The Impact of the Fungal Priority Pathogens List on Medical Mycology: A Northern European Perspective.

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.

Invasive Fungal Diseases in Adult Patients in Intensive Care Unit (FUNDICU): 2024 consensus definitions from ESGCIP, EFISG, ESICM, ECMM, MSGERC, ISAC, and ISHAM.

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.

Circulatory Inflammatory Proteins as Early Diagnostic Biomarkers for Invasive Aspergillosis in Patients with Hematologic Malignancies-an Exploratory Study.

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.

Microbiological Diagnosis of Pulmonary Aspergillus Infections.

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.

Performance of the beta-glucan test for the diagnosis of invasive fusariosis and scedosporiosis: a meta-analysis.

The (1→3)-ß-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.

IF and IS are severe fungal infections for which diagnosis is often delayed. This meta-analysis shows that beta-glucan testing in serum had a sensitivity of about 80% for IF/IS and could detect the disease earlier compared to conventional diagnostic tests.

Implementation of Lateral Flow Assays for the Diagnosis of Invasive Aspergillosis in European Hospitals: A Survey from Belgium and a Literature Review of Test Performances in Different Patient Populations.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Clinical Impact of Polymerase Chain Reaction-Based Aspergillus and Azole Resistance Detection in Invasive Aspergillosis: A Prospective Multicenter Study.

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).

Performance of the JF5-Based Galactomannoprotein EIA Compared to the Lateral Flow Device and the Galactomannan EIA in Serum and Bronchoalveolar Lavage Fluid.

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 0.5 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.

Toxoplasmosis after allogeneic haematopoietic cell transplantation: experience using a PCR-guided pre-emptive approach.

OBJECTIVES: Prophylaxis with trimethoprim-sulphamethoxazole (TMP-SMZ) is recommended in Toxoplasma-seropositive allogeneic haematopoietic cell transplant (HCT) recipients to prevent reactivation, but it is associated with numerous side effects. We report our experience of a pre-emptive approach guided by a polymerase chain reaction (PCR) in patients not receiving prophylaxis. METHODS: In this retrospective, single-centre experience, seropositive recipients and seronegative recipients receiving a graft from a seropositive donor were screened by PCR for the presence of Toxoplasma gondii DNA in peripheral blood until at least 6 months after transplantation. Confirmed PCR positivity triggered a pre-emptive anti-Toxoplasma therapy. Cases of Toxoplasma reactivation (using the European Society for Blood and Marrow Transplantation definitions) were compared with four controls (without reactivation), matched in time and recipient serostatus, to identify risk factors for reactivation by multivariate analysis. RESULTS: From November 2001 to August 2020, 1455 consecutive adult patients (59 cases and 1396 controls) were screened. The overall 1-year cumulative incidence of toxoplasmosis was 4.1% and the 1-year cumulative incidence in the seropositive recipients was 8.8%. Reactivation was associated with second transplant (OR 2.51, 95%CI 1.28-4.94, p 0.011), myeloablative conditioning (OR 2.24, 95%CI 1.17-4.41, p 0.011), total body irradiation (OR 2.29, 95%CI 1.17-4.44, p 0.010), acute graft-versus-host disease (GvHD) (OR 2.27, 95%CI 1.26-4.08, p 0.008) and use of high-dose corticosteroids (OR 2.08, 95%CI 1.14-3.78, p 0.018). In multivariate analysis only acute GvHD remained significant (adjusted OR 2.54, 95%CI 1.16-5.71, p 0.021). CONCLUSIONS: A PCR-based pre-emptive approach might serve as an acceptable alternative for patients unable to start with or to continue TMP-SMZ prophylaxis. Acute GvHD was identified as the single independent predictor for reactivation.

Lung microbiota predict invasive pulmonary aspergillosis and its outcome in immunocompromised patients.

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.

Serum amyloid P component is an essential element of resistance against Aspergillus fumigatus.

Serum amyloid P component (SAP, also known as Pentraxin 2; APCS gene) is a component of the humoral arm of innate immunity involved in resistance to bacterial infection and regulation of tissue remodeling. Here we investigate the role of SAP in antifungal resistance. Apcs-/- mice show enhanced susceptibility to A. fumigatus infection. Murine and human SAP bound conidia, activate the complement cascade and enhance phagocytosis by neutrophils. Apcs-/- mice are defective in vivo in terms of recruitment of neutrophils and phagocytosis in the lungs. Opsonic activity of SAP is dependent on the classical pathway of complement activation. In immunosuppressed mice, SAP administration protects hosts against A. fumigatus infection and death. In the context of a study of hematopoietic stem-cell transplantation, genetic variation in the human APCS gene is associated with susceptibility to invasive pulmonary aspergillosis. Thus, SAP is a fluid phase pattern recognition molecule essential for resistance against A. fumigatus.

Genetic Variation in PFKFB3 Impairs Antifungal Immunometabolic Responses and Predisposes to Invasive Pulmonary Aspergillosis.

Activation of immune cells in response to fungal infection involves the reprogramming of their cellular metabolism to support antimicrobial effector functions. Although metabolic pathways such as glycolysis are known to represent critical regulatory nodes in antifungal immunity, it remains undetermined whether these are differentially regulated at the interindividual level. In this study, we identify a key role for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in the immunometabolic responses to Aspergillus fumigatus. A genetic association study performed in 439 recipients of allogeneic hematopoietic stem cell transplantation (HSCT) and corresponding donors revealed that the donor, but not recipient, rs646564 variant in the PFKFB3 gene increased the risk of invasive pulmonary aspergillosis (IPA) after transplantation. The risk genotype impaired the expression of PFKFB3 by human macrophages in response to fungal infection, which was correlated with a defective activation of glycolysis and the ensuing antifungal effector functions. In patients with IPA, the risk genotype was associated with lower concentrations of cytokines in the bronchoalveolar lavage fluid samples. Collectively, these findings demonstrate the important contribution of genetic variation in PFKFB3 to the risk of IPA in patients undergoing HSCT and support its inclusion in prognostic tools to predict the risk of fungal infection in this clinical setting. IMPORTANCE The fungal pathogen Aspergillus fumigatus can cause severe and life-threatening forms of infection in immunocompromised patients. Activation of glycolysis is essential for innate immune cells to mount effective antifungal responses. In this study, we report the contribution of genetic variation in the key glycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) to the risk of invasive pulmonary aspergillosis (IPA) after allogeneic hematopoietic stem cell transplantation. The PFKFB3 genotype associated with increased risk of infection was correlated with an impairment of the antifungal effector functions of macrophages in vitro and in patients with IPA. This work highlights the clinical relevance of genetic variation in PFKFB3 to the risk of IPA and supports its integration in risk stratification and preemptive measures for patients at high risk of IPA.

Performance of Existing Definitions and Tests for the Diagnosis of Invasive Fungal Diseases other than Invasive Candidiasis and Invasive Aspergillosis in Critically Ill, Adult Patients: A Systematic Review with Qualitative Evidence Synthesis.

The Fungal Infections Definitions in Intensive Care Unit (ICU) patients (FUNDICU) project aims to provide standard sets of definitions for invasive fungal diseases (IFDs) in critically ill, adult patients, including invasive aspergillosis (IA), invasive candidiasis (IC), Pneumocystis jirovecii pneumonia (PJP), and other non-IA, non-IC IFDs. The first step of the project was the conduction of separated systematic reviews of the characteristics and applicability to critically ill, adult patients outside classical populations at risk (hematology patients, solid organ transplant recipients) of available definitions and diagnostic tests for IFDs. We report here the results of two systematic reviews exploring the performance of available definitions and tests, for PJP and for other non-IA, non-IC IFDs. Starting from 2585 and 4584 records for PJP and other IFDs, respectively, 89 and 61 studies were deemed as eligible for full-text evaluation. However, only two studies for PJP and no studies for other IFDs met the FUNDICU protocol criteria for inclusion in qualitative synthesis. Currently, there is no sufficient solid data for directly evaluating the performance of existing definitions and laboratory tests for the diagnosis of PJP and other non-IA, non-IC IFDs in critically ill adult patients outside classical populations at risk.

Defining Galactomannan Positivity in the Updated EORTC/MSGERC Consensus Definitions of Invasive Fungal Diseases.

The consensus definitions of invasive fungal diseases from the EORTC/MSGERC were recently revised and updated. They now include consensus cutoff values for the galactomannan test that support the diagnosis of probable invasive aspergillosis. In this supplement article, we provide a rationale for these proposed thresholds based on the test's characteristics and performance in different patient populations and in different specimen types.

Prospective Evaluation of the Turbidimetric ß-D-Glucan Assay and 2 Lateral Flow Assays on Serum in Invasive Aspergillosis.

BACKGROUND: Invasive aspergillosis (IA) remains a potentially lethal disease and requires timely diagnosis and initiation of antifungal therapy. Recently, the IMMY lateral flow assay (LFA), the OLM Diagnostics lateral flow device (LFD), and the Wako turbidimetric ß-d-glucan assay have been approved for use as a diagnostic aid. However, their performance in diagnosing IA on serum samples from at-risk patients and the added value to the existing detection of serum galactomannan remain to be investigated. METHODS: We prospectively collected serum samples from 239 hematology patients and evaluated the diagnostic performance of these 3 assays while using the 2019 EORTC/MSG definitions (study number S59863/S61797, NCT03004092). RESULTS: We identified 5 cases of proven IA, 36 cases of probable IA, and 188 controls. The LFA had the highest negative predictive value (NPV) and sensitivity (0.90 and 0.49, respectively) while galactomannan detection had the highest positive predictive value and specificity (0.93 and 0.99, respectively). Sensitivity was not significantly different between both tests. When used in combination, the highest NPV was seen in patients with a negative LFA and a negative ß-d-glucan test. The sensitivity of the LFD was significantly lower than the LFA. After omitting serum galactomannan from the definitions to control for incorporation bias, the sensitivity of the LFA outperformed galactomannan detection (0.41 vs 0.31, P = .046). CONCLUSIONS: The LFA is a fast and effective alternative to serum galactomannan detection for the diagnosis of IA and is especially useful for centers with low sample throughputs. The addition of the Wako ß-D-glucan assay further improves the diagnostic performance.

The Aspergillus Lateral Flow Assay for the Diagnosis of Invasive Aspergillosis: an Update.

PURPOSE OF REVIEW: To review the data on the Aspergillus lateral flow assay for the diagnosis of invasive Aspergillosis. RECENT FINDINGS: Aspergillus spp. cause a wide spectrum of disease with invasive aspergillosis (IA) as its most severe manifestation. Early and reliable diagnosis of disease is crucial to decrease associated morbidity and mortality, and enable prompt initiation of treatment for IA. Most recently, non-culture-based tests, such as Aspergillus galactomannan (GM), have been useful in early identification and treatment of patients with IA. However, cost, turnaround time, and variable performance indifferent populations at risk for IA remain significant drawbacks to the use of this test. Several diagnostic tests for IA have been developed, including the sona Aspergillus GM Lateral flow assay (GM-LFA) rapid test. SUMMARY: The GM-LFA has shown excellent performance for the diagnosis of IA in patients with hematologic malignancy and may be a viable option for settings where ELISA GM testing is not feasible. Further evaluation of the GM-LFA in the non-hematology setting is ongoing, including in solid organ transplant recipients and patients in the intensive care unit.

Variable Correlation between Bronchoalveolar Lavage Fluid Fungal Load and Serum-(1,3)-ß-d-Glucan in Patients with Pneumocystosis-A Multicenter ECMM Excellence Center Study.

Pneumocystis jirovecii pneumonia is a difficult invasive infection to diagnose. Apart from microscopy of respiratory specimens, two diagnostic tests are increasingly used including real-time quantitative PCR (qPCR) of respiratory specimens, mainly in bronchoalveolar lavage fluids (BAL), and serum ß-1,3-d-glucan (BDG). It is still unclear how these two biomarkers can be used and interpreted in various patient populations. Here we analyzed retrospectively and multicentrically the correlation between BAL qPCR and serum BDG in various patient population, including mainly non-HIV patients. It appeared that a good correlation can be obtained in HIV patients and solid organ transplant recipients but no correlation can be observed in patients with hematologic malignancies, solid cancer, and systemic diseases. This observation reinforces recent data suggesting that BDG is not the best marker of PCP in non-HIV patients, with potential false positives due to other IFI or bacterial infections and false-negatives due to low fungal load and low BDG release.

Point of care aspergillus testing in intensive care patients.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) is an increasingly recognized complication in intensive care unit (ICU) patients, especially those with influenza, cirrhosis, chronic obstructive pulmonary disease, and other diseases. The diagnosis can be challenging, especially in the ICU, where clinical symptoms as well as imaging are mostly nonspecific. Recently, Aspergillus lateral flow tests were developed to decrease the time to diagnosis of IPA. Several studies have shown promising results in bronchoalveolar lavage fluid (BALf) from hematology patients. We therefore evaluated a new lateral flow test for IPA in ICU patients. METHODS: Using left-over BALf from adult ICU patients in two university hospitals, we studied the performance of the Aspergillus galactomannan lateral flow assay (LFA) by IMMY (Norman, OK, USA). Patients were classified according to the 2008 EORTC-MSG definitions, the AspICU criteria, and the modified AspICU criteria, which incorporate galactomannan results. These internationally recognized consensus definitions for the diagnosis of IPA incorporate patient characteristics, microbiology and radiology. The LFA was read out visually and with a digital reader by researchers blinded to the final clinical diagnosis and IPA classification. RESULTS: We included 178 patients, of which 55 were classified as cases (6 cases of proven and 26 cases of probable IPA according to the EORTC-MSG definitions, and an additional 23 cases according to the modified AspICU criteria). Depending on the definitions used, the sensitivity of the LFA was 0.88-0.94, the specificity was 0.81, and the area under the ROC curve 0.90-0.94, indicating good overall test performance. CONCLUSIONS: In ICU patients, the LFA performed well on BALf and can be used as a rapid screening test while waiting for other microbiological results.

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.

Phagosomal removal of fungal melanin reprograms macrophage metabolism to promote antifungal immunity.

In response to infection, macrophages adapt their metabolism rapidly to enhance glycolysis and fuel specialized antimicrobial effector functions. Here we show that fungal melanin is an essential molecule required for the metabolic rewiring of macrophages during infection with the fungal pathogen Aspergillus fumigatus. Using pharmacological and genetic tools, we reveal a molecular link between calcium sequestration by melanin inside the phagosome and induction of glycolysis required for efficient innate immune responses. By remodeling the intracellular calcium machinery and impairing signaling via calmodulin, melanin drives an immunometabolic signaling axis towards glycolysis with activation of hypoxia-inducible factor 1 subunit alpha (HIF-1α) and phagosomal recruitment of mammalian target of rapamycin (mTOR). These data demonstrate a pivotal mechanism in the immunometabolic regulation of macrophages during fungal infection and highlight the metabolic repurposing of immune cells as a potential therapeutic strategy.