Novel antifungals and treatment approaches to tackle resistance and improve outcomes of invasive fungal disease.

SUMMARYFungal infections are on the rise, driven by a growing population at risk and climate change. Currently available antifungals include only five classes, and their utility and efficacy in antifungal treatment are limited by one or more of innate or acquired resistance in some fungi, poor penetration into "sequestered" sites, and agent-specific side effect which require frequent patient reassessment and monitoring. Agents with novel mechanisms, favorable pharmacokinetic (PK) profiles including good oral bioavailability, and fungicidal mechanism(s) are urgently needed. Here, we provide a comprehensive review of novel antifungal agents, with both improved known mechanisms of actions and new antifungal classes, currently in clinical development for treating invasive yeast, mold (filamentous fungi), Pneumocystis jirovecii infections, and dimorphic fungi (endemic mycoses). We further focus on inhaled antifungals and the role of immunotherapy in tackling fungal infections, and the specific PK/pharmacodynamic profiles, tissue distributions as well as drug-drug interactions of novel antifungals. Finally, we review antifungal resistance mechanisms, the role of use of antifungal pesticides in agriculture as drivers of drug resistance, and detail detection methods for antifungal resistance.

Profiling Bacteria in the Lungs of Patients with Severe Influenza Versus COVID-19 with or without Aspergillosis.

RATIONALE: The influence of the lung bacterial microbiome, including potential pathogens, in patients with influenza- or COVID-19-associated pulmonary aspergillosis (IAPA or CAPA) is yet to be explored. OBJECTIVES: To explore the composition of the lung bacterial microbiome and its association with viral and fungal infection, immunity and outcome in severe influenza versus COVID-19 with or without aspergillosis. METHODS: We performed a retrospective study in mechanically ventilated influenza and COVID-19 patients with or without invasive aspergillosis in whom bronchoalveolar lavage (BAL) for bacterial culture (with or without PCR) was obtained within two weeks after ICU admission. Additionally, 16S rRNA gene sequencing data and viral and bacterial load of BAL samples from a subset of these patients, and of patients requiring non-invasive ventilation, were analyzed. We integrated 16S rRNA gene sequencing data with existing immune parameter datasets. MEASUREMENTS AND MAIN RESULTS: Potential bacterial pathogens were detected in 20% (28/142) of influenza and 37% (104/281) of COVID-19 patients, while aspergillosis was detected in 38% (54/142) of influenza and 31% (86/281) of COVID-19 patients. A significant association between bacterial pathogens in BAL and 90-day mortality was found only in influenza patients, particularly IAPA patients. COVID-19 but not influenza patients showed increased pro-inflammatory pulmonary cytokine responses to bacterial pathogens. CONCLUSIONS: Aspergillosis is more frequently detected in lungs of severe influenza patients than bacterial pathogens. Detection of bacterial pathogens associates with worse outcome in influenza patients, particularly in those with IAPA, but not in COVID-19 patients. The immunological dynamics of tripartite viral-fungal-bacterial interactions deserve further investigation. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Nationwide quality assurance of high-throughput diagnostic molecular testing during the SARS-CoV-2 pandemic: role of the Belgian National Reference Centre.

Since the onset of the coronavirus disease (COVID-19) pandemic in Belgium, UZ/KU Leuven has played a crucial role as the National Reference Centre (NRC) for respiratory pathogens, to be the first Belgian laboratory to develop and implement laboratory developed diagnostic assays for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and later to assess the quality of commercial kits. To meet the growing demand for decentralised testing, both clinical laboratories and government-supported high-throughput platforms were gradually deployed across Belgium. Consequently, the role of the NRC transitioned from a specialised testing laboratory to strengthening capacity and coordinating quality assurance. Here, we outline the measures taken by the NRC, the national public health institute Sciensano and the executing clinical laboratories to ensure effective quality management of molecular testing throughout the initial two years of the pandemic (March 2020 to March 2022).

Evaluation and implementation of optimized antimicrobial dosing strategies in obese and underweight patients.

PURPOSE: We aimed to develop and implement dosing recommendations for antimicrobials in obese and underweight patients within an academic hospital, and assess their impact on antibiotic prescribing. METHODS: A multi-step approach project was performed. First, obese and underweight patient prevalence and antimicrobial prescription frequency was determined in a point prevalence study. Second and third, a literature review and e-survey provided dosing evidence. Fourth, a consensus meeting was organized to formulate dosing recommendations. Fifth, these were implemented in our clinical validation service as six clinical rules continuously screening patients' records for potentially inappropriate prescriptions (PIPs). Uptake was evaluated by documenting the number of advices and acceptance rate. Last, an interrupted time series analysis (ITS) compared pre- and post-implementation periods to measure the impact of the intervention on residual PIPs/day. A residual PIP was defined as a PIP which persisted up to 48 h. RESULTS: First, 41% of 15.896 hospitalized patients received antimicrobials over 20 days; of which 12% were obese and 9% underweight. Antibiotics were predominantly prescribed according to standard dosing regimens, adjusted to renal function. Next, six dosing recommendations, derived from literature, survey, and consensus, were implemented. In the fifth step, during an 18-week period, 219 advices were given, with 86% acceptance rate. Last, in the ITS analysis, at preintervention, a median of 75% residual PIPs/day existed, reduced to 0% postintervention. Use of clinical rules resulted in a significant immediate 84% relative reduction in residual PIPs (95% CI 0.55-0.94). CONCLUSION: After conducting a literature review, e-survey, and seeking consensus from a panel of experts, dosing recommendations for antimicrobial treatment in both obese and underweight patients were developed. These recommendations have been successfully implemented into clinical practice, addressing the specific needs of these patient populations.

COVID-19 Outcomes in Lung Transplant Recipients Following Pre-Exposure Prophylaxis With Tixagevimab-Cilgavimab During the Omicron BA.5 Surge: A Single Center Analysis.

Lung transplant (LTx) recipients are at high risk for COVID-19 related morbidity and mortality. Data regarding pre-exposure prophylaxis (PrEP) with tixagevimab-cilgavimab in this population are scarce. We therefore evaluated COVID-19 breakthrough infections and COVID-19 related complications after PrEP in a retrospective single-center study, including 264 LTx recipients who received PrEP between June 2022 and December 2022, when Omicron BA.5 was the dominant circulating SARS-CoV-2 variant. PrEP was indicated for fully vaccinated patients with poor seroconversion (anti-S <260 BAU/mL). COVID-19 breakthrough infection after PrEP occurred in 11.0% within the first 3 months, increasing to 17.4% within 6 months. Hospitalization rate rose from 27.6% to 52.9% (p = 0.046), while ICU admissions and COVID-19 mortality remained low, respectively occurring in 6.5% and 4.3% of patients with breakthrough infection within 6 months. COVID-19 breakthrough infection and associated hospitalization remained an important problem during the Omicron BA.5 surge in fully vaccinated LTx recipients with deficient seroconversion, despite PrEP with tixagevimab-cilgavimab. However, ICU admissions and COVID-19 mortality were low. Waning of neutralizing effects of PrEP and changing circulating SARS-CoV-2 variants may explain increases in COVID-19 infections and hospitalizations over time after PrEP, highlighting the need for novel, long-term effective PrEP strategies in these high-risk patients.

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.

First detection of triazole-resistant aspergillus fumigatus harbouring the TR34/L98H Cyp51A mutation in Burkina Faso.

BACKGROUND: Triazole-resistant Aspergillus fumigatus (TRAF) isolates are a growing public health problem with worldwide distribution. Epidemiological data on TRAF is limited in Africa, particularly in West Africa. OBJECTIVES: This study aimed to screen for the environmental presence of TRAF isolates in the indoor air of two hospitals in Burkina Faso. MATERIALS AND METHODS: Air samples were collected in wards housing patients at risk for invasive aspergillosis, namely infectious diseases ward, internal medicine ward, nephrology ward, pulmonology ward, medical emergency ward and paediatric ward. Sabouraud Dextrose Agar supplemented with triazoles was used to screen the suspected TRAF isolates and EUCAST method to confirm the resistance of suspected isolates. Sequencing of cyp51A gene was used to identify the resistance mechanism of confirmed TRAF isolates. RESULTS: Of the 198 samples collected and analysed, 67 showed growth of A. fumigatus isolates. The prevalence of TRAF isolates was 3.23% (4/124). One TRAF isolate exhibited a pan-triazole resistance. Sequencing of cyp51A gene identified the TR34/L98H mutation for this pan-triazole resistant isolate. This study showed for the first time the circulation of the pan-azole resistant isolate harbouring the TR34/L98H mutation in Burkina Faso. CONCLUSIONS: These findings emphasise the need to map these TRAF isolates in all parts of Burkina Faso and to establish local and national continuous surveillance of environmental and clinical TRAF isolates in this country.

Insights from Three Pan-European Multicentre Studies on Invasive Candida Infections and Outlook to ECMM Candida IV.

Invasive candidiasis and candidemia remain a significant public health concern. The European Confederation of Medical Mycology (ECMM) conducted three pan-European multicentre studies from 1997 to 2022 to investigate various aspects of invasive Candida infections. These studies revealed shifting trends in Candida species distribution, with an increase of non-albicans Candida species as causative pathogens, increasing rates of antifungal resistance, and persistently high mortality rates. Despite advancements in antifungal treatment, the persistently high mortality rate and increasing drug resistance, as well as limited drug access in low-income countries, underscore the need for continued research and development in the treatment of Candida infections. This review aims to summarize the findings of the three completed ECMM Candida studies and emphasize the importance of continued research efforts. Additionally, it introduces the upcoming ECMM Candida IV study, which will focus on assessing candidemia caused by non-albicans Candida species, including Candida auris, investigating antifungal resistance and tolerance, and evaluating novel treatment modalities on a global scale.

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

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

Guidelines for the management of Toxoplasma gondii infection and disease in patients with haematological malignancies and after haematopoietic stem-cell transplantation: guidelines from the 9th European Conference on Infections in Leukaemia, 2022.

Patients with haematological malignancies might develop life-threatening toxoplasmosis, especially after allogeneic haematopoietic stem-cell transplantation (HSCT). Reactivation of latent cysts is the primary mechanism of toxoplasmosis following HSCT; hence, patients at high risk are those who were seropositive before transplantation. The lack of trimethoprim-sulfamethoxazole prophylaxis and various immune status parameters of the patient are other associated risk factors. The mortality of toxoplasma disease-eg, with organ involvement-can be particularly high in this setting. We have developed guidelines for managing toxoplasmosis in haematology patients, through a literature review and consultation with experts. In allogeneic HSCT recipients seropositive for Toxoplasma gondii before transplant, because T gondii infection mostly precedes toxoplasma disease, we propose weekly blood screening by use of quantitative PCR (qPCR) to identify infection early as a pre-emptive strategy. As trimethoprim-sulfamethoxazole prophylaxis might fail, prophylaxis and qPCR screening should be combined. However, PCR in blood can be negative even in toxoplasma disease. The duration of prophylaxis should be a least 6 months and extended during treatment-induced immunosuppression or severe CD4 lymphopenia. If a positive qPCR test occurs, treatment with trimethoprim-sulfamethoxazole, pyrimethamine-sulfadiazine, or pyrimethamine-clindamycin should be started, and a new sample taken. If the second qPCR test is negative, clinical judgement is recommended to either continue or stop therapy and restart prophylaxis. Therapy must be continued until a minimum of two negative PCRs for infection, or for at least 6 weeks for disease. The pre-emptive approach is not indicated in seronegative HSCT recipients, after autologous transplantation, or in non-transplant haematology patients, but PCR should be performed with a high level of clinical suspicion.

The value of point-of-care tests for the detection of SARS-CoV-2 RNA or antigen in bronchoalveolar lavage fluid.

BACKGROUND: Transmission of SARS-CoV-2 from donor to recipient is a clinically relevant risk for developing severe COVID-19 after lung transplantation (LTx). This risk of iatrogenic transmission can be reduced by timely detection of viral RNA or antigen in samples of bronchoalveolar lavage (BAL) fluid obtained at the time of lung procurement. We aimed to retrospectively evaluate the detection of SARS-CoV-2 RNA or antigen in BAL fluid samples using three point-of-care tests (POCTs). METHODS: BAL fluid samples came from patients hospitalized in an intensive care unit during the COVID-19 pandemic. These pandemic samples were scored as positive or negative for SARS-CoV-2 by a RT-qPCR comparator assay for orf1ab. Three commercially available POCTs were then evaluated: cobas SARS-CoV-2 & Influenza A/B assay with the cobas Liat RT-qPCR system (Roche Diagnostics), ID NOW COVID-19 and COVID-19 2.0 (Abbott), and SARS-CoV-2 Rapid Antigen Test (RAT) (Roche Diagnostics). Samples from the pre-pandemic era served as negative controls. RESULTS: We analyzed a total of 98 BAL fluid samples, each from a different patient: 58 positive pandemic samples (orf1ab Ct<38), 20 putatively negative pandemic samples (orf1ab Ct≥38), and 20 pre-pandemic samples. Univariate logistic regression shows that the probability of detection was highest for cobas Liat, followed by ID NOW, and then RAT. Of clinical relevance, cobas Liat detected SARS-CoV-2 RNA in 30 of the 31 positive pandemic samples that were collected within 10 days after RT-qPCR diagnosis of SARS-CoV-2 infection. None of the 20 pre-pandemic samples had a false-positive result for any POCT. CONCLUSIONS: POCTs enable the detection of SARS-CoV-2 RNA or antigen in BAL fluid samples and may provide additional information to decide if donor lungs are suitable for transplantation. Detection of respiratory pathogens with POCTs at the time of donor lung procurement is a potential strategy to increase safety in LTx by preventing iatrogenic transmission and severe postoperative infections.

Influenza-associated and COVID-19-associated pulmonary aspergillosis in critically ill patients.

Influenza-associated pulmonary aspergillosis (IAPA) and COVID-19-associated pulmonary aspergillosis (CAPA) are increasingly recognised as important complications in patients requiring intensive care for severe viral pneumonia. The diagnosis can typically be made in 10-20% of patients with severe influenza or COVID-19, but only when appropriate diagnostic tools are used. Bronchoalveolar lavage sampling for culture, galactomannan testing, and PCR forms the cornerstone of diagnosis, whereas visual examination of the tracheobronchial tract during bronchoscopy is required to detect invasive Aspergillus tracheobronchitis. Azoles are the first-choice antifungal drugs, with liposomal amphotericin B as an alternative in settings where azole resistance is prevalent. Despite antifungal therapy, IAPA and CAPA are associated with poor outcomes, with fatality rates often exceeding 50%. In this Review, we discuss the mechanistic and clinical aspects of IAPA and CAPA. Moreover, we identify crucial knowledge gaps and formulate directions for future research.

Lower respiratory tract single-cell RNA sequencing and neutrophil extracellular trap profiling of COVID-19-associated pulmonary aspergillosis: a single centre, retrospective, observational study.

BACKGROUND: COVID-19-associated pulmonary aspergillosis (CAPA) is a severe superinfection with the fungus Aspergillus affecting patients who are critically ill with COVID-19. The pathophysiology and the role of neutrophil extracellular traps (NETs) in this infection are largely unknown. We aimed to characterise the immune profile, with a focus on neutrophils and NET concentrations, of critically ill patients with COVID-19, with or without CAPA. METHODS: We conducted a single-centre, retrospective, observational study in two patient cohorts, both recruited at University Hospitals Leuven, Belgium. We included adults aged 18 years or older who were admitted to the intensive care unit because of COVID-19 between March 31, 2020, and May 18, 2021, and who were included in the previous Contagious trial (NCT04327570). We investigated the immune cellular landscape of CAPA versus COVID-19 only by performing single-cell RNA sequencing (scRNA-seq) on bronchoalveolar lavage fluid. Bronchoalveolar lavage immune cell fractions were compared between patients with CAPA and patients with COVID-19 only. Additionally, we determined lower respiratory tract NET concentrations using biochemical assays in patients aged 18 years and older who were admitted to the intensive care unit because of severe COVID-19 between March 15, 2020, and Dec 31, 2021, for whom bronchoalveolar lavage was available in the hospital biobank. Bronchoalveolar lavage NET concentrations were compared between patients with CAPA and patients with COVID-19 only and integrated with existing data on immune mediators in bronchoalveolar lavage and 90-day mortality. FINDINGS: We performed scRNA-seq of bronchoalveolar lavage on 43 samples from 39 patients, of whom 36 patients (30 male and six female; 14 with CAPA) were included in downstream analyses. We performed bronchoalveolar lavage NET analyses in 59 patients (46 male and 13 female), of whom 26 had CAPA. By scRNA-seq, patients with CAPA had significantly lower neutrophil fractions than patients with COVID-19 only (16% vs 33%; p=0·0020). The remaining neutrophils in patients with CAPA preferentially followed a hybrid maturation trajectory characterised by expression of genes linked to antigen presentation, with enhanced transcription of antifungal effector pathways. Patients with CAPA also showed depletion of mucosal-associated invariant T cells, reduced T helper 1 and T helper 17 differentiation, and transcriptional defects in specific aspects of antifungal immunity in macrophages and monocytes. We observed increased formation of NETs in patients with CAPA compared with patients with COVID-19 only (DNA complexed with citrullinated histone H3 median 15 898 ng/mL [IQR 4588-86 419] vs 7062 ng/mL [775-14 088]; p=0·042), thereby explaining decreased neutrophil fractions by scRNA-seq. Low bronchoalveolar lavage NET concentrations were associated with increased 90-day mortality in patients with CAPA. INTERPRETATION: Qualitative and quantitative disturbances in monocyte, macrophage, B-cell, and T-cell populations could predispose patients with severe COVID-19 to develop CAPA. Hybrid neutrophils form a specialised response to CAPA, and an adequate neutrophil response to CAPA is a major determinant for survival in these patients. Therefore, measuring bronchoalveolar lavage NETs could have diagnostic and prognostic value in patients with CAPA. Clinicians should be wary of aspergillosis when using immunomodulatory therapy that might inhibit NETosis to treat patients with severe COVID-19. FUNDING: Research Foundation Flanders, KU Leuven, UZ Leuven, VIB, the Fundação para a Ciência e a Tecnologia, the European Regional Development Fund, la Caixa Foundation, the Flemish Government, and Horizon 2020.

Attributable mortality of candidemia - Results from the ECMM Candida III multinational European Observational Cohort Study.

INTRODUCTION: Despite antifungal advancements, candidaemia still has a high mortality rate of up to 40%. The ECMM Candida III study in Europe investigated the changing epidemiology and outcomes of candidaemia for better understanding and management of these infections. METHODS: In this observational cohort study, participating hospitals enrolled the first ten consecutive adults with blood culture-proven candidemia. Collected data included patient demographics, risk factors, hospital stay duration (follow-up of 90 days), diagnostic procedures, causative Candida spp., management details, and outcome. Controls were included in a 1:1 fashion from the same hospitals. The matching process ensured similarity in age (10-year range), primary underlying disease, hospitalization in intensive care versus non-ICU ward, and major surgery within 2 weeks before candidemia between cases and controls. Overall and attributable mortality were described, and a survival probability for cases and controls was performed. RESULTS: One hundred seventy-one pairs consisting of patients with candidemia and matched controls from 28 institutions were included. In those with candidemia, overall mortality was 40.4%. Attributable mortality was 18.1% overall but differed between causative Candida species (7.7% for Candida albicans, 23.7% for Candida glabrata/Nakaseomyces glabratus, 7.7% for Candida parapsilosis and 63.6% for Candida tropicalis). Regarding risk factors, the presence of a central venous catheter, total parenteral nutrition and acute or chronic renal disease were significantly more common in cases versus controls. Duration of hospitalization, and especially that of ICU stay, was significantly longer in candidemia cases (20 (IQR 10-33) vs 15 days (IQR 7-28); p = 0.004). CONCLUSIONS: Although overall and attributable mortality in this subgroup analysis of matched case/control pairs remains high, the attributable mortality appears to have decreased in comparison to historical cohorts. This decrease may be driven by improved prognosis of Candida albicans and Candida parapsilosis candidemia; whereas candidemia due to other Candida spp. exhibits a much higher attributable mortality.