Mucormycosis is an emerging, life-threatening human infection caused by Mucorales fungi1-3. Metabolic disorders uniquely predispose an ever-expanding group of patients to mucormycosis through poorly understood mechanisms1,2,4,5, suggesting that uncharacterized host metabolic effectors may confer protective immunity against this infection. Here we uncover a master regulatory role of albumin in host defence against Mucorales through the modulation of fungal pathogenicity. Our initial studies identified severe hypoalb uminaemia as a prominent metabolic abnormality and an independent biomarker of poor mucormycosis outcome across three distinct cohorts of patients with mucormycosis. Notably, purified albumin selectively inhibits Mucorales growth among a range of pathogens, and albumin-deficient mice display susceptibility specifically to mucormycosis. The antifungal activity of albumin is mediated by the release of bound free fatty acids (FFAs). Albumin prevents FFA oxidation, which otherwise abolishes their antifungal properties, and sera from patients with mucormycosis display high levels of oxidized FFAs. Physiologically, albumin-bound FFAs suppress the expression of key virulence factors by inhibiting protein synthesis, the reby rendering Mucorales avirulent in vivo. Overall, we identify a host defence mechanism that directs the pathogen to suppress its pathogenicity program in response to unfavourable metabolic cues regulated by albumin. These findings have major implications for the pathogenesis and management of mucormycosis.

Invasive aspergillosis (IA) crude mortality has shown a sustained reduction over the past decades, demonstrated in randomized controlled clinical trials of new antifungal agents and across large population surveys. New diagnostic tools and integrated management approaches have driven faster, more targeted initiation of appropriate antifungal therapy. In parallel, improvements in the identification of periods at highest risk for IA and in practices for management of the underlying disease processes predisposing to immunosuppression, including immunomodulatory therapies, have progressed. Given the highly complex and interconnected relationship between the underlying disease and its treatment and the predisposition to IA that the underlying disease creates, it is difficult to separate out which mortality improvements could be attributable to improved management of IA and which to better management of the underlying disease. The reductions in IA mortality have been sustained despite increases in the number of older, more vulnerable patients with more severe underlying disease undergoing treatment for acute haematological disorders and haematopoietic cell transplantation. This gradual and subtle move to a higher risk, more co-morbid patient population may have obscured any impact from the management developments other than antifungal therapy over this period, including better fungal diagnosis and supportive care. The overwhelming single factor contributing to a reduction in IA mortality over the past years appears to have been the routine adoption of mould-active antifungals, azoles in particular. Any impact of consensus definitions used to classify disease, improvements in diagnostic tools and earlier targeted strategies, remains difficult to measure based on available data. However, recently, the use of mould-active azoles has become threatened by the emergence of azole resistance in Aspergillus fumigatus, the frequent co-occurrence of Aspergillus species and Mucorales species, and difficult to handle drug-drug interactions, thereby fuelling an ongoing search for novel antifungal agents.

Trichoderma species are emerging as pathogens, causing invasive fungal infections, particularly in immunocompromised individuals. We report the case of a 61-year-old neutropenic female with hepatosplenic T-cell lymphoma and profound neutropenia, who developed a breakthrough infection with Trichoderma longibrachiatum while receiving liposomal amphotericin B for probable invasive pulmonary aspergillosis. Despite combination antifungal therapy the patient ultimately succumbed to multiple organ failure. Trichoderma longibrachiatum and Aspergillus fumigatus were identified as causative fungal pathogens. Antifungal susceptibility testing of the T. longibrachiatum isolate revealed resistance to isavuconazole but susceptibility to amphotericin B, voriconazole, itraconazole and olorofim.

BACKGROUND:Genetic predisposition plays a major role in the development of invasive pulmonary aspergillosis (IPA). The risk and course of IPA vary significantly among patients, yet continuously new genetic mechanisms that influence individual antifungal immune responses are being discovered. While genetic variability in interleukin (IL)-1 family cytokines is recognized as an important cause of disease susceptibility, it is unclear whether and how less-studied IL-1 family members, such as the IL-36 cytokine subfamily, are genetically regulated and influence the risk of infection. METHODS:We analyzed how genetic variants in the IL36 loci associate with the risk of IPA in 328 eligible recipients of allogeneic hematopoietic stem cell transplants and their corresponding donors. The functional consequences of relevant genetic variants were investigated using clinical samples and in vitro infection models. RESULTS:We report that recipient, but not donor, single-nucleotide polymorphisms (SNPs) rs895497 in IL36A and rs4849142 in IL36B increase the risk of IPA after transplantation. The strongest contribution of these SNPs to infection risk was observed in a combined analysis of transplant pairs. IL-36β was expressed in both human type II-like alveolar epithelial cells and macrophages following Aspergillus fumigatus infection. The risk genotype was associated with impaired production of IL-1β in bronchoalveolar lavage fluid samples from infected patients, as well as in fungal-stimulated macrophages. Moreover, macrophages harboring the risk genotype exhibited impaired fungicidal activity. CONCLUSIONS:Our findings suggest that genotype-specific mechanisms mediated by IL-36β act on the nonhematopoietic compartment, can impair antifungal immune responses in macrophages, and ultimately predispose to transplant recipient susceptibility to IPA.

As microbiological tests play an important role in our diagnostic algorithms and clinical approach towards patients at-risk for pulmonary aspergillosis, a good knowledge of the diagnostic possibilities and especially their limitations is extremely important. In this review, we aim to reflect critically on the available microbiological diagnostic modalities for diagnosis of pulmonary aspergillosis and formulate some future prospects. Timely start of adequate antifungal treatment leads to a better patient outcome, but overuse of antifungals should be avoided. Current diagnostic possibilities are expanding, and are mainly driven by enzyme immunoassays and lateral flow device tests for the detection of Aspergillus antigens. Most of these tests are directed towards similar antigens, but new antibodies towards different targets are under development. For chronic forms of pulmonary aspergillosis, anti- Aspergillus IgG antibodies and precipitins remain the cornerstone. More studies on the possibilities and limitations of molecular testing including targeting resistance markers are ongoing. Also, metagenomic next-generation sequencing is expanding our future possibilities. It remains important to combine different test results and interpret them in the appropriate clinical context to improve performance. Test performances may differ according to the patient population and test results may be influenced by timing, the tested matrix, and prophylactic and empiric antifungal therapy. Despite the increasing armamentarium, a simple blood or urine test for the diagnosis of aspergillosis in all patient populations at-risk is still lacking. Research on diagnostic tools is broadening from a pathogen focus on biomarkers related to the patient and its immune system.

Fungal diseases represent a considerable global health concern, affecting >1 billion people annually. In response to this growing challenge, the World Health Organization introduced the pivotal fungal priority pathogens list (FPPL) in late 2022. The FPPL highlights the challenges in estimating the global burden of fungal diseases and antifungal resistance (AFR), as well as limited surveillance capabilities and lack of routine AFR testing. Furthermore, training programs should incorporate sufficient information on fungal diseases, necessitating global advocacy to educate health care professionals and scientists. Established international guidelines and the FPPL are vital in strengthening local guidance on tackling fungal diseases. Future iterations of the FPPL have the potential to refine the list further, addressing its limitations and advancing our collective ability to combat fungal diseases effectively. Napp Pharmaceuticals Limited (Mundipharma UK) organized a workshop with key experts from Northern Europe to discuss the impact of the FPPL on regional clinical practice.

Objectives Invasive aspergillosis (IA) is a major cause of mortality in immunocompromised patients and it is difficult to diagnose because of the lack of reliable highly sensitive diagnostics. We aimed to identify circulating immunological markers that could be useful for an early diagnosis of IA. Methods We collected longitudinally serum samples from 33 cases with probable/proven IA and two matched control cohorts without IA (one with microbiological and clinical evidence of bacterial or viral non-fungal pneumonia and one without evidence of infection, all matched for neutropenia, primary underlying disease, and receipt of corticosteroids/other immunosuppressants) at a tertiary university hospital. In addition, samples from an independent cohort (n = 20 cases of proven/probable IA and 20 matched controls without infection) were obtained. A panel of 92 circulating proteins involved in inflammation was measured by proximity extension assay. A random forest model was used to predict the development of IA using biomarkers measured before diagnosis. Results While no significant differences were observed between IA cases and infected controls, concentrations of 30 inflammatory biomarkers were different between cases and non-infected controls, of which nine were independently replicated: PD-L1, MMP-10, Interleukin(IL)-10, IL-15RA, IL-18, IL-18R1, CDCP1, CCL19 and IL-17C. From the differential abundance analysis of serum samples collected more than 10 days before diagnosis and at diagnosis, increased IL-17C concentrations in IA patients were replicated in the independent cohort. Conclusions An increased circulating concentration of IL-17C was detected both in the discovery and independent cohort, both at the time of diagnosis and in samples 10 days before the diagnosis of IA, suggesting it should be evaluated further as potential (early) biomarker of infection.

Mucormycosis is an emerging, life-threatening human infection caused by fungi of the order Mucorales. Metabolic disorders uniquely predispose an ever-expanding group of patients to mucormycosis via poorly understood mechanisms. Therefore, it is highly likely that uncharacterized host metabolic effectors confer protective immunity against mucormycosis. Here, we uncover a master regulatory role of albumin in host defense against Mucorales through the modulation of the fungal pathogenicity program. Our initial studies identified severe hypoalbuminemia as a prominent metabolic abnormality and a biomarker of poor outcome in independent cohorts of mucormycosis patients. Strikingly, we found that purified albumin selectively inhibits Mucorales growth among a range of human pathogens, and albumin-deficient mice display susceptibility specifically to mucormycosis. The antifungal activity of albumin is mediated by the release of bound free fatty acids (FFAs). Importantly, albumin prevents FFA oxidation, which results in loss of their antifungal properties. A high degree of FFA oxidation is found in the sera of patients with mucormycosis. Physiologically, albumin-bound FFAs blocks the expression of the mycotoxin mucoricin and renders Mucorales avirulent in vivo. Overall, we discovered a novel host defense mechanism that directs the pathogen to suppress its growth and the expression of virulence factors in response to unfavorable metabolic cues regulated by albumin. These findings have major implications for the pathogenesis and management of mucormycosis.

Purpose The aim of this document was to develop standardized research definitions of invasive fungal diseases (IFD) in non-neutropenic, adult patients without classical host factors for IFD, admitted to intensive care units (ICUs). Methods After a systematic assessment of the diagnostic performance for IFD in the target population of already existing definitions and laboratory tests, consensus definitions were developed by a panel of experts using the RAND/UCLA appropriateness method. Results Standardized research definitions were developed for proven invasive candidiasis, probable deep-seated candidiasis, proven invasive aspergillosis, probable invasive pulmonary aspergillosis, and probable tracheobronchial aspergillosis. The limited evidence on the performance of existing definitions and laboratory tests for the diagnosis of IFD other than candidiasis and aspergillosis precluded the development of dedicated definitions, at least pending further data. The standardized definitions provided in the present document are aimed to speed-up the design, and increase the feasibility, of future comparative research studies.

The (1→3)-β-D-glucan (BDG) is a component of the fungal cell wall that can be detected in serum and used as an adjunctive tool for the diagnosis of invasive mold infections (IMI) in patients with hematologic cancer or other immunosuppressive conditions. However, its use is limited by modest sensitivity/specificity, inability to differentiate between fungal pathogens, and lack of detection of mucormycosis. Data about BDG performance for other relevant IMI, such as invasive fusariosis (IF) and invasive scedosporiosis/lomentosporiosis (IS) are scarce. The objective of this study was to assess the sensitivity of BDG for the diagnosis of IF and IS through systematic literature review and meta-analysis. Immunosuppressed patients diagnosed with proven or probable IF and IS, with interpretable BDG data were eligible. A total of 73 IF and 27 IS cases were included. The sensitivity of BDG for IF and IS diagnosis was 76.7% and 81.5%, respectively. In comparison, the sensitivity of serum galactomannan for IF was 27%. Importantly, BDG positivity preceded the diagnosis by conventional methods (culture or histopathology) in 73% and 94% of IF and IS cases, respectively. Specificity was not assessed because of lacking data. In conclusion, BDG testing may be useful in patients with suspected IF or IS. Combining BDG and galactomannan testing may also help differentiating between the different types of IMI.

Abstract Background As mortality of invasive mould infections (IMI) remains high, there is a need for improved biomarkers for timely diagnosis and patient stratification. Various moulds have been shown to induce T-helper cell (Th) 1, Th2, Th9 and Th17 subsets resulting not in potent anti-Aspergillus T-cells effector mechanisms, and in elevated serum levels of cytokines such as IFN-γ and IL-6, IL-8, IL-15 and IL-17. The ECMM study “Immunologic Markers for Treatment Monitoring and Diagnosis in Invasive Mold Infection” aimed to identify circulating immunological markers that could be useful for an early diagnosis of IMI. Methods We collected longitudinal serum samples from 33 cases with probable/proven IMI and two matched control cohorts without IMI, and from an independent validation cohort with 20 cases and 20 matched controls (Figure 1, Table 1). None of the patients received mold active prophylaxis. A panel of 92 circulating proteins involved in inflammation was measured using a targeted proteomics platform (Olink Proteomics AB (Uppsala, Sweden)) and protein concentrations were compared. Correction for multiple testing was performed by Benjamini-Hochberg method. Figure 1 Overview of sample collection Table 1 An overview of the main characteristics of and differences between the derivation cohort and the validation cohort, including characteristics of all cases and all controls. Results Abundance analysis on the day of diagnosis revealed 30 differentially regulated proteins. Nine were replicated in an independent cohort: MMP-10, IL-18, IL-18-R1, IL10, CDCP1 and IL-17C. In the analysis of serum samples collected more than 10 days before diagnosis, six proteins showed higher concentrations in the cases compared to the controls (CCL20, IL-6, IL-17C, CCL23, CXCL5, CXCL1 and CX3CL1), while nine proteins had lower concentrations (Figure 2). The higher IL-17C concentration could be replicated in the independent validation cohort (Figure 3). When comparing cases only to infected controls this difference in expression was not significant anymore after correcting for multiple testing. Figure 2 Figure 3 Conclusion We found a consistent higher expression of IL-17C in cases with IMI versus non-infected controls, both at the time of diagnosis and in samples 10 days before the diagnosis of IMI. IL-17C, known for its pro-inflammatory function at the epithelial sites, activates expression of other proinflammatory cytokines and of chemokines such as CXCL1/2/3 and CCL20, and could present an early biomarker for IMI. Abundance analysis of the primary cohort (a) at time of diagnosis, and (b) at more than 10 days before diagnosis, comparing cases with non infected controls. Abundance analysis of the validation cohort (a) at time of diagnosis, and (b) at more than 10 days before diagnosis, comparing cases with non infected controls. Table 2-1 Table 2 Clinical and microbiological information of the 33 cases included in the primary cohort and the 20 cases included in the validation cohort PART 1. Disclosures Martin Hoenigl, n/a, Astellas: Grant/Research Support|Euroimmun: Grant/Research Support|F2G: Grant/Research Support|Gilead: Grant/Research Support|Immy: Grant/Research Support|MSD: Grant/Research Support|Mundipharma: Grant/Research Support|Pfizer: Grant/Research Support|Pulmocide: Grant/Research Support|Scynexis: Grant/Research Support

Diagnosis of invasive aspergillosis is based on a combination of criteria, of which the detection of Aspergillus galactomannan (GM) often is decisive. To date, the most commonly used method to determine GM is an enzyme-linked immune assay (EIA). But since a few years lateral flow assays (LFAs) were introduced, providing the possibility for rapid single sample testing. More and more LFAs are entering the market, but, although often being equated, all use their own antibodies, procedures and interpretation criteria. A recent European survey revealed that about 24–33% of laboratories implemented a lateral flow assay on-site. We conducted a survey at 81 Belgian hospital laboratories regarding the implementation of LFAs in their centre. In addition, we performed an extensive review of all publicly available studies on the performance of lateral flow assays to diagnose invasive aspergillosis. Response rate to the survey was 69%. Of the 56 responding hospital laboratories, 6 (11%) used an LFA. The Soña Aspergillus galactomannan LFA (IMMY, Norman, Oklahoma, USA) was used in 4/6 centres, while two centres used the QuicGM (Dynamiker, Tianjin, China) and one centre used the FungiXpert Aspergillus Galactomannan Detection K-set LFA (Genobio [Era Biology Technology], Tianjin, China). One centre used 2 distinct LFAs. In 3/6 centres, the sample is sent to another lab for confirmation with GM-EIA when the LFA result is positive and in 2/6 when the LFA results is negative. In one centre, a confirmatory GM-EIA is always performed in house. In three centres the LFA result is used as a complete substitute for GM-EIA. Available LFA performance studies are very diverse and results vary in function of the study population and type of LFA. Apart from the IMMY and OLM LFA, only very limited performance data are available. From two out of three LFAs used in Belgium, no clinical performance studies are published in literature. A large variety of LFAs are used in Belgian Hospitals, some of which no clinical validation studies are published. These results do likely have implications for other parts of Europe and for the rest of the world as well. Due to the variable performance of LFA tests and the limited validation data available, each laboratory must check the available performance information of the specific test considered for implementation. In addition, laboratories should perform an implementation verification study.

Abstract Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Background Early diagnosis of invasive aspergillosis (IA) is an important factor to improve survival, but it remains challenging. QuicGM Aspergillus Galactomannan Ag Lateral Flow Assay (LFA) is based on a fluorescence immunochromatography test using a monoclonal antibody to identify Aspergillus galactomannan antigen in serum and bronchoalveolar lavage fluid. Methods In this study, we analyzed the diagnostic performance of QuicGM in 120 serum samples of high-risk hematology patients [41 cases of proven (n = 4) and probable (n = 37) IA and 79 equally high-risk controls] and compared it to performance of the galactomannan (GM) [Platelia Aspergillus enzyme immunoassay (ELISA) (Bio-Rad, Marnes-la-Coquette, France)]. Probable cases defined by a positive Biorad GM in serum were also included in the cohort. Results Using the same cut-off of 0.5, QuicGM was positive in 22 cases versus GM in 21 cases. However, QuicGM was false positive in 11 controls with GM only in 1 control. These false positive samples remained positive upon repeated testing with the QuicGM. The test results of QuicGM and GM correlated well with a Spearman's correlation coefficient of 0.80 (P <.001). McNemar test showed no significant difference between both tests (P = 1) and Cohen's kappa coefficient was 0.65 (CI 0.49-0.81), suggesting a substantial strength of agreement. QuicGM sensitivity (using 0.5 cutoff) was 54% versus 51% for GM. Specificity was 86% for QuicGM versus 99% for GM. Positive predictive value (PPV) was only 67% for QuicGM versus 95% for GM. Combining GM and QuicGM in serum increased sensitivity to 59%. Youden index of QuicGM in the cohort was 0.39. Using this cutoff, sensitivity was 66% and specificity was 80%. When using a higher cutoff of 0.6 for the QuicGM, specificity increased to 94% and PPV to 78%, at the cost of a decrease in sensitivity to 44%. Discussion Including probable cases defined by a positive Biorad GM in serum could have led to a bias in favor of the GM. With the cutoff suggested by the company, there was a notable decrease in specificity and PPV for the QuicGM. The need to adjust this cut-off must be questioned. With the implementation of LFAs in the EORTC-MSGERG criteria, one usually strives for the same cutoff of 0.5, to assure a practical approach. However, we must remain thoughtful about slight differences in these tests. They all have their own IgG and IgM built. Caution is needed more in the setting of diagnosis than in screening. More knowledge must be obtained regarding how different hospital laboratories have implemented the LFAs. Conclusion Diagnostic performances of QuicGM and Biorad GM correlate well. Further research is necessary regarding the optimal cutoff index.

Early diagnosis of invasive aspergillosis is an important factor to improve survival but remains challenging. The detection of Aspergillus antigens is included in the consensus case definitions of the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycoses Study Group as a criterion of "probable" invasive aspergillosis. JF5, a mouse IgG3 monoclonal antibody detecting an Aspergillus mannoprotein, has already been implemented as a lateral flow device (LFD). Now, also a JF5-based enzyme-linked immunosorbent assay (EIA) is commercialized (Aspergillus specific galactomannoprotein [GP] EIA, Euroimmun Medizinische Labordiagnostika AG). In this study, we analyzed the diagnostic performance of GP in 63 bronchoalveolar lavage fluid (BALf) samples and 224 serum samples and compared it to performance of the galactomannan (GM) (Platelia Aspergillus enzyme immunoassay (EIA) (Bio-Rad, Marnes-la-Coquette, France)) and the JF5-based LFD (AspLFD; OLM Diagnostics, Newcastle Upon Tyne, United Kingdom). The diagnostic performance of GP and GM correlated well with both having high specificity. With an optimized cutoff threshold for positivity of 0.4-deviating from the 05 threshold recommended by the manufacturer-sensitivity of GP in serum is not significantly different than that of GM. However, in BALf sensitivity of GP is significantly less than for GM.

Abstract Background Invasive aspergillosis (IA) by a triazole-resistant Aspergillus fumigatus is associated with high mortality. Real-time resistance detection will result in earlier initiation of appropriate therapy. Methods In a prospective study, we evaluated the clinical value of the AsperGenius polymerase chain reaction (PCR) assay in hematology patients from 12 centers. This PCR assay detects the most frequent cyp51A mutations in A. fumigatus conferring azole resistance. Patients were included when a computed tomography scan showed a pulmonary infiltrate and bronchoalveolar fluid (BALf) sampling was performed. The primary end point was antifungal treatment failure in patients with azole-resistant IA. Results Of 323 patients enrolled, complete mycological and radiological information was available for 276 (94%), and probable IA was diagnosed in 99/276 (36%). Sufficient BALf for PCR testing was available for 293/323 (91%). Aspergillus DNA was detected in 116/293 (40%) and A. fumigatus DNA in 89/293 (30%). The resistance PCR was conclusive in 58/89 (65%) and resistance detected in 8/58 (14%). Two had a mixed azole-susceptible/azole-resistant infection. In the 6 remaining patients, treatment failure was observed in 1. Galactomannan positivity was associated with mortality (P = .004) while an isolated positive Aspergillus PCR was not (P = .83). Conclusions Real-time PCR-based resistance testing may help to limit the clinical impact of triazole resistance. In contrast, the clinical impact of an isolated positive Aspergillus PCR on BALf seems limited. The interpretation of the EORTC/MSGERC PCR criterion for BALf may need further specification (eg, minimum cycle threshold value and/or PCR positive on >1 BALf sample).

Rationale Recent studies have revealed that the lung microbiota of critically ill patients is altered and predicts clinical outcomes. The incidence of invasive fungal infections, namely, invasive pulmonary aspergillosis (IPA), in immunocompromised patients is increasing, but the clinical significance of variations in lung bacterial communities is unknown. Objectives To define the contribution of the lung microbiota to the development and course of IPA. Methods and measurements We performed an observational cohort study to characterise the lung microbiota in 104 immunocompromised patients using bacterial 16S ribosomal RNA gene sequencing on bronchoalveolar lavage samples sampled on clinical suspicion of infection. Associations between lung dysbiosis in IPA and pulmonary immunity were evaluated by quantifying alveolar cytokines and chemokines and immune cells. The contribution of microbial signatures to patient outcome was assessed by estimating overall survival. Main results Patients diagnosed with IPA displayed a decreased alpha diversity, driven by a markedly increased abundance of the Staphylococcus , Escherichia , Paraclostridium and Finegoldia genera and a decreased proportion of the Prevotella and Veillonella genera. The overall composition of the lung microbiome was influenced by the neutrophil counts and associated with differential levels of alveolar cytokines. Importantly, the degree of bacterial diversity at the onset of IPA predicted the survival of infected patients. Conclusions Our results reveal the lung microbiota as an understudied source of clinical variation in patients at risk of IPA and highlight its potential as a diagnostic and therapeutic target in the context of respiratory fungal diseases.