Between March 25, 2020 and May 2, 2020, all patients who died from ARDS related to COVID-19 were prospectively included in four intensive care units (ICUs) in two centers of a French tertiary hospital in Strasbourg. During their ICU stay, patients were treated in accordance with standard practice and ventilated with protective ventilation to reduce the risk of ventilator induced lung injury (VILI).13 There was no exclusion criterion. Trained intensivists performed lung biopsies at bedside within the hour following death. The area to be biopsied was selected based on the lung injuries on the most recent imaging and the procedure was carried out under ultrasound guidance. All lung biopsies were analyzed at the hospital's Department of Pathology for histopathologic evaluation. Medical conditions and medications, current medical course, and antemortem diagnostic findings were recorded.

The study was approved by the local ethics committee of the University Hospital of Strasbourg (reference CE-2020-34). Approval for the post-mortem biopsies was obtained from the biomedical agency and the French ministry of research.

Tissue samples for histopathologic analysis were immediately fixed in 4% neutral buffered formalin for 24h, which ensures inactivation of the virus, and then processed via standard procedure to slides stained with hematoxylin–eosin (HE), Masson's trichrome, PAS and Perls staining. All the samples were blindly analyzed by an experienced pathologist, trained in pulmonary pathology.

Microscopic examination included a systematic search of the histologic criteria described at the 3 phases of diffuse alveolar damage (DAD), the histological hallmark for ARDS. Capillary congestion, intra-alveolar edema, loose fibrin deposition and hyaline membranes were considered as exudative changes. Type II pneumocyte hyperplasia, alveolar septa thickening with fibroblast proliferation and organizing fibroblastic tissue in air spaces were considered as features of the proliferative/organizing phase. Prominent interstitial collagenous fibrosis with architectural remodeling and/or honeycomb-like change defined the fibrotic phase. Extensive intra-alveolar fibrin deposition forming fibrin “balls” in alveolar ducts and alveoli, coexisting with intraluminal loose connective tissue polyps were histologic criteria of acute fibrinous and organizing pneumonia (AFOP).

Interstitial and intra-alveolar inflammatory infiltrate was analyzed using an automated chromogenic multiplexed immunohistochemistry assay on the Discovery Ultra automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA)14 with four antibodies targeting CD4, CD8, CD20 and CD68. Presence of SARS-CoV-2 virus in the lung tissue sections was evaluated by in situ hybridization (ISH) using RNAscope technology with a SARS-Cov-2 spike probe to COVID-19 coronavirus15 and by immunohistochemistry with a polyclonal Sars nucleocapsid protein antibody (200-401-A50; Rockland Immunochemicals, Inc., USA).16,17

The aim of this study is descriptive. No formal hypothesis was implemented to drive the sample size calculation and the maximum number of patients who met the inclusion criteria was included. Descriptive data were expressed as mean (SD) or median (IQR) for continuous variables and categorical variables as number (%). Tests were two-sided with significance set at α less than 0.05. Differences between groups were considered to be significant at a P value of<0.05. Students’ t-test was performed to compare two groups sampled from normal distributions with equal variances. One-way analysis of variance (one-way ANOVA) was used to compare means of more than two groups. Statistical analyses were performed with GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA).

A Total of 183 patients were admitted in ICU during this period. Twenty-two patients were included in this study. Median age was 66 [63–74] years. 16/22 (73%) patients were male. Predominant medical history of these patients was overweight and obesity. Few patients were active smokers. Table 1 provides an overview of initial laboratory results. Table 2 provides an overview of ventilatory parameters at admission and at time of death.

Median SAPS II and SOFA scores on admission in ICU were respectively 58 [47–67] and 11 [8–15]. The median duration of mechanical ventilation was 17 [8–24] days. 20/22 patients (91%) received neuromuscular blocking drugs (NMBD) with a median of 8 [6–15] days. 11/22 patients (50%) received inhaled nitric oxide. 13/22 patients (59%) developed ventilator-associated pneumonia (VAP). 11/22 (50%) patients received corticosteroids for a median of 9 [7–10] days. Median dynamic respiratory system compliance at death was 21 [15–24]mL/cmH2O. 5/22 patients (23%) received veno-venous extracorporeal membrane oxygenation (VV-ECMO). 8/22 patients (36%) developed a pulmonary embolism.

Patients were stratified according to the delay since the beginning of clinical criteria for ARDS related to COVID-19: 4 (18%) patients with severe pneumonia for less than 1 week, 11 (50%) patients between 1 and 3 weeks, and 7 (32%) patients for more than 3 weeks.

Regarding the four patients who died during the first week, one died of cardiac arrest induced by tension pneumothorax and three had limit in therapeutic effort (one because of Ewing Sarcoma and multiple myeloma, one because of acute promyelocytic leukemia with stroke and one because of multiple organ failure due to COVID-19 and a staphylococcus aureus VAP).

The average number of lung tissue probes obtained by needle biopsies from different lobes was 9.4. As expected, histologic features of exudative, proliferative and fibrotic phase of DAD (Fig. 1 shows different histological findings) were observed. Time to onset of histological lesions was closely related to the duration of evolution of ARDS related to COVID-19 (Fig. 2). Exudative changes were present in 3 of the 4 patients (75%) within the first week of ARDS related to COVID-19, 8 of the 11 patients (72%) within the second and third weeks, and none of the 7 patients with more than 3 weeks of evolution (p<0.01; Fig. 2). Intra-alveolar loose fibrin exudate and hyaline membranes gradually decreased over weeks (p=0.006 and p=0.01 respectively; Fig. 3).

Figure 1.

Microscopic lung findings in Covid-19. (A) Loose intra-alveolar fibrin exudates (arrow), admixed with inflammatory cells (star). Masson's trichrome. (B) Intra-alveolar “fibrin balls” (arrow). Hematoxylin-eosin staining. (C) Intra-alveolar loose fibrous plugs (star) of organizing pneumonia. Masson's trichrome. (D) Endotheliitis (arrow) in a small arterial vessel. Hematoxylin–eosin staining. The scale bar corresponds to 50μm for A, B and C, and 100μm for D.

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Figure 2.

Time to onset and evolution of histological lesions noted over time (exudative phase, proliferative phase and end-stage fibrosis) in 22 patients with ARDS related to COVID-19.

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Figure 3.

Relation between acute fibrinous and organizing pneumonia (AFOP) features, intra-alveolar loose fibrin exudate, hyaline membranes and duration of evolution of 22 severe ARDS related to COVID-19.

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By contrast, the incidence of proliferative changes increased over time and were reported in 2/4 patients (50%) within the first week, 8/11 patients (73%) within the 1–3 weeks, and 6/7 patients (88%) after the third week (Fig. 2). Hyperplasia of type II pneumocytes mostly with cytologic atypia was noted in 17 of the 19 patients (89%) presenting proliferative changes.

The presence of end-stage fibrosis increased over time: the pattern appeared in none of the 4 patients with disease for less than 1 week, in 3 of the 11 patients (27%) with disease for 1–3 weeks, and in all (100%) of the patients with a disease of more than 3 weeks (p<0.01; Fig. 2). Patients with end-stage fibrosis had been on mechanical ventilation for a longer period of time than patients without fibrosis (Table 3). It should be noted that histological criteria of more than one phase coexisted in 16 of 22 patients (73%).

No differences in histologic findings were observed between patients who received steroids and those who did not (Table 4). Regarding patients who developed a VAP and those who did not, there was significantly more fibrosis in the patients who developed VAP (Table 5).

Features of AFOP were found in 10 patients (45%) (Table 6). They began to appear during the second–third week in 36% of the patients, with a large number of fibrin balls; and were found in 86% of the patients after the third week (p<0.02; Fig. 3), showing remnants of fibrin plugs associated with loose fibrous polyps of organizing pneumonia (OP).

Interstitial inflammatory infiltrate was found in all patients regardless of the duration of ventilation. It was mostly mild to moderate, and composed of lymphocytes with some monocytes. Plasma cells were present after the third week. Neutrophils were scarce. Endo-alveolar infiltrate was present in 19/22 (86%) of patients and almost exclusively composed by macrophages.

Immune cell subpopulations were analyzed in 12/22 cases (from day 1 to day 25 after ventilation) using an automated chromogenic multiplexed immunohistochemistry (IHC) assay with four antibodies (Fig. 4). In all cases only rare scattered interstitial CD20+ B-cells were observed. The infiltrate consisted of T-cells, with a mixture of CD4+ and CD8+ lymphocytes. There were slightly more CD4+ than CD8+ lymphocytes in 6/12 (50%), and slightly more CD8+ than CD4+ lymphocytes in 6/12 (50%) of patients. CD68+ macrophages were mostly located in alveolar spaces.

Figure 4.

Detection of SARS-CoV-2 and characterization of inflammation. (A) Edema of alveolar walls and atypical pneumocytes with enlarged nuclei (arrow). Hematoxylin–eosin staining. (B) SARS-Cov-2 mRNA detection in atypical pneumocytes by in situ hybridization. (C) Viral nucleocapsid protein detection in atypical pneumocytes by immunohistochemistry. (D) Multiplex chromogenic detection by DAB staining (brown) of CD20+ lymphocytes, purple staining of CD8+ lymphocytes, teal staining (blue) of CD4+ lymphocytes and yellow staining of macrophages. CD4+ lymphocytes outnumber CD8+ lymphocytes in this case. The scale bar corresponds to 50μm.

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The presence of the SARS-CoV2—tested by immunohistochemistry (IHC)—was only found in 2/22 patients (9%) (Fig. 4). Both died early, during the first week of ICU stay, after 4 and 7 days of symptomatic illness.

As IHC, RNA scope allowed detection of SARS-CoV-2 viral RNA only in the two same patients (9%) (Fig. 4).

Both viral RNA and protein were located in atypical pneumocytes and in some intra-alveolar macrophages. No staining of endothelial cells could be identified.

Although RT-PCR testing for the virus in tracheobronchial secretions was positive up until death in the majority of our patients (20/22 patients)—using the two methods (IHC and RNAscope)—no trace of the virus was found in lung tissue of patients who died after the first week.

Even if 8/22 patients (36%) developed a pulmonary embolism, only two of the 22 patients had recent thrombosis of small pulmonary arteries and one patient had endotheliitis in lung vessels (Fig. 1). To note, all patients received heparin therapy. Six received prophylactic anticoagulation (27.3%) and 16 therapeutic anticoagulation (72.7%). Other common findings were diffuse small vessel platelet aggregation, intra-alveolar hemorrhage or areas of hemorrhagic necrosis of the lung parenchyma.