This prospective observational MIA cohort in patients with COVID-19 describes the clinical, radiological, histopathological, microbiological, and immunological differences between different disease stages. Patients with mild-moderate disease had fewer abnormalities on CT, did not show DAD in lung biopsies and had a less pronounced cytokine response compared to patients with severe-critical disease. However, differences between patients with severe-critical disease and post-acute disease were rather limited. Radiology showed equally high prevalence of crazy paving and bilateral consolidation. Histologically, DAD was present equally in both groups with > 60% of patients with post-acute COVID-19 in early exudative or mid proliferative stage. Immunologically, no significant differences in cytokine responses, except IFNy, were seen.
In none of the four radiologically confirmed COVID-19 patients we could confirm post-mortem the diagnosis of COVID-19 infection, despite elaborate investigations. This emphasises the need to reconsider the diagnosis of COVID-19 if the molecular confirmation in lacking, and to optimize the diagnostic strategy for both COVID-19 and alternative diagnoses.
Furthermore, despite having a disease onset of > 28 days, most patients with post-acute COVID-19 had a radiological, histological, and immunological profile of acute respiratory distress syndrome. One may postulate that patients included in this cohort i.e., hospitalized patients that died, had an aberrant disease course that did not show a dampening of the acute lung damage and failed to progress to a more chronic phase 11, 12. Even in patients with the longest disease duration (> 32 days) no trend toward chronic disease was observed. This confirms the idea of acute inflammation as an important cause of mortality 13, 14. Although groups are small, this may have important implications for treatment of these patients, as they may benefit, even at such a long disease duration, from (prolonged) treatments like those given to patients with severe-critical disease.
A remarkable finding was the difference in composition of the inflammatory infiltrate, with macrophages still being present in a relatively high percentage in the post-acute disease. Macrophages play an important and dual pro- and anti-inflammatory role in ARDS. Literature suggests that the M1 type is present in earlier phases, releasing pro-inflammatory cytokines, while the M2 type is present in later phases to eliminate apoptotic cells, thereby possibly contributing to fibrosis 15. Histopathological fibrosis was seen in a high percentage of patients with severe-critical and post-acute disease. It must be noted that not all patients with fibrosis showed typical DAD features. Some patients showed mild to moderate fibrosis thought to be pre-existent, without any other signs of DAD.
Next, megakaryocytes were easily found in a large proportion of patients. It has been reported that an increased number of pulmonary megakaryocytes, responsible for production of platelets, can be seen in lung tissue of COVID-19 patients with DAD. This is thought to underline the relation with embolic/thrombotic events reported in COVID-19 patients 16. Nevertheless, a low number of thrombi and vasculitis were seen in this cohort, much lower than commonly reported in the literature 17, 18. One possible reason is that we performed MIA instead of full autopsies and obtained tissue cores of approximately 5X30mm, which has a higher chance of ‘sampling error’, especially to catch relatively large structures like thrombi and larger vessels.
We found profound abnormalities in the lungs and liver, but no distinctive COVID-related findings were found in the other organs that were investigated. In other autopsy series, various abnormalities were described in virtually all organs. However, in these studies, complete autopsies were performed with more extensive tissue sampling and higher yield 12, 19. We collected per protocol lung biopsies from radiological abnormal and normal tissue, however “radiologically normal” lung tissue was limited, and disease involvement of all lobes was most frequent. This was confirmed histologically as biopsies labelled “radiologically normal” had only in 3/44 (6.8%) cases no or few abnormalities.
No significant differences in cytokine responses, except IFNy, were found between disease stages. However, IP-10, IL-6, and GM-CSF levels tended to be higher in patients with severe-critical disease compared to mild-moderate disease. These are pro-inflammatory cytokines, typically seen during cytokine storm and related to hyperinflammation-induced severe disease. Histopathological findings in the lung could support this hypothesis: lymphocytes and plasma cells were more prominent in patients with severe-critical disease.
Moreover, a trend towards decreased cytokine levels was seen in post-acute COVID-19. Even though this decrease did not return to levels as low as during mild-moderate disease, this, together with the finding that macrophages and T-lymphocytes were seen more prominently in the lung during severe-critical disease, could point to an increased immune response followed by a start to return to normal. Patients with post-acute COVID-19 did not have leukopenia shortly before death, and therefore this observation cannot be explained by immune exhaustion.
Of course, these interpretations should be assessed with caution because of small groups, differences were not significant, and cytokines were measured in plasma which is not necessarily representative of findings in the lung. Furthermore, this is a cohort of the most ill patients. We do not know if these findings truly reflect COVID-19 or reflect other complications during their disease.
IFNα levels tended to be higher in patients with mild-moderate disease. IFNα is a type I interferon and has been shown to be an important anti-viral response cytokine related to COVID-19 severity. An impaired IFN type I response was previously reported to be present in patients that developed critical disease 20. Moreover, patients with inborn errors in type I IFN are at risk of developing life-threatening COVID-19 21. In addition, the observed trend for higher IP-10 levels in IgM negative patients points toward an impaired immune response. Our findings could indicate that mild disease patients but not patients with severe-critical or post-acute disease, had an adequate antiviral response and therefore did not develop typical COVID-19 pneumonia and may have died of other causes than the virus or hyperinflammation.
Almost 30% of our patients had a bacterial pneumonia, which is higher than the 14% co- or sur-infection rate in mixed wards/ICU settings reported in the literature 22. This may be explained by selection bias, i.e., the most severely ill patients were included in this cohort. Nevertheless, restricted antibiotic use should be propagated from an antimicrobial stewardship perspective.
Our study has limitations. Although MIA is a good alternative to conventional autopsies, tissue sample size is relatively small, which limits a good overview of all architectural abnormalities that might be present and increase sampling error. Also, certain crucial organs like the brain have not been sampled. Moreover, during (ICU) admission, patients generally develop multiple problems, leading to organ damage and changes in inflammatory response. It is therefore impossible to disentangle what abnormalities have been caused by SARS CoV-2 and what not.
In conclusion, we found that patients that die during different COVID-19 disease stages show certain distinct clinical, radiological, pathological, and immunological features. Patients with mild-moderate disease showed relatively few pathological abnormalities with a probable adequate immune response and did not die of but with COVID-19. Patients with severe-critical disease showed extensive pulmonary abnormalities, typically reflecting an overreactive and distorted immune response, and mostly died because of COVID-19 pneumonia. Lastly, patients with post-acute disease, despite some trends towards a dampened immunological response, mostly have similar clinical, radiological, and histopathological features compared to severe-critical disease.
The study of pathophysiological changes during different disease stages remains important to elucidate the mechanisms of this new disease. The distinct features during different disease stages show that a tailored and personal management of COVID-19 patients is necessary.