parvum subtypes were found: IIaA15G1R1 and IIaA15G2R1. The molecular characterization of Cryptosporidium isolates is an important step in improving our understanding of the epidemiology and transmission of the infection.

What do Pneumocystis organisms tell us about the phylogeography of their hosts? The case of the woodmouse Apodemus sylvaticus in continental Europe and western Mediterranean islands.

Pneumocystis fungi represent a highly diversified biological group with numerous species, which display a strong host-specificity suggesting a long co-speciation process. In the present study, the presence and genetic diversity of Pneumocystis organisms was investigated in 203 lung samples from woodmice (Apodemus sylvaticus) collected on western continental Europe and Mediterranean islands. The presence of Pneumocystis DNA was assessed by nested PCR at both large and small mitochondrial subunit (mtLSU and mtSSU) rRNA loci. Direct sequencing of nested PCR products demonstrated a very high variability among woodmouse-derived Pneumocystis organisms with a total number of 30 distinct combined mtLSU and mtSSU sequence types. However, the genetic divergence among these sequence types was very low (up to 3.87%) and the presence of several Pneumocystis species within Apodemus sylvaticus was considered unlikely. The analysis of the genetic structure of woodmouse-derived Pneumocystis revealed two distinct groups. The first one comprised Pneumocystis from woodmice collected in continental Spain, France and Balearic islands. The second one included Pneumocystis from woodmice collected in continental Italy, Corsica and Sicily. These two genetic groups were in accordance with the two lineages currently described within the host species Apodemus sylvaticus. Pneumocystis organisms are emerging as powerful tools for phylogeographic studies in mammals.

Complementation of a manganese-dependent superoxide dismutase-deficient yeast strain with Pneumocystis carinii sod2 gene.

Manganese-dependent superoxide dismutase (MnSOD) is one of the key enzymes involved in the cellular defense against oxidative stress. Previously, the Pneumocystis carinii sod2 gene (Pcsod2) was isolated and characterized. Based on protein sequence comparison, Pcsod2 was suggested to encode a putative MnSOD protein likely to be targeted into the mitochondrion. In this work, the Pcsod2 was cloned and expressed as a recombinant protein in EG110 Saccharomyces cerevisiae strain lacking the MnSOD-coding gene (Scsod2) in order to investigate the function and subcellular localization of P. carinii MnSOD (PcMnSOD). The Pcsod2 gene was amplified by PCR and cloned into the pYES2.1/V5-His-TOPO(®) expression vector. The recombinant construct was then transformed into EG110 strain. Once its expression had been induced, PcMnSOD was able to complement the growth defect of EG110 yeast cells that had been exposed to the redox-cycling compound menadione. N-term sequencing of the PcMnSOD protein allowed identifying the cleavage site of a mitochondrial targeting peptide. Immune-colocalization of PcMnSOD and yeast CoxIV further confirmed the mitochondrial localization of the PcMnSOD. Heterologous expression of PcMnSOD in yeast indicates that Pcsod2 encodes an active MnSOD, targeted to the yeast mitochondrion that allows the yeast cells to grow in the presence of reactive oxygen species (ROS).

Antibody and cytokine responses to Giardia excretory/secretory proteins in Giardia intestinalis-infected BALB/c mice.

The humoral and cellular responses against excretory/secretory proteins and soluble extracts of Giardia intestinalis were evaluated in the course of experimental G. intestinalis infection in BALB/c mice. Production of IgG1, IgG2a, IgA, and IgE antibodies against excreted/secreted proteins and soluble extract was detected after infection by G. intestinalis. Specific IgA antibody against E/S proteins and soluble extract form intestinal fluids in infected mice was detected by ELISA. The Western blotting identified proteins of 30, 58, 63, and 83 kDa for IgA and IgG, respectively. High proliferation rate in vitro of spleen cell and secretion of interleukin-4 (IL-4) at 21 days p.i. after stimulation with excreted/secreted proteins and low proliferative response in the presence of soluble extract in infected BALB/c mice was observed. High production of interferon gamma (IFN-γ) and interleukin-5 (IL-5) at the time of decreasing cyst output (14-21 days p.i.) in infected mice was recorded, suggesting the important role of these cytokines in the control of the infection. Interestingly, progressive and gradual increase of the interleukin-10 after stimulation with both preparations was recorded from 7 days until 28 days after infection, indicating the possible regulatory effect of these antigens on the immune response during Giardia infection. Therefore, the infection by Giardia duodenalis stimulates a mixed response Th1 and Th2, mainly stimulated by excretory/secretory antigens. The immunogenicity of these antigens may be a suitable for identification of the proteins related with the effective immune response in the course of infection by G. duodenalsis.

Cryptosporidium parvum-induced ileo-caecal adenocarcinoma and Wnt signaling in a mouse model.

Cryptosporidium species are apicomplexan protozoans that are found worldwide. These parasites constitute a large risk to human and animal health. They cause self-limited diarrhea in immunocompetent hosts and a life-threatening disease in immunocompromised hosts. Interestingly, Cryptosporidium parvum has been related to digestive carcinogenesis in humans. Consistent with a potential tumorigenic role of this parasite, in an original reproducible animal model of chronic cryptosporidiosis based on dexamethasone-treated or untreated adult SCID mice, we formerly reported that C. parvum (strains of animal and human origin) is able to induce digestive adenocarcinoma even in infections induced with very low inoculum. The aim of this study was to further characterize this animal model and to explore metabolic pathways potentially involved in the development of C. parvum-induced ileo-caecal oncogenesis. We searched for alterations in genes or proteins commonly involved in cell cycle, differentiation or cell migration, such as ß-catenin, Apc, E-cadherin, Kras and p53. After infection of animals with C. parvum we demonstrated immunohistochemical abnormal localization of Wnt signaling pathway components and p53. Mutations in the selected loci of studied genes were not found after high-throughput sequencing. Furthermore, alterations in the ultrastructure of adherens junctions of the ileo-caecal neoplastic epithelia of C. parvum-infected mice were recorded using transmission electron microscopy. In conclusion, we found for the first time that the Wnt signaling pathway, and particularly the cytoskeleton network, seems to be pivotal for the development of the C. parvum-induced neoplastic process and cell migration of transformed cells. Furthermore, this model is a valuable tool in understanding the host-pathogen interactions associated with the intricate infection process of this parasite, which is able to modulate host cytoskeleton activities and several host-cell biological processes and remains a significant cause of infection worldwide.

Aerially transmitted human fungal pathogens: what can we learn from metagenomics and comparative genomics? / Hongos de transmisión aérea patógenos para el ser humano: conocimientos adquiridos a partir de su metagenómica y genómica comparativa

In the last few decades, aerially transmitted human fungal pathogens have been increasingly recognized to impact the clinical course of chronic pulmonary diseases, such as asthma, cystic fibrosis or chronic obstructive pulmonary disease. Thanks to recent development of culture-free high-throughput sequencing methods, the metagenomic approaches are now appropriate to detect, identify and even quantify prokaryotic or eukaryotic microorganism communities inhabiting human respiratory tract and to access the complexity of even low-burden microbe communities that are likely to play a role in chronic pulmonary diseases. In this review, we explore how metagenomics and comparative genomics studies can alleviate fungal culture bottlenecks, improve our knowledge about fungal biology, lift the veil on cross-talks between host lung and fungal microbiota, and gain insights into the pathogenic impact of these aerially transmitted fungi that affect human beings. We reviewed metagenomic studies and comparative genomic analyses of carefully chosen microorganisms, and confirmed the usefulness of such approaches to better delineate biology and pathogenesis of aerially transmitted human fungal pathogens. Efforts to generate and efficiently analyze the enormous amount of data produced by such novel approaches have to be pursued, and will potentially provide the patients suffering from chronic pulmonary diseases with a better management. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012) (AU)

En las últimas décadas se ha reconocido cada vez más la influencia de los hongos patógenos para el ser humano, y cuya transmisión es aérea, en el curso clínico de afecciones pulmonares crónicas, como el asma, la fibrosis quística o la enfermedad pulmonar obstructiva crónica. Gracias al desarrollo reciente de métodos de secuenciación de alto rendimiento, que no requieren cultivo, en la actualidad los análisis metagenómicos permiten detectar, identificar e incluso cuantificar comunidades de microorganismos procariotas o eucariotas que habitan en las vías respiratorias del ser humano, y acceder a la complejidad de las comunidades microbianas cuya población es de baja densidad, que posiblemente desempeñan un papel en las enfermedades pulmonares crónicas. En la presente revisión examinamos cómo los estudios metagenómicos y genómicos comparativos pueden ayudar a superar los obstáculos de los cultivos de hongos, mejorar nuestros conocimientos sobre la biología fúngica, desvelar el diálogo cruzado (crosstalk) entre el pulmón del huésped y la microbiota fúngica asociada, y adquirir información sobre la influencia patogénica de estos hongos transmitidos por el aire que afectan al ser humano. Revisamos los estudios metagenómicos y los análisis genómicos comparativos de microorganismos cuidadosamente seleccionados, y confirmamos la utilidad de estas estrategias para definir mejor la biología y la patogenia de hongos de transmisión aérea que son patógenos para el ser humano. Los esfuerzos por generar y analizar eficientemente la ingente cantidad de datos obtenidos con estos nuevos métodos deberán continuar, y es posible que ofrezcan un mejor tratamiento de los pacientes portadores de enfermedades pulmonares crónicas.Este manuscrito forma parte de la serie de artículos presentados en el «V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi» (Oaxaca, México, 2012) (AU)

Histoplasma capsulatum and Pneumocystis spp. co-infection in wild bats from Argentina, French Guyana, and Mexico.

BACKGROUND: Histoplasma capsulatum and Pneumocystis organisms cause host infections primarily affecting the lung tissue. H. capsulatum is endemic in the United States of America and Latin American countries. In special environments, H. capsulatum is commonly associated with bat and bird droppings. Pneumocystis-host specificity has been primarily studied in laboratory animals, and its ability to be harboured by wild animals remains as an important issue for understanding the spread of this pathogen in nature. Bats infected with H. capsulatum or Pneumocystis spp. have been found, with this mammal serving as a probable reservoir and disperser; however, the co-infection of bats with both of these microorganisms has never been explored. To evaluate the impact of H. capsulatum and Pneumocystis spp. infections in this flying mammal, 21 bat lungs from Argentina (AR), 13 from French Guyana (FG), and 88 from Mexico (MX) were screened using nested-PCR of the fragments, employing the Hcp100 locus for H. capsulatum and the mtLSUrRNA and mtSSUrRNA loci for Pneumocystis organisms. RESULTS: Of the 122 bats studied, 98 revealed H. capsulatum infections in which 55 of these bats exhibited this infection alone. In addition, 51 bats revealed Pneumocystis spp. infection of which eight bats exhibited a Pneumocystis infection alone. A total of 43 bats (eight from AR, one from FG, and 34 from MX) were found co-infected with both fungi, representing a co-infection rate of 35.2% (95% CI = 26.8-43.6%). CONCLUSION: The data highlights the H. capsulatum and Pneumocystis spp.co-infection in bat population's suggesting interplay with this wild host.

Aerially transmitted human fungal pathogens: what can we learn from metagenomics and comparative genomics?

In the last few decades, aerially transmitted human fungal pathogens have been increasingly recognized to impact the clinical course of chronic pulmonary diseases, such as asthma, cystic fibrosis or chronic obstructive pulmonary disease. Thanks to recent development of culture-free high-throughput sequencing methods, the metagenomic approaches are now appropriate to detect, identify and even quantify prokaryotic or eukaryotic microorganism communities inhabiting human respiratory tract and to access the complexity of even low-burden microbe communities that are likely to play a role in chronic pulmonary diseases. In this review, we explore how metagenomics and comparative genomics studies can alleviate fungal culture bottlenecks, improve our knowledge about fungal biology, lift the veil on cross-talks between host lung and fungal microbiota, and gain insights into the pathogenic impact of these aerially transmitted fungi that affect human beings. We reviewed metagenomic studies and comparative genomic analyses of carefully chosen microorganisms, and confirmed the usefulness of such approaches to better delineate biology and pathogenesis of aerially transmitted human fungal pathogens. Efforts to generate and efficiently analyze the enormous amount of data produced by such novel approaches have to be pursued, and will potentially provide the patients suffering from chronic pulmonary diseases with a better management. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

Growth and airborne transmission of cell-sorted life cycle stages of Pneumocystis carinii.

Pneumocystis organisms are airborne opportunistic pathogens that cannot be continuously grown in culture. Consequently, the follow-up of Pneumocystis stage-to-stage differentiation, the sequence of their multiplication processes as well as formal identification of the transmitted form have remained elusive. The successful high-speed cell sorting of trophic and cystic forms is paving the way for the elucidation of the complex Pneumocystis life cycle. The growth of each sorted Pneumocystis stage population was followed up independently both in nude rats and in vitro. In addition, by setting up a novel nude rat model, we attempted to delineate which cystic and/or trophic forms can be naturally aerially transmitted from host to host. The results showed that in axenic culture, cystic forms can differentiate into trophic forms, whereas trophic forms are unable to evolve into cystic forms. In contrast, nude rats inoculated with pure trophic forms are able to produce cystic forms and vice versa. Transmission experiments indicated that 12 h of contact between seeder and recipient nude rats was sufficient for cystic forms to be aerially transmitted. In conclusion, trophic- to cystic-form transition is a key step in the proliferation of Pneumocystis microfungi because the cystic forms (but not the trophic forms) can be transmitted by aerial route from host to host.

Diamidines versus Monoamidines as Anti-Pneumocystis Agents: An in Vivo Study.

Some compounds articulated around a piperazine or an ethylenediamine linker have been evaluated in vitro to determine their activity in the presence of a 3T6 fibroblast cell line and an axenic culture of Pneumocystis carinii, respectively. The most efficient antifungal derivatives, namely N,N'-bis(benzamidine-4-yl)ethane-1,2-diamine (compound 6, a diamidine) and N-(benzamidine-4-yl)-N'-phenylethane-1,2-diamine (compound 7, a monoamidine), exhibited no cytotoxicity and were evaluated in vivo in a rat model. Only the diamidine 6 emerged as a promising hit for further studies.

Molecular epidemiology of Blastocystis in Lebanon and correlation between subtype 1 and gastrointestinal symptoms.

Blastocystis is the most common eukaryotic parasite in the intestinal tract of humans. Because of its potential impact in public health, we acquired the first data concerning the prevalence of this parasite and the frequency of the Blastocystis subtypes (STs) in the Lebanese population. In this study, fecal samples from 220 Lebanese symptomatic and asymptomatic patients were collected and a total of 42 patients (19%) were identified as positive for this parasite by direct-light microscopy of smears. Among these, 36 Blastocystis isolates were genotyped using partial small subunit ribosomal RNA gene sequencing. The ST distribution in the present Lebanese population was as follows: ST3 (33.3%), ST2 (33.3%), ST1 (30.6%), and ST4 (2.8%). These data were compared with those available in other Middle Eastern and neighboring countries. Finally, ST1 was significantly more prevalent among symptomatic patients of this Lebanese population.

Molecular detection of Histoplasma capsulatum in the lung of a free-ranging common noctule (Nyctalus noctula) from France using the Hcp100 gene.

Histoplasma capsulatum is a dimorphic fungus that is widely distributed in the tropical or subtropical areas of the world and infects several mammalian hosts, mainly bats. Infective propagules grow in bat and bird droppings. A specific molecular marker, a highly sensitive fragment of a co-activator protein-coding gene (Hcp100), was used to detect H. capsulatum in lung samples of wild and captive bats from France using a nested polymerase chain reaction. To determine whether bats in France are potential carriers of H. capsulatum, 83 bats were sampled from two regions in France. Sixty-one specimens belonging to the Pteropus rodricensis (n = 45) and Rousettus aegyptiacus (n = 16) species were collected from a zoologic park (La Palmyre, western France). Twenty-two specimens were recovered from the Natural History Museum (Bourges) including the species Plecotus austriacus (n = 1), Pipistrellus pipistrellus (n = 3), and Nyctalus noctula (n = 18). From the lung DNA samples of 83 dead bats, only one sample of an N. noctula bat from Bourges amplified the H. capsulatum Hcp100 marker. The amplified product was sequenced and revealed a high similarity to the G217B H. capsulatum reference strain sequence that was deposited in the GenBank database. This finding suggests that H. capsulatum is an environmental pathogen in France that may infect bats.

Transcriptomic insights into the oxidative response of stress-exposed Aspergillus fumigatus.

NADPH-oxidase mediated production of Reactive Oxygen Species (ROS) by alveolar macrophages and neutrophils is a critical mechanism for immune defence against Aspergillus fumigatus. Fungal oxidative stress response includes enzymatic response by superoxide dismutases (SOD), catalases, and enzymes from the thioredoxin and glutathione systems, which are regulated by the transcription factor Yap1. Secondary metabolites are also involved in defense against ROS. Some of the secondary metabolite clusters are controlled by the transcriptional regulator LaeA. The redundancy of antioxidant systems, and the variable impact of SOD or catalase gene deletions on in vitro oxidative stress sensitivity and in vivo virulence suggest a complex regulation of oxidative stress response in A. fumigatus, making high-throughput approaches, such as microarray or next generation sequencing (NGS), highly relevant to study their respective role. These approaches have been widely applied to A fumigatus, in order to characterize its metabolic response to different stresses mimicking in vivo conditions (such as antifungals, or neutrophils), or to transcription factor deletion (including LaeA). In some studies, oxidative stress response process and antioxidant enzymes have been identified as key metabolic pathways. However, oxidative stress response has not been analyzed systematically and a further data analysis could be helpful to clarify the role of A. fumigatus antioxidant systems and, potentially, to identify new drug targets. In this review, we synthesized available A. fumigatus microarrays and NGS data, focusing on the role of antioxidant systems. We analyzed the different methodologies that were used for transcriptomic analysis, and we compared biological processes and antioxidant system modulations in A. fumigatus exposed to stress.