Clinical, imaging, serological, and histopathological features of pulmonary post-acute sequelae after mild COVID-19 (PASC)

BackgroundA significant proportion of patients experience prolonged pulmonary, cardiocirculatory or neuropsychiatric symptoms after Coronavirus disease 2019 (COVID-19), termed post-acute sequelae of COVID (PASC). Lung manifestations of PASC include cough, dyspnea on exertion and persistent radiologic abnormalities and have been linked to viral persistence, ongoing inflammation and immune dysregulation. So far, there is limited data on lung histopathology and tissue-based immune cell subtyping in PASC. Methods51 unvaccinated patients (median age, 40 years; 43% female) with a median of 17 weeks (range, 2-55 weeks) after mild SARS-CoV-2 infection (without hospitalization) underwent full clinical evaluation including high-resolution computed tomography (HR-CT) and transbronchial biopsy. We used RT-PCR/FISH and immunohistochemistry (nucleocapsid/spike/CD3/CD4/CD8) for residual SARS-CoV-2 detection and T lymphocyte subtyping, respectively. We assessed interstitial fibrosis and macrophage profiles by transmission electron microscopy (TEM) and immunofluorescence multiplex staining, while cytokine profiling in bronchoalveolar lavage (BAL) fluid was performed by legendplex immunoassay. ResultsDyspnea on exertion was the leading symptom of pulmonary PASC in our cohort. In 16% and 42.9% of patients, FEV1 and MEF50 were [≤] 80% and 35.3% showed low attenuation volume (LAV) in >5% of lung area, in line with airflow obstruction. There was a significant correlation between oxygen pulse and time since COVID (p=0.009). Histopathologically, PASC manifested as organizing pneumonia (OP), fibrinous alveolitis and increased CD4+ T cell infiltrate predominantly around airways (bronchiolitis), while the residual virus components were detectable in only a single PASC patient (2%). T cell infiltrates around small airways were inversely correlated with time since COVID, however, this trend failed to reach statistical significance. We identified discrete interstitial fibrosis and a pro-fibrotic macrophage subtype (CD68/CD163/S100A9) as well as significantly elevated interleukin 1{beta} in BAL fluid from PASC patients (p=0.01), but H-scores for fibrotic macrophage population did not correlate with severity of clinical symptoms or T cell infiltration. InterpretationWe show decreased FEV1/MEF50 and increased LAV in line with obstructive lung disease due to CD4+ T cell-predominant bronchiolitis as well as evidence of pro-fibrotic signaling in a subset of unvaccinated PASC patients. Since our results point towards self-limiting inflammation of small airways without detectable viral reservoirs, it remains unclear whether pulmonary symptoms in PASC are SARS-CoV-2-specific or represent a general response to viral infection. Still, evidence of pro-fibrotic signaling should warrant clincal follow-up and further research into possible long-time fibrotic remodeling in PASC patients. Key pointsO_LIDyspnea on exertion is the leading clinical manifestation of PASC in the lung C_LIO_LIa minority of pts have significantly impaired lung function (FVC/TLC[≤]80% or DLCO[≤]70%) in spiroergometry and/or radiologic abnormalities, oxygen pulse seems to normalize over time O_LI16% and 42.9% of pts have FEV1 and MEF50[≤]80% and 35.3% have LAV>5% of lung area, in line with airflow obstruction due to bronchiolitis C_LI C_LIO_LIResidual virus was not detectable in the lung tissue of all but one PASC patient (2%) C_LIO_LIHistologically, PASC may manifest as T cell-mediated bronchiolitis, OP and fibrinous alveolitis C_LIO_LIThere is evidence of fibrotic remodeling (ultrastructural interstitial fibrosis, pro-fibrotic macrophage subpopulation, pro-fibrotic cytokine IL-1{beta} in BAL) but this did not correlate with the degree of T cell infiltrate/bronchiolitis C_LI

Fatal cases after Omicron BA.1 and BA.2 infection: Diffuse alveolar damage occurs only in a minority - results of an autopsy study

Compared with previously prevalent variants of SARS-CoV-2, the Omicron lineages BA.1 and BA.2 are known to be associated with mild clinical courses. In addition, well-established animal models do not develop severe diseases. To address whether the supposedly fatal cases after Omicron-BA.1/2 infection show the known COVID-19 organ alterations, especially in the lungs, 23 full and 3 partial autopsies in the deceased with known Omicron BA.1/2 infections have been consecutively performed. Viral RNA was determined by RT-qPCR and RNA-in situ hybridization. The lineages were analyzed by whole genome sequencing or S-gene analysis. Despite high viral loads in almost all nasopharyngeal swabs and in 13 lung tissue samples, death caused by COVID-19-associated diffuse alveolar damage (DAD) in the acute and organizing stages was found in only eight cases (31%). This rate is significantly lower compared to previous studies, including non-Omicron variants, where rates of 92% and 69% for non-vaccinated and fully vaccinated vaccines were observed. It is of special interest that neither vaccination status nor known risk factors (i.e., age, comorbidities, obesity, immuno-suppression) were significantly associated with a direct cause of death by COVID-19. Only the reason for the hospital admission of the patients due to COVID-19-related symptoms showed a significant correlation with directly COVID-19-caused deaths (P < 0.001). DAD still occurred in the Omicron BA.1/BA.2 era of the SARS-CoV-2 pandemic but at a considerably lower frequency than seen with previous variants of concern. In our study, none of the known risk factors discriminated the cases with COVID-19-caused death from those that had COVID-19 infections but died due to a different disease. Therefore, the hosts genomics might play a key role in this regard. Further studies are urgently needed to elucidate the existence of a genomic mechanism as a risk factor for a fatal course.

Plasmacytoid dendritic cells regulate megakaryocyte and platelet homeostasis

Platelet homeostasis is essential for vascular integrity and immune defense. While the process of platelet formation by fragmenting megakaryocytes (thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of megakaryocytes by their progenitor cells (megakaryopoiesis) remains unclear. Here we use intravital 2 photon microscopy to track individual megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as crucial bone marrow niche cells that regulate megakaryopoiesis. pDCs monitor the bone marrow for platelet-producing megakaryocytes and deliver IFN- to the megakaryocytic niche to trigger local on-demand proliferation of megakaryocyte progenitors. This fine-tuned coordination between thrombopoiesis and megakaryopoiesis is crucial for megakaryocyte and platelet homeostasis in steady state and stress. However, uncontrolled pDC function within the megakaryocytic niche is detrimental. Accordingly, we show that pDCs activated by SARS-CoV2 drive inappropriate megakaryopoiesis associated with thrombotic complications. Together, we uncover a hitherto unknown megakaryocytic bone marrow niche maintained by the constitutive delivery of pDC-derived IFN-.

Assessing and improving the validity of COVID-19 autopsy studies - a multi center approach to establish essential standards for immunohistochemical and ultrastructural analyses

BackgroundAutopsy studies have provided valuable insights into the pathophysiology of COVID-19. Controversies remain whether the clinical presentation is due to direct organ damage by SARS-CoV-2 or secondary effects, e.g. by an overshooting immune response. SARS-CoV-2 detection in tissues by RT-qPCR and immunohistochemistry (IHC) or electron microscopy (EM) can help answer these questions, but a comprehensive evaluation of these applications is missing. MethodsWe assessed publications using IHC and EM for SARS-CoV-2 detection in autopsy tissues. We systematically evaluated commercially available antibodies against the SARS-CoV-2 spike protein and nucleocapsid, dsRNA, and non-structural protein Nsp3 in cultured cell lines and COVID-19 autopsy tissues. In a multicenter study, we evaluated specificity, reproducibility, and inter-observer variability of SARS-CoV-2 nucleocapsid staining. We correlated RT-qPCR viral tissue loads with semiquantitative IHC scoring. We used qualitative and quantitative EM analyses to refine criteria for ultrastructural identification of SARS-CoV-2. FindingsPublications show high variability in the detection and interpretation of SARS-CoV-2 abundance in autopsy tissues by IHC or EM. In our study, we show that IHC using antibodies against SARS-CoV-2 nucleocapsid yields the highest sensitivity and specificity. We found a positive correlation between presence of viral proteins by IHC and RT-qPCR-determined SARS-CoV-2 viral RNA load (r=-0.83, p-value <0.0001). For EM, we refined criteria for virus identification and also provide recommendations for optimized sampling and analysis. 116 of 122 publications misinterpret cellular structures as virus using EM or show only insufficient data. We provide publicly accessible digitized EM and IHC sections as a reference and for training purposes. InterpretationSince detection of SARS-CoV-2 in human autopsy tissues by IHC and EM is difficult and frequently incorrect, we propose criteria for a re-evaluation of available data and guidance for further investigations of direct organ effects by SARS-CoV-2. Key messagesO_LIDetection of SARS-CoV-2 proteins by IHC in autopsy tissues is less sensitive in comparison to SARS-CoV-2 RNA detection by RT-qPCR. C_LIO_LIFor determination of SARS-CoV-2 protein positive cells by IHC in autopsy tissues, detection of spike protein is less sensitive than nucleocapsid protein. C_LIO_LICorrect identification of SARS-CoV-2 particles in human samples by EM is limited to the respiratory system. C_LIO_LIInterpretation of IHC and EM should follow substantiated consensus criteria to enhance accuracy. C_LIO_LIExisting datasets describing SARS-CoV-2 presence in human autopsy tissues need to be critically re-evaluated. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/22269205v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@eafd97org.highwire.dtl.DTLVardef@1aed770org.highwire.dtl.DTLVardef@1c21ab9org.highwire.dtl.DTLVardef@68a101_HPS_FORMAT_FIGEXP M_FIG C_FIG

High viral loads: what drives fatal cases of COVID-19 in vaccinees? an autopsy study

BackgroundThe rate of SARS-CoV-2 breakthrough infections in vaccinees is becoming an increasingly serious issue. ObjectiveTo determine the causes of death, histological organ alteration, and viral spread in relation to demographic, clinical-pathological, viral variants, and vaccine types. DesignComprehensive retrospective observational cohort study. Setting: Consecutive cases from four German academic medical centers. PatientsDeceased with proven SARS-CoV-2 infection after vaccination who died between January and November 2021. Collections of 29 vaccinees which were analyzed and compared to 141 nonvaccinated control cases. ResultsAutopsies were performed on 16 partially and 13 fully vaccinated individuals. Most patients were elderly and suffered from several relevant comorbidities. Real-time RT-PCR (RT-qPCR) identified a significantly increased rate of generalized viral dissemination within the organism in vaccinated cases versus nonvaccinated cases (45% vs. 16%, respectively; P = 0.008). Vaccinated cases also showed high viral loads, reaching Ct values below 10, especially in the upper airways and lungs. This was accompanied by high rates of pulmonal bacterial or mycotic superinfections and the occurrence of immunocompromising factors such as malignancies, immunosuppressive drug intake, or decreased immunoglobulin levels. All these findings were particularly accentuated in partially vaccinated patients compared to fully vaccinated individuals. A fatal course after vaccination occurred in only 14% of all COVID-19 deceased in Augsburg. LimitationsRestricted number of cases ConclusionsFatal cases of COVID-19 in vaccinees were rare and often associated with severe comorbidities or other immunosuppressive conditions. Interestingly, we observed striking virus dissemination in our case study, which may indicate a decreased ability to eliminate the virus in patients with an impaired immune system. However, the potential role of antibody-dependent enhancement must also be ruled out in future studies. Funding sourceThis work was supported by the German Registry of COVID-19 Autopsies (www.DeRegCOVID.ukaachen.de) and funded by the Federal Ministry of Health (ZMVI1-2520COR201), the Federal Ministry of Education and Research within the framework of the network of university medicine (DEFEAT PANDEMICs, 01KX2021), and the German Federal Ministry of Food and Agriculture through the Federal Office for Agriculture and Food (project ZooSeq, grant number 2819114019).

Contamination of personal protective equipment during COVID-19 autopsies

Confronted with an emerging infectious disease, the medical community faced relevant concerns regarding the performance of autopsies of COVID-19 deceased at the beginning of the pandemic. This attitude has changed, and autopsies are now recognized as indispensable tools for elucidating COVID-19; despite this, the true risk of infection for autopsy staff is still debated. To elucidate the rate of SARS-CoV-2 contamination in personal protective equipment (PPE), swabs were taken at nine locations of the PPE of one physician and an assistant each from 11 full autopsies performed at four different centers. Further samples were obtained for three minimally invasive autopsies (MIA) conducted at a fifth center. Lung/bronchus swabs of the deceased served as positive controls. SARS-CoV-2 RNA was detected by RT-qPCR. In 9/11 full autopsies PPE samples were tested RNA positive with PCR, in total 21% of all PPE samples taken. The main contaminated parts of the PPE were the gloves (64% positive), the aprons (50% positive), and the upper sides of shoes (36% positive) while for example the fronts of safety goggles were only positive in 4.5% of the samples and all face masks were negative. In MIA, viral RNA was observed in one sample from a glove, but not in other swabs. Infectious virus isolation in cell culture was performed in RNA positive swabs from full autopsies. Of all RNA positive PPE samples, 21% of the glove samples were positive for infectious virus taken in 3/11 full autopsies. In conclusion, in >80% of autopsies, PPE was contaminated with viral RNA. In >25% of autopsies, PPE was found to be even contaminated with infectious virus, signifying a potential risk of infection among autopsy staff. Adequate PPE and hygiene measures, including appropriate waste deposition, are therefore mandatory to enable safe work environment.

Complement activation induces excessive T cell cytotoxicity in severe COVID-19

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathogenesis, and it remains unclear if T cells also contribute to disease pathology. Here, we combined single-cell transcriptomics and proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated, CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Age-dependent generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. The proportion of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a correlated with clinical outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.

Multisystemic cellular tropism of SARS-CoV-2 in autopsies of COVID-19 patients

BackgroundMultiorgan tropism of SARS-CoV-2 has previously been shown for several major organs. MethodsWe have comprehensively analyzed 25 different formalin-fixed paraffin-embedded (FFPE) tissues/organs from autopsies of fatal COVID-19 cases (n=8), using detailed histopathological assessment, detection of SARS-CoV-2 RNA using polymerase chain reaction and RNA in situ hybridization, viral protein using immunohistochemistry, and virus particles using transmission electron microscopy. Finally, we confirmed these findings in an independent external autopsy cohort (n=9). FindingsSARS-CoV-2 RNA was mainly localized in epithelial cells, endothelial and mesenchymal cells across all organs. Next to lung, trachea, kidney, heart, or liver, viral RNA was also found in tonsils, salivary glands, oropharynx, thyroid, adrenal gland, testicles, prostate, ovaries, small bowel, lymph nodes, skin and skeletal muscle. Viral RNA was predominantly found in cells expressing ACE2, TMPRSS2, or both. The SARS-CoV-2 replicating RNA was also detected in these organs. Immunohistochemistry and electron microscopy were not suitable for reliable and specific SARS-CoV-2 detection in autopsies. The findings were validated using in situ hybridization on external COVID-19 autopsy samples. Finally, apart from the lung, correlation of virus detection and histopathological assessment did not reveal any specific alterations that could be attributed to SARS-CoV-2. InterpretationSARS-CoV-2 could be observed in virtually all organs, colocalizing with ACE2 and TMPRSS2 mainly in epithelial but also in mesenchymal and endothelial cells, and viral replication was found across all organ systems. Apart from the respiratory tract, no specific (histo-)morphologic alterations could be assigned to the SARS-CoV-2 infection. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSSARS-CoV-2 has been shown to infect the respiratory tract and affect several other major organs. However, on a cellular level, the localization of SARS-CoV-2 and its targets ACE2 and TMPRSS2 have not been described comprehensively. Added value of this studyWe have analyzed tissue SARS-CoV-2 RNA using RT-PCR and visualized its localization together with ACE2 and TMPRSS2 using in situ hybridization (ISH) in 25 different autopsy tissues. SARS-CoV-2 sense and antisense RNA were detected in 16 tissues/organs, mainly in epithelial cells and, to a lesser extent, in endothelial or stromal cells. Detection of viral protein using immunohistochemistry or viral particles using transmission electron microscopy did not yield specific results. Interestingly, apart from the respiratory tract and specifically the lungs, we have not found a specific pathology that would be associated with extrapulmonary viral spread. Implications of all the available evidenceWe provide a recommendation on using these methods in autopsy diagnostics for SARS-CoV-2. Our data extend the current hypothesis of severe COVID-19 being multisystemic diseases. Our data also provide clear evidence of infection and replication of SARS-CoV-2 in the endothelial cell across all organs, extending the hypothesis on the (micro)vascular involvement in COVID-19.

SARS-CoV-2 RNA screening in routine pathology specimens

Virus detection methods are important to cope with the SARS-CoV-2 pandemics. Apart from the lung, SARS-CoV-2 was detected in multiple organs in severe cases. Less is known on organ tropism in patients developing mild or no symptoms, and some of such patients might be missed in symptom-indicated swab testing. Here we tested and validated several approaches and selected the most reliable RT-PCR protocol for the detection of SARS-CoV-2 RNA in patients routine diagnostic formalin-fixed and paraffin-embedded (FFPE) specimens available in pathology, to assess a) organ tropism in samples from COVID-19-positive patients, b) unrecognized cases in selected tissues from negative or not-tested patients during a pandemic peak, and c) retrospectively, pre-pandemic lung samples. We identified SARS-CoV-2 RNA in four samples from confirmed COVID-19 patients, in two gastric biopsies, one colon resection, and one pleural effusion specimen, while all other specimens, particularly from patients with mild COVID-19 disease course, were negative. In the pandemic peak cohort, we identified one previously unrecognized COVID-19 case in tonsillectomy samples. All pre-pandemic lung samples were negative. In conclusion, SARS-CoV-2 RNA detection in FFPE pathology specimens can potentially improve surveillance of COVID-19, allow retrospective studies, and advance our understanding of SARS-CoV-2 organ tropism and effects.

Antemortem vs. postmortem histopathological and ultrastructural findings in paired transbronchial biopsies and lung autopsy samples from three patients with confirmed SARS-CoV-2 infection

BackgroundAcute respiratory distress syndrome (ARDS) is the major cause of death in coronavirus disease 2019 (COVID-19). Multiple autopsy-based reports of COVID-19 lung pathology describe diffuse alveolar damage (DAD), organizing pneumonia (OP) and fibrotic change, but data on early pathological changes as well as during progression of the disease are rare. Research questionComparison of histopathological and ultrastructural findings in paired transbronchial biopsies (TBBs) and autopsy material from three patients with confirmed SARS-CoV-2-infection. MethodsWe prospectively enrolled 3 patients with confirmed SARS-CoV-2 infection. Full clinical evaluation was performed including high-resolution computed tomography (HR-CT). We took TBBs at different time points during the disease and autopsy tissue samples after the patients death. ResultsSARS-CoV-2 was detected by RT-PCR and/or FISH in all TBBs. Lung histology revealed pneumocyte hyperplasia and capillary congestion in one patient who died short after hospital admission with detectable virus in 1/2 autopsy samples from the lung. SARS-CoV-2 was detected in 2/2 autopsy samples from a patient with a fulminant course of the disease and very short latency between biopsy and autopsy, both showing widespread DAD. In a third patient with a prolonged course, i.e. five weeks of ICU treatment with ECMO, autopsy samples showed extensive interstitial fibrosis without detectable virus by RT-PCR and/or FISH. InterpretationWe report the course of COVID-19 in paired TBB and autopsy samples from three patients at an early stage, in rapidly progressive and in a prolonged disease course. Our findings illustrate vascular, organizing and fibrotic patterns of COVID-19-induced lung injury and suggest an early spread of SARS-CoV-2 from the upper airways to the lung periphery with diminishing viral load during disease.