Fumigatus Infection Research Articles

Citizen science reveals landscape-scale exposures to multiazole-resistant Aspergillus fumigatus bioaerosols.

Using a citizen science approach, we identify a country-wide exposure to aerosolized spores of a human fungal pathogen, Aspergillus fumigatus, that has acquired resistance to the agricultural fungicide tebuconazole and first-line azole clinical antifungal drugs. Genomic analysis shows no distinction between resistant genotypes found in the environment and in patients, indicating that at least 40% of azole-resistant A. fumigatus infections are acquired from environmental exposures. Hotspots and coldspots of aerosolized azole-resistant spores were not stable between seasonal sampling periods. This suggests a high degree of atmospheric mixing resulting in an estimated per capita cumulative annual exposure of 21 days (±2.6). Because of the ubiquity of this measured exposure, it is imperative that we determine sources of azole-resistant A. fumigatus to reduce treatment failure in patients with aspergillosis.

Lung microbiota changes during pulmonary Aspergillus fumigatus infection in rats

Since the realization that the lungs are not sterile but are normally inhabited by various bacterial species, studies have been conducted to define healthy lung microbiota and to investigate whether it changes during lung diseases, infections, and inflammation. Using next-generation sequencing, we investigated bacterial microbiota from whole lungs in two rat strains (previously shown to differ in gut microbiota composition) in a healthy state and during pulmonary infection caused by the opportunistic fungus Aspergillus fumigatus. No differences in alpha diversity indices and microbial composition between DA and AO rats before infection were noted. Fungal infection caused dysbiosis in both rat strains, characterized by increased alpha diversity indices and unchanged beta diversity. The relative abundance of genera and species was increased in DA but decreased in AO rats during infection. Changes in lung microbiota coincided with inflammation (in both rat strains) and oxidative stress (in DA rats). Disparate response of lung microbiota in DA and AO rats to pulmonary fungal infection might render these two rat strains differentially susceptible to a subsequent inflammatory insult.

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.

Loss of the mammalian G-protein coupled receptor, G2A, modulates severity of invasive pulmonary aspergillosis.

Aspergillus fumigatus is a well-known opportunistic pathogen that causes a range of diseases including the often-fatal disease, invasive pulmonary aspergillosis (IPA), in immunocompromised populations. The severity of IPA is dependent on both host- and pathogen-derived signaling molecules that mediate host immunity and fungal growth. Oxylipins are bioactive oxygenated fatty acids known to influence host immune response and Aspergillus developmental programs. Aspergillus synthesizes 8-HODE and 5,8-diHODE that have structural similarities to 9-HODE and 13-HODE, which are known ligands of the host G-protein-coupled receptor G2A (GPR132). Oxylipins were extracted from infected lung tissue to assess fungal oxylipin production and the Pathhunter β-arrestin assay was used to assess agonist and antagonist activity by fungal oxylipins on G2A. An immunocompetent model of A. fumigatus infection was used to assess changes in survival and immune responses for G2A-/- mice. Here we report that Aspergillus oxylipins are produced in lung tissue of infected mice and in vitro ligand assays suggest 8-HODE is a G2A agonist and 5,8-diHODE is a partial antagonist. To address the hypothesis that G2A could be involved in the progression of IPA, we assessed the response of G2A-/- mice to A. fumigatus infection. G2A-/- mice showed a survival advantage over wild-type mice; this was accompanied by increased recruitment of G2A-/- neutrophils and increased levels of inflammatory markers in A. fumigatus-infected lungs. We conclude that G2A suppresses host inflammatory responses to Aspergillus fumigatus although it remains unclear if fungal oxylipins are involved in G2A activities.

COVID-19-associated pulmonary aspergillosis in immunocompetent patients: a virtual patient cohort study.

The opportunistic fungus Aspergillus fumigatus infects the lungs of immunocompromised hosts, including patients undergoing chemotherapy or organ transplantation. More recently however, immunocompetent patients with severe SARS-CoV2 have been reported to be affected by COVID-19 Associated Pulmonary Aspergillosis (CAPA), in the absence of the conventional risk factors for invasive aspergillosis. This paper explores the hypothesis that contributing causes are the destruction of the lung epithelium permitting colonization by opportunistic pathogens. At the same time, the exhaustion of the immune system, characterized by cytokine storms, apoptosis, and depletion of leukocytes may hinder the response to A. fumigatus infection. The combination of these factors may explain the onset of invasive aspergillosis in immunocompetent patients. We used a previously published computational model of the innate immune response to infection with Aspergillus fumigatus. Variation of model parameters was used to create a virtual patient population. A simulation study of this virtual patient population to test potential causes for co-infection in immunocompetent patients. The two most important factors determining the likelihood of CAPA were the inherent virulence of the fungus and the effectiveness of the neutrophil population, as measured by granule half-life and ability to kill fungal cells. Varying these parameters across the virtual patient population generated a realistic distribution of CAPA phenotypes observed in the literature. Computational models are an effective tool for hypothesis generation. Varying model parameters can be used to create a virtual patient population for identifying candidate mechanisms for phenomena observed in actual patient populations.

Efficient Generation of Multiple Seamless Point Mutations Conferring Triazole Resistance in Aspergillus fumigatus.

Aspergillus fumigatus is a common human fungal pathogen that can cause a range of diseases. Triazoles are used to treat A. fumigatus infections, but resistance is increasing due to mutations in genes such as cyp51A, hmg1 and overexpression of efflux pumps. Verifying the importance of these mutations is time-consuming, and although the use of CRISPR-Cas9 methods has shortened this process, it still relies on the construction of repair templates containing a selectable marker. Here, employing in vitro-assembled CRISPR-Cas9 along with a recyclable selectable marker, we devised a quick and easy way to effectively and seamlessly introduce mutations conferring triazole resistance in A. fumigatus. We used it to introduce, alone and in combination, triazole resistance-conferring mutations in cyp51A, cyp51B and hmg1. With the potential to seamlessly introduce genes imparting resistance to additional existing and novel antifungals, toxic metals, and environmental stressors, this technique can considerably improve the ability to introduce dominant mutations in A. fumigatus.

The Novel Monoclonal IgG1-Antibody AB90-E8 as a Diagnostic Tool to Rapidly Distinguish Aspergillus fumigatus from Other Human Pathogenic Aspergillus Species.

In most cases, invasive aspergillosis (IA) is caused by A. fumigatus, though infections with other Aspergillus spp. with lower susceptibilities to amphotericin B (AmB) gain ground. A. terreus, for instance, is the second leading cause of IA in humans and of serious concern because of its high propensity to disseminate and its in vitro and in vivo resistance to AmB. An early differentiation between A. fumigatus and non-A. fumigatus infections could swiftly recognize a potentially ineffective treatment with AmB and lead to the lifesaving change to a more appropriate drug regime in high-risk patients. In this study, we present the characteristics of the monoclonal IgG1-antibody AB90-E8 that specifically recognizes a surface antigen of A. fumigatus and the closely related, but not human pathogenic A. fischeri. We show immunostainings on fresh frozen sections as well as on incipient mycelium picked from agar plates with tweezers or by using the expeditious tape mount technique. All three methods have a time advantage over the common procedures currently used in the routine diagnosis of IA and outline the potential of AB90-E8 as a rapid diagnostic tool.

Exploring the intersection of Aspergillus fumigatus biofilms, infections, immune response and antifungal resistance.

Aspergillus fumigatus is an opportunistic pathogen that primarily affects the lungs and frequently elicits an allergic immune response in human hosts via inhalation of its airborne asexual spores (conidia). In immunocompromised individuals, the conidia of this fungus can germinate in the lung and result in severe systemic infections characterised by widespread tissue and organ damage. Conversely, in healthy hosts, the innate immune system is instrumental in eliminating the conidia and preventing disease progression. As with numerous other pathogenic fungi, A. fumigatus possesses a set of virulence factors that facilitate its infective mechanism and the circumvention of immune defences in susceptible hosts. The intrinsic capacity of A. fumigatus to form complex 3D-structured biofilms, both on biotic and abiotic surfaces, represents a key determinant of its evasion of the host immune system and resistance to antifungal drugs. This review delineates the pivotal role of A. fumigatus biofilm structure and function as a significant virulence factor in pathogenic infections, such as aspergilloma and invasive pulmonary aspergillosis (IPA). Additionally, we discuss the importance for the development of novel antifungal drugs as drug-resistant strains continue to evolve. Furthermore, co-infections of A. fumigatus with other nosocomial pathogens have a substantial impact on patient's health outcomes. In this context, we provide a brief overview of COVID-19-associated pulmonary aspergillosis (CAPA), a recently documented condition that has gained attention due to its associated high degree of severity.

HapE and hmg1 Mutations Are Drivers of cyp51A-Independent Pan-Triazole Resistance in an Aspergillus fumigatus Clinical Isolate.

Aspergillus fumigatus is a ubiquitous environmental mold that can cause severe disease in immunocompromised patients and chronic disease in individuals with underlying lung conditions. Triazoles are the most widely used class of antifungal drugs to treat A. fumigatus infections, but their use in the clinic is threatened by the emergence of triazole-resistant isolates worldwide, reinforcing the need for a better understanding of resistance mechanisms. The predominant mechanisms of A. fumigatus triazole resistance involve mutations affecting the promoter region or coding sequence of the target enzyme of the triazoles, Cyp51A. However, triazole-resistant isolates without cyp51A-associated mutations are frequently identified. In this study, we investigate a pan-triazole-resistant clinical isolate, DI15-105, that simultaneously carries the mutations hapEP88L and hmg1F262del, with no mutations in cyp51A. Using a Cas9-mediated gene-editing system, hapEP88L and hmg1F262del mutations were reverted in DI15-105. Here, we show that the combination of these mutations accounts for pan-triazole resistance in DI15-105. To our knowledge, DI15-105 is the first clinical isolate reported to simultaneously carry mutations in hapE and hmg1 and only the second with the hapEP88L mutation. IMPORTANCE Triazole resistance is an important cause of treatment failure and high mortality rates for A. fumigatus human infections. Although Cyp51A-associated mutations are frequently identified as the cause of A. fumigatus triazole resistance, they do not explain the resistance phenotypes for several isolates. In this study, we demonstrate that hapE and hmg1 mutations additively contribute to pan-triazole resistance in an A. fumigatus clinical isolate lacking cyp51-associated mutations. Our results exemplify the importance of and the need for a better understanding of cyp51A-independent triazole resistance mechanisms.

Aspergillus fumigatus transcription factor ZfpA regulates hyphal development and alters susceptibility to antifungals and neutrophil killing during infection.

Hyphal growth is essential for host colonization during Aspergillus infection. The transcription factor ZfpA regulates A. fumigatus hyphal development including branching, septation, and cell wall composition. However, how ZfpA affects fungal growth and susceptibility to host immunity during infection has not been investigated. Here, we use the larval zebrafish-Aspergillus infection model and primary human neutrophils to probe how ZfpA affects A. fumigatus pathogenesis and response to antifungal drugs in vivo. ZfpA deletion promotes fungal clearance and attenuates virulence in wild-type hosts and this virulence defect is abrogated in neutrophil-deficient zebrafish. ZfpA deletion also increases susceptibility to human neutrophils ex vivo while overexpression impairs fungal killing. Overexpression of ZfpA confers protection against the antifungal caspofungin by increasing chitin synthesis during hyphal development, while ZfpA deletion reduces cell wall chitin and increases caspofungin susceptibility in neutrophil-deficient zebrafish. These findings suggest a protective role for ZfpA activity in resistance to the innate immune response and antifungal treatment during A. fumigatus infection.

Uncovering alterations in the murine interferon response due to dsRNA mycovirus during Aspergillus fumigatus infection

Abstract Aspergillus fumigatus is a mold that causes an array of diseases, the most severe form being invasive aspergillosis which occurs in highly immunocompromised individuals. We demonstrated that tissue-resident alveolar macrophages are critical for maintaining host resistance against Aspergillus fumigatus due to their role in the early type I/III interferon response. Specifically, CD11c-Cre x Mavs fl/flmice have a decreased interferon response and survival outcomes when challenged with A. fumigatus, which can be functionally restored via adoptive transfer of wild-type SiglecF +alveolar macrophages. Why alveolar macrophages are potent inducers of the type I/III interferon response after Aspergillus fumigatus challenge remains elusive. Here, we demonstrate that TGF-beta metabolically programs alveolar macrophages to be potent interferon inducers via enhanced dependency on oxidative phosphorylation and peroxisome biogenesis. The type I/III interferon responses were partially related to the inability of alveolar macrophages to recognize mycoviral double-stranded RNA (dsRNA). Aspergillus fumigatus strains infected with polymycovirus 1 (PmV1) are shown to induce a robust type I/III interferon responses both in vitro and in vivo. Critically, Mda5/MAVS-dependent interferon response can still be induced in a fungal dsRNA independent manner, which warrant further explorations. In this study we identified key metabolic traits of alveolar macrophages which enable robust Mda5/MAVS signaling necessary for initiating the Aspergillus fumigatus induced interferon response, which maintains host resistance against this ubiquitous mold, through both fungal dsRNA-dependent and -independent mechanisms.

TNFα-Mediated Damaging Inflammation During Aspergillus fumigatusPulmonary Infection is Regulated by Type I Interferon Receptor 2 (IFNAR2)

Abstract Invasive pulmonary aspergillosis (IPA), caused by the fungus Aspergillus fumigatus, generally occurs in patients with suppressed or non-functioning immune systems. The recent increase in the number of cases of influenza and SARS-Cov-2-infected patients acquiring IPA suggests that severe respiratory viral infections may create a suppressed lung immune environment that allows for fungal infections to occur. We recently found that differential type I interferon (IFN) signaling, a classic anti-viral host response, regulates susceptibility to IPA. Specifically, we found that absence of the type I IFN receptor 2 (IFNAR2) subunit of the heterodimeric IFNAR1/2 receptor, leads to increased damage and inflammation in response to A. fumigatus lung infection, while absence of IFNAR1 did not. Although the Ifnar2 −/−mice had higher morbidity, we unexpectedly found that Ifnar2 −/−mice killed more A. fumigatus by 24 hours post-infection compared to WT and Ifnar1 −/−mice. However, this early clearance of A. fumigatus did not prevent invasive disease from developing in the Ifnar2 −/−mice as infection progressed, suggesting that the early damage responses were creating a favorable environment for invasive growth. By altering the inflamed environment of the Ifnar2 −/−mice early during infection, via neutralization of TNFα, we were able to reverse the damage and morbidity in these mice back to WT levels. Further, our results suggest that altered myeloid effector cell responses in Ifnar2 −/−mice during A. fumigatus infection contribute to the damage that occurs. Together, our results expand our understanding of how anti-viral mechanisms, via IFNAR2, are creating a permissive environment for fungal infections to occur through regulation of damage. Supported by grants from NIH/NIGMS (INBRE P20GM103474), NIH/NIAID (K22AI153671), AAI Careers in Immunology Fellowship, Parker B. Francis Fellowship

Succinylation modification provides new insights for the treatment of immunocompromised individuals with drug-resistant Aspergillus fumigatus infection.

Invasive Aspergillus fumigatus infection poses a serious threat to global human health, especially to immunocompromised individuals. Currently, triazole drugs are the most commonly used antifungals for aspergillosis. However, owing to the emergence of drug-resistant strains, the effect of triazole drugs is greatly restricted, resulting in a mortality rate as high as 80%. Succinylation, a novel post-translational modification, is attracting increasing interest, although its biological function in triazole resistance remains unclear. In this study, we initiated the screening of lysine succinylation in A. fumigatus. We discovered that some of the succinylation sites differed significantly among strains with unequal itraconazole (ITR) resistance. Bioinformatics analysis showed that the succinylated proteins are involved in a broad range of cellular functions with diverse subcellular localizations, the most notable of which is cell metabolism. Further antifungal sensitivity tests confirmed the synergistic fungicidal effects of dessuccinylase inhibitor nicotinamide (NAM) on ITR-resistant A. fumigatus. In vivo experiments revealed that treatment with NAM alone or in combination with ITR significantly increased the survival of neutropenic mice infected with A. fumigatus. In vitro experiments showed that NAM enhanced the killing effect of THP-1 macrophages on A. fumigatus conidia. Our results suggest that lysine succinylation plays an indispensable role in ITR resistance of A. fumigatus. Dessuccinylase inhibitor NAM alone or in combination with ITR exerted good effects against A. fumigatus infection in terms of synergistic fungicidal effect and enhancing macrophage killing effect. These results provide mechanistic insights that will aid in the treatment of ITR-resistant fungal infections.

Surrogate infection model predicts optimal alveolar macrophage number for clearance of Aspergillus fumigatus infections

The immune system has to fight off hundreds of microbial invaders every day, such as the human-pathogenic fungus Aspergillus fumigatus. The fungal conidia can reach the lower respiratory tract, swell and form hyphae within six hours causing life-threatening invasive aspergillosis. Invading pathogens are continuously recognized and eliminated by alveolar macrophages (AM). Their number plays an essential role, but remains controversial with measurements varying by a factor greater than ten for the human lung. We here investigate the impact of the AM number on the clearance of A. fumigatus conidia in humans and mice using analytical and numerical modeling approaches. A three-dimensional to-scale hybrid agent-based model (hABM) of the human and murine alveolus allowed us to simulate millions of virtual infection scenarios, and to gain quantitative insights into the infection dynamics for varying AM numbers and infection doses. Since hABM simulations are computationally expensive, we derived and trained an analytical surrogate infection model on the large dataset of numerical simulations. This enables reducing the number of hABM simulations while still providing (i) accurate and immediate predictions on infection progression, (ii) quantitative hypotheses on the infection dynamics under healthy and immunocompromised conditions, and (iii) optimal AM numbers for combating A. fumigatus infections in humans and mice.

Rapid and sustained T cell-based immunotherapy against invasive fungal disease via a combined two step procedure.

Aspergillus fumigatus (Asp) infections constitute a major cause of morbidity and mortality in patients following allogeneic hematopoietic stem cell transplantation (HSCT). In the context of insufficient host immunity, antifungal drugs show only limited efficacy. Faster and increased T-cell reconstitution correlated with a favorable outcome and a cell-based therapy approach strongly indicated successful clearance of fungal infections. Nevertheless, complex and cost- or time-intensive protocols hampered their implementation into clinical application. To facilitate the clinical-scale manufacturing process of Aspergillus fumigatus-specific T cells (ATCs) and to enable immediate (within 24 hours) and sustained (12 days later) treatment of patients with invasive aspergillosis (IA), we adapted and combined two complementary good manufacturing practice (GMP)-compliant approaches, i) the direct magnetic enrichment of Interferon-gamma (IFN-γ) secreting ATCs using the small-scale Cytokine Secretion Assay (CSA) and ii) a short-term in vitro T-cell culture expansion (STE), respectively. We further compared stimulation with two standardized and commercially available products: Asp-lysate and a pool of overlapping peptides derived from different Asp-proteins (PepMix). For the fast CSA-based approach we detected IFN-γ+ ATCs after Asp-lysate- as well as PepMix-stimulation but with a significantly higher enrichment efficiency for stimulation with the Asp-lysate when compared to the PepMix. In contrast, the STE approach resulted in comparably high ATC expansion rates by using Asp-lysate or PepMix. Independent of the stimulus, predominantly CD4+ helper T cells with a central-memory phenotype were expanded while CD8+ T cells mainly showed an effector-memory phenotype. ATCs were highly functional and cytotoxic as determined by secretion of granzyme-B and IFN-γ. For patients with IA, the immediate adoptive transfer of IFN-γ+ ATCs followed by the administration of short-term in vitro expanded ATCs from the same donor, might be a promising therapeutic option to improve the clinical outcome.

Mortality Causes in Captive Cantabrian capercaillie (Tetrao urogallus cantabricus) in Spain.

The Cantabrian capercaillie (Tetrao urogallus cantabricus) is one of the most severely threatened subspecies of capercaillie. Its current population range is restricted to a small area of the Cantabrian Mountains (northwestern Spain), with only around 200 individuals remaining. As part of the national strategy for the conservation of the subspecies, the Cantabrian capercaillie Captive Breeding Center of Sobrescobio opened in 2009. Here, we use the information provided by the necropsies performed in this facility on 29 individuals (11 males, 13 females and 5 undetermined; 16 chicks and 13 adults) in order to describe the main mortality causes of captive-bred Cantabrian capercaillies. After necropsy, tissue samples were taken for evaluation using standard methods in histology and microbiology. The majority of the captive animals (18/29, 62.07%) died due to infectious diseases, mainly due to Escherichia coli, Clostridium perfringens, or Aspergillus fumigatus infection. The remaining 11 animals died due to stress-related processes (i.e., rupture of the heart apex and cardiomyopathy or neurogenic shock) (8/29, 27.59%), duodenal obstruction and coelomitis (1/29, 3.45%), perforation of the proventriculus and heart with a briar branch (1/29, 3.45%) or euthanasia due to a valgus leg deformity that prevented proper animal welfare (1/29, 3.45%). Young animals (i.e., younger than 2 months) died mainly due to infectious diseases (14/16, 87.5%), while stress-related causes were responsible for most adult deaths (7/13, 53.85%). We additionally report that two free-ranging adult males died due to exertional myopathy. This study provides relevant information for reducing mortality in captive capercaillies and improving both living conditions in captivity and the adaptation of these animals to the wild.

Serum Cytokine Biomarkers for Use in Diagnosing Pulmonary Tuberculosis versus Chronic Pulmonary Aspergillosis.

Aspergillus fumigatus-induced chronic pulmonary aspergillosis (CPA), the most common pulmonary tuberculosis (TB) sequela, tends to occur after pulmonary infection with the intracellular pathogen Mycobacterium tuberculosis (Mtb). Timely and accurate detection of A. fumigatus infection of pulmonary TB patients would undoubtedly greatly improve patient prognosis. Currently, the galactomannan (GM) antigen test is commonly used to detect A. fumigatus infection but has poor sensitivity that renders this assay inadequate for use in clinical practice. Given the fact CPA and TB induce different host immune responses, we evaluated serum cytokine level profiles of CPA, TB patients and patients with both diseases (CPA-TB) for multiple cytokines and cytokine combinations. The results revealed significantly higher serum levels of numerous proinflammatory cytokines, including IL-1β, IL-6, IL-8, IL-12p70, IFN-α, IFN-γ and TNF-α, in peripheral blood of CPA-TB patients versus that of TB patients. IL-8 levels alone provided the best discriminatory performance for distinguishing between TB and either CPA-TB patients (AUC = 0.949) or CPA patients (AUC = 0.964). Moreover, both IL-8 and TNF-α (AUC = 0.996) levels could be used to distinguish between TB and CPA-TB patients. Likewise, IL-8, TNF-α and IL-6 levels together could be used to distinguish between CPA-TB and TB patients. In this study, multiple cytokines were identified that may serve as potential biomarkers for use in detecting TB patients with CPA. Furthermore, our results should enhance understanding of how immune system dysfunctions influence susceptibility to Mtb and/or A. fumigatus infections.

Targeting Sterylglucosidase A to Treat Aspergillus fumigatus Infections.

Invasive fungal infections are a leading cause of death in immunocompromised patients. Current therapies have several limitations, and innovative antifungal agents are critically needed. Previously, we identified the fungus-specific enzyme sterylglucosidase as essential for pathogenesis and virulence of Cryptococcus neoformans and Aspergillus fumigatus (Af) in murine models of mycoses. Here, we developed Af sterylglucosidase A (SglA) as a therapeutic target. We identified two selective inhibitors of SglA with distinct chemical scaffolds that bind in the active site of SglA. Both inhibitors induce sterylglucoside accumulation and delay filamentation in Af and increase survival in a murine model of pulmonary aspergillosis. Structure-activity relationship (SAR) studies identified a more potent derivative that enhances both in vitro phenotypes and in vivo survival. These findings support sterylglucosidase inhibition as a promising antifungal approach with broad-spectrum potential. IMPORTANCE Invasive fungal infections are a leading cause of death in immunocompromised patients. Aspergillus fumigatus is a fungus ubiquitously found in the environment that, upon inhalation, causes both acute and chronic illnesses in at-risk individuals. A. fumigatus is recognized as one of the critical fungal pathogens for which a substantive treatment breakthrough is urgently needed. Here, we studied a fungus-specific enzyme, sterylglucosidase A (SglA), as a therapeutic target. We identified selective inhibitors of SglA that induce accumulation of sterylglucosides and delay filamentation in A. fumigatus and increase survival in a murine model of pulmonary aspergillosis. We determined the structure of SglA, predicted the binding poses of these inhibitors through docking analysis, and identified a more efficacious derivative with a limited SAR study. These results open several exciting avenues for the research and development of a new class of antifungal agents targeting sterylglucosidases.

Fungal Integrated Histomolecular Diagnosis Using Targeted Next-Generation Sequencing on Formalin-Fixed Paraffin-Embedded Tissues.

Histopathology is the gold standard for fungal infection (FI) diagnosis, but it does not provide a genus and/or species identification. The objective of the present study was to develop targeted next-generation sequencing (NGS) on formalin-fixed tissue samples (FTs) to achieve a fungal integrated histomolecular diagnosis. Nucleic acid extraction was optimized on a first group of 30 FTs with Aspergillus fumigatus or Mucorales infection by macrodissecting the microscopically identified fungal-rich area and comparing Qiagen and Promega extraction methods through DNA amplification by A. fumigatus and Mucorales primers. Targeted NGS was developed on a second group of 74 FTs using three primer pairs (ITS-3/ITS-4, MITS-2A/MITS-2B, and 28S-12-F/28S-13-R) and two databases (UNITE and RefSeq). A prior fungal identification of this group was established on fresh tissues. Targeted NGS and Sanger sequencing results on FTs were compared. To be valid, the molecular identifications had to be compatible with the histopathological analysis. In the first group, the Qiagen method yielded a better extraction efficiency than the Promega method (100% and 86.7% of positive PCRs, respectively). In the second group, targeted NGS allowed fungal identification in 82.4% (61/74) of FTs using all primer pairs, in 73% (54/74) using ITS-3/ITS-4, in 68.9% (51/74) using MITS-2A/MITS-2B, and in 23% (17/74) using 28S-12-F/28S-13-R. The sensitivity varied according to the database used (81% [60/74] using UNITE compared to 50% [37/74] using RefSeq [P = 0.000002]). The sensitivity of targeted NGS (82.4%) was higher than that of Sanger sequencing (45.9%; P < 0.00001). To conclude, fungal integrated histomolecular diagnosis using targeted NGS is suitable on FTs and improves fungal detection and identification.

Epidemiological trends in nontuberculous mycobacterial infection among people with cystic fibrosis in Germany

ObjectivesPeople with cystic fibrosis (pwCF) are at risk for infection with nontuberculous mycobacteria (NTM). The epidemiology and screening practice of NTM among pwCF in Germany are largely unknown and require investigation. MethodsWe analyzed the data of the German Cystic Fibrosis Registry from 2016 to 2020 for NTM. The annual prevalence and incidence of any NTM, Mycobacterium abscessus complex (MABC), Mycobacterium avium complex (MAC), Mycobacterium gordonae, and other mycobacteria were determined and correlated to patient characteristics. Patients with incident MABC and MAC infection were compared. ResultsThe annual NTM prevalence and incidence remained stable between 7.53% and 8.76%, as well as 3.31% and 4.95%, respectively, among the approximately 6000 registry participants. MABC was the most common NTM, whereas only the prevalence of MAC increased slightly. In each year, only about one-third of all patients were screened for NTM. An association between NTM infections and Aspergillus fumigatus infection and/or allergic bronchopulmonary aspergillosis was observed. On average, patients with incident MAC infection were older than patients with MABC infection. ConclusionThe NTM burden in pwCF in Germany remained unchanged between 2016 and 2020. MABC was the dominant species detected, whereas only MAC infections increased with time and patient age. The previously observed association of Aspergillus fumigatus and NTM was reaffirmed. Awareness of NTM needs to be improved.