A Pathology-based Case Series of Influenza- and COVID-19-associated Pulmonary Aspergillosis: The Proof Is in the Tissue.

Rationale: Invasive pulmonary aspergillosis has emerged as a frequent coinfection in severe coronavirus disease (COVID-19), similarly to influenza, yet the clinical invasiveness is more debated. Objectives: We investigated the invasive nature of pulmonary aspergillosis in histology specimens of influenza and COVID-19 ICU fatalities in a tertiary care center. Methods: In this monocentric, descriptive, retrospective case series, we included adult ICU patients with PCR-proven influenza/COVID-19 respiratory failure who underwent postmortem examination and/or tracheobronchial biopsy during ICU admission from September 2009 until June 2021. Diagnosis of probable/proven viral-associated pulmonary aspergillosis (VAPA) was made based on the Intensive Care Medicine influenza-associated pulmonary aspergillosis and the European Confederation of Medical Mycology (ECMM) and the International Society for Human and Animal Mycology (ISHAM) COVID-19-associated pulmonary aspergillosis consensus criteria. All respiratory tissues were independently reviewed by two experienced pathologists. Measurements and Main Results: In the 44 patients of the autopsy-verified cohort, 6 proven influenza-associated and 6 proven COVID-19-associated pulmonary aspergillosis diagnoses were identified. Fungal disease was identified as a missed diagnosis upon autopsy in 8% of proven cases (n = 1/12), yet it was most frequently found as confirmation of a probable antemortem diagnosis (n = 11/21, 52%) despite receiving antifungal treatment. Bronchoalveolar lavage galactomannan testing showed the highest sensitivity for VAPA diagnosis. Among both viral entities, an impeded fungal growth was the predominant histologic pattern of pulmonary aspergillosis. Fungal tracheobronchitis was histologically indistinguishable in influenza (n = 3) and COVID-19 (n = 3) cases, yet macroscopically more extensive at bronchoscopy in influenza setting. Conclusions: A proven invasive pulmonary aspergillosis diagnosis was found regularly and with a similar histological pattern in influenza and in COVID-19 ICU case fatalities. Our findings highlight an important need for VAPA awareness, with an emphasis on mycological bronchoscopic work-up.

Triazole resistance in Aspergillus fumigatus isolates in Africa: a systematic review.

Emergence of triazole resistance has been observed in Aspergillus fumigatus over the past decade including Africa. This review summarizes the current published data on the epidemiology and reported mechanisms of triazole-resistant Aspergillus fumigatus (TRAF) in both environmental and clinical isolates from Africa. Searches on databases Medline, PubMed, HINARI, Science Direct, Scopus and Google Scholar on triazole resistance published between 2000 and 2021 from Africa were performed. Isolate source, antifungal susceptibility using internationally recognized methods, cyp51A mechanism of resistance and genotype were collected. Eleven published African studies were found that fitted the search criteria; these were subsequently analyzed. In total this constituted of 1686 environmental and 46 clinical samples. A TRAF prevalence of 17.1% (66/387) and 1.3% (5/387) was found in respectively environmental and clinical settings in African studies. Resistant to itraconazole, voriconazole, and posaconazole was documented. Most of the triazole-resistant isolates (30/71, 42.25%) were found to possess the TR34/L98H mutation in the cyp51A-gene; fewer with TR46/Y121F/T289A (n = 8), F46Y/M172V/E427K (n = 1), G54E (n = 13), and M172V (n = 1) mutations. African isolates with the TR34/L98H, TR46/Y121F/T289A and the G54E mutations were closely related and could be grouped in one of two clusters (cluster-B), whereas the cyp51A-M172V mutation clustered with most cyp51A-WT strains (cluster-A). A single case from Kenya shows that TR34/L98H from environmental and clinical isolates are closely related. Our findings highlight that triazole resistance in environmental and clinical A. fumigatus is a cause for concern in a number of African countries. There is need for epidemiological surveillance to determine the true burden of the problem in Africa. LAY SUMMARY: Emergence of triazole resistance has been observed in Aspergillus fumigatus. TRAF was found from environmental (17.1%) and clinical (1.3%) settings in Africa. We highlighted that triazole resistance in environmental and clinical A. fumigatus is a cause for concern in a number of African countries.

Stable prevalence of triazole-resistance in Aspergillus fumigatus complex clinical isolates in a Belgian tertiary care center from 2016 to 2020.

BACKGROUND: Prevalence reports of triazole-resistance in Aspergillus fumigatus differ between countries and centers and may likewise vary over time. Continuous local surveillance programs to establish the evolving epidemiology of triazole-resistance in A. fumigatus are crucial to guide therapeutic recommendations. Here, we determined the prevalence of triazole-resistance in A. fumigatus complex culture-positive patients at the tertiary care center University Hospitals Leuven in Belgium in clinical isolates from 2016 to 2020. METHODS: All A. fumigatus complex isolates cultured from UZ Leuven patients between 2016 and 2020 were screened for triazole-resistance. Confirmation of resistance to voriconazole, posaconazole and itraconazole was performed with the European Committee for Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. Mutations in the cyp51A gene in triazole-resistant isolates were determined by sequencing. Patients were classified as susceptible or resistant cases based on their isolate's susceptibility phenotype. RESULTS: We screened 2494 A. fumigatus complex isolates from 1600 patients (320 ± 38 [SD] patients per year). The prevalence of triazole-resistance in patients was 8.3% (28/337), 6.7% (26/386), 7.0% (21/301), 7.1% (21/294) and 7.4% (21/282) in 2016, 2017, 2018, 2019 and 2020 respectively, with an overall triazole-resistance prevalence of 7.1% (85/1192; 95% CI 6.6-7.7%). The TR34/L98H mutation was the most prevalent (83.0%, 78/94) with most isolates displaying resistance to all triazole antifungals tested (94.8%, 74/78). CONCLUSION: The prevalence of triazole-resistance in A. fumigatus has remained stable from 2016 to 2020 in our center ranging between 6.7 and 8.3%, with an overall five-year prevalence of 7.1%. The environmentally associated cyp51A gene mutations were most prevalent amongst triazole-resistant isolates and conferred resistance to all antifungals tested in 73% of the isolates.

Prevalence of voriconazole-resistant invasive aspergillosis and its impact on mortality in haematology patients.

BACKGROUND: Increasing resistance of Aspergillus fumigatus to triazoles in high-risk populations is a concern. Its impact on mortality is not well understood, but rates from 50% to 100% have been reported. OBJECTIVES: To determine the prevalence of voriconazole-resistant A. fumigatus invasive aspergillosis (IA) and its associated mortality in a large multicentre cohort of haematology patients with culture-positive IA. METHODS: We performed a multicentre retrospective study, in which outcomes of culture-positive haematology patients with proven/probable IA were analysed. Patients were stratified based on the voriconazole susceptibility of their isolates (EUCAST broth microdilution test). Mycological and clinical data were compared, along with survival at 6 and 12 weeks. RESULTS: We identified 129 A. fumigatus culture-positive proven or probable IA cases; 103 were voriconazole susceptible (79.8%) and 26 were voriconazole resistant (20.2%). All but one resistant case harboured environment-associated resistance mutations in the cyp51A gene: TR34/L98H (13 cases) and TR46/Y121F/T289A (12 cases). Triazole monotherapy was started in 75.0% (97/129) of patients. Mortality at 6 and 12 weeks was higher in voriconazole-resistant cases in all patients (42.3% versus 28.2%, P = 0.20; and 57.7% versus 36.9%, P = 0.064) and in non-ICU patients (36.4% versus 21.6%, P = 0.16; and 54.4% versus 30.7%; P = 0.035), compared with susceptible ones. ICU patient mortality at 6 and 12 weeks was very high regardless of triazole susceptibility (75.0% versus 66.7%, P = 0.99; and 75.0% versus 73.3%, P = 0.99). CONCLUSIONS: A very high prevalence of voriconazole resistance among culture-positive IA haematology patients was observed. The overall mortality at 12 weeks was significantly higher in non-ICU patients with voriconazole-resistant IA compared with voriconazole-susceptible IA.

Gliotoxin and bis(methylthio)gliotoxin are not reliable as biomarkers of invasive aspergillosis.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) remains a life-threatening opportunistic infection, but can be difficult to diagnose. New biomarkers are therefore needed. Gliotoxin (GT), a secondary metabolite of Aspergillus fumigatus, and bis(methylthio)gliotoxin (bmGT), a degradation product of GT, have been proposed as potential biomarkers. However, these findings have yet to be confirmed. OBJECTIVES: To identify the diagnostic potential of GT and bmGT in serum and bronchoalveolar lavage fluid (BALf) in haematology patients compared to galactomannan (GM). MATERIALS AND METHODS: We prospectively collected culture supernatant, serum and BALf from patients with culture-positive IPA and measured GT and bmGT concentrations using ultra high-performance liquid chromatography-quadrupole time of flight mass spectrometry. Galactomannan was detected using a commercially available enzyme immunoassay. RESULTS: We included 18 patients with proven (n = 6) and probable (n = 12) IPA, all with positive cultures for Aspergillus fumigatus. BmGT was only detected in serum from one patient (5.6%), whereas GM was positive (optical density ≥ 0.5) in 11/18 patients (61.1%, P = 0.002). We could not find GT in any serum sample. In BALf, bmGT was detected in 8/18 patients (44.4%) and GT in 9/18 patients (50%), compared to GM (optical density ≥ 1.0) in all patients (100%). CONCLUSIONS: Gliotoxin and bis(methylthio)gliotoxin had a very poor performance for diagnosing IPA. As other biomarkers are more sensitive and easier to detect, we would not recommend serum or BALf GT/bmGT to be used in the diagnosis of IPA.

Triazole resistance surveillance in Aspergillus fumigatus.

Triazole resistance is an increasing concern in the opportunistic mold Aspergillus fumigatus. Resistance can develop through exposure to azole compounds during azole therapy or in the environment. Resistance mutations are commonly found in the Cyp51A-gene, although other known and unknown resistance mechanisms may be present. Surveillance studies show triazole resistance in six continents, although the presence of resistance remains unknown in many countries. In most countries, resistance mutations associated with the environment dominate, but it remains unclear if these resistance traits predominately migrate or arise locally. Patients with triazole-resistant aspergillus disease may fail to antifungal therapy, but only a limited number of cohort studies have been performed that show conflicting results. Treatment failure might be due to diagnostic delay or due to the limited number of alternative treatment options. The ISHAM/ECMM Aspergillus Resistance Surveillance working group was set up to facilitate surveillance studies and stimulate international collaborations. Important aims are to determine the resistance epidemiology in countries where this information is currently lacking, to gain more insight in the clinical implications of triazole resistance through a registry and to unify nomenclature through consensus definitions.

Sensitive bioluminescence imaging of cryptococcosis in Galleria mellonella improves antifungal screening under in vivo conditions.

Cryptococcus neoformans is an environmental yeast that primarily affects immunocompromised individuals, causing respiratory infections and life-threatening meningoencephalitis. Treatment is complicated by limited antifungal options, with concerns such as adverse effects, dose-limiting toxicity, blood-brain barrier permeability, and resistance development, emphasizing the critical need to optimize and expand current treatment options against invasive cryptococcosis. Galleria mellonella larvae have been introduced as an ethical intermediate for in vivo testing, bridging the gap between in vitro antifungal screening and mouse studies. However, current infection readouts in G. mellonella are indirect, insensitive, or invasive, which hampers the full potential of the model. To address the absence of a reliable non-invasive method for tracking infection, we longitudinally quantified the cryptococcal burden in G. mellonella using bioluminescence imaging (BLI). After infection with firefly luciferase-expressing C. neoformans, the resulting bioluminescence signal was quantitatively validated using colony-forming unit analysis. Longitudinal comparison of BLI to health and survival analysis revealed increased sensitivity of BLI in discriminating cryptococcal burden during early infection. Furthermore, BLI improved the detection of treatment efficacy using first-line antifungals, thereby benchmarking this model for antifungal testing. In conclusion, we introduced BLI as a real-time, quantitative readout of cryptococcal burden in G. mellonella over time, enabling more sensitive and reliable antifungal screening.

Damping excessive viral-induced IFN-γ rescues the impaired anti-Aspergillus host immune response in influenza-associated pulmonary aspergillosis.

BACKGROUND: Influenza-associated pulmonary aspergillosis (IAPA) is a severe fungal superinfection in critically ill influenza patients that is of incompletely understood pathogenesis. Despite the use of contemporary therapies with antifungal and antivirals, mortality rates remain unacceptably high. We aimed to unravel the IAPA immunopathogenesis as a means to develop adjunctive immunomodulatory therapies. METHODS: We used a murine model of IAPA to investigate how influenza predisposes to the development of invasive pulmonary aspergillosis. Immunocompetent mice were challenged with an intranasal instillation of influenza on day 0 followed by an orotracheal inoculation with Aspergillus 4 days later. Mice were monitored daily for overall health status, lung pathology with micro-computed tomography (µCT) and fungal burden with bioluminescence imaging (BLI). At endpoint, high parameter immunophenotyping, spatial transcriptomics, histopathology, dynamic phagosome biogenesis assays with live imaging, immunofluorescence staining, specialized functional phagocytosis and killing assays were performed. FINDINGS: We uncovered an early exuberant influenza-induced interferon-gamma (IFN-γ) production as the major driver of immunopathology in IAPA and delineated the molecular mechanisms. Specifically, excessive IFN-γ production resulted in a defective Th17-immune response, depletion of macrophages, and impaired killing of Aspergillus conidia by macrophages due to the inhibition of NADPH oxidase-dependent activation of LC3-associated phagocytosis (LAP). Markedly, mice with partial or complete genetic ablation of IFN-γ had a restored Th17-immune response, LAP-dependent mechanism of killing and were fully protected from invasive fungal infection. INTERPRETATION: Together, these results identify exuberant viral induced IFN-γ production as a major driver of immune dysfunction in IAPA, paving the way to explore the use of excessive viral-induced IFN-γ as a biomarker and new immunotherapeutic target in IAPA. FUNDING: This research was funded by the Research Foundation Flanders (FWO), project funding under Grant G053121N to JW, SHB and GVV; G057721N, G0G4820N to GVV; 1506114 N to KL and GVV; KU Leuven internal funds (C24/17/061) to GVV, clinical research funding to JW, Research Foundation Flanders (FWO) aspirant mandate under Grant 1186121N/1186123 N to LS, 11B5520N to FS, 1SF2222N to EV and 11M6922N/11M6924N to SF, travel grants V428023N, K103723N, K217722N to LS. FLvdV was supported by a Vidi grant of the Netherlands Association for Scientific Research. FLvdV, JW, AC and GC were supported by the Europeans Union's Horizon 2020 research and innovation program under grant agreement no 847507 HDM-FUN. AC was also supported by the Fundação para a Ciência e a Tecnologia (FCT), with the references UIDB/50026/2020, UIDP/50026/2020, PTDC/MED-OUT/1112/2021 (https://doi.org/10.54499/PTDC/MED-OUT/1112/2021), and 2022.06674.PTDC (http://doi.org/10.54499/2022.06674.PTDC); and the "la Caixa" Foundation under the agreement LCF/PR/HR22/52420003 (MICROFUN).

Bioluminescence Imaging, a Powerful Tool to Assess Fungal Burden in Live Mouse Models of Infection.

Aspergillus fumigatus and Cryptococcus neoformans species infections are two of the most common life-threatening fungal infections in the immunocompromised population. Acute invasive pulmonary aspergillosis (IPA) and meningeal cryptococcosis are the most severe forms affecting patients with elevated associated mortality rates despite current treatments. As many unanswered questions remain concerning these fungal infections, additional research is greatly needed not only in clinical scenarios but also under controlled preclinical experimental settings to increase our understanding concerning their virulence, host-pathogen interactions, infection development, and treatments. Preclinical animal models are powerful tools to gain more insight into some of these needs. However, assessment of disease severity and fungal burden in mouse models of infection are often limited to less sensitive, single-time, invasive, and variability-prone techniques such as colony-forming unit counting. These issues can be overcome by in vivo bioluminescence imaging (BLI). BLI is a noninvasive tool that provides longitudinal dynamic visual and quantitative information on the fungal burden from the onset of infection and potential dissemination to different organs throughout the development of disease in individual animals. Hereby, we describe an entire experimental pipeline from mouse infection to BLI acquisition and quantification, readily available to researchers to provide a noninvasive, longitudinal readout of fungal burden and dissemination throughout the course of infection development, which can be applied for preclinical studies into pathophysiology and treatment of IPA and cryptococcosis in vivo.

Microcomputed Tomography to Visualize and Quantify Fungal Infection Burden and Inflammation in the Mouse Lung Over Time.

Pulmonary mycoses are an important threat for immunocompromised patients, and although current treatments are effective, they suffer from multiple limitations and fail to further reduce mortality. With the increasing immunocompromised population and increased antifungal resistance, fungal infection research is more relevant than ever. In preclinical respiratory fungal infection research, animal models are indispensable. However, too often researchers still rely on endpoint measurements to assess fungal burden while the dynamics of disease progression are left undiscovered. To open up this "black box", microcomputed tomography (µCT) can be implemented to longitudinally visualize lung pathology in a noninvasive way and to quantify µCT-image derived biomarkers. That way, disease onset, progression, and responsiveness to treatment can be followed up with high resolution spatially and temporally in individual mice, increasing statistical power. Here, we describe a general method for the use of low-dose high-resolution µCT to longitudinally visualize and quantify lung pathology in mouse models of respiratory fungal infections, applied to mouse models of aspergillosis and cryptococcosis.

Powerful and Real-Time Quantification of Antifungal Efficacy against Triazole-Resistant and -Susceptible Aspergillus fumigatus Infections in Galleria mellonella by Longitudinal Bioluminescence Imaging.

Aspergillus fumigatus is an environmental mold that causes life-threatening respiratory infections in immunocompromised patients. The plateaued effectiveness of antifungal therapy and the increasing prevalence of triazole-resistant isolates have led to an urgent need to optimize and expand the current treatment options. For the transition of in vitro research to in vivo models in the time- and resource-consuming preclinical drug development pipeline, Galleria mellonella larvae have been introduced as a valuable in vivo screening intermediate. Despite the high potential of this model, the current readouts of fungal infections in G. mellonella are insensitive, irreproducible, or invasive. To optimize this model, we aimed for the longitudinal quantification of the A. fumigatus burden in G. mellonella using noninvasive bioluminescence imaging (BLI). Larvae were infected with A. fumigatus strains expressing a red-shifted firefly luciferase, and the substrate dosage was optimized for the longitudinal visualization of the fungal burden without affecting larval health. The resulting photon flux was successfully validated for fungal quantification against colony forming units (CFU) analyses, which revealed an increased dynamic range from BLI detection. Comparison of BLI to survival rates and health index scores additionally revealed improved sensitivity for the early discrimination of differences in fungal burdens as early as 1 day after infection. This was confirmed by the improved detection of treatment efficacy against triazole-susceptible and -resistant strains. In conclusion, we established a refined G. mellonella aspergillosis model that enables the noninvasive real-time quantification of A. fumigatus by BLI. This model provides a quick and reproducible in vivo system for the evaluation of treatment options and is in line with 3Rs recommendations. IMPORTANCE Triazole-resistant Aspergillus fumigatus strains are rapidly emerging, and resistant infections are difficult to treat, causing mortality rates of up to 88%. The recent WHO priority list underscores A. fumigatus as one of the most critical fungal pathogens for which innovative antifungal treatment should be (urgently) prioritized. Here, we deliver a Galleria mellonella model for triazole-susceptible and -resistant A. fumigatus infections combined with a statistically powerful quantitative, longitudinal readout of the A. fumigatus burden for optimized preclinical antifungal screening. G. mellonella larvae are a convenient invertebrate model for in vivo antifungal screenings, but so far, the model has been limited by variable and insensitive observational readouts. We show that bioluminescence imaging-based fungal burden quantification outperforms these readouts in reliability, sensitivity, and time to the detection of treatment effects in both triazole-susceptible and -resistant infections and can thus lead to better translatability from in vitro antifungal screening results to in vivo confirmation in mouse and human studies.

Performances of disk diffusion method for determining triazole susceptibility of Aspergillus species: Systematic review.

The therapeutic management of invasive aspergillosis should be guided by antifungal susceptibility testing (AFST). The disk diffusion (DD) method due to its simplicity and low cost could be an appropriate alternative to the reference methods (CLSI, EUCAST) which are not suitable for AFST in routine clinical microbiology laboratories, particularly in resource-constrained settings. This review summarizes the available data on the performance of the DD method in determining triazole susceptibility profile of Aspergillus species. The published articles on the performance of DD method for determining triazole susceptibility of Aspergillus spp. were systematically searched on major medical databases and Google Scholar. We identified 2725 articles of which 13 met the inclusion criteria. The overall average agreement value obtained between DD and CLSI broth microdilution (CLSI-BMD) methods for the itraconazole 10 µg disk (70.75%) was low especially when the medium used was not Mueller-Hinton (MH) agar. In contrast average agreement for the voriconazole 1 µg disk and the posaconazole 5 µg disk were > 94% regardless of media used. The correlation coefficient values between the DD and CLSI-BMD methods on MH agar were acceptable (≥ 0.71) for the itraconazole 10 µg disk and posaconazole 5 µg disk and good (≥ 0.80) for the voriconazole 1 and 10 µg disk. The reproducibility of the DD method regardless to the medium used was ≥ 82%. This systematic review shows that the disk diffusion method could be a real alternative for triazole antifungals susceptibility testing of Aspergillus spp.

Quantitative PCR Effectively Quantifies Triazole-Susceptible and Triazole-Resistant Aspergillus fumigatus in Mixed Infections.

Increasing resistance to triazole antifungals in Aspergillus fumigatus is worrisome because of the associated high mortality of triazole-resistant A. fumigatus (TRAF) infections. While most studies have focused on single triazole-susceptible (WT) or TRAF infections, reports of TRAF cases developing mixed WT and TRAF infections have been described in several studies. However, the prevalence of mixed infections and their responses to current recommended therapies are unknown and could be inappropriate, leading to poor clinical outcomes. To address the urgent need for tools to diagnose, monitor disease development and therapy efficacies in mixed infection settings where quantification of WT versus TRAF is key, this study developed a novel qPCR assay to differentiate WT and TRAF harboring the cyp51A-TR34/L98H mutation. The proposed assay successfully quantified A. fumigatus and discriminated TRAF-TR34 in vitro and in vivo, which was achieved by increasing the yield of extracted DNA through improved homogenization and specific primers targeting the WT-sequence or TR34-insertion and a TaqMan-probe directed to A. fumigatus. The here-developed qPCR assay overcomes sensitivity issues of methodologies such as CFU counts, providing specific, reproducible, and reliable quantitative information to study and follow up the (interplay and individual) effects of mixed A. fumigatus infections on disease development and treatment responses.

Longitudinal multimodal imaging-compatible mouse model of triazole-sensitive and -resistant invasive pulmonary aspergillosis.

Invasive pulmonary aspergillosis (IPA) caused by the mold Aspergillus fumigatus is one of the most important life-threatening infections in immunocompromised patients. The alarming increase of isolates resistant to the first-line recommended antifungal therapy urges more insights into triazole-resistant A. fumigatus infections. In this study, we systematically optimized a longitudinal multimodal imaging-compatible neutropenic mouse model of IPA. Reproducible rates of pulmonary infection were achieved through immunosuppression (sustained neutropenia) with 150 mg/kg cyclophosphamide at day -4, -1 and 2, and an orotracheal inoculation route in both sexes. Furthermore, increased sensitivity of in vivo bioluminescence imaging for fungal burden detection, as early as the day after infection, was achieved by optimizing luciferin dosing and through engineering isogenic red-shifted bioluminescent A. fumigatus strains, one wild type and two triazole-resistant mutants. We successfully tested appropriate and inappropriate antifungal treatment scenarios in vivo with our optimized multimodal imaging strategy, according to the in vitro susceptibility of our luminescent fungal strains. Therefore, we provide novel essential mouse models with sensitive imaging tools for investigating IPA development and therapy in triazole-susceptible and triazole-resistant scenarios.

First Investigative Study of Azole-Resistant Aspergillus fumigatus in the Environment in Burkina Faso.

Azole-resistant Aspergillus fumigatus (ARAF) strains have been reported on all continents, however, limited data exist on these strains in Africa, while several factors, mainly environmental ones, suggest their presence on this continent. This study aimed to assess the environmental prevalence of ARAF strains in Burkina Faso, a country situated in the West African region where data on ARAF is non-existent. In total, 120 environmental samples (soil) were collected and analyzed. Samples were screened for resistance using three azole-containing agar plates; one without azole antifungal (growth control) and two supplemented with either itraconazole (4 mg/L) or voriconazole (2 mg/L). The EUCAST susceptibility testing method was used to confirm the azole-resistant phenotype of A. fumigatus sensu-stricto isolates. Mutations in the cyp51A gene were determined by sequencing. Of the 120 samples, 51 positive samples showed growth of A. fumigatus isolates on control medium. One ARAF (2%; 1/51) isolate was found amongst A. fumigatus positive samples and harbored the F46Y/M172V/E427K cyp51A mutations. No TR34/L98H or TR46/Y121F/T289A mutations were observed. Our study described the first A. fumigatus isolate resistant to an azole antifungal in Burkina Faso.

Triazole-Resistance in Environmental Aspergillus fumigatus in Latin American and African Countries.

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.

Early oseltamivir reduces risk for influenza-associated aspergillosis in a double-hit murine model.

Invasive pulmonary aspergillosis (IPA) is a life-threatening fungal infection occurring mainly in immunocompromised patients. We recently identified IPA as an emerging co-infection with high mortality in critically ill, but otherwise immunocompetent influenza patients. The neuraminidase inhibitor oseltamivir is the current standard-of-care treatment in hospitalized influenza patients; however, its efficacy in influenza-associated pulmonary aspergillosis (IAPA) is not known. Therefore, we have established an imaging-supported double-hit mouse model to investigate the therapeutic effect of oseltamivir on the development of IAPA. Immunocompetent mice received intranasal instillation influenza A or PBS followed by orotracheal inoculation with Aspergillus fumigatus 4 days later. Oseltamivir treatment or placebo was started at day 0, day 2, or day 4. Daily monitoring included micro-computed tomography and bioluminescence imaging of pneumonia and fungal burden. Non-invasive biomarkers were complemented with imaging, molecular, immunological, and pathological analysis. Influenza virus-infected immunocompetent mice developed proven airway IPA upon co-infection with Aspergillus fumigatus, whereas non-influenza-infected mice fully cleared Aspergillus, confirming influenza as a risk factor for developing IPA. Longitudinal micro-CT showed pulmonary lesions after influenza infection worsening after Aspergillus co-infection, congruent with bioluminescence imaging and histology confirming Aspergillus pneumonia. Early oseltamivir treatment prevented severe influenza pneumonia and mitigated the development of IPA and associated mortality. A time-dependent treatment effect was consistently observed with imaging, molecular, and pathological analyses. Hence, our findings underscore the importance of initiating oseltamivir as soon as possible, to suppress influenza infection and mitigate the risk of potentially lethal IAPA disease.

Hmg1 Gene Mutation Prevalence in Triazole-Resistant Aspergillus fumigatus Clinical Isolates.

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.

Functional Characterization of Clinical Isolates of the Opportunistic Fungal Pathogen Aspergillus nidulans.

Aspergillus nidulans is an opportunistic fungal pathogen in patients with immunodeficiency, and virulence of A. nidulans isolates has mainly been studied in the context of chronic granulomatous disease (CGD), with characterization of clinical isolates obtained from non-CGD patients remaining elusive. This study therefore carried out a detailed biological characterization of two A. nidulans clinical isolates (CIs), obtained from a patient with breast carcinoma and pneumonia and from a patient with cystic fibrosis that underwent lung transplantation, and compared them to the reference, nonclinical FGSC A4 strain. Both CIs presented increased growth in comparison to that of the reference strain in the presence of physiologically relevant carbon sources. Metabolomic analyses showed that the three strains are metabolically very different from each other in these carbon sources. Furthermore, the CIs were highly susceptible to cell wall-perturbing agents but not to other physiologically relevant stresses. Genome analyses identified several frameshift variants in genes encoding cell wall integrity (CWI) signaling components. Significant differences in CWI signaling were confirmed by Western blotting among the three strains. In vivo virulence studies using several different models revealed that strain MO80069 had significantly higher virulence in hosts with impaired neutrophil function than the other strains. In summary, this study presents detailed biological characterization of two A. nidulanssensu stricto clinical isolates. Just as in Aspergillus fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits. Further studies are required to fully characterize A. nidulans strain-specific virulence traits and pathogenicity.IMPORTANCE Immunocompromised patients are susceptible to infections with opportunistic filamentous fungi from the genus Aspergillus Although A. fumigatus is the main etiological agent of Aspergillus species-related infections, other species, such as A. nidulans, are prevalent in a condition-specific manner. A. nidulans is a predominant infective agent in patients suffering from chronic granulomatous disease (CGD). A. nidulans isolates have mainly been studied in the context of CGD although infection with A. nidulans also occurs in non-CGD patients. This study carried out a detailed biological characterization of two non-CGD A. nidulans clinical isolates and compared the results to those with a reference strain. Phenotypic, metabolomic, and genomic analyses highlight fundamental differences in carbon source utilization, stress responses, and maintenance of cell wall integrity among the strains. One clinical strain had increased virulence in models with impaired neutrophil function. Just as in A. fumigatus, strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits.