Towards Understanding COVID-19: Molecular Insights,Co-infections,Associated Disorders,and Aging

Statistical analysis and machine learning

The data included in the Table 2 and in part of the Table 3 were statistically analyzed as described [23]. Specifically, metabolites and therapeutic compounds with over 80% missing ratios in a particular patient group were removed for the metabolomics dataset containing endogenous metabolites while full proteomics features were used for the subsequent statistical analysis. Missing values were imputed with the minimal value and zero in metabolomics and proteomics dataset respectively. Log2 fold-change (log2 FC) was calculated on the mean of the same patient group for each pair of comparing groups. Two-sided unpaired Welch’s t test was performed for each pair of comparing groups and adjusted p values were calculated using Benjamini & Hochberg correction. The statistical significantly changed proteins or metabolites were selected using the criteria of adjust p value less than 0.05 indicated and absolute log2 FC larger than 0.25. From the training cohort, authors selected important protein and metabolite features with mean decrease accuracy larger than 3 using random forest. In the random forest analysis, a thousand trees were built using R package randomForest (version 4.6.14) with 10-fold cross validation, and this was repeated for 100 times. The normalized additive predicting probability was computed as the final predicting probability. The larger probability for the binary classification was adopted as the predictive label. For validation in the test cohort 2 (C3) generated by targeted proteomics and metabolomics, z-score normalization was applied before running the model validation. Those selected important features were used for the random forest analysis on the independent test cohort. The authors also ran the randomForest analysis with omics features after z-score normalization and got same classification results [23].

Statistical tests, p values

In the reported studies discussed here, a significant test result means p < 0.05 [24].

Those dying with, but not of, COVID-19 may still be infectious

Accumulating evidence shows that microbial co-infection increases the risk of disease severity in humans. There have been few studies about SARS-CoV-2 co-infection with other pathogens. In the retrospective study by Zhu et al., 257 laboratory-confirmed COVID-19 patients in Jiangsu Province, China were enrolled from January 22 to February 2, 2020 [45]. They were re-confirmed by real-time RT-PCR and tested for 39 respiratory pathogens. In total, 24 respiratory pathogens were found among the patients, and 242 (94.2%) patients were co-infected with one or more pathogens. Bacterial co-infections were dominant in all COVID-19 patients, Streptococcus pneumonia was the most common, followed by Klebsiella pneumonia and Haemophilus influenzae. Most co-infections occurred within 1–4 days of onset of COVID-19 disease. In addition, the proportion of viral co-infections, fungal co-infections and bacterial-fungal co-infections were the highest in severe COVID-19 cases. There were 10, 22, 20, and 13 pathogens found in symptomatic, mild, moderate, and severe/critical cases, respectively. S. pneumoniae, K. pneumoniae, H. influenzae, fungi Aspergillus, EB virus, Escherichia coli, and Staphylococcus aureus were simultaneously found in four clinical groups. Moraxella catarrhalis and Acinetobacter baumannii were found in asymptomatic category, mild category and moderate category [45]. The 81 patients (31.5 %) had viral co-infection, 236 (91.8 %) had bacterial co-infection, and 60 (23.3 %) had fungal co-infection [45].

Lacy et al. (2020) [46] presented a case (Snohomish County Medical Examiner’s Office, Everett, WA, USA) of sudden unexpected death due to COVID-19 as a means of illustrating common autopsy findings, as well as diagnostic and biosafety considerations. The authors also review and summarize the current COVID-19 literature in an effort to provide practical evidence-based biosafety guidance for medical examiner-coroner offices encountering COVID-19 at autopsy. While in some cases, a history of fever and/or respiratory distress (e.g., cough or shortness of breath) may suggest the diagnosis, epidemiologic studies indicate that the majority of individuals infected with COVID-19 develop mild to no symptoms. Those dying with—but not of—COVID-19 may still be infectious, however [46]. Swabs of the right and left main bronchi were procured. The reverse transcriptase–polymerase chain reaction (RT-PCR) results for SARS-CoV-2 returned as positive 11.5 hours after submission. Negative influenza results returned several days later. Bacterial cultures were positive for methicillin-sensitive Staphylococcus aureus and Streptococcus viridans. The lack of acute histologic inflammation in the lungs was observed. Based on the autopsy and investigative findings, and in accordance with US National Vital Statistics certification guidelines, the cause of death was determined to be acute respiratory distress syndrome (ARDS) due to viral pneumonia due to COVID-19. Other essential contributory factors included type 2 diabetes mellitus, hypertension, and obesity. In this case [46], the lungs were moderately heavy and edematous (right: 818 g, left: 705 g). Hilar and mediastinal lymph nodes appeared enlarged. The heart exhibited moderate coronary atherosclerosis in each of the main coronary distributions, but there were no occlusions or critical stenoses. The myocardium was free of obvious infarcts. There was moderate infrarenal aortic atherosclerosis. The kidneys were finely granular and had focal cortical scars. The brain exhibited hydrocephalus ex vacuo.

Therefore, the co-infections with Staphylococcus aureus and Streptococcus viridans are not defined as a cause of death or contributory factors in this case [46]. The authors interpret that “given the lack of acute histologic inflammation in the lungs, the bacterial culture results were interpreted as being most likely contaminants or postmortem artifact” [46]. However, the review by Bauer et al. (2006) reveal that ARDS is frequently associated with bacterial infections [47]. Moreover, the Japanese case of 34-year-old woman with ARDS due to methicillin-resistant Staphylococcus aureus sepsis in hyper-IgE syndrome was reported by Sato and colleagues [48].

Hereby death cause of COVID-19 virus-positive cases is uncertain. Thus, the statistics of certification defining the cause of death due to COVID-19 is a contentious issue. In other words, COVID-19 death can be due to different causes of related or unrelated underlying diseases and conditions. Correlation of corona virus positivity and death in a low percentage of population might have even just been noise. Of course, correlation is not causation; the shift toward death with corona virus positivity could have been due to any number of other issues, such as co-infections, and/or other overt, covert and inadvertent conditions.

Goursaud et al. reported COVID-19 necrotizing pneumonia and extracorporeal membrane oxygenation as a challenge for anticoagulation [49]. High levels of anticoagulation must be considered with caution in the most severe patients with SARS-CoV-2 necrotizing pneumonia [49].

COVID-19 and bacterial co-infections: biogenic amines in Staphylococcus aureus, necrotizing pneumonia

Necrotizing pneumonia has been described in COVID-19 fatal case in France [49]. Necrotizing pneumonia is known to be associated with fungal or bacterial co-infection, especially Staphylococcus aureus, which is one of the major causes of death following virus influenza infection [50]. In the genus Staphylococcus, certain species are capable of producing trace biogenic amines through the activity of staphylococcal aromatic amino acid decarboxylase (SadA). SadA decarboxylates aromatic amino acids to produce trace biogenic amines. SadA-expressing staphylococci were prevalent in the gut of most probands, where they are part of the human intestinal microflora. Furthermore, sadA-expressing staphylococci showed increased adherence to human colorectal adenocarcinoma HT-29 cells and 2- to 3-fold increased internalization. Internalization and adherence was also increased in a sadA mutant in the presence of tryptamine [17, 18]. Of note, tryptamine can induce necrosis [16]. Therefore, tryptamine and other trace biogenic amines can play a role in necrotizing pneumonia.

Bacterial meningitis is an inflammation of the meninges which covers and protects the brain and the spinal cord. Such inflammation is mostly caused by blood-borne bacteria that cross the blood-brain barrier (BBB) and finally invade the brain parenchyma. Pathogens such as Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae are the main etiological causes of bacterial meningitis. Furthermore, clinical studies have shown indications of meningitis-caused dementia [51].

Twelve different biogenic amines formation in 58 isolates of Streptococcus thermophilus from home-made natural yogurt were investigated in histidine (HDB) and lysine decarboxylase broth (LDB). Tryptamine produced at mg/L concentrations in both HDB and LDB [52]. In HDB, tryptamine was produced at 1–100 mg/L in 47 S. thermophilus isolates while in LDB, 7 isolates produced 101–500 mg/L tryptamine [52]. HDB and LDB contained 1 g peptone, 0.5 g Lab-Lemco powder, 2.5 mg NaCl, 4.01 g L-histidine monohydrochloride monohydrate or L-lysine monohydrochloride, and 2.5 mg pyridoxal HCl in 500 mL distilled water; the pH was adjusted to 5.5 with 1 M KOH or 6% trichloroacetic acid.

These data suggest that S. thermophilus strains can use histidine and lysine decarboxylases for production of tryptamine, phenylethylamine, tyramine, serotonin, and dopamine or HDB and LDB that included peptone and Lab-Lemco powder contained sufficient amount of tryptophan (16 mg/L), to be decarboxylated by tryptophan decarboxylase. The authors concluded that the other biogenic amines are likely to have been produced from the amino acids in peptone and beef extract [52]. Two isolates of S. thermophilus produced significant amounts of tyramine, 504 and 539 mg/L while tyrosine was calculated at 14 mg/L. The formation of dopamine by S. thermophilus ranged from 1.17 to 2160 mg/L. Tryptamine was not determined in 10 HDB and 14 LDB cultivated S. thermophiles isolates.

Therefore, tryptamine unlikely derived from HDB or LDB alone. However, some strains could metabolize tryptamine and other biogenic amines. Thus, the biogenic amines content in HDB and LDB should be estimated and controlled.

Tryptamine was produced by Klebsiella pneumoniae in HDB and by K. pneumoniae and Pseudo-monas putida in LDB [53].

Food industries consider S. thermophilus as non-pathogenic.

In 2018, tryptamine (0 to 120.18±4.29 mg/kg) and other biogenic amines were reported to be produced by bacterial strains isolated from the popular fermented soybean product from 15 different regions in China [54]. Some of these strains were able to degrade biogenic amines [54].

Of note, in this study published by the journal Scientific Reports (Nature Publishing Group) [54], the data on content of biogenic amines in soybean extracts are provided, but no data available on a content of biogenic amines in the media for cultivation of bacterial isolates for the detection of biogenic amines. Meanwhile, LB medium composed of NaCl, 10 g/L Tryptone, 10 g/L, and Yeast Extract, 5 g/L and used for bacteria cultivation may contain biogenic amines that can be degraded by some bacterial strains thus effecting results on biogenic amines analysis.

The seven tryptamine-producing bacteria were isolated in South Korea from commercial salted and fermented sand lance (Ammodytes personatus) fish sauces using an L-tryptophan decarboxylating medium. These tryptamine-producing bacteria, identified using an API kit and 16S rRNA analysis, included Lysinibacillus xylanilyticus (one strain), Lysinibacillus fusiformis (four strains), and Staphylococcus epidermidis (two strains). Lysinibacillus spp. produced the highest levels of tryptamine in culture broth containing 0.5% L-tryptophan, compared with 1.0% and 2.0% preparations. After 72 h of incubation, Staphylococcus epidermidis produced the highest levels of tryptamine (60.50μg/mL and 664.86μg/mL) in culture broth containing 2.0% L-tryptophan [55]. Thus, Staphylococcus strains producing high amount of tryptamine can originate from food including Korean fish sauces.

Staphylococcus aureus strain HZW450 isolated (China: Hangzhou, Submitted 11-APR-2017, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital College of Medicine Zhejiang University, China) from pus of a patient with impetigo (highly contagious skin infection) possess pyridoxal-depen-dent decarboxylase (GenBank: ATH56183.1; 472 amino acids; pyridoxal-deC, DOPA decarboxylase family). High amino acid sequence identity to pyridoxal-deC (3e-157, 99.08%) presents in the nasal sample (anterior nares) of a male participant (emb|ODTW01004449.1; Submitted 01-SEP-2017) from the dbGaP study “HMP Core Microbiome Sampling Protocol (USA control set). Hangzhou, the capital of Zhejiang province in China, was confronted with the pandemic of a novel coronavirus (COVID-19) that originated in Wuhan, Hubei province. According to the Health Commission of Zhejiang Province, the 6 cases were first reported on January 19, 2020, and the cumulative cases reached 169 as of February 20, 2020. The situation in Hangzhou was once rather severe—it was the top-ranking city with respect to the number of confirmed cases in Zhejiang province at the beginning of the epidemic [56]. Therefore, the bacterial Staphylococcus aureus strain possessing gene for DOPA decarboxylase was isolated from human sample in China before the first cases of COVID-19 reported. Similar gene presents in the human sample in the US.

McDanel and colleagues reported in 2016 that Staphylococcus aureus is a common cause of respiratory infections, including pneumonia, and can lead to necrotizing pneumonia and death. Influenza complicated by S. aureus co-infection can progress rapidly to death within a week of symptom onset [57]. Increased mortality rates associated with Staphylococcus aureus and influenza co-infection was investigated in Maryland and Iowa in the US [57]. In this study, the researchers retrospectively analyzed data for 195 respiratory infection patients who had positive Staphyloccocus aureus cultures and who were hospitalized in two hospitals in Iowa and Maryland during 2003–2009. Odds for death for patients who also had influenza-positive test results were > 4 times higher than for those who had negative influenza test results [57]. This dataset did not include information about variables such as influenza vaccination status, mechanical ventilation, co-infection with organisms other than influenza and S. aureus, and whether the pneumonia was necrotizing [57]. The characterization of biogenic amine production by S. aureus clinical isolates is notavailable.

Up to 75% of those infected with influenza that go on to acquire pneumonia, are confirmed to have bacterial co-infection [50].

The mouse model used by Jia et al. (2018) to explore coinfection-induced death [58]. To minimize the impact of the pathogens themselves on coinfection-related mortality, various coinfection sequences and time points were planned using non-lethal doses of influenza virus (PR8) and S. aureus (MRSA). The results indicated that mortality is related to the sequence and timing of infection with both pathogens. Specifically, mortality was highest (> 80%) in mice infected with MRSA [d, d-1, d-2, and d-3 groups from day 0 to day 3 post-infection with influenza virus, which was higher than that in other co-infected groups [58].

Despite invasive aspergillosis being reported in immunocompetent severe COVID-19 patients [59, 60], other pathogen profiles are not yet esta-blished, and descriptive clinico-microbiological studies are still warranted [49]. Fungi Aspergillus niger transformed tryptamine into 5-hydroxyindole-3-acetamide [61].

Sharifipour and colleagues [62] reported (September 2020) the study of nineteen critically ill patients admitted to ICU wards in two referral hospitals for coronavirus in Qom, Iran. These patients were enrolled in the study of bacterial infections in COVID-19 patients [62]. Patients were given antibiotics such as ceftriaxone and azithromycin before admission to the ICUs. Inclusion criteria were being infected by COVID-19, hospitalized, intubated, and mechanically ventilated > 48 h in ICUs [62]. Of nineteen COVID-19 patients, 11 (58%) patients were male and 8 (42%) were female, with a mean age of ∼67 years old. The 16 of 19 patients had diabetes. The average ICU length of stay was ∼15 days and at the end of the study, 18 cases (95%) expired and only was 1 case (5%) discharged. In total, all patients were found positive for bacterial infections, including seventeen Acinetobacter baumannii (90%) and two Staphylococcus aureus (10%) strains. There was no difference in the bacteria species detected in any of the sampling points. Seventeen of 17 strains of Acinetobacter baumannii were resistant to the evaluated antibiotics [62]. Overall, it is important to limit the risk of infection and the spread of these resistant strains through controlling nosocomial infections accurately and bringing secondary infections caused by resistant bacteria that can increase the mortality rate in COVID-19 critical patients into attention[62].

Taking into account that high proportion of COVID-positive patients had preexisting diabetes (16 of 19 COVID-positive patients had diabetes in Iran [62]), it is worth to look into possibility that diabetic patients could be infected during insulin administration. Grissinger reported in 2017 that “wrong patient” insulin pen injections are alarmingly frequent in the US [63]. The insulin pen sharing could lead to pathogen transmission. The large-scale potential exposures to blood-borne pathogens caused by using insulin pens for multiple patients after changing the needle include: 2,114 patients at a Texas Army medical center in 2009; 2,345 patients at a Wisconsin clinic in 2011; 716 patients at a New York Veterans Affairs medical center in 2013; 1,915 patients at a New York general hospital in 2013; 3,149 patients at a Connecticut hospital in 2014 [63].

During the 1918–1919 pandemic, the bacteria most often recovered from the sputum, lungs, and blood of pneumonia patients, alive or dead, were common colonizers of the upper respiratory tracts of healthy persons, i.e., Hemophilus influenzae, Streptococcus pneumoniae, S. pyogenes, and/or Staphylococcus aureus. Brundage and Shanks (2008) hypothesize that infections with the pandemic influenza strain generally caused self-limited (rarely fatal) illnesses that enabled colonizing strains of bacteria to produce highly lethal pneumonias [64]. They suggest opportunities for prevention and treatment during the next pandemic with bacterial vaccines and antimicrobial drugs, particularly if a pandemic strain-specific vaccine is unavailable or inaccessible to isolated, crowded, or medically underserved populations [64].

Morens, Taubenberger, and Fauci of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD reported in 2008 that the majority of deaths in the 1918–1919 influenza pandemic likely resulted directly from secondary bacterial pneumonia caused by common upper respiratory–tract bacteria. Less substantial data from the subsequent 1957 and 1968 pandemics are consistent with these findings [65]. In this study of the postmortem samples from people who died of influenza during 1918–1919, the authors revealed the uniformly exhibited severe changes indicative of bacterial pneumonia. Bacteriologic and histopathologic results from published autopsy series clearly and consistently implicated secondary bacterial pneumonia caused by common upper respiratory–tract bacteria in most influenza fatalities [65].

Wunderink and Waterer [66] reported in 2014 that pneumonia is sometimes referred to as the forgotten killer. The World Health Organization estimates that lower respiratory tract infection is the most common infectious cause of death in the world (the third most common cause overall), with almost 3.5 million deaths yearly. Together, pneumonia and influenza constitute the ninth leading cause of death in the US, resulting in 50,000 estimated deaths in 2010. This number is probably underestimated, since deaths from sepsis (for which pneumonia is the most common source) and deaths attributed to other conditions (e.g., cancer and AD) for which pneumonia is the terminal event are coded separately [66]. Clinical features suggesting community-acquired methicillin-resistant Staphylococcus aureus (MRSA) pneumonia are: cavitary infiltrate or necrosis; rapidly increasing pleural effusion; gross hemoptysis (not just blood-streaked); concurrent influenza; neutropenia; erythematous rash; skin pustules; young, previously healthy patient; severe pneumonia during summer months [66]. According to European Centre for Disease Prevention and Control since 31 December 2019 and as of 02 November 2020, 46 597 299 cases of COVID-19 (in accordance with the applied case definitions and testing strategies in the affected countries) have been reported, including 1,201,162 deaths (COVID-19 situation update worldwide, as of November 2, 2020 https://www.ecdc.europa.eu/en/geographical-distribution-2019-ncov-cases).

The first New York case was reported in early March 2020 and by the first week in April, the New York Presbyterian Hospital system, including Columbia and Weill Cornell, had close to 2,500 in-patients with over 700 patients on ventilators [67]. The standard of care for COVID-19 was supportive care only with essential morbidity and mortality recognized. An emerging crisis without proven treatments COVID-19 is a mild illness in more than 80% of people infected, but about 15% will require hospitalization and about 5% intensive care unit support.

Epidemiology, clinical course, and outcomes of critically ill adults with COVID-19 in New York City were reported by Cummings et al. [68] and by Goyal et al. [69]. The authors concluded that critical illness among patients hospitalized with COVID-19 in New York City is common and associated with a high frequency of invasive mechanical ventilation, extrapulmonary organ dysfunction, and substantial in-hospital mortality [68]. The incidence of bacterial superinfection in the setting was unknown early in the outbreak in the study reported by Cummings et al. [68]. In the study by Goyal et al., bacteremia (the presence of bacteria in the bloodstream) was revealed in 19/338, 4.8% (all), 15 (11.9%) of invasive mechanical ventilation (N = 130); 4 (1.8%) of no invasive mechanical ventilation (N = 263). The viral co-infection was revealed in 4/393, 1.0% (all), 2 (1.5%) in invasive mechanical ventilation and 2 (0.8%) in no invasive mechanical ventilation [69]. In-hospital treatment included hydroxychloroquine, remdesivir, and oral corticosteroids [69]. This report characterizes the first 393 consecutive patients with COVID-19 who were admitted to two hospitals in New York City [69]. Of note, no treatment with antibiotics indicated for the COVID-positive patients. Among these patients, obesity reported for 35.8%, diabetes - 25.2%, hypertension-50.1%, chronic obstructive pulmonary disease (COPD)-5.1%, asthma-12.5%, coronary artery disease-13.7%, death-10.2%. Infiltrates on initial chest radiograph (during hospital stay) were revealed in 75.3% of the COVID-19 patients, and no data provided on the infectious agents in the infiltrates. All these diseases including COPD [70] and asthma [71] was found to be associated with increased risk of dementia [1], whereas nursing home patients have been disproportionately affected by COVID-19. It has been reported that one-fourth of all COVID-19 deaths nationwide occurred in nursing homes and other long-term care facilities [72]. Analyses of Connecticut, New Jersey, and New York indicate that nursing homes with higher percentages of Medicaid patients were more likely to have COVID-19 deaths [72]. Among the 1,162 nursing homes in this study, 184 (15.8%) had 6 or more COVID-19 associated deaths [72].

Therefore based on this study [69] bacteremia and to a lesser extent viral co-infection in COVID-19 patients are related to Invasive Mechanical Ventilation in New York City Hospitals.

Sen-Crowe et al. reported (2020) that in Florida, the overall and July, 2020 case-fatality ratios (CFRs) remain low at 1.4% and 0.90%, respectively [73]. California reports a CFR of 1.8% since the onset of the pandemic and 1.1% in July 2020. The state of Texas reports an CFR of 1.4% overall and in the past month (July 2020) [73].

Postmortem studies of COVID-19 patients all over the world

Pomara, Li Volti, and Cappello published the article in 2020 [74]: COVID-19 Deaths: Are We Sure It Is Pneumonia? Please, Autopsy, Autopsy, Autopsy!

Salerno and collogues from Italy published the review article in 2020 “No Autopsies on COVID-19 Deaths: A Missed Opportunity and the Lockdown of Science” [75]. The authors of this review state that despite the increasing number of published studies on COVID-19, in all the examined studies the lack of a well-defined pathophysiology of death among patients who died following COVID-19 infection is evident. Autopsy should be considered mandatory to define the exact cause of death, thus providing useful clinical and epidemiologic information as well as pathophysiological insights to further provide therapeutic tools. A total of 50 articles met the inclusion criteria. However, only 7 of these studies reported autopsy-based data. The analysis of the main data from the selected studies concerns the complete analysis of 12,954 patients, of whom 2,269 died (with a mortality rate of 17.52%). Laboratory confirmation of COVID-19 infection was obtained in all cases and comorbidities were fully reported in 46 studies. The most common comorbidities were: cardiovascular diseases (hypertension and coronary artery disease), metabolic disorders (diabetes, overweight, or obesity), respiratory disorders (chronic obstructive pulmonary disease), and cancer. The most common reported complications were: ARDS, acute kidney injury, cardiac injury, liver insufficiency, and septic shock. Only 7 papers reported histological investigations. Nevertheless, only two complete autopsies are described, and the cause of death was listed as COVID-19 in only one of them. The lack of postmortem investigation did not allow a definition of the exact cause of death to determine the pathways of this infection. Based on the few histopathological findings reported in the analyzed studies, it seems to be a clear alteration of the coagulation system: frequently prothrombotic activity with consequent thromboembolism was described in COVID-19 patients.

The article published by Sperhake [76] provides an overview of the status of the autopsy series published worldwide and shows the path taken by the city of Hamburg in Germany, where autopsies are ordered by the health authorities in the interests of disease control. The risk of infection posed by SARS-CoV-2-positive deceased persons may be overestimated [76].

Most patients with COVID-19 are asymptomatic or experience only mild symptoms, including fever, dry cough, and shortness of breath. However, some individuals deteriorate rapidly and develop ARDS. The most common histopathologic correlate of ARDS is diffuse alveolar damage (DAD), characterized by hyaline membrane formation in the alveoli in the acute stage, and interstitial widening by edema and fibroblast proliferation in the organizing stage. Barton and colleagues reported that DAD has a long list of potential etiologies, including infection, vaping-associated pulmonary injury, oxygen toxicity, drug toxicity, toxic inhalants or ingestants, shock, severe trauma, sepsis, irradiation, and acute exacerbations of usual interstitial pneumonia [77].

Schaller et al., 2020 recent report of examinations conducted in Germany states that “because there are still insufficient data on cause of death, we describe postmortem examinations in a case series of patients with COVID-19” [78]. Between April 4 and April 19, 2020, the serial postmortem examinations were conducted in patients with proven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection who died at the University Medical Center Augsburg (Germany). Autopsies were conducted according to published best practice. Specimens from lung, heart, liver, spleen, kidney, brain, pleural effusion, and cerebrospinal fluid (CSF) were assessed. Postmortem nasopharyngeal, tracheal, bronchial swabs, pleural effusion, and CSF were tested for SARS-CoV-2 by reverse transcriptase–polymerase chain reaction. In this postmortem evaluation of 10 patients with COVID-19, acute and organizing diffuse alveolar damage and SARS-CoV-2 persistence in the respiratory tract were the predominant histopathologic findings and constituted the leading cause of death in patients with and without invasive ventilation. Periportal liver lymphocyte infiltration was considered unspecific inflammation. Whether myoepicardial alterations represented systemic inflammation or early myocarditis is unclear; criteria for true myocarditis were not met. Central nervous system involvement by COVID-19 could not be detected. This study has limitations, including the small number of cases from a single center and missing proof of direct viral organ infection[78].

Pulmonary postmortem findings in a series of COVID-19 cases were revealed in northern Italy two-center descriptive study [79]. The predominant pattern of lung lesions in patients with COVID-19 patients is diffuse alveolar damage, as described in patients infected with severe acute respiratory syndrome and Middle East respiratory syndrome coronaviruses. Hyaline membrane formation and pneumocyte atypical hyperplasia are frequent. Importantly, the presence of platelet–fibrin thrombi in small arterial vessels is consistent with coagulopathy, which appears to be common in patients with COVID-19 [79].

Tyramine infusion in twelve healthy active males causes a local noradrenaline release in the leg. The increase in noradrenaline was associated with an increase in clot microstructure formation [80]. Biogenic amines tyramine, tryptamine and β-phenyl-etylamine are secondary metabolites of microbial shikimate pathway produced by human microbiome [1, 2].

In China reports, COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is associated with coagulopathy causing venous and arterial thrombosis [81, 82]. Recent data from the pandemic epicenter in Wuhan, China, reported neurological complications in 36% of 214 patients with COVID-19; acute cerebrovascular disease (mainly ischemic stroke) was more common among 88 patients with severe COVID-19 than those with non-severe disease (5.7% versus 0.8%) [83].

However, the mechanisms, phenotype, and optimal management of ischemic stroke associated with COVID-19 remain uncertain [84]. Beyrouti and other researchers in London, UK described the demographic, clinical, radiological, and laboratory characteristics of six consecutive patients assessed April 1–16, 2020 at the National Hospital for Neurology and Neurosurgery, Queen Square, London, UK, with acute ischemic stroke and COVID-19 (confirmed by reverse-transcriptase PCR (RT-PCR)). All six patients had large vessel occlusion with markedly elevated D-dimer levels (≥1000μg/L). Three patients had multiterritory infarcts, two had concurrent venous thrombosis, and, in two, ischemic strokes occurred despite therapeutic anticoagulation [84].

In New Orleans, autopsies were performed on ten African American decedents aged 44–78 years with cause of death attributed to COVID-19, reflective of the dominant demographic of deaths following COVID-19 diagnosis [85]. Important findings include the presence of thrombosis and microangiopathy in the small vessels and capillaries of the lungs, with associated hemorrhage, that contributed to death. Features of diffuse alveolar damage, including hyaline membranes, were present, even in patients who had not been ventilated. Cardiac findings included individual cell necrosis without lymphocytic myocarditis. There was no evidence of secondary pulmonary infection by microorganisms [85].

Solomon et al. [86] reported that neurologic symptoms, including headache, altered mental status, and anosmia, occur in many patients with COVID-19. The neuropathological findings were from autopsies of 18 consecutive patients with SARS-CoV-2 infection who died in a single teaching hospital between April 14 and April 29, 2020 in Massachusetts, USA [86]. All the patients had nasopharyngeal swab samples that were positive for SARS-CoV-2 on qualitative reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assays. Histo-pathological examination of brain specimens ob-tained from 18 patients who died 0 to 32 days after the onset of symptoms of COVID-19 showed only hypoxic changes and did not show encephalitis or other specific brain changes referable to the virus. The virus was detected at low levels in six brain sections obtained from 5 patients; these levels were not consistently related to the interval from the onset of symptoms to death. Positive tests may have been due to in situ virions or viral RNA from blood [86].

The autopsy findings of 21 COVID-19 patients hospitalized at the University Hospital Basel and at the Cantonal Hospital Baselland, Switzerland were reported by Menter et al. [87]. The primary cause of death was respiratory failure with exudative diffuse alveolar damage and massive capillary congestion, often accompanied by microthrombi despite anticoagulation. Ten cases showed superimposed bronchopneumonia. Further findings included pulmonary embolism (n = 4), alveolar hemorrhage (n = 3), and vasculitis (n = 1). Pathologies in other organ systems were predominantly attributable to shock; three patients showed signs of generalized and five of pulmonary thrombotic microangiopathy. Six patients were diagnosed with senile cardiac amyloidosis upon autopsy. Most patients suffered from one or more comorbidities (hypertension, obesity, cardiovascular diseases, and diabetes mellitus). Additionally, there was an overall predominance of males and individuals with blood group A (81% and 65%, respectively) [87]. Myocardial hypertrophy observed in 71% COVID-19 cases Liver pathologies such as liver steatosis was observed in 41% and liver shock necrosis in 29% of COVID-19 cases. The cytoplasm of kidney podocytes, endothelial cells and proximal tubular epithelial cells contained multiple vesicles revealed by electron microscopy [87]. At higher magnification, the vesicles contain double membranes. Similar multiple vesicles were earlier observed in AD brain and in tryptamine-treated human neuronal cells by Paley [4]. In summary, the findings provide an insight into the complexity of COVID-19 pathophysiology. SARS-CoV-2 substantially contributed to fatality in all cases, but the authors postulate a multifactorial cause of death, with COVID-19 as a contributory factor in multimorbid patients [87]. Major findings that imply an impaired microcirculation include pulmonary capillarostasis and the presence of microthrombi in the lungs and kidneys despite anticoagulation. The findings corroborate clinical and epidemiological data on cardiovascular morbidity and disease outcome and add amyloid transthyretin (ATTR) amyloidosis as a risk factor. Of note, ATTR is thyroxine-binding prealbumin while serum thyroxine was lower in severe COVID-19 compared to nonsevere cases and healthy individuals [23].

Barton et al. (2020) report the findings of two complete autopsies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive individuals who died in Oklahoma in March 2020 [77]. A 77-year-old obese man with a history of hypertension, splenectomy, and 6 days of fever and chills died while being transported for medical care. He tested positive for SARS-CoV-2 on postmortem nasopharyngeal and lung parenchymal swabs. Autopsy revealed diffuse alveolar damage, chronic inflammation, and edema in the bronchial mucosa. A 42-year-old obese man with a history of myotonic dystrophy developed abdominal pain followed by fever, shortness of breath, and cough. Postmortem nasopharyngeal swab was positive for SARS-CoV-2; lung parenchymal swabs were negative. Autopsy showed acute bronchopneumonia with evidence of aspiration. Neither autopsy revealed viral inclusions, mucus plugging in airways, eosinophils, or myocarditis [77]. Bacterial cultures (aerobic/anaerobic) of the lung tissue from the 42-year-old patient grew nontoxigenic Escherichia coli, Candida tropicalis, and Proteus mirabilis. In complete autopsy of the 77-year-old patient, the hepatic centrilobular steatosis and remote cholecystectomy and in the 42-year-old, hepatic cirrhosis and advanced remote cholecystectomy were revealed. Coronary artery disease was found in both patients.

Ackermann et al. reported in 2020 that the lungs from patients with COVID-19 showed distinctive vascular features, consisting of severe endothelial injury associated with the presence of intracellular virus and disrupted cell membranes. Histologic analysis of pulmonary vessels in patients with COVID-19 showed widespread thrombosis with microangiopathy. Alveolar capillary microthrombi were 9 times as prevalent in patients with COVID-19 as in patients with influenza (p < 0.001). In lungs from patients with COVID-19, the amount of new vessel growth—predominantly through a mechanism of intussusceptive angiogenesis—was 2.7 times as high as that in the lungs from patients with influenza (p < 0.001) [88]. In this study, pulmonary autopsy specimens from seven patients who died from respiratory failure caused by SARS-CoV-2 infection were compared with lungs from seven patients who died from pneumonia caused by influenza A virus subtype H1N1—a strain associated with the 1918 and 2009 influenza pandemics [88].

Archer et al. described in 2020 the differences between COVID-19 pneumonia, acute respiratory distress syndrome, and high altitude pulmonary edema (HAPE) [89]. Although 80% of people with COVID-19 have a minor, acute respiratory infection, the mortality ranges from 2% to 7%. Patients with COVID-19 pneumonia may decompensate because of hypoxemic respiratory failure. Autopsy data show inflammation, diffuse alveolar damage, alveolar fluid accumulation, and occasional hyaline membranes, consistent ARDS [89]. Recently, HAPE physiology was proposed to explain the edema and hypoxemia in COVID-19 pneumonia.

Fried et al reported 4 cases (positive SARS-CoV-2 testing) that illustrate a variety of cardiovascular presentations of COVID-19 infection [90]. Case 1: A 64-year-old woman is presented with COVID-19 pneumonia and differential diagnosis included myopericarditis and cardiac amyloidosis. Case 2: A 38-year-old man with a history of type 2 diabetes mellitus presented with 1 week of cough, pleuritic chest pain, and progressive shortness of breath to an outside hospital. The patient had a bradycardic arrest lasting 6 minutes. Case 3: A 64-year-old woman with a nonischemic cardiomyopathy, atrial fibrillation, hypertension, and diabetes mellitus presented with a nonproductive cough and shortness of breath for 2 days. On arrival, she was afebrile, with blood pressure 153/120 mm Hg, heart rate 100 bpm, and oxygen saturation 88%. Case 4: A 51-year-old man with a history of heart transplantation in 2007 and renal transplantation in 2010 presented with intermittent fever, dry cough, and shortness of breath for 9 days. He denied any recent travel or sick contacts. His outpatient immunosuppression included tacrolimus 5 mg twice daily, mycophenolate mofetil 250 mg twice daily, and prednisone 5 mg daily[90].

In our study, the congophilic cerebral angiopathy associated with angiogenesis was revealed in blood vessels of tryptamine-treated mouse [91]. Thus two biogenic amines produced by human gut microbiome tryptamine [91] and tyramine [80] can induce thrombosis, microangiopathy and angiogenesis similar to those revealed in COVID-19 patients [77, 85, 88].

Pneumonia in elderly before COVID-19 era

Cacciatore et al. (2017) presented analysis of Italian cases of pneumonia observed in elderly [92] before COVID-19 was discovered. The data of this study are suitable for comparison with pneumonia associated with COVID-19 positivity. The authors discuss several important aspects of pneumonia in the elderly, which is a common and severe problem. In this review, the authors analyze the state of the art for pneumonia in the elderly. Several aspects are discussed: 1) how common is the disease; signs and symptoms in the elderly; 2) the elderly must always be hospitalized and which is the best place - ICU or medical ward?; 3) the role of comorbidities; 4) etiology and pathogenesis; medical treatment - when and how to start; 5) antibiotic resistance; 6) antibiotics in hospital-acquired and ventilator-related pneumonia; 7) assisted non-invasive ventilation; 8) the treatment in the terminally ill elderly patient [92]. Pneumonia is a frequent illness highly conditioning morbidity and mortality in the elderly population. In this age group, similarly, in all racial groups, it is the fourth leading cause of death and the leading cause of death from infectious disease. Pneumonia often is the terminal event that complicates a long-term illness (dementia, cancer, as prolonged immobilization syndrome). Authors distinguish different types of pneumonia depending on the environment in which they develop: community acquired pneumonia (CAP) or pneumonia community, nursing home acquired pneumonia or hospital acquired pneumonia or in hospital development and ventilator-associated pneumonia [92]. Data derived from an observational study conducted by the authors in the Emergency Department of Cardarelli Hospital in Naples, between January 1, 2015 and October 15, 2015, identified 3,255 patients with respiratory infection diagnosis on 72,134 accesses to the Emergency Department. The incident rate of respiratory infection is 45/1,000 patients of which 1,559/72,134 corresponding to an incidence rate of 22/1,000 with a diagnosis of pneumonia, increasing to 77.4/1,000 for patients with 65 years and over. In elderly patients hospitalized for pneumonia, the mortality rate is about 20%. If pneumonia develops in patients already hospitalized for other conditions, mortality increase to 50–70%, and is higher in women than in men. Pneumonia is the most common form of hospital-acquired infection. Data from European Center Disease Control (ECDC), published in 2015, related to a survey conducted on 231,459 patients hospitalized between 2011 and 2012, identified 13829 patients (6.0%) with hospital infection [92]. For comparison, the estimated COVID-19 positive is 8.408% in Florida currently and 0.36% COVID-19 positive were hospitalized residents of total Floridapopulation.

Therefore, the incidence of COVID-19-related pneumonia from March 2020 to February 2021 in Florida is essentially lower than the incidence of pneumonia in Europe in the period before COVID-19.

Jackson et al. reported in 2004 [93] that the overall rate of community-acquired pneumonia ranged from 18.2 cases per 1,000 person-years among persons aged 65–69 years to 52.3 cases per 1,000 person-years among those aged ≥85 years. In this US population, 59.3% of all pneumonia episodes were treated on an outpatient basis. In multivariate analysis, risk factors for community-acquired pneumonia included age, male sex, chronic obstructive pulmonary disease, asthma, diabetes mellitus, congestive heart failure, and smoking. On the basis of these data, the authors estimate that roughly 915,900 cases of community-acquired pneumonia occur annually among seniors in the US and that approximately 1 of every 20 persons aged > or = 85 years will have a new episode of community-acquired pneumonia each year [93].

CDC estimates that influenza was associated with more than 35.5 million illnesses, more than 16.5 million medical visits, 490,600 hospitalizations, and 34,200 deaths during the 2018–2019 influenza season. This burden was similar to estimated burden during the 2012–2013 influenza season (Centers for Disease C, Prevention. Estimated influenza illnesses and hospitalizations averted by influenza vaccination – United States, 2012–13 influenza season. MMWR Morb Mortal Wkly Rep 2013; 62(49): 997–1000).

Other necrotizing infections

It was discussed here that COVID-19 positivity can be associated with necrotizing pneumonia [49]. Moreover, cardiac necrosis [85] and liver shock necrosis [87] were found in COVID-19 patients. Thereby, the other necrotizing medical conditions can be related to necrosis in different organs of COVID-19 patients.

Erichsen Andersson et al. presented in 2018 the analysis of necrotizing fasciitis (NF) experienced by survivors and family in Nordic multi-center study [94]. Necrotizing soft tissue infection is the most serious of all soft tissue infections. The patient’s life is dependent on prompt diagnosis and aggressive treatment. Diagnostic delays are related to increased morbidity and mortality, and the risk of under- or missed diagnosis is high due to the rarity of the condition [94].

Molecular characterization of methicillin-resistant Staphylococcus aureus from a fatal case of NF in an extremely low-birth-weight infant was characterized by Orii et al. (2010) in Japan [95].

Peker et al reported (2010) a case of invasive and mortal NF caused by Staphylococcus epidermidis in an infant with extremely low birthweight in Turkey [96].

The case of multifocal NF complicating a single vaccine injection was reported by Perbet et al. in France [97]. Injection of hepatitis B vaccine of a 16-year-old immunocompetent woman developed into rapidly spreading multifocal NF of the right arm and the thighs, with septic shock. Treatment with antimicrobial therapy and surgical debridements allowed amputation to be avoided with a favourable outcome. The etiological agent was a methicillin-sensitive Staphylococcus aureus isolate [97].

The complete genome sequence of a highly virulent methicillin-sensitive Staphylococcus aureus strain, MCRF184, belonging to sequence type 45 was reported by Aswani et al. (2016) [98]. This staphylococcal strain was isolated in Wisconsin, USA from a surgical biopsy specimen of a patient with necrotizing fasciitis [98].

One of most recent case of necrotizing enterocolitis, medical condition that lead to premature birth was recently reported by Kim and Seo (2020) [99].

Challenges in diagnosing necrotizing enterocolitis were outlined in another report (2020) [100]. Staphylococcus is related to necrotizing enterocolitis. Particularly, necrotizing enterocolitis with epidemic Staphylococcus in a neonatal intensive care unit was reported in 1984 [101]. In 2012, Lemyre et al. [102] reported that a decrease in the number of cases of necrotizing enterocolitis is associated with the enhancement of infection prevention. This report describes control measures during a Staphylococcus aureus outbreak in a neonatal intensive care unit (2012) [102].

The severity of acute necrotizing pancreatitis ranges from self-limited to rapidly progressive illness leading to multiple organ failure. Ala-Kokko et al. of Oulu University Hospital, Finland reported that Staphylococcus epidermidis in pancreatic necrosis was associated with increased mortality in the study of 67 consecutive patients [24]. Mechanical ventilation (p = 0.002), surgical management (p = 0.028), open packing surgical management (p = 0.03), renal replacement therapy (p < 0.001), use of inotropic drugs (p = 0.012), and Staphylococcus epidermidis growth (p = 0.029) in infected pancreatic tissue were all associated with mortality [24]. While most complicating infections in animal pancreatic studies, hospitalized patients, and in pancreatic infection are caused by a few species of bacteria, normal intestinal flora consists of more than 400 species. In the natural course of severe acute pancreatitis, cultures of infected pancreatic necroses yield monomicrobial flora in 60–87% of cases; in these studies, polymicrobial flora was confirmed in only 13–40% of cases. A preponderance of Gram negative aerobic bacteria is usually present (Escherichia coli, Pseudomonas spp, Proteus, Klebsiella spp) which suggests an enteric origin, but Gram positive bacteria (Staphylococcus aureus, Streptococcus faecalis, Enterococcus), anaerobes, and, occasionally, fungi have also been found [103].

A short temporal history of pancreatitis was highly associated with pancreatic cancer in analysis of the data from two pooled case-control studies of pancreatic cancer (n = 4,515) in the San Francisco Bay Area and the M.D. Anderson Cancer Center, suggesting that pancreatitis may be an early manifestation of pancreatic cancer in some individuals [104]. Acute pancreatitis is linked to pancreatic cancer. Sadr-Azodi et al. (2018) reported a population-based cohort study including all Swedish residents diagnosed with a first-time episode of acute pancreatitis between 1997 and 2013 and corresponding matched pancreatitis-free individuals from the general population [105]. Overall, 49,749 individuals with acute pancreatitis and 138,750 matched individuals without acute pancreatitis were followed up for 1,192,134 person-years (median 5.3 years). A total of 769 individuals developed pancreatic cancer, of whom 536 (69.7%) had a history of acute pancreatitis. The risk of pancreatic cancer was substantially increased during the first few years after a diagnosis of acute pancreatitis but declined gradually over time, reaching a level comparable to the pancreatitis-free population after > 10 years of follow-up. In those with non-gallstone-related acute pancreatitis, the risk of pancreatic cancer declined to a level comparable to the pancreatitis-free population only when follow-up time was censored for a second episode of acute pancreatitis or a diagnosis of chronic pancreatitis. Increasing number of recurrent episodes of acute pancreatitis was associated with increased risk of pancreatic cancer. These findings imply a delay in the diagnosis of pre-existing pancreatic cancer, if clinically presented as acute pancreatitis. Any association between non-gallstone-related acute pancreatitis and pancreatic cancer in the long-term (> 10 years) could be mediated through recurrent acute pancreatitis or chronic pancreatitis [105].

Case of fatal myocardial necrosis caused by Staphylococcus lugdunensis and Cytomegalovirus in a patient with scleroderma was reported by Pirilä et al. in Finland (2006) [106]. At the autopsy extensive necrosis of the myocardium was seen, with an almost complete absence of inflammatory cells and the presence of bacterial structures identified as Staphylococcus lugdunensis by PCR.

Paley (2021) [2] suggests that tryptamine, an inducer of necrosis and a metabolic product of some Staphylococcus strains, can be implicated in necrotizing diseases such as necrotizing pneumonia. Here I discuss other necrotizing diseases and medical conditions: necrotizing fasciitis, necrotizing enterocolitis, necrotizing pancreatitis and myocardial necrosis in connection with community-acquired or hospital-acquired (nosocomial) Staphylococcus infections in different countries. I recommend the measurement of necrosis-inducing tryptamine and other biogenic amines for characterization of bacterial clinical isolates especially from cases of necrotizing diseases and conditions.

Coagulase-negative staphylococci (CNS) are the predominant bacteria found in fermented foods worldwide. Because of this, food-derived CNS are used as starters for cheese and meat fermentation, and have been investigated for use as starters in soybean fermentation. Although food-derived CNS are generally considered non-pathogenic, their safety remains uncertain following the isolation of CNS from skin infections in humans and animals, and because they belong to the same genus as the highly pathogenic species Staphylococcus aureus. The food-derived CNS produce tryptamine and other biogenic amines [107].

Taken together the data support that SARS-CoV-2 can be a contributory factor in all cases with COVID-19 of a multifactorial cause of death in multimorbid patients.