Metagenomic Next-Generation Sequencing of Plasma for Diagnosis of COVID-19-Associated Pulmonary Aspergillosis.

Timely diagnosis remains an unmet need in non-neutropenic patients at risk for aspergillosis, including those with COVID-19-associated pulmonary aspergillosis (CAPA), which in its early stages is characterized by tissue-invasive growth of the lungs with limited angioinvasion. Currently available mycological tests show limited sensitivity when testing blood specimens. Metagenomic next-generation sequencing (mNGS) to detect microbial cell-free DNA (mcfDNA) in plasma might overcome some of the limitations of conventional diagnostics. A two-center cohort study involving 114 COVID-19 intensive care unit patients evaluated the performance of plasma mcfDNA sequencing for the diagnosis of CAPA. Classification of CAPA was performed using the European Confederation for Medical Mycology (ECMM)/International Society for Human and Animal Mycoses (ISHAM) criteria. A total of 218 plasma samples were collected between April 2020 and June 2021 and tested for mcfDNA (Karius test). Only 6 patients were classified as probable CAPA, and 2 were classified as possible, while 106 patients did not fulfill CAPA criteria. The Karius test detected DNA of mold pathogens in 12 samples from 8 patients, including Aspergillus fumigatus in 10 samples from 6 patients. Mold pathogen DNA was detected in 5 of 6 (83% sensitivity) cases with probable CAPA (A. fumigatus in 8 samples from 4 patients and Rhizopus microsporus in 1 sample), while the test did not detect molds in 103 of 106 (97% specificity) cases without CAPA. The Karius test showed promising performance for diagnosis of CAPA when testing plasma, being highly specific. The test detected molds in all but one patient with probable CAPA, including cases where other mycological tests from blood resulted continuously negative, outlining the need for validation in larger studies.

COVID-19-Associated Pulmonary Aspergillosis Isolates Are Genomically Diverse but Similar to Each Other in Their Responses to Infection-Relevant Stresses.

Secondary infections caused by the pulmonary fungal pathogen Aspergillus fumigatus are a significant cause of mortality in patients with severe coronavirus disease 19 (COVID-19). Even though epithelial cell damage and aberrant cytokine responses have been linked to susceptibility to COVID-19-associated pulmonary aspergillosis (CAPA), little is known about the mechanisms underpinning copathogenicity. Here, we analyzed the genomes of 11 A. fumigatus isolates from patients with CAPA in three centers from different European countries. CAPA isolates did not cluster based on geographic origin in a genome-scale phylogeny of representative A. fumigatus isolates. Phenotypically, CAPA isolates were more similar to the A. fumigatus A1160 reference strain than to the Af293 strain when grown in infection-relevant stresses, except for interactions with human immune cells wherein macrophage responses were similar to those induced by the Af293 reference strain. Collectively, our data indicate that CAPA isolates are genomically diverse but are more similar to each other in their responses to infection-relevant stresses. A larger number of isolates from CAPA patients should be studied to better understand the molecular epidemiology of CAPA and to identify genetic drivers of copathogenicity and antifungal resistance in patients with COVID-19. IMPORTANCE Coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) has been globally reported as a life-threatening complication in some patients with severe COVID-19. Most of these infections are caused by the environmental mold Aspergillus fumigatus, which ranks third in the fungal pathogen priority list of the WHO. However, little is known about the molecular epidemiology of Aspergillus fumigatus CAPA strains. Here, we analyzed the genomes of 11 A. fumigatus isolates from patients with CAPA in three centers from different European countries, and carried out phenotypic analyses with a view to understanding the pathophysiology of the disease. Our data indicate that A. fumigatus CAPA isolates are genomically diverse but are more similar to each other in their responses to infection-relevant stresses.

Isavuconazole plasma concentrations in critically ill patients during extracorporeal membrane oxygenation.

BACKGROUND: Isavuconazole is an antifungal drug used for treatment of invasive fungal infections. Critically ill COVID-19 and influenza patients require extracorporeal membrane oxygenation (ECMO) in cases with severe acute respiratory distress syndrome and have risk factors for invasive pulmonary aspergillosis. Little is known about isavuconazole plasma concentrations during ECMO. OBJECTIVES: To determine isavuconazole plasma concentrations in seven patients treated with intravenous isavuconazole under ECMO and the influence of the ECMO circuit immediately after the first isavuconazole dose. METHODS: Critically ill patients treated with isavuconazole (standard doses) and ECMO were included in this study. Sixty-four blood samples used for measurement of isavuconazole concentrations were collected at several timepoints starting 2 h after the first isavuconazole dose up to 168 h. An additional 27 blood samples were drawn from the inflow and outflow line of the membrane oxygenator to assess any potential isavuconazole clearance effect of the ECMO oxygenation device and the lines. RESULTS: Median isavuconazole trough levels above 1 µg/mL (min. 0.83, max. 1.73) or 2 µg/mL (min. 0.84, max. 2.97) were achieved 24 h or 96 h after the first dose of isavuconazole. The isavuconazole plasma concentrations pre (inflow line) and post (outflow line) the membrane oxygenator were directly correlated (ρ = 0.987, R2 = 0.994, P < 0.001). Post membrane oxygenator isavuconazole concentrations were directly correlated to contemporaneous samples obtained from the arterial lines of patients (ρ = 0.942, R2 = 0.945, P < 0.001). CONCLUSIONS: Isavuconazole concentrations might be influenced by the higher volume of distribution due to ECMO therapy, but were not altered by the ECMO oxygenator and achieved median plasma concentrations >1 µg/mL 24 h after the first loading dose.

Prognostic Impact of Bronchoalveolar Lavage Fluid Galactomannan and Aspergillus Culture Results on Survival in COVID-19 Intensive Care Unit Patients: a Post Hoc Analysis from the European Confederation of Medical Mycology (ECMM) COVID-19-Associated Pulmonary Aspergillosis Study.

Critically ill patients with coronavirus disease 2019 (COVID-19) may develop COVID-19-associated pulmonary aspergillosis (CAPA), which impacts their chances of survival. Whether positive bronchoalveolar lavage fluid (BALF) mycological tests can be used as a survival proxy remains unknown. We conducted a post hoc analysis of a previous multicenter, multinational observational study with the aim of assessing the differential prognostic impact of BALF mycological tests, namely, positive (optical density index of ≥1.0) BALF galactomannan (GM) and positive BALF Aspergillus culture alone or in combination for critically ill patients with COVID-19. Of the 592 critically ill patients with COVID-19 enrolled in the main study, 218 were included in this post hoc analysis, as they had both test results available. CAPA was diagnosed in 56/218 patients (26%). Most cases were probable CAPA (51/56 [91%]) and fewer were proven CAPA (5/56 [9%]). In the final multivariable model adjusted for between-center heterogeneity, an independent association with 90-day mortality was observed for the combination of positive BALF GM and positive BALF Aspergillus culture in comparison with both tests negative (hazard ratio, 2.53; 95% CI confidence interval [CI], 1.28 to 5.02; P = 0.008). The other independent predictors of 90-day mortality were increasing age and active malignant disease. In conclusion, the combination of positive BALF GM and positive BALF Aspergillus culture was associated with increased 90-day mortality in critically ill patients with COVID-19. Additional study is needed to explore the possible prognostic value of other BALF markers.

Aspergillus Lateral Flow Assay with Digital Reader for the Diagnosis of COVID-19-Associated Pulmonary Aspergillosis (CAPA): a Multicenter Study.

This multicenter study evaluated the IMMY Aspergillus Galactomannan Lateral Flow Assay (LFA) with automated reader for diagnosis of pulmonary aspergillosis in patients with COVID-19-associated acute respiratory failure (ARF) requiring intensive care unit (ICU) admission between 03/2020 and 04/2021. A total of 196 respiratory samples and 148 serum samples (n = 344) from 238 patients were retrospectively included, with a maximum of one of each sample type per patient. Cases were retrospectively classified for COVID-19-associated pulmonary aspergillosis (CAPA) status following the 2020 consensus criteria, with the exclusion of LFA results as a mycological criterion. At the 1.0 cutoff, sensitivity of LFA for CAPA (proven/probable/possible) was 52%, 80% and 81%, and specificity was 98%, 88% and 67%, for bronchoalveolar lavage fluid (BALF), nondirected bronchoalveolar lavage (NBL), and tracheal aspiration (TA), respectively. At the 0.5 manufacturer's cutoff, sensitivity was 72%, 90% and 100%, and specificity was 79%, 83% and 44%, for BALF, NBL and TA, respectively. When combining all respiratory samples, the receiver operating characteristic (ROC) area under the curve (AUC) was 0.823, versus 0.754, 0.890 and 0.814 for BALF, NBL and TA, respectively. Sensitivity and specificity of serum LFA were 20% and 93%, respectively, at the 0.5 ODI cutoff. Overall, the Aspergillus Galactomannan LFA showed good performances for CAPA diagnosis, when used from respiratory samples at the 1.0 cutoff, while sensitivity from serum was limited, linked to weak invasiveness during CAPA. As some false-positive results can occur, isolated results slightly above the recommended cutoff should lead to further mycological investigations.

The Antifungal Pipeline: Fosmanogepix, Ibrexafungerp, Olorofim, Opelconazole, and Rezafungin.

The epidemiology of invasive fungal infections is changing, with new populations at risk and the emergence of resistance caused by the selective pressure from increased usage of antifungal agents in prophylaxis, empiric therapy, and agriculture. Limited antifungal therapeutic options are further challenged by drug-drug interactions, toxicity, and constraints in administration routes. Despite the need for more antifungal drug options, no new classes of antifungal drugs have become available over the last 2 decades, and only one single new agent from a known antifungal class has been approved in the last decade. Nevertheless, there is hope on the horizon, with a number of new antifungal classes in late-stage clinical development. In this review, we describe the mechanisms of drug resistance employed by fungi and extensively discuss the most promising drugs in development, including fosmanogepix (a novel Gwt1 enzyme inhibitor), ibrexafungerp (a first-in-class triterpenoid), olorofim (a novel dihyroorotate dehydrogenase enzyme inhibitor), opelconazole (a novel triazole optimized for inhalation), and rezafungin (an echinocandin designed to be dosed once weekly). We focus on the mechanism of action and pharmacokinetics, as well as the spectrum of activity and stages of clinical development. We also highlight the potential future role of these drugs and unmet needs.

Antifungal prophylaxis for prevention of COVID-19-associated pulmonary aspergillosis in critically ill patients: an observational study.

BACKGROUND: Coronavirus disease 19 (COVID-19)-associated pulmonary aspergillosis (CAPA) emerged as important fungal complications in patients with COVID-19-associated severe acute respiratory failure (ARF). Whether mould active antifungal prophylaxis (MAFP) can prevent CAPA remains elusive so far. METHODS: In this observational study, we included all consecutive patients admitted to intensive care units with COVID-19-associated ARF between September 1, 2020, and May 1, 2021. We compared patients with versus without antifungal prophylaxis with respect to CAPA incidence (primary outcome) and mortality (secondary outcome). Propensity score adjustment was performed to account for any imbalances in baseline characteristics. CAPA cases were classified according to European Confederation of Medical Mycology (ECMM)/International Society of Human and Animal Mycoses (ISHAM) consensus criteria. RESULTS: We included 132 patients, of whom 75 (57%) received antifungal prophylaxis (98% posaconazole). Ten CAPA cases were diagnosed, after a median of 6 days following ICU admission. Of those, 9 CAPA cases were recorded in the non-prophylaxis group and one in the prophylaxis group, respectively. However, no difference in 30-day ICU mortality could be observed. Thirty-day CAPA incidence estimates were 1.4% (95% CI 0.2-9.7) in the MAFP group and 17.5% (95% CI 9.6-31.4) in the group without MAFP (p = 0.002). The respective subdistributional hazard ratio (sHR) for CAPA incidence comparing the MAFP versus no MAFP group was of 0.08 (95% CI 0.01-0.63; p = 0.017). CONCLUSION: In ICU patients with COVID-19 ARF, antifungal prophylaxis was associated with significantly reduced CAPA incidence, but this did not translate into improved survival. Randomized controlled trials are warranted to evaluate the efficacy and safety of MAFP with respect to CAPA incidence and clinical outcomes.

Soluble urokinase plasminogen activator receptor (suPAR) predicts critical illness and kidney failure in patients admitted to the intensive care unit.

Soluble urokinase plasminogen activator receptor (suPAR) is an inflammatory biomarker and risk factor for kidney diseases, with a potential prognostic value in critically ill patients. In this monocentric prospective study, we measured plasma suPAR levels immediately after ICU admission in unselected 237 consecutive patients using a turbidimetric assay. Primary objective was the prognostic value for ICU- and 28-day mortality. Secondary objectives were association with sequential organ failure assessment (SOFA) score, coagulation and inflammation markers, AKI-3 and differences in prespecified subgroups. Median suPAR levels were 8.0 ng/mL [25th-75th percentile 4.3-14.4], with lower levels in ICU survivors than non-survivors (6.7 vs. 11.6 ng/mL, p < 0.001). SuPAR levels were higher in COVID-19, kidney disease, moderate-to-severe liver disease, and sepsis. ICU mortality increased by an odds ratio (OR) of 4.7 in patients with the highest compared to lowest quartile suPAR. Kaplan-Meier overall survival estimates at 3 months were 63% and 49%, in patients with suPAR below/above 12 ng/mL (log-rank p = 0.027). Due to an observed interaction between SOFA score and suPAR, we performed a random forest method identifying cutoffs. ICU mortality was 53%, 17% and 2% in patients with a SOFA score > 7, SOFA ≤ 7 & suPAR > 8 ng/mL, and SOFA score ≤ 7 & suPAR ≤ 8 ng/mL, respectively. suPAR was a significant predictor for AKI-3 occurrence (OR per doubling 1.89, 95% CI: 1.20-2.98; p = 0.006). suPAR levels at ICU admission may offer additional value for risk stratification especially in ICU patients with moderate organ dysfunction as reflected by a SOFA score ≤ 7.

Risk factors and outcome of pulmonary aspergillosis in critically ill coronavirus disease 2019 patients-a multinational observational study by the European Confederation of Medical Mycology.

OBJECTIVES: Coronavirus disease 2019 (COVID-19) -associated pulmonary aspergillosis (CAPA) has emerged as a complication in critically ill COVID-19 patients. The objectives of this multinational study were to determine the prevalence of CAPA in patients with COVID-19 in intensive care units (ICU) and to investigate risk factors for CAPA as well as outcome. METHODS: The European Confederation of Medical Mycology (ECMM) conducted a multinational study including 20 centres from nine countries to assess epidemiology, risk factors and outcome of CAPA. CAPA was defined according to the 2020 ECMM/ISHAM consensus definitions. RESULTS: A total of 592 patients were included in this study, including 11 (1.9%) patients with histologically proven CAPA, 80 (13.5%) with probable CAPA, 18 (3%) with possible CAPA and 483 (81.6%) without CAPA. CAPA was diagnosed a median of 8 days (range 0-31 days) after ICU admission predominantly in older patients (adjusted hazard ratio (aHR) 1.04 per year; 95% CI 1.02-1.06) with any form of invasive respiratory support (HR 3.4; 95% CI 1.84-6.25) and receiving tocilizumab (HR 2.45; 95% CI 1.41-4.25). Median prevalence of CAPA per centre was 10.7% (range 1.7%-26.8%). CAPA was associated with significantly lower 90-day ICU survival rate (29% in patients with CAPA versus 57% in patients without CAPA; Mantel-Byar p < 0.001) and remained an independent negative prognostic variable after adjusting for other predictors of survival (HR 2.14; 95% CI 1.59-2.87, p ≤ 0.001). CONCLUSION: Prevalence of CAPA varied between centres. CAPA was significantly more prevalent among older patients, patients receiving invasive ventilation and patients receiving tocilizumab, and was an independent strong predictor of ICU mortality.

Serum Lateral Flow assay with digital reader for the diagnosis of invasive pulmonary aspergillosis: A two-centre mixed cohort study.

BACKGROUND: Detection of galactomannan (GM) from bronchoalveolar lavage fluid (BALF) or serum is broadly used for diagnosis of invasive aspergillosis (IA), although the sensitivity of GM from serum is lower in non-neutropenic patients. We evaluated the Aspergillus galactomannan Lateral Flow assay (LFA) with digital readout from serum in a mixed cohort of patients. METHODS: We performed a retrospective two-centre study evaluating the LFA from serum of patients with clinical suspicion of IA obtained between 2015 and 2021 at the University of California San Diego and the Medical University of Graz. The sensitivity and specificity was calculated for proven/probable aspergillosis versus no aspergillosis. Correlation with same-sample GM was calculated using Spearman correlation analysis and kappa statistics. RESULTS: In total, 122 serum samples from 122 patients were analysed, including proven IA (n = 1), probable IA or coronavirus-associated pulmonary aspergillosis (CAPA) (n = 27), and no IA/CAPA/non-classifiable (n = 94). At a 0.5 ODI cut-off, the sensitivity and specificity of the LFA was 78.6% and 80.5%. Spearman correlation analysis showed a strong correlation between serum LFA ODI and serum GM ODI (ρ 0.459, p < .0001). Kappa was 0.611 when both LFA and GM were used with a 0.5 ODI cut-off, showing substantial agreement (p < .001). DISCUSSION: The LFA with digital read out from serum showed good performance for the diagnosis of probable/proven aspergillosis, with substantial agreement to GM from serum. Like the LFA from BALF, the LFA from serum may serve as a more rapid test compared to conventional GM, particularly in settings where GM is not readily available.

Admission levels of Soluble Urokinase Plasminogen Activator Receptor (suPAR) are Associated with the Development of Severe Complications in Hospitalised COVID-19 Patients: A Prospective Cohort Study.

OBJECTIVE: To examine the association between plasma levels of the soluble urokinase plasminogen activator receptor (suPAR) and the incidence of severe complications of COVID-19. METHODS: 403 RT-PCR-confirmed COVID-19 patients were recruited and prospectively followed-up at a major hospital in the United Arab Emirates. The primary endpoint was time from admission until the development of a composite outcome, including acute respiratory distress syndrome (ARDS), intensive care unit (ICU) admission, or death from any cause. Patients discharged alive were considered as competing events to the primary outcome. Competing risk regression was used to quantify the association between suPAR and the incidence of the primary outcome. RESULTS: 6.2% of patients experienced ARDS or ICU admission, but none died. Taking into account competing risk, the incidence of the primary outcome was 11.5% (95% confidence interval [CI], 6.7-16.3) in patients with suPAR levels >3.91 ng/mL compared to 2.9% (95% CI, 0.4-5.5) in those with suPAR ≤3.91 ng/mL. Also, an increase by 1 ng/mL in baseline suPAR resulted in a 58% rise in the hazard of developing the primary outcome (hazard ratio 1.6, 95% CI, 1.2-2.1, p = 0.003). CONCLUSION: suPAR has an excellent prognostic utility in predicting severe complications in hospitalised COVID-19 patients.

Autopsy Proven Pulmonary Mucormycosis Due to Rhizopus microsporus in a Critically Ill COVID-19 Patient with Underlying Hematological Malignancy.

Viral infections can cause acute respiratory distress syndrome (ARDS), consequently leading to susceptibility for secondary pulmonary infections. Over the past few weeks, a number of studies have reported on secondary pulmonary aspergillosis complicating severe COVID-19. We report the case of a 53-year old male patient with secondary acute myeloid leukemia (AML) who suffered from COVID-19 ARDS and was diagnosed postmortem with mucormycosis.

Aspergillus fumigatus and Its Allergenic Ribotoxin Asp f I: Old Enemies but New Opportunities for Urine-Based Detection of Invasive Pulmonary Aspergillosis Using Lateral-Flow Technology.

Invasive pulmonary aspergillosis (IPA) caused by Aspergillus fumigatus is a life-threatening lung disease of immunocompromised patients. Diagnosis currently relies on non-specific chest CT, culture of the fungus from invasive lung biopsy, and detection of the cell wall carbohydrate galactomannan (GM) in serum or in BAL fluids recovered during invasive bronchoscopy. Urine provides an ideal bodily fluid for the non-invasive detection of pathogen biomarkers, with current urine-based immunodiagnostics for IPA focused on GM. Surrogate protein biomarkers might serve to improve disease detection. Here, we report the development of a monoclonal antibody (mAb), PD7, which is specific to A. fumigatus and related species in the section Fumigati, and which binds to its 18 kDa ribotoxin Asp f I. Using PD7, we show that the protein is secreted during hyphal development, and so represents an ideal candidate for detecting invasive growth. We have developed a lateral-flow device (Afu-LFD®) incorporating the mAb which has a limit of detection of ~15 ng Asp f I/mL urine. Preliminary evidence of the test's diagnostic potential is demonstrated with urine from a patient with acute lymphoid leukaemia with probable IPA. The Afu-LFD® therefore provides a potential novel opportunity for non-invasive urine-based detection of IPA caused by A. fumigatus.

Invasive pulmonary aspergillosis complicating COVID-19 in the ICU - A case report.

It is not yet known, if critically ill COVID-19 patients are prone to fungal infections. We report a 69-year-old patient without typical risk factors for invasive pulmonary aspergillosis (IPA), who developed IPA two weeks after onset of symptoms. Our report shows that IPA may occur in critically ill COVID-19 patients.