A Pathology-based Case Series of Influenza- and COVID-19-associated Pulmonary Aspergillosis: The Proof Is in the Tissue.

RATIONALE: Invasive pulmonary aspergillosis has emerged as a frequent coinfection in severe COVID-19, similarly to influenza; yet the clinical invasiveness is more debated. OBJECTIVES: We investigated the invasive nature of pulmonary aspergillosis in histology specimens of influenza and COVID-19 intensive care unit (ICU) fatalities in a tertiary care center. METHODS: In this monocentric, descriptive, retrospective case series we included adult ICU patients with PCR-proven influenza/COVID-19 respiratory failure that underwent postmortem examination and/or tracheobronchial biopsy during ICU admission from September 2009 until June 2021. Diagnosis of probable/proven viral-associated pulmonary aspergillosis (VAPA) was made based on the ICM-IAPA and ECMM/ISHAM-CAPA consensus criteria. All respiratory tissues were independently reviewed by two experienced pathologists. MEASUREMENTS AND MAIN RESULTS: In the 44 patients of the autopsy-verified cohort, 6 proven influenza-associated and 6 proven COVID-19-associated pulmonary aspergillosis diagnoses were identified. Fungal disease was identified as missed-diagnosis upon autopsy in 8% of proven cases (n=1/12), yet most frequently found as confirmation of probable antemortem diagnosis (n=11/21, 52%) despite receiving antifungal treatment. Bronchoalveolar lavage galactomannan testing showed highest sensitivity for VAPA diagnosis. Among both viral entities, an impeded fungal growth was the predominant histologic pattern of pulmonary aspergillosis. Fungal tracheobronchitis was histologically indistinguishable in influenza (n=3) and COVID-19 (n=3) cases, yet macroscopically more extensive at bronchoscopy in influenza setting. CONCLUSIONS: Proven invasive pulmonary aspergillosis diagnosis was found regularly and with a similar histological pattern in influenza and in COVID-19 ICU case-fatalities. Our findings highlight an important need for VAPA awareness with an emphasis on mycological bronchoscopic work-up. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).

Visualising SARS-CoV-2 infection of the lung in deceased COVID-19 patients.

BACKGROUND: SARS-CoV-2 is a single-stranded positive-sense RNA virus. Several negative-sense SARS-CoV-2 RNA species, both full-length genomic and subgenomic, are produced transiently during viral replication. Methodologies for rigorously characterising cell tropism and visualising ongoing viral replication at single-cell resolution in histological sections are needed to assess the virological and pathological phenotypes of future SARS-CoV-2 variants. We aimed to provide a robust methodology for examining the human lung, the major target organ of this RNA virus. METHODS: A prospective cohort study took place at the University Hospitals Leuven in Leuven, Belgium. Lung samples were procured postmortem from 22 patients who died from or with COVID-19. Tissue sections were fluorescently stained with the ultrasensitive single-molecule RNA in situ hybridisation platform of RNAscope combined with immunohistochemistry followed by confocal imaging. FINDINGS: We visualised perinuclear RNAscope signal for negative-sense SARS-CoV-2 RNA species in ciliated cells of the bronchiolar epithelium of a patient who died with COVID-19 in the hyperacute phase of the infection, and in ciliated cells of a primary culture of human airway epithelium that had been infected experimentally with SARS-CoV-2. In patients who died between 5 and 13 days after diagnosis of the infection, we detected RNAscope signal for positive-sense but not for negative-sense SARS-CoV-2 RNA species in pneumocytes, macrophages, and among debris in the alveoli. SARS-CoV-2 RNA levels decreased after a disease course of 2-3 weeks, concomitant with a histopathological change from exudative to fibroproliferative diffuse alveolar damage. Taken together, our confocal images illustrate the complexities stemming from traditional approaches in the literature to characterise cell tropism and visualise ongoing viral replication solely by the surrogate parameters of nucleocapsid-immunoreactive signal or in situ hybridisation for positive-sense SARS-CoV-2 RNA species. INTERPRETATION: Confocal imaging of human lung sections stained fluorescently with commercially available RNAscope probes for negative-sense SARS-CoV-2 RNA species enables the visualisation of viral replication at single-cell resolution during the acute phase of the infection in COVID-19. This methodology will be valuable for research on future SARS-CoV-2 variants and other respiratory viruses. FUNDING: Max Planck Society, Coronafonds UZ/KU Leuven, European Society for Organ Transplantation.

Lung epithelial and myeloid innate immunity in influenza-associated or COVID-19-associated pulmonary aspergillosis: an observational study.

BACKGROUND: Influenza-associated pulmonary aspergillosis (IAPA) and COVID-19-associated pulmonary aspergillosis (CAPA) affect about 15% of critically ill patients with influenza or COVID-19, respectively. These viral-fungal coinfections are difficult to diagnose and are associated with increased mortality, but data on their pathophysiology are scarce. We aimed to explore the role of lung epithelial and myeloid innate immunity in patients with IAPA or CAPA. METHODS: In this observational study, we retrospectively recruited patients who had been admitted to the intensive care unit (ICU) of University Hospitals Leuven, Belgium, requiring non-invasive or invasive ventilation because of severe influenza or COVID-19, with or without aspergillosis, between Jan 1, 2011, and March 31, 2021, whose bronchoalveolar lavage samples were available at the hospital biobank. Additionally, biobanked in vivo tracheobronchial biopsy samples from patients with IAPA or CAPA and invasive Aspergillus tracheobronchitis admitted to ICUs requiring invasive ventilation between the same dates were collected from University Hospitals Leuven, Hospital Network Antwerp (Belgium), and Amiens-Picardie University Hospital (France). We did nCounter gene expression analysis of 755 genes linked to myeloid innate immunity and protein analysis of 47 cytokines, chemokines, and growth factors on the bronchoalveolar lavage samples. Gene expression data were used to infer cell fractions by use of CIBERSORTx, to perform hypergeometric enrichment pathway analysis and gene set enrichment analysis, and to calculate pathway module scores for the IL-1ß, TNF-α, type I IFN, and type II IFN (IFNγ) pathways. We did RNAScope targeting influenza virus or SARS-CoV-2 RNA and GeoMx spatial transcriptomics on the tracheobronchial biopsy samples. FINDINGS: Biobanked bronchoalveolar lavage samples were retrieved from 166 eligible patients, of whom 40 had IAPA, 52 had influenza without aspergillosis, 33 had CAPA, and 41 had COVID-19 without aspergillosis. We did nCounter gene expression analysis on bronchoalveolar lavage samples from 134 patients, protein analysis on samples from 162 patients, and both types of analysis on samples from 130 patients. We performed RNAScope and spatial transcriptomics on the tracheobronchial biopsy samples from two patients with IAPA plus invasive Aspergillus tracheobronchitis and two patients with CAPA plus invasive Aspergillus tracheobronchitis. We observed a downregulation of genes associated with antifungal effector functions in patients with IAPA and, to a lesser extent, in patients with CAPA. We found a downregulated expression of several genes encoding proteins with functions in the opsonisation, recognition, and killing of conidia in patients with IAPA versus influenza only and in patients with CAPA versus COVID-19 only. Several genes related to LC3-associated phagocytosis, autophagy, or both were differentially expressed. Patients with CAPA had significantly lower neutrophil cell fractions than did patients with COVID-19 only. Patients with IAPA or CAPA had downregulated IFNγ signalling compared with patients with influenza only or COVID-19 only, respectively. The concentrations of several fibrosis-related growth factors were significantly elevated in the bronchoalveolar lavage fluid from patients with IAPA versus influenza only and from patients with CAPA versus COVID-19 only. In one patient with CAPA, we visualised an active or very recent SARS-CoV-2 infection disrupting the epithelial barrier, facilitating tissue-invasive aspergillosis. INTERPRETATION: Our results reveal a three-level breach in antifungal immunity in IAPA and CAPA, affecting the integrity of the epithelial barrier, the capacity to phagocytise and kill Aspergillus spores, and the ability to destroy Aspergillus hyphae, which is mainly mediated by neutrophils. The potential of adjuvant IFNγ in the treatment of IAPA and CAPA should be investigated. FUNDING: Research Foundation Flanders, Coronafonds, the Max Planck Society, the Fundação para a Ciência e a Tecnologia, the European Regional Development Fund, "la Caixa" Foundation, and Horizon 2020.

Potential importance of early treatment of SARS-CoV-2 infection in intestinal transplant patient: A case report.

BACKGROUND: Predispositions for severe coronavirus disease 2019 (COVID-19) are age, immunosuppression, and co-morbidity. High levels of maintenance immunosuppression render intestinal transplant (ITx) patients vulnerable for severe COVID-19. COVID-19 also provokes several gastroenterological pathologies which have not been discussed in ITx, so far. CASE SUMMARY: During the second European COVID-19 wave in November 2020, an ITx recipient was admitted to the hospital because of electrolyte disturbances due to dehydration. Immunosuppression consisted of tacrolimus, azathioprine, and low-dose corticosteroids. During hospitalization, she tested positive on screening COVID-19 nasopharyngeal polymerase chain reaction swab, while her initial test was negative. She was initially asymptomatic and had normal inflammatory markers. Tacrolimus levels were slightly raised, as Azathioprine was temporarily halted. Due to elevated D-dimers at that time, prophylactic low-molecular weight heparin was started. Seven days after the positive test, dyspnea, anosmia, and C-reactive protein increase (25 mg/L) were noted. Remdesivir was administered during 5 d in total. High stomal output was noted in two consecutive days and several days thereafter. To exclude infection or rejection, an ileoscopy and biopsy were performed and excluded these. Four weeks later, she was discharged from the hospital and remains in good health since then. CONCLUSION: Early eradication of severe acute respiratory syndrome coronavirus 2 in ITx recipients may be warranted to prevent acute rejection provocation by it.