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Abstract Cryptococcal meningitis is a high-mortality infection. Adding 5-fluorocytosine (5-FC) to its treatment improves outcomes, but resistance to 5-FC presents a significant challenge. We conducted whole-genome sequencing on seven C. neoformans isolates with varying 5-FC susceptibility, along with proteomic and in silico analyses. Our findings indicate that mutations in genes of the pyrimidine salvage pathway are responsible for 5-FC resistance. Specifically, we identified an E64G missense mutation in the FUR1 gene, a large deletion in the FCY1 gene, and a point mutation in FCY1 leading to a truncated protein. The proteomic data indicated that these mutations resulted in the absence or reduction of crucial enzymes in resistant isolates. Genetic transformations confirmed the association between these mutations and 5-FC resistance. Resistance to 5-FC can develop during treatment and is closely tied to mutations in key metabolic enzymes. Understanding in vivo resistance development is crucial for combating resistance and enhancing patient outcomes.

ABSTRACTThe present paper includes a meta-analysis of literature data on 318 species of fungi belonging to 34 orders in their response to 8 antifungal agents (amphotericin B, caspofungin, fluconazole, itraconazole, ketoconazole, posaconazole, terbinafine, and voriconazole). Main trends of MIC results at the ordinal level were visualized. European Committee on Antimicrobial Susceptibility Testing and Clinical & Laboratory Standards Institute (CLSI) clinical breakpoints were used as the staff gauge to evaluate MIC values ranging from resistance to susceptibility, which were subsequently compared with a phylogenetic tree of the fungal kingdom. Several orders (Hypocreales, Microascales, and Mucorales) invariably showed resistance. Also the basidiomycetous orders Agaricales, Polyporales, Sporidiales, Tremellales, and Trichosporonales showed relatively high degrees of azole multi-resistance, while elsewhere in the fungal kingdom, including orders with numerous pathogenic and opportunistic species, that is, Onygenales, Chaetothyiales, Sordariales, and Malasseziales, in general were susceptible to azoles. In most cases, resistance vs susceptibility was consistently associated with phylogenetic distance, members of the same order showing similar behavior.IMPORTANCEA kingdom-wide the largest set of published wild-type antifungal data comparison were analyzed. Trends in resistance in taxonomic groups (monophyletic clades) can be compared with the phylogeny of the fungal kingdom, eventual relationships between fungus–drug interaction and evolution can be described.

ABSTRACT Fungal rhinosinusitis (FRS) is a common problem worldwide, with an increasing burden in arid climate regions. Aspergillus species are the most common causative agents involved. In the present study, we investigated the prevalence, molecular characterization, and antifungal susceptibility of opportunists causing FRS in Sudan on the basis of strains collected over a period of 5 years. β-Tubulin and calmodulin sequencing were used for species identification, and antifungal susceptibility profiles were evaluated by the protocol of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Predominant species belonged to the Aspergillus flavus complex (n = 244), A. terreus complex (n = 16), A. fumigatus complex (n = 7), and other fungi (n = 17). Molecular identification of 94 strains of Aspergillus revealed the following species: A. flavus (n = 88), A. terreus (n = 1), A. citrinoterreus (n = 2), A. fumigatus (n = 1), A. caespitosus (n = 1), and A. sydowii (n = 1). Several A. flavus and an A. fumigatus isolates showed reduced susceptibility to azoles (minimum inhibitory concentrations above the clinical breakpoints or epidemiological cutoff values). Despite several mutations revealed in cyp51A of these isolates, none could be directly linked to azole resistance. Molecular identification of fungi causing FRS is useful to identify cryptic species and for epidemiologic studies. IMPORTANCE Fungal rhinosinusitis (FRS) is a significant clinical problem in arid regions. This study provides new insights into the prevalence, etiology, and antifungal susceptibility of FRS pathogens in Sudan, where the disease burden is high. Aspergillus species, particularly the A. flavus complex, were identified as the primary FRS pathogens in the region, with some evidence of antifungal resistance. The molecular identification of fungal species causing FRS is useful for detecting antifungal resistance, identifying cryptic species, and characterizing the epidemiology of the disease. The emergence of Azole resistance Aspergilli in Sudan highlights the need for continued surveillance and appropriate use of antifungal agents. These findings have important implications for clinical management, public health policy, and future research on FRS. Publishing this study in Microbiology Spectrum would enable other researchers and clinicians to build on these findings, ultimately improving the diagnosis, treatment, and prevention of FRS.

Aspergillus luchuensis, an industrially important member of Aspergillus species belonging to section Nigri used in fermentation in East Asia, was isolated from an immunocompromised patient with probable invasive pulmonary aspergillosis who failed voriconazole therapy in China. This isolate showed non-wild-type susceptibility to itraconazole, voriconazole, isavuconazole, and posaconazole. A G1378A mutation in cyp51A, resulting in the G441S amino acid substitution, which is the homolog to G448S conferring triazole-resistance in A. fumigatus, was detected in the A. luchuensis isolate.

ABSTRACT Germination of inhaled Aspergillus fumigatus conidia is a necessary sequitur for infection. Germination of conidia starts with the breaking of dormancy, which is initiated by an increase of the cellular perimeter in a process termed isotropic growth. This swelling phase is followed by polarized growth, resulting in the formation of a germ tube. The multinucleate tubular cells exhibit tip growth from the hyphae, after which lateral branches emerge to form the mycelial network. The regulatory mechanisms governing conidial germination are not well defined. In this study, we identified a novel role for the transcription factor SltA in the orchestration of germination and hyphal development. Conidia lacking sltA fail to appropriately regulate isotropic growth and begin to swell earlier and subsequently switch to polarized growth faster. Additionally, hyphal development is distorted in a ∆sltA isolate as hyphae are hyper-branching and wider, and show branching at the apical tip. ∆sltA conidia are more tolerant to cell wall stressors on minimal medium compared to the wild-type (WT) strain. A transcriptome analysis of different stages of early growth was carried out to assess the regulatory role of SltA. Null mutants generated for three of the most dysregulated genes showed rapid germ tube emergence. Distinct from the phenotype observed for ∆sltA, conidia from these strains lacked defects in isotropic growth, but switched to polarized growth faster. Here, we characterize and describe several genes in the regulon of SltA, highlighting the complex nature of germination. IMPORTANCE Aspergillus fumigatus is the main human fungal pathogen causing aspergillosis. For this fungus, azoles are the most commonly used antifungal drugs for treatment of aspergillosis. However, the prevalence of azole resistance is alarmingly increasing and linked with elevated mortality. Germination of conidia is crucial within its asexual life cycle and plays a critical role during the infection in the human host. Precluding germination could be a promising strategy considering the role of germination in Aspergillus spp. pathogenicity. Here, we identify a novel role for SltA in appropriate maintenance of dormancy, germination, and hyphal development. Three genes in the regulon of SltA were also essential for appropriate germination of conidia. With an expanding knowledge of germination and its different morphotypes, more advances can be made toward potential anti-germination targets for therapy.

Whole genome sequencing (WGS) is widely used for outbreak analysis of bacteriology and virology but is scarcely used in mycology. Here, we used WGS for genotyping Aspergillus fumigatus isolates from a potential Aspergillus outbreak in an intensive care unit (ICU) during construction work. After detecting the outbreak, fungal cultures were performed on all surveillance and/or patient respiratory samples. Environmental samples were obtained throughout the ICU. WGS was performed on 30 isolates, of which six patient samples and four environmental samples were related to the outbreak, and twenty samples were unrelated, using the Illumina NextSeq 550. A SNP-based phylogenetic tree was created from outbreak samples and unrelated samples. Comparative analysis (WGS and short tandem repeats (STRs), microsatellite loci analysis) showed that none of the strains were related to each other. The lack of genetic similarity suggests the accumulation of Aspergillus spores in the hospital environment, rather than a single source that supported growth and reproduction of Aspergillus fumigatus. This supports the hypothesis that the Aspergillus outbreak was likely caused by release of Aspergillus fumigatus spores during construction work. Indeed, no new Aspergillus cases were observed in the ICU after cessation of construction. This study demonstrates that WGS is a suitable technique for examining inter-strain relatedness of Aspergillus fumigatus in the setting of an outbreak investigation.

Aspergillus fumigatus has been found to coinfect patients with severe SARS-CoV-2 virus infection, leading to COVID-19-associated pulmonary aspergillosis (CAPA). The CAPA all-cause mortality rate is approximately 50% and may be complicated by azole resistance. Genomic epidemiology can help shed light on the genetics of A. fumigatus causing CAPA, including the prevalence of resistance-associated alleles. We present a population genomic analysis of 21 CAPA isolates from four European countries with these isolates compared against 240 non-CAPA A. fumigatus isolates from a wider population. Bioinformatic analysis and antifungal susceptibility testing were performed to quantify resistance and identify possible genetically encoded azole-resistant mechanisms. The phylogenetic analysis of the 21 CAPA isolates showed that they were representative of the wider A. fumigatus population with no obvious clustering. The prevalence of phenotypic azole resistance in CAPA was 14.3% (n = 3/21); all three CAPA isolates contained a known resistance-associated cyp51A polymorphism. The relatively high prevalence of azole resistance alleles that we document poses a probable threat to treatment success rates, warranting the enhanced surveillance of A. fumigatus genotypes in these patients. Furthermore, potential changes to antifungal first-line treatment guidelines may be needed to improve patient outcomes when CAPA is suspected.

Olorofim (F901318) is a new antifungal currently under clinical development that shows both in vitro and in vivo activity against a number of filamentous fungi including Aspergillus fumigatus. In this study we screened A. fumigatus isolates for intrinsic olorofim-resistant A. fumigatus and evaluated the ability of A. fumigatus to acquire an olorofim-resistant phenotype. No intrinsic resistance was found in 975 clinical A. fumigatus isolates. However, we found that isolates with increased olorofim MICs (> 8 mg/L) could be selected using a high number of conidia and olorofim exposure under laboratory conditions. Assessment of the frequency of acquired olorofim resistance development of A. fumigatus was shown to be higher than for voriconazole but lower than for itraconazole. Sequencing the PyrE gene of isogenic isolates with olorofim MICs of >8 mg/L identified various amino acid substitutions with a hotspot at locus G119. Olorofim was shown to have reduced affinity to mutated target protein dihydroorotate dehydrogenase (DHODH) and the effect of these mutations were proven by introducing the mutations directly in A. fumigatus. We then investigated whether G119 mutations were associated with a fitness cost in A. fumigatus. These experiments showed a small but significant reduction in growth rate for strains with a G119 V substitution, while strains with a G119C substitution did not exhibit a reduction in growth rate. These in vitro findings were confirmed in an in vivo pathogenicity model

IntroductionThis study was intended to investigate the clinical features and predisposing factors of fungal keratitis (FK), as well as molecular identification and antifungal susceptibility of causative agents in Tehran, Iran.MethodsThis cross-sectional study was carried out from April 2019 to May 2021. All fungi isolates were identified using conventional methods and were confirmed by DNA-PCR-based molecular assays. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) was used to identify yeast species. Minimum inhibitory concentrations (MIC) of eight antifungal agents were assessed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) microbroth dilution reference method. ResultsFungal etiology was confirmed in 86 (7.23%) of 1189 corneal ulcers. A significant predisposing factor for FK was ocular trauma caused by plant materials. Therapeutic penetrating keratoplasty (PKP) was required in 60.4% of cases. The predominant fungal species isolated was Fusarium spp. (39.5%) followed by Aspergillus spp. (32.5%) and Candida spp. (16.2%).DiscussionThe MIC results indicate that amphotericin B may be appropriate for treating FK caused by Fusarium species. FK caused by Candida spp. can be treated with flucytosine, voriconazole, posaconazole, miconazole, and caspofungin. In developing countries such as Iran, corneal infection due to filamentous fungi is a common cause of corneal damage. In this region, fungal keratitis is observed primarily within the context of agricultural activity and subsequent ocular trauma. Fungal keratitis can be managed better with understanding the ”local“ etiologies and antifungal susceptibility patterns.

We performed an observational study to investigate intensive care unit incidence, risk factors, and outcomes of coronavirus disease–associated pulmonary aspergillosis (CAPA). We found 10%–15% CAPA incidence among 823 patients in 2 cohorts. Several factors were independently associated with CAPA in 1 cohort and mortality rates were 43%–52%.

Abstract Background Pseudallescheria keratitis is rare but important type of fungal keratitis because of the inherently resistance of the organism to many existing antifungal agents. Methods Slit-lamp and confocal microscopy were used for clinical examinations. Fungal isolates were identified based on morphological characteristics and DNA sequence of the internal transcribed spacer region (ITS). In vitro antifungal susceptibility testing for fungal isolates was performed according to the Clinical and Laboratory Standards Institute (CLSI, M38-A2). Result All patients had a history of ocular trauma. In clinical examination hypopion were seen in three patients. The main antifungal medications were topical voriconazole. After treatment the visual acuity of all patients improved in 2–3 weeks. Conclusion All four patients of Pseudallescheria keratitis had similar clinical features. Accurate and rapid identification of species should be helpful in treating p. boydii keratitis.

The opportunistic pathogen Aspergillus fumigatus is found on all continents and thrives in soil and agricultural environments. Its ability to readily adapt to novel environments and to produce billions of spores led to the spread of azole-resistant A. fumigatus across the globe, posing a threat to many immunocompromised patients, including critically ill patients with severe influenza or COVID-19. In our study, we sought to compare the adaptational response to azoles from A. fumigatus isolates that differ in azole susceptibility and genetic background. To gain more insight into how short-term adaptation to stressful azole compounds is managed through gene expression, we conducted an RNA-sequencing study on the response of A. fumigatus to itraconazole and the newest clinically approved azole, isavuconazole. We observed many similarities in ergosterol biosynthesis up-regulation across isolates, with the exception of the pan-azole-resistant isolate, which showed very little differential regulation in comparison to other isolates. Additionally, we found differential regulation of membrane efflux transporters, secondary metabolites, iron metabolism, and various stress response and cell signaling mechanisms.

ABSTRACT Aspergillus fumigatus is the major filamentous fungal pathogen in humans. The gold standard treatment of A. fumigatus is based on azole drug use, but the appearance of azole-resistant isolates is increasing at an alarming rate. The cyp51A gene encodes the enzymatic target of azole drugs, and azole-resistant alleles of cyp51A often have an unusual genetic structure containing a duplication of a 34- or 46-bp region in the promoter causing enhanced gene transcription. These tandem repeats are called TR34 and TR46 and produce duplicated binding sites for the SrbA and AtrR transcription factors. Using site-directed mutagenesis, we demonstrate that both the SrbA (sterol response element [SRE]) and AtrR binding sites (AtrR response element [ATRE]) are required for normal cyp51A gene expression. Loss of either the SRE or ATRE from the distal 34-bp repeat of the TR34 promoter (further 5′ from the transcription start site) caused loss of expression of cyp51A and decreased voriconazole resistance. Surprisingly, loss of these same binding sites from the proximal 34- or 46-bp repeat led to increased cyp51A expression and voriconazole resistance. These data indicate that these duplicated regions in the cyp51A promoter function differently. Our findings suggest that the proximal 34- or 46-bp repeat in cyp51A recruits a corepressor that requires multiple factors to act while the distal repeat is free of this repression and provides the elevated cyp51A expression caused by these promoter duplications. IMPORTANCE Aspergillus fumigatus is the most common human filamentous fungal pathogen. Azole drugs are the current therapy of choice for A. fumigatus, but the prevalence of azole resistance is increasing. The main genetic alteration causing azole resistance is a change in the cyp51A gene, which encodes the target of these drugs. Azole-resistant cyp51A alleles routinely contain duplications in their promoter regions that cause increased gene transcription. Here, we demonstrate that clinical isolates containing a 34- or 46-bp duplication in the cyp51A promoter required the presence of the transcription factor-encoding atrR gene to exhibit elevated azole resistance. Eliminations of transcription factor binding sites in the cyp51A gene have differential actions on expression of the resulting mutant allele. These data dissect the molecular inputs to cyp51A transcription and reveal a complicated function of the promoter of this gene that is critical in azole resistance.

Coronavirus disease 2019 (COVID‐19), caused by the novel coronavirus SARS-CoV-2, emerged in Wuhan, China, in December 2019 and rapidly spread around the world. Invasive aspergillosis has been reported as a complication of severe influenza pneumonia among intensive care patients. Similarities between COVID-19 and influenza pneumonia, together with limited published case series, suggest that aspergillosis may be an important complication of COVID-19. This report describes a case of ventilator-associated pneumonia involving Aspergillus flavus in a patient with COVID-19 from Buenos Aires, Argentina.

Histoplasmosis, caused by the thermally dimorphic fungus Histoplasma spp., is a disease with a broad clinical spectrum, presenting from asymptomatic/flu-like symptoms to progressive disseminated disease in people with immunosuppression. In recent years, the concept of histoplasmosis as a disease restricted to the American continent has changed, as now histoplasmosis is reported in many regions around the world. In Latin America, histoplasmosis represents a threat, especially in people with advanced HIV disease (AHD). Diagnosis of histoplasmosis in people living with HIV (PLHIV) is challenging due to the low index of suspicion of the disease, non-specificity of signs and symptoms, and limited access to specific laboratory testing, while the diagnostic delay is significantly associated with mortality. In the last decade, novel diagnostic tests have been developed for the rapid detection of histoplasmosis, such as commercial kits for antigen detection. Furthermore, advocacy groups were created that presented histoplasmosis as a public health problem, with emphasis on patients at risk of progressive disseminated disease. This review aims to discuss the impact of histoplasmosis associated with AHD in Latin America and the strategies employed to tackle histoplasmosis, from the implementation of laboratory testing to disease advocacy and public health interventions.

IMPORTANCE:. Despite high mortality rates of COVID-19-associated pulmonary aspergillosis (CAPA) in the ICU, antifungal prophylaxis remains a subject of debate. We initiated nebulized conventional amphotericin B (c-AmB) as antifungal prophylaxis in COVID-19 patients on invasive mechanical ventilation (IMV). OBJECTIVES:. To assess the CAPA incidence in COVID-19 patients on IMV treated with and without nebulized c-AmB as antifungal prophylaxis. DESIGN, SETTING, AND PARTICIPANTS:. Retrospective cohort study of consecutive COVID-19 patients admitted to our adult 17-bed ICU in a university-affiliated general hospital in Ede, The Netherlands, between January 25, 2021, and July 9, 2021. Patients not requiring IMV or transferred from or to another ICU were excluded. From April 9, 2021, daily nebulized amphotericin B in all patients on IMV was initiated. MAIN OUTCOMES AND MEASURES:. Bronchoscopy with bronchoalveolar lavage (BAL) was performed in case of positive cultures for Aspergillus from the respiratory tract and/or unexplained respiratory deterioration. Incidence of probable and proven CAPA was compared between patients treated with and without nebulized antifungal prophylaxis using Pearson chi-square test. RESULTS:. A total of 39 intubated COVID-19 patients could be analyzed, of which 16 were treated with antifungal prophylaxis and 23 were not. Twenty-six patients underwent bronchoscopy with BAL. In patients treated with antifungal prophylaxis, the incidence of probable/proven CAPA was significantly lower when compared with no antifungal prophylaxis (27% vs 67%; p = 0.047). Incidence of tracheobronchial lesions and positive Aspergillus cultures and BAL-galactomannan was significantly lower in patients treated with antifungal prophylaxis (9% vs 47%; p = 0.040, 9% vs 53%; p = 0.044, and 20% vs 60%; p = 0.047, respectively). No treatment-related adverse events and no case of proven CAPA were encountered in patients receiving antifungal prophylaxis. CONCLUSIONS AND RELEVANCE:. Nebulization of c-AmB in critically ill COVID-19 patients on IMV is safe and may be considered as antifungal prophylaxis to prevent CAPA. However, a randomized controlled trial to confirm this is warranted.

A sexual cycle was described in 2009 for the opportunistic fungal pathogen Aspergillus fumigatus, opening up for the first time the possibility of using techniques reliant on sexual crossing for genetic analysis. The present study was undertaken to evaluate whether the technique ‘bulk segregant analysis’ (BSA), which involves detection of differences between pools of progeny varying in a particular trait, could be applied in conjunction with next-generation sequencing to investigate the underlying basis of monogenic traits in A. fumigatus. Resistance to the azole antifungal itraconazole was chosen as a model, with a dedicated bioinformatic pipeline developed to allow identification of SNPs that differed between the resistant progeny pool and resistant parent compared to the sensitive progeny pool and parent. A clinical isolate exhibiting monogenic resistance to itraconazole of unknown basis was crossed to a sensitive parent and F1 progeny used in BSA. In addition, the use of backcrossing and increasing the number in progeny pools was evaluated as ways to enhance the efficiency of BSA. Use of F1 pools of 40 progeny led to the identification of 123 candidate genes with SNPs distributed over several contigs when aligned to an A1163 reference genome. Successive rounds of backcrossing enhanced the ability to identify specific genes and a genomic region, with BSA of progeny (using 40 per pool) from a third backcross identifying 46 genes with SNPs, and BSA of progeny from a sixth backcross identifying 20 genes with SNPs in a single 292 kb region of the genome. The use of an increased number of 80 progeny per pool also increased the resolution of BSA, with 29 genes demonstrating SNPs between the different sensitive and resistant groupings detected using progeny from just the second backcross with the majority of variants located on the same 292 kb region. Further bioinformatic analysis of the 292 kb region identified the presence of a cyp51A gene variant resulting in a methionine to lysine (M220K) change in the CYP51A protein, which was concluded to be the causal basis of the observed resistance to itraconazole. The future use of BSA in genetic analysis of A. fumigatus is discussed.

We investigated the prevalence of azole resistance of Aspergillus fumigatus isolates in the Netherlands by screening clinical A. fumigatus isolates for azole resistance during 2013–2018. We analyzed azole-resistant isolates phenotypically by in vitro susceptibility testing and for the presence of resistance mutations in the Cyp51A gene. Over the 6-year period, 508 (11%) of 4,496 culture-positive patients harbored an azole-resistant isolate. Resistance frequency increased from 7.6% (95% CI 5.9%–9.8%) in 2013 (58/760 patients) to 14.7% (95% CI 12.3%–17.4%) in 2018 (112/764 patients) (p = 0.0001). TR34/L98H (69%) and TR46/Y121F/T289A (17%) accounted for 86% of Cyp51A mutations. However, the mean voriconazole MIC of TR34/L98H isolates decreased from 8 mg/L (2013) to 2 mg/L (2018), and the voriconazole-resistance frequency was 34% lower in 2018 than in 2013 (p = 0.0001). Our survey showed changing azole phenotypes in TR34/L98H isolates, which hampers the use of current PCR-based resistance tests.

In 2018, we conducted a cross-sectional study to investigate azole resistance in environmental Aspergillus fumigatus isolates obtained from different agricultural fields in China. Using 63 soil cores, we cultured for azole-resistant A. fumigatus and characterized isolates by their cyp51A gene type, short tandem repeat genotype, and mating type. Of 206 A. fumigatus isolates, 21 (10.2%) were azole resistant. Nineteen of 21 had mutations in their cyp51A gene (5 TR34/L98H, 8 TR34/L98H/S297T/F495I, 6 TR46/Y121F/T289A). Eighteen were cultured from soil samples acquired from strawberry fields, suggesting this soil type is a potential hotspot for azole resistance selection. Twenty resistant isolates were mating type MAT1-1, suggesting asexual sporulation contributed to their evolution. Prochloraz, difenoconazole, and tebuconazole were the most frequently detected fungicides in soil samples with azole-resistant fungus. Our study results suggest that managing the fungicides used in agriculture will help contain the problem of antifungal drug resistance in clinics.

With population genetic evidence of recombination ongoing in the natural Aspergillus fumigatus population and a sexual cycle demonstrated in the laboratory the question remained what the natural niche for A. fumigatus sex is. Composting plant-waste material is a known substrate of A. fumigatus to thrive and withstand temperatures even up to 70°C. Previous studies have shown indirect evidence for sexual reproduction in these heaps but never directly demonstrated the sexual structures due to technical limitations. Here, we show that flower bulb waste material from stockpiles undergoing composting can provide the conditions for sexual reproduction. Direct detection of ascospore structures was shown in agricultural flower bulb waste material by using a grid-based detection assay. Furthermore, we demonstrate that ascospores can germinate after exposure to 70°C for up to several days in contrast to asexual conidia that are unable to survive a two-hour heat shock. This indicates a sufficient time frame for ascospores to survive and escape composting stockpiles. Finally, sexual crosses with cleistothecium and viable ascospore formation could successfully be performed on flower bulb waste material. Recombination of A. fumigatus can now be explained by active sexual reproduction in nature as we show in this study that flower bulb waste material provides an environmental niche for sex.

Azole resistance is a major concern for treatment of infections with Aspergillus fumigatus. Environmental resistance selection is a main route for Aspergillus spp. to acquire azole resistance. We investigated the presence of environmental hotspots for resistance selection in the Netherlands on the basis of the ability of A. fumigatus to grow and reproduce in the presence of azole fungicide residues. We identified 3 hotspots: flower bulb waste, green waste material, and wood chippings. We recovered azole-resistant A. fumigatus from these sites; all fungi contained cyp51A tandem repeat–mediated resistance mechanisms identical to those found in clinical isolates. Tebuconazole, epoxiconazole, and prothioconazole were the most frequently found fungicide residues. Stockpiles of plant waste contained the highest levels of azole-resistant A. fumigatus, and active aerobic composting reduced Aspergillus colony counts. Preventing plant waste stockpiling or creating unfavorable conditions for A. fumigatus to grow in stockpiles might reduce environmental resistance burden.

Abstract Background The prevalence of azole resistance in clinical and environmental Aspergillus fumigatus isolates is rising over the past decades, but the molecular basis of the development of antifungal drug resistance is not well understood. This study focuses on the role of phenotypic plasticity in the evolution of azole resistance in A. fumigatus. When A. fumigatus is challenged with a new stressful environment, phenotypic plasticity may allow A. fumigatus to adjust their physiology to still enable growth and reproduction, therefore allowing the establishment of genetic adaptations through natural selection on the available variation in the mutational and recombinational gene pool. To investigate these short-term physiological adaptations, we conducted time series transcriptome analyses on three clinical A. fumigatus isolates, during incubation with itraconazole. Results After analysis of expression patterns, we identified 3955, 3430, 1207, and 1101 differentially expressed genes (DEGs), after 30, 60, 120 and 240 min of incubation with itraconazole, respectively. We explored the general functions in these gene groups and we identified 186 genes that were differentially expressed during the whole time series. Additionally, we investigated expression patterns of potential novel drug-efflux transporters, genes involved in ergosterol and phospholipid biosynthesis, and the known MAPK proteins of A. fumigatus. Conclusions Our data suggests that A. fumigatus adjusts its transcriptome quickly within 60 min of exposure to itraconazole. Further investigation of these short-term adaptive phenotypic plasticity mechanisms might enable us to understand how the direct response of A. fumigatus to itraconazole promotes survival of the fungus in the patient, before any “hard-wired” genetic mutations arise.

Objectives: Patients receiving remission induction therapy for acute myeloid leukaemia (AML) are at high risk of developing invasive fungal disease (IFD). Newer therapies with targeted antileukemic agents and the emergence of azole resistance pose a challenge to the strategy of primary antifungal prophylaxis. We report the experience of a diagnostic-driven care pathway (DCP) for the management of IFD in these patients, using only culture-directed mould inactive prophylaxis. Methods: Retrospectively, we used a single-centre study of consecutive patients receiving intensive chemotherapy for myeloid malignancies between 2014 and 2021. DCP consisted of serial cultures and serum galactomannan (sGM) screening, CT imaging, and bronchoscopy to direct targeted antifungal treatment. IFD was classified according to the 2020 EORTC/MSGERC criteria. Results: A total of 192 patients with myeloid malignancies received 300 courses of intensive chemotherapy. There were 14 cases of invasive yeast infections and 18 of probable/proven invasive mould disease (IMD). The incidence of probable/proven IMD during the first cycle of remission-induction chemotherapy was 4.6% (n = 9). sGM remained negative in all cases of invasive aspergillosis (IA), with positive mycology findings in bronchoalveolar lavage. All-cause mortality was 9.4% (n = 18) 100 days after starting chemotherapy and was comparable between patients with or without IFD. The fungal-related mortality was 1% (n = 2). Conclusion: Diagnostic-driven based management without universal mould active prophylaxis is a feasible strategy in the management of IFD and limits unnecessary antimould treatment during intensive chemotherapy. The poor performance of serial serum galactomannan screening in detecting IA warrants further investigation.

Summary: Influenza-associated pulmonary aspergillosis (IAPA) has been reported increasingly since the advent of use of neuraminidase (NA) inhibitors following the 2009 influenza pandemic. We hypothesize that blocking host NA modulates the immune response against Aspergillus fumigatus. We demonstrate that NA influences the host response against A. fumigatus in vitro and that oseltamivir increases the susceptibility of mice to pulmonary aspergillosis. Oseltamivir impairs the mouse splenocyte and human peripheral blood mononuclear cell (PBMC) killing capacity of A. fumigatus, and adding NA restores this defect in PBMCs. Furthermore, the sialic acid-binding receptor SIGLEC15 is upregulated in PBMCs stimulated with A. fumigatus. Silencing of SIGLEC15 decrease PBMC killing of A. fumigatus. We provide evidence that host NA activity and sialic acid recognition are important for anti-Aspergillus defense. NA inhibitors might predispose individuals with severe influenza to invasive aspergillosis. These data shed light on the pathogenesis of invasive fungal infections and may identify potential therapeutic targets.

Chronic inflammation and immune dysfunction play a key role in the development of non-AIDS–related comorbidities. The aim of our study was to characterize the functional phenotype of immune cells in people living with HIV (PLHIV). We enrolled a cross-sectional cohort study of PLHIV on stable antiretroviral therapy and healthy controls. We assessed ex vivo cytokine production capacity and transcriptomics of monocytes and T cells upon bacterial, fungal, and viral stimulation. PLHIV exhibited an exacerbated proinflammatory profile in monocyte-derived cytokines, but not in lymphocyte-derived cytokines. Particularly, the production of the IL-1β to imiquimod, E. coli LPS, and Mycobacterium tuberculosis was increased, and this production correlated with plasma concentrations of high-sensitivity C-reactive protein and soluble CD14. This increase in monocyte responsiveness remained stable over time in subsequent blood sampling after more than 1 year. Transcriptome analyses confirmed priming of the monocyte IL-1β pathway, consistent with a monocyte-trained immunity phenotype. Increased plasma concentrations of β-glucan, a well-known inducer of trained immunity, were associated with increased innate cytokine responses. Monocytes of PLHIV exhibited a sustained proinflammatory immune phenotype with priming of the IL-1β pathway. Training of the innate immune system in PLHIV likely plays a role in long-term HIV complications and provides a promising therapeutic target for inflammation-related comorbidities.

Aspergillus fumigatus is a saprobic fungus that causes a range of pulmonary diseases, some of which are characterised by fungal persistence such as is observed in cystic fibrosis (CF) patients. Creation of genetic variation is critical for A. fumigatus to adapt to the lung environment, but biofilm formation, especially in CF patients, may preclude mutational supply in A. fumigatus due to its confinement to the hyphal morphotype. We tested our hypothesis that genetic variation is created through parasexual recombination in chronic biofilms by phenotypic and genetic analysis of A. fumigatus isolates cultured from different origins. As diploids are the hallmark of parasex, we screened 799 A. fumigatus isolates obtained from patients with CF, chronic pulmonary lung disease and acute invasive aspergillosis, and from the environment for spore size. Benomyl sensitivity, nuclear content measurements through fluorescence-activated cell sorting and scanning electron microscopy were used to confirm the diploid state of large size spores. Whole genome sequencing was used to characterise diploid-associated genetic variation. We identified 11 diploids in isolates recovered from six of 11 (55%) CF patients and from one of 24 (4%) chronic aspergillosis patients, but not in 368 isolates from patients with acute Aspergillus infection and the environment. Diploid formation was associated with accumulation of mutations and variable haploid offspring including a voriconazole-resistant isolate. Parasexual recombination allows A. fumigatus to adapt and persist in CF patients, and plays a role in azole resistance development. Our findings are highly significant for understanding the genetics and biology of A. fumigatus in the human lung.

ABSTRACT This article is to alert medical mycologists and infectious disease specialists of recent name changes of medically important species of the filamentous mold Fusarium. Fusarium species can cause localized and life-threating infections in humans. Of the 70 Fusarium species that have been reported to cause infections, close to one-third are members of the Fusarium solani species complex (FSSC), and they collectively account for approximately two-thirds of all reported Fusarium infections. Many of these species were recently given scientific names for the first time by a research group in the Netherlands, but they were misplaced in the genus Neocosmospora. In this paper, we present genetic arguments that strongly support inclusion of the FSSC in Fusarium. There are potentially serious consequences associated with using the name Neocosmospora for Fusarium species because clinicians need to be aware that fusaria are broadly resistant to the spectrum of antifungals that are currently available.

Studies on the occurrence of fungal communities in the marine environment are still scarce, but mycotic infections in cetaceans are increasingly reported. Fungal disease following infection with Aspergillus species is most frequently reported, with the respiratory tract commonly affected in cetaceans and other taxa, like humans and birds. Infection with Aspergillus spp. is dependent on the characteristics of the fungus as well as the hosts immune status, with dissemination into other organs being relatively common. Along the southern North Sea, harbor porpoise (Phocoena phocoena) strandings increased significantly since 2005 and necropsies to determine causes of death have been conducted since 2008. Here we describe the post-mortem findings in stranded, free-ranging harbor porpoises on the Dutch coast which were diagnosed with fungal disease, to determine the prevalence of mycotic infections, and to compare them to those described in other species. A total of 18/754 (2.4%) harbor porpoises showed lesions compatible with localized or disseminated fungal disease as confirmed by histological examination. The respiratory tract was most commonly affected (67%), followed by the central nervous system (CNS, 33%), and auditory system (AS, 17%). Aspergillosis was confirmed for 11/18, by fungal growth (as A. fumigatus species complex, n = 9) and PCR (as Aspergillus spp., n = 1, and as A. fumigatus sensu strictu by sequence analysis, n = 1). One live stranded and euthanized animal presented partial hemiplegia of the blowhole and therefore an MRI was conducted, which resulted in a unique image of the extensive, fungus-induced lesion in the left cerebellar hemisphere, deforming and displacing the brainstem, and additionally affected the AS. The gross- and histologic lesions in the 18 porpoises diagnosed with fungal disease were similar to changes described in other mammalian species. The prevalence of fungal disease in free-living harbor porpoises is lower than seen in captive and rehabilitated animals, suggesting that captivity increases the risk to develop mycotic infections. Finally, fungal infection in the CNS and AS are usually considered consequences of vascular dissemination originating from pulmonary foci. However, only 1/7 cases with otitis and/or encephalitis demonstrated pulmonary aspergillosis, suggesting a different pathogenesis.

Introduction: Recognizing fungal keratitis based on the clinical presentation is challenging. Topical therapy may be initiated with antibacterial agents and corticosteroids, thus delaying the fungal diagnosis. As a consequence, the fungal infection may progress ultimately leading to more severe infection and blindness. We noticed an increase of fungal keratitis cases in the Netherlands, especially caused by Fusarium species, which prompted us to conduct a retrospective cohort study, aiming to describe the epidemiology, clinical management, and outcome.Materials and Methods: As fungi are commonly sent to the Dutch mycology reference laboratory for identification and in vitro susceptibility testing, the fungal culture collection was searched for Fusarium isolates from corneal scrapings, corneal swabs, and from contact lens (CL) fluid, between 2005 and 2016. All Fusarium isolates had been identified up to species level through sequencing of the ITS1-5.8S-ITS2 region of the rDNA and TEF1 gene. Antifungal susceptibility testing was performed according to the EUCAST microbroth dilution reference method. Antifungal agents tested included amphotericin B, voriconazole, and natamycin. In addition, susceptibility to the antisepticum chlorhexidine was tested. Ophthalmologists were approached to provide demographic and clinical data of patients identified through a positive culture.Results: Between 2005 and 2016, 89 cases of Fusarium keratitis from 16 different hospitals were identified. The number of cases of Fusarium keratitis showed a significant increase over time (R2 = 0.9199), with one case in the first 5 years (2005–2009) and multiple cases from 2010 and onwards. The male to female ratio was 1:3 (p = 0.014). Voriconazole was the most frequently used antifungal agent, but treatment strategies differed greatly between cases including five patients that were treated with chlorhexidine 0.02% monotherapy. Keratitis management was not successful in 27 (30%) patients, with 20 (22%) patients requiring corneal transplantation and seven (8%) requiring enucleation or evisceration. The mean visual acuity (VA) was moderately impaired with a logMAR of 0.8 (95% CI 0.6–1, Snellen equivalent 0.16) at the time of Fusarium culture. Final average VA was within the range of normal vision [logMAR 0.2 (95% CI 0.1–0.3), Snellen equivalent 0.63]. CL wear was reported in 92.9% of patients with Fusarium keratitis. The time between start of symptoms and diagnosis of fungal keratitis was significantly longer in patients with poor outcome as opposed to those with (partially) restored vision; 22 vs. 15 days, respectively (mean, p = 0.024). Enucleation/evisceration occurred in patients with delayed fungal diagnosis of more than 14 days after initial presentation of symptoms. The most frequently isolated species was F. oxysporum (24.7%) followed by F. solani sensu stricto (18%) and F. petroliphilum (9%). The lowest MICs were obtained with amphotericin B followed by natamycin, voriconazole, and chlorhexidine.Conclusion: Although Fusarium keratitis remains a rare complication of CL wear, we found a significant increase of cases in the Netherlands. The course of infection may be severe and fungal diagnosis was often delayed. Antifungal treatment strategies varied widely and the treatment failure rate was high, requiring transplantation or even enucleation. Our study underscores the need for systematic surveillance of fungal keratitis and a consensus management protocol.

Background: Critically ill COVID-19 patients have proven to be at risk for developing invasive fungal infections. However, the incidence and impact of possible/probable COVID-19-associated pulmonary aspergillosis (CAPA) in severe COVID-19 patients varies between cohorts. We aimed to assess the incidence, risk factors, and clinical outcome of invasive pulmonary aspergillosis in a regional cohort of COVID-19 intensive care patients. Methods: We performed a regional, multicentre, retrospective cohort study in the intensive care units (ICUs) in North Brabant, The Netherlands. We included adult patients with rt-PCR-confirmed SARS-CoV-2 infection (COVID-19), requiring mechanical ventilation for acute respiratory distress syndrome. Demographics, clinical course, biomarker value, and treatment outcomes were compared between the groups with possible/probable CAPA from the main study centre and the regional centres, and without signs of CAPA from the main study centre as controls. The primary aim was to assess the regional impact of possible/probable CAPA in COVID-19 ICU patients, measured as all-cause mortality at 30 days after ICU admission. Secondary outcomes were risk factors for developing CAPA, based on underlying host factors and to identify the value of the mycological arguments for the diagnosing of CAPA. Results: Between 1 March and 30 April 2020, we included 123 patients with severe COVID-19: 29 patients (30.9%) in the main ICU with possible/probable CAPA, and 65 (69.1%) with no signs of CAPA; 29 patients in the regional ICUs with signs of CAPA. Patients’ characteristics and risk factors did not differ for CAPA and non-CAPA patients. Patients with COPD and/or chronic steroid medication developed CAPA more frequently, although this was not statistically significant. CAPA patients were admitted to the ICU earlier, had lower PF-ratios, and more often required renal replacement therapy. All-cause 30-day mortality was significantly higher in mechanically ventilated COVID-19 patients with possible/probable CAPA 39.7% (23/58) compared to patients without evidence for CAPA 16.9% (11/65) (OR 3.2 [95% CI 1.4–7.4] p = 0.005). Conclusion: The high incidence of possible and probable CAPA in critically ill COVID-19 patients is alarming. The increase in 30-day mortality in CAPA highlights the need for active surveillance and management strategies in critically ill COVID-19 patients.

Tuberculosis produces two clinical manifestations: active and latent (non-apparent) disease. The latter is estimated to affect one-third of the world population and constitutes a source of continued transmission should the disease emerge from its hidden state (reactivation). Methods to diagnose latent TB have been evolving and aim to detect the disease in people who are truly infected with M. tuberculosis, versus those where other mycobacteria, or even other pathologies not related to TB, are present. The current use of proteomic and transcriptomic approaches may lead to improved detection methods in the coming years.

We collected sputum samples and cough plates from 15 cystic fibrosis patients in the Netherlands who were colonized with Aspergillus fumigatus; we recovered A. fumigatus of the same genotype in cough aerosols and sputum samples from 2 patients. The belief that transmission of A. fumigatus from cystic fibrosis patients does not occur should be reconsidered.

We investigated azole resistance in Aspergillus fumigatus in a tertiary reference hospital in the Netherlands during 1994–2016. The 5-year patient-adjusted proportion of resistance increased from 0.79% for 1996–2001 to 4.25% for 2002–2006, 7.17% for 2007–2011, and 7.04% for 2012–2016. However, we observed substantial variation between years.

Effective management and treatment of fungal diseases is hampered by poor diagnosis, limited options for antifungal therapy, and the emergence of antifungal drug resistance. An understanding of molecular mechanisms contributing to resistance is essential to optimize the efficacy of currently available antifungals. In this perspective, one of the oldest antifungals, 5-fluorocytosine (5-FC), has been the focus of recent studies applying advanced genomic and transcriptomic techniques to decipher the order of events at the molecular level that lead to resistance. These studies have highlighted the complexity of resistance and provided new insights that are reviewed in the present paper.

In order to successfully infect or colonize human hosts or survive changing environments, Aspergillus fumigatus needs to adapt through genetic changes or phenotypic plasticity. The genomic changes are based on the capacity of the fungus to produce genetic variation, followed by selection of the genotypes that are most fit to the new environment. Much scientific work has focused on the metabolic plasticity, biofilm formation or the particular genetic changes themselves leading to adaptation, such as antifungal resistance in the host. Recent scientific work has shown advances made in understanding the natural relevance of parasex and how both the asexual and sexual reproduction can lead to tandem repeat elongation in the target gene of the azoles: the cyp51A gene. In this review, we will explain how the fungus can generate genetic variation that can lead to adaptation. We will discuss recent advances that have been made in the understanding of the lifecycle of A. fumigatus to explain the differences observed in speed and type of mutations that are generated under different environments and how this can facilitate adaptation, such as azole-resistance selection.

Invasive aspergillosis due to azole-resistant Aspergillus fumigatus is difficult to manage. We describe a case of azole-resistant invasive aspergillosis in a female bottlenose dolphin, who failed to respond to voriconazole and posaconazole therapy. As intravenous therapy was precluded, high dose posaconazole was initiated aimed at achieving trough levels exceeding 3 mg/l. Posaconazole serum levels of 3–9.5 mg/l were achieved without significant side-effects. Follow-up bronchoscopy and computed tomography showed complete resolution of the lesions.

Azole-resistance in Aspergillus fumigatus is an emerging worldwide threat as it precludes the use of one of the 3 major classes of antifungal drugs to treat chronic and invasive aspergillosis [1]. In addition to the well-known environmental emergence of azole-resistant A. fumigatus strains, associated with the use of fungicides in agriculture [2,3], the development of in-host resistance, facilitated by medical antifungal use, has been described [4]. Investigations involving linked sets of (isogenic) clinical isolates of A. fumigatus sequentially recovered from individual patients, are extremely important in order to improve our understanding of how azole resistance develops in-host. Here we present the whole genome sequences of 13 clinical isogenic A. fumigatus isolates. These isolates were cultured from a single patient suffering from invasive aspergillosis over a period of 2 years. This patient underwent a wide range of antifungal therapies and the resultant isolates acquired multiple azole resistance in-host during the course of infection. The data presented here is related to our research paper titled “In-host microevolution of Aspergillus fumigatus: a phenotypic and genotypic analysis” which describes the phenotypic characterisation of these clinical isolates [5]. The raw sequence data was deposited in the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra), under BioProject ID number PRJNA528395.

For various studies in the clinic as well as the environment, it is essential to be able to selectively isolate Aspergillus fumigatus from samples containing bacteria as well as various other fungi (mainly Mucorales). Six agar media were compared for effectiveness in selectively isolating Aspergillus fumigatus from agricultural plant waste, woodchip waste, green waste, soil, grass and air samples collected in The Netherlands at a 48 °C incubation. The Flamingo Medium incubated at 48 °C, provided the most effective condition for the isolation of A. fumigatus from environmental samples, since it effectively inhibited the growth of competing fungi (mainly Mucorales) present in the environmental samples. Flamingo Medium reduced the number of colonies of Mucorales species by 95% and recovered an average of 20−30% more A. fumigatus colonies compared to the other media. We further confirmed that Flamingo Medium can inhibit the growth of clinical Mucorales, which occasionally present in patient’s tissue and can also be used for clinical applications. We suggest the use of Flamingo Medium as an efficient method for the study of A. fumigatus from important environmental niches for which there is increasing interest. Additionally, it can also be used in the clinic to isolate A. fumigatus especially from tissue contaminated with Mucorales.

Aspergillus spp. is an opportunistic human pathogen that may cause a spectrum of pulmonary diseases. In order to establish infection, inhaled conidia must germinate, whereby they break dormancy, start to swell, and initiate a highly polarized growth process. To identify critical biological processes during germination, we performed a cross-platform, cross-species comparative analysis of germinating A. fumigatus and A. niger conidia using transcriptional data from published RNA-Seq and Affymetrix studies. A consensus co-expression network analysis identified four gene modules associated with stages of germination. These modules showed numerous shared biological processes between A. niger and A. fumigatus during conidial germination. Specifically, the turquoise module was enriched with secondary metabolism, the black module was highly enriched with protein synthesis, the darkgreen module was enriched with protein fate, and the blue module was highly enriched with polarized growth. More specifically, enriched functional categories identified in the blue module were vesicle formation, vesicular transport, tubulin dependent transport, actin-dependent transport, exocytosis, and endocytosis. Genes important for these biological processes showed similar expression patterns in A. fumigatus and A. niger, therefore, they could be potential antifungal targets. Through cross-platform, cross-species comparative analysis, we were able to identify biologically meaningful modules shared by A. fumigatus and A. niger, which underscores the potential of this approach.

Triazole-resistance has been reported increasingly in Aspergillus fumigatus. An international expert team proposed to avoid triazole monotherapy for the initial treatment of invasive aspergillosis in regions with >10% environmental-resistance, but this prevalence is largely unknown for most American and African countries. Here, we screened 584 environmental samples (soil) from urban and rural locations in Mexico, Paraguay, and Peru in Latin America and Benin and Nigeria in Africa for triazole-resistant A. fumigatus. Samples were screened using triazole-containing agars and confirmed as triazole-resistant by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth dilution reference method. Isolates were further characterized by cyp51A sequencing and short-tandem repeat typing. Fungicide presence in samples was likewise determined. Among A. fumigatus positive samples, triazole-resistance was detected in 6.9% (7/102) of samples in Mexico, 8.3% (3/36) in Paraguay, 9.8% (6/61) in Peru, 2.2% (1/46) in Nigeria, and none in Benin. Cyp51A gene mutations were present in most of the triazole-resistant isolates (88%; 15/17). The environmentally-associated mutations TR34/L98H and TR46/Y121F/T289A were prevalent in Mexico and Peru, and isolates harboring these mutations were closely related. For the first time, triazole-resistant A. fumigatus was found in environmental samples in Mexico, Paraguay, Peru, and Nigeria with a prevalence of 7–10% in the Latin American countries. Our findings emphasize the need to establish triazole-resistance surveillance programs in these countries.

Fungal keratitis is difficult to treat, especially Fusarium keratitis. In vitro studies show that chlorhexidine could be an interesting option as monotherapy. We describe a case series of four patients (four eyes) with Fusarium keratitis at Radboud University Medical Center (Nijmegen, the Netherlands). The patients were treated with chlorhexidine 0.02% eye drops. The in vitro activity of eight antifungals and chlorhexidine was determined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. We also reviewed the literature on the use of chlorhexidine in the treatment of fungal keratitis. Topical chlorhexidine was well tolerated, and all patients showed complete resolution of the keratitis upon treatment with chlorhexidine. A PubMed search of the available literature was conducted (last search 8 March 2020) and yielded two randomized clinical trials (natamycin versus chlorhexidine) and one case report addressing the treatment of fungal keratitis with chlorhexidine. Chlorhexidine was found to be safe with regard to toxicity and to be superior to natamycin in the clinical trials. Chlorhexidine showed in vitro fungicidal activity against Fusarium and clinical effectiveness in our cases, supporting further clinical evaluation. Advantages of chlorhexidine are its topical application, its general availability, its low costs, its broad-spectrum activity, and its fungicidal mechanism of action at low concentrations.

Invasive fungal diseases (IFDs) often represent complicated infections in complex patient populations. The Center of Expertise in Mycology Radboudumc/CWZ (EMRC) organizes a biweekly multidisciplinary mycology meeting to discuss patients with severe fungal infections and to provide comprehensive advice regarding diagnosis and treatment. Here, we describe the patient population discussed at these meetings during a one-year period with regards to their past medical history, diagnosis, microbiological and other diagnostic test results and antifungal therapy. The majority of patients discussed were adults (83.1%), 62.5% of whom suffered from pulmonary infections or signs/symptoms, 10.9% from otorhinolaryngeal infections and/or oesophagitis, 9.4% from systemic infections and 9.4% from central nervous system infections. Among children, 53.8% had pulmonary infections or signs/symptoms, 23.1% systemic fungal infections and 23.1% other, miscellaneous fungal infections. 52.5% of adult patients with pulmonary infections/symptoms fulfilled diagnostic criteria for chronic pulmonary aspergillosis (CPA). Culture or polymerase chain reaction (PCR) demonstrated fungal pathogens in 81.8% of patients, most commonly Aspergillus. A multidisciplinary mycology meeting can be a useful addition to the care for patients with (I)FDs and can potentially aid in identifying healthcare and research needs regarding the field of fungal infections. The majority of patients discussed at the multidisciplinary meetings suffered from pulmonary infections, predominantly CPA.

Azole-resistant Aspergillus fumigatus isolates recovered at high frequency from patients, harbor mutations that are associated with variation of promoter length in the cyp51A gene. Following the discovery of the TR34/L98H genotype, new variations in tandem repeat (TR) length and number of repeats were identified, as well as additional single nucleotide polymorphisms (SNPs) in the cyp51A gene, indicating that the diversity of resistance mutations in A. fumigatus is likely to continue to increase. Investigating the development routes of TR variants is critical to be able to design preventive interventions. In this study, we tested the potential effects of azole exposure on the selection of TR variations, while allowing haploid A. fumigatus to undergo asexual reproduction. Through experimental evolution involving voriconazole (VOR) exposure, an isolate harboring TR343/L98H evolved from a clinical TR34/L98H ancestor isolate, confirmed by whole genome sequencing. TR343/L98H was associated with increased cyp51A expression and high VOR and posaconazole MICs, although additional acquired SNPs could also have contributed to the highly azole-resistant phenotype. Exposure to medical azoles was found to select for TR343, thus supporting the possibility of in-host selection of TR34 variants.

The disease caused by the new SARS-CoV-2, known as Coronavirus disease 2019 (COVID-19), was first identified in China in December 2019 and rapidly spread around the world. Coinfections with fungal pathogens in patients with COVID-19 add challenges to patient care. We conducted a literature review on fungal coinfections in patients with COVID-19. We describe a report of a patient with disseminated histoplasmosis who was likely infected with SARS-CoV-2 and experienced COVID-19 during hospital care in Buenos Aires, Argentina. This patient presented with advanced HIV disease, a well-known factor for disseminated histoplasmosis; on the other hand, we suspected that COVID-19 was acquired during hospitalization but there is not enough evidence to support this hypothesis. Clinical correlation and the use of specific Histoplasma and COVID-19 rapid diagnostics assays were key to the timely diagnosis of both infections, permitting appropriate treatment and patient care.

Recently, mutations in the 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase-encoding gene (hmg1), a gene involved in ergosterol production, were associated with triazole-resistance in Aspergillus fumigatus. In this study, we determined the prevalence and characteristics of hmg1 mutations in a collection of clinical triazole-resistant A. fumigatus isolates collected during 2001–2019 from two international mycology reference centers: the Belgian National Reference Center for Mycosis and the Center of Expertise in Mycology Radboudumc/CWZ. Clinical isolates with and without cyp51A gene mutations and randomly selected wild-type (WT) controls were included. Isolates were characterized by in vitro susceptibility testing, cyp51A and hmg1 sequencing, and short tandem repeat typing. Available clinical records were analyzed for previous triazole exposure. In 23 isolates (24%) of the 95 triazole-resistant A. fumigatus isolates, hmg1 gene mutations were observed; including 5/23 (22%) isolates without cyp51A gene mutations and 18/72 (25%) with cyp51A mutations. Four previously described hmg1 gene mutations (E105K, G307R/D, G466V, and S541G) and two novel mutations (W273S and L304P) were found; 4/23 (17%) in the sterol-sensing-domain region. No triazole-antifungal exposure was reported in 75% (9/12) of patients harboring an isolate with hmg1 gene mutations. Three of 39 WT isolates (8%) contained a hmg1 gene mutation; E105K (2-isolates) and S541G. Hmg1 gene mutations were predominantly found in A. fumigatus with cyp51A mutations with voriconazole MICs ≥ 8 mg/L.

Cystic fibrosis (CF) can be complicated by fungal infection of the respiratory tract. Fungal detection rates in CF sputa are highly dependent on the culture protocol and incubation conditions and thus may lead to an underestimation of the true prevalence of fungal colonization. We conducted a prospective study to evaluate the additional value of mucolytic pre-treatment, increased inoculum (100 μL), additional fungal culture media (Sabouraud agar; SAB, Medium B+, Scedosporium selective agar; SceSel+ and Dichloran-Glycerol agar; DG18) and longer incubation time (3 weeks) compared with our current protocol. Using the new protocol, we prospectively analyzed 216 expectorated sputum samples from adult and pediatric CF patients (n = 77) and compared the culture yield to a three year retrospective cohort that used direct 10 μL loop inoculation on SAB with 5 days incubation (867 sputum samples/103 patients). Detection rates for molds increased from 42% to 76% (p < 0.0001). Twenty-six percent of cultures were polymicrobial in the prospective cohort as opposed to 4.7% in the retrospective cohort (p < 0.0001). Colonization rate with A. fumigatus increased from 36% to 57%. SAB and DG18 showed the highest detection rates for all molds (SAB 58.6%; DG18 56.9%) and DG18 had the best performance for molds other than A. fumigatus. The larger sample volume and longer incubation also contributed to the increased recovery of molds. The introduction of a modified fungal culture protocol leads to a major increase in detection rate and the diversity of molds, which influences fungal epidemiology and may have implications for treatment decisions.

Antimicrobial peptides and proteins (AMPs) provide an important line of defence against invading microorganisms. However, the activity of AMPs against the human fungal pathogen Aspergillus fumigatus remains poorly understood. Therefore, the aim of this study was to characterise the anti-Aspergillus activity of specific human AMPs, and to determine whether A. fumigatus can possess resistance to specific AMPs, as a result of in-host adaptation. AMPs were tested against a wide range of clinical isolates of various origins (including cystic fibrosis patients, as well as patients with chronic and acute aspergillosis). We also tested a series of isogenic A. fumigatus isolates obtained from a single patient over a period of 2 years. A range of environmental isolates, obtained from soil in Scotland, was also included. Firstly, the activity of specific peptides was assessed against hyphae using a measure of fungal metabolic activity. Secondly, the activity of specific peptides was assessed against germinating conidia, using imaging flow cytometry as a measure of hyphal growth. We showed that lysozyme and histones inhibited hyphal metabolic activity in all the A. fumigatus isolates tested in a dose-dependent fashion. In addition, imaging flow cytometry revealed that histones, β-defensin-1 and lactoferrin inhibited the germination of A. fumigatus conidia.

Invasive aspergillosis (IA) is a deep tissue infection with a high mortality occurring mostly in immunocompromised patients. To investigate the pathology of patients with IA it may be important to determine the genotype of the invasive isolate of Aspergillus, however available tissues for study are often formalin fixed paraffin embedded (FFPE). Although DNA has been successfully isolated from such tissues for species identification, genotyping of Aspergillus species on such tissues has not yet been performed. In this study we aimed to determine the genotype of Aspergillus fumigatus in FFPE tissue and serum samples from five patients with invasive aspergillosis using nine highly polymorphic short tandem repeat (STRAf) loci. FFPE lung and bronchial biopsies from all patients were successfully typed. By comparing the latter result with non-FFPE materials from non-sterile samples such as sputum, bronchoalveolar lavage and lung abscess, we found identical genotypes within three patients, while the two other patients had a dominant genotype shared among all sample types. Genotyping of serum samples was successful in two serum samples with galactomannan ratios of 4 and 5.6, but failed in serum samples with galactomannan levels

Aspergillus diseases are often caused by Aspergillus fumigatus. Azoles are the mainstay of therapy, but the management of aspergillosis is hampered by the emergence of azole resistance. Rapid detection of azole resistance might benefit treatment outcome by early treatment modifications. However, the yield of fungal culture in invasive aspergillosis is low and susceptibility testing requires several days to be completed. To overcome the low yield of fungal cultures and slow detection of resistance, it is possible to use molecular tools directly on clinical specimens in order to rapidly detect molecular markers of azole resistance. Molecular tools to detect resistant markers in the Cyp51A gene can be expected to be less sensitive compared to molecular tools to detect Aspergillus DNA as the Cyp51A gene is a single copy gene and the target for Aspergillus DNA is often a multi-copy gene. In this mini-review, we summarize the current molecular tools for detection of azole-resistant A. fumigatus directly in clinical material. Several in-house PCR assays have been applied directly on clinical material. Furthermore, two assays are commercial available; the AsperGenius and MycoGENIE. The amplification of resistance markers was successful in 70–100% of samples that were positive for Aspergillus DNA in BAL samples using the AsperGenius assay. Despite using several samples per patient, amplification of resistance markers was only successful in 33–57% of patients with Aspergillus DNA in blood. Furthermore, several sequence based methods have been applied with the benefit of the ability to detect other Cyp51A gene alterations.

ABSTRACT Cryptococcosis is a major fungal disease caused by members of the Cryptococcus gattii and Cryptococcus neoformans species complexes. After more than 15 years of molecular genetic and phenotypic studies and much debate, a proposal for a taxonomic revision was made. The two varieties within C. neoformans were raised to species level, and the same was done for five genotypes within C. gattii. In a recent perspective (K. J. Kwon-Chung et al., mSphere 2:e00357-16, 2017, https://doi.org/10.1128/mSphere.00357-16 ), it was argued that this taxonomic proposal was premature and without consensus in the community. Although the authors of the perspective recognized the existence of genetic diversity, they preferred the use of the informal nomenclature “C. neoformans species complex” and “C. gattii species complex.” Here we highlight the advantage of recognizing these seven species, as ignoring these species will impede deciphering further biologically and clinically relevant differences between them, which may in turn delay future clinical advances.