The Impact of Initial COVID-19 Episode Inflammation Among Adults on Mortality Within 12 Months Post-hospital Discharge

Authors: Arch G. Mainous III1,2*Benjamin J. Rooks1 and Frank A. Orlando1 May 12, 2022 Frontiers in Medicine

Background: Inflammation in the initial COVID-19 episode may be associated with post-recovery mortality. The goal of this study was to determine the relationship between systemic inflammation in COVID-19 hospitalized adults and mortality after recovery from COVID-19.

Methods: An analysis of electronic health records (EHR) for patients from 1 January, 2020 through 31 December, 2021 was performed for a cohort of COVID-19 positive hospitalized adult patients. 1,207 patients were followed for 12 months post COVID-19 episode at one health system. 12-month risk of mortality associated with inflammation, C-reactive protein (CRP), was assessed in Cox regressions adjusted for age, sex, race and comorbidities. Analyses evaluated whether steroids prescribed upon discharge were associated with later mortality.

Results: Elevated CRP was associated other indicators of severity of the COVID-19 hospitalization including, supplemental oxygen and intravenous dexamethasone. Elevated CRP was associated with an increased mortality risk after recovery from COVID-19. This effect was present for both unadjusted (HR = 1.60; 95% CI 1.18, 2.17) and adjusted analyses (HR = 1.61; 95% CI 1.19, 2.20) when CRP was split into high and low groups at the median. Oral steroid prescriptions at discharge were found to be associated with a lower risk of death post-discharge (adjusted HR = 0.49; 95% CI 0.33, 0.74).

Discussion: Hyperinflammation present with severe COVID-19 is associated with an increased mortality risk after hospital discharge. Although suggestive, treatment with anti-inflammatory medications like steroids upon hospital discharge is associated with a decreased post-acute COVID-19 mortality risk.

Introduction

The impact of coronavirus disease 2019 (COVID-19) has been immense. In terms of directly measured outcomes, as of February, 2022, worldwide more than 5.9 million people have died from directly linked COVID-19 episodes. More than 950,000 direct deaths from COVID-19 have been documented in the United States (1). Some evidence has suggested that some patients with COVID-19 may be at risk for developing health problems after the patient has recovered from the initial episode (24). Common sequelae that have been noted are fatigue, shortness-of-breath, and brain fog. Perhaps more concerningly, in addition to these symptoms, several studies have shown that following recovery from the initial COVID-19 episode, some patients are at risk for severe morbidity and mortality (58). Patients who have recovered from COVID-19 are at increased risk for hospitalization and death within 6–12 months after the initial episode. This morbidity and mortality is typically not listed or considered as a COVID-19 linked hospitalization or death in the medical records and thus are underreported as a post-acute COVID-19 sequelae.

The reason for this phenomenon of severe outcomes as post-acute sequelae of COVID-19 is not well understood. Early in COVID-19 episode, the disease is primarily driven by the replication of SARS-CoV-2. COVID-19 also exhibits a dysregulated immune/inflammatory response to SARS-CoV-2 that leads to tissue damage. The downstream impact of the initial COVID-19 episode is consistently higher in people with more severe acute infection (569). Cytokine storm, hyperinflammation, and multi-organ failure have also been indicated in patients with a severe COVID-19 episode (10). Cerebrospinal fluid samples indicate neuroinflammation during acute COVID-19 episodes (11). Moreover, even 40–60 days post-acute COVID-19 infection there is evidence of a significant remaining inflammatory response in patients (12). Thus, it could be hypothesized that the hyperinflammation that some COVID-19 patients have during the initial COVID-19 episode creates a systemic damage to multiple organ systems (1314). Consequently, that hyperinflammation and the corresponding systemic damage to multiple organ systems may lead to severe post-acute COVID-19 sequelae.

Following from this hyperinflammation, the use of steroids as anti-inflammatory treatments among patients with high inflammation during the initial COVID-19 episode may do more than just help in the initial episode but may act as a buffer to the downstream morbidity and mortality from the initial COVID-19 episode (1415).

The purpose of this study was to examine the relationship between substantial systemic inflammation, as measured by C-reactive protein (CRP), with post-acute COVID-19 sequelae among patients hospitalized with COVID-19. This 12-month mortality risk was examined in a longitudinal cohort of patients who tested positive for COVID-19 as determined by Polymerase Chain Reaction (PCR) testing within a large healthcare system.

Methods

The data for this project comes from a de-identified research databank containing electronic health records (EHR) of patients tested for or diagnosed with COVID-19 in any setting in the University of Florida (UF) Health system. Usage of the databank for research is not considered human subjects research, and IRB review was not required to conduct this study.

Definition of Cohort

The cohort for this study consisted of all adult patients aged 18 and older who were tested for COVID-19 between January 01, 2020 and December 31, 2021 within the UF Health system, in any encounter type (ambulatory, Emergency Department, inpatient, etc.). Although a patient in the cohort could have had a positive test administered in any of these settings, a patient was only included into the cohort if that patient experienced a hospitalization for COVID-19. Since this study included data from the early stages of the pandemic before consistent coding standards for documenting COVID-19 in the EHR had been established, a patient was considered to have been hospitalized for COVID-19 if they experienced any hospitalization within 30 days of a positive test for COVID-19. The databank contained EHR data for all patients in the cohort current through December 31, 2021. COVID-19 diagnosis was validated by PCR. Baseline dates for COVID-19 positive patients were established at the date of their earliest recorded PCR-confirmed positive COVID-19 test. Each patient was only included once in the analysis. For patients with multiple COVID-19 tests, if at least one test gave a positive result, the patient was classified as COVID-19 positive, and the date of their earliest positive COVID-19 test result was used as their baseline date. Patients who did not have a positive COVID-19 test were not included in the analysis. Patients were tested in the context of seeking care for COVID-19; the tests were not part of general screening and surveillance.

Only patients with at least 365 days of follow-up time after their baseline date were retained in the cohort. Patients with more than 365 days of follow-up were censored at 365 days. The cohort was also left censored at the 30-day mark post-hospital discharge to ensure that health care utilization was post-acute and not part of the initial COVID-19 episode of care (e.g., not a readmission).

Inflammation

C-reactive protein (CRP) was used as the measure of inflammation in this study. The UF Health laboratory measured CRP in serum using latex immunoturbidimetry assay. CRP measures were sourced from patient EHR data. The cohort was restricted to only include patients with at least one CRP measurement within their initial COVID-19 episode of care (between the date of their initial positive COVID-19 test and the left-hand censoring date). For patients with multiple measurements of CRP, the maximum value available was used.

Steroids

Intravenous dexamethasone during their initial COVID-19 hospitalization was assessed. Prescriptions for oral steroids (tablets of dexamethasone) that were prescribed either at or post-hospital discharge for their initial COVID-19 episode of care were included into the analysis. Prescriptions were identified using RxNorm codes available in each patient’s EHR.

Severity of Initial COVID-19 Hospitalization

We also measured the severity of the initial episode of COVID-19 hospitalization. This severity should track with the level of inflammation in the initial COVID-19 episode. We used the National Institutes of Health’s “Therapeutic Management of Hospitalized Adults With COVID-19” disease severity levels and definitions (16). The recommendations are based on four ascending levels: hospitalized but does not require supplemental oxygen, hospitalized and requires supplemental oxygen, hospitalized and requires supplemental oxygen through a high-flow device or noninvasive ventilation, hospitalized and requires mechanical ventilation or extracorporeal membrane oxygenation. For this study, because of the general conceptual model of severity moving from no supplemental oxygen to supplemental oxygen to mechanical ventilation, we collapsed the two supplemental non-mechanical ventilation oxygen into one intermediate category of severity.

Outcome Variables

The primary outcome investigated in this study was the 365-day all-cause mortality. Mortality data was sourced both from EHR data and the Social Security Death Index (SSDI), allowing for the assessment of deaths which occurred outside of UF’s healthcare system. When conflicting dates of death were observed between the EHR and SSDI, the date recorded in the patient’s medical record was used. Patients who died within their 365-day follow-up window were censored at the date of their recorded death. The cause of death was not available in the EHR based database and was not routinely and reliably reported in either the SSDI or EHR. We were unable to estimate the cause of death.

Comorbidities

Comorbidities and demographic variables which could potentially confound the association between inflammation represented by CRP and mortality post-acute COVID-19 were collected at baseline for each member of the cohort. Demographic variables included patient age, race, ethnicity, and sex. The Charlson Comorbidity Index was also calculated, accounting for the conditions present for each patient at their baseline. The Charlson Comorbidity Index was designed to be used to predict 1-year mortality and is a widely used measure to account for comorbidities (17).

Analysis

CRP was evaluated using descriptive statistics. We performed a median split of the CRP levels and defined elevated inflammation as a CRP level at or above the median and levels below the median as low inflammation. Additionally, as a way to examine greater separation between high and low inflammation, we segmented CRP levels into tertiles and categorized elevated inflammation as the top tertile and compared it to the first tertile by chi-square tests.

CRP level was also cross classified by severity of COVID-19 hospitalization and associations between the two variables were assessed using one-way ANOVA tests.

Kaplan-Meier curves comparing the survival probabilities of the high and low inflammation groups were created and compared using a log-rank test. Hazard ratios for the risk of death for post-acute COVID-19 complications by COVID-19 status were determined using Cox proportional hazard models. We obtained hazard ratios for mortality based on tertile and median splits of CRP. These analyses were then modified to control for age, sex, race, ethnicity, and the Charlson Comorbidity Index.

Additional analyses stratified by use of steroids were performed to compare the strength of the association between inflammation and death. The proportional hazards assumption was confirmed by inspection of the Schoenfeld residual plots for each variable included in the models and testing of the time-dependent beta coefficients. Analyses were conducted using the survival package in R v4.0.5.

Results

A total of 1,207 patients were included in the final cohort (Table 1). The characteristics of the patients are featured in Table 1. The mean CRP rises with the severity of illness in these COVID-19 inpatients. The mean CRP in the lowest severity (no supplemental oxygen) is 59.4 mg/L (SD = 61.8 mg/L), while the mean CRP in the intermediate severity group (supplemental oxygen) is 126.9 mg/L (SD = 98.6 mg/L), and the mean CRP in the highest severity group (ventilator or ECMO) is 201.2 mg/L (SD = 117.0 mg/L) (p < 0.001). Similarly, since dexamethasone is only recommended for the most severe patients with COVID-19, patients with dexamethasone had higher CRP (158.8 mg/L; SD = 114.9 mg/L) than those not on Dexamethasone (102.8 mg/L; SD = 90/8 mg/L) (p < 0.001).TABLE 1

Table 1. Characteristics of the patients in the cohort.

Figure 1 presents the Kaplan-Meier curves comparing the risk of mortality by inflammation over time. A log-rank test indicated there was a statistically significant difference in survival probabilities between the two groups (p = 0.002).FIGURE 1

Figure 1. All-cause mortality Kaplan-Meier curve comparing individuals with median or greater vs. below median C-reactive protein levels. Log rank test = p.002.

Table 2 shows the relationship between levels of inflammation and mortality post-recovery from COVID-19. In both unadjusted and adjusted analyses, elevated inflammation has a significantly increased risk compared to those with low inflammation in the initial COVID-19 episode. This finding of higher inflammation during the initial COVID-19 hospitalization and increased mortality risk after recovery was similar when CRP was split at the median and when the third tertile of CRP was compared to the first tertile of CRP. The proportional hazards assumption was met when the Schoenfeld plots.TABLE 2

Table 2. All-cause mortality hazard ratios by inflammation and steroid use.

We examined the hypothesized relationship that potentially decreasing inflammation in COVID-19 patients with an initial severe episode may have beneficial downstream effects on post-acute COVID-19 sequelae. Oral steroid prescriptions at discharge among these hospitalized COVID-19 patients were found to be associated with a lower risk of death post-discharge (Table 2).

Discussion

The results of this study reaffirm the importance of post-acute COVID-19 sequelae. This study is the first to show the impact of inflammation in the initial COVID-19 hospitalization episode on downstream mortality after the patient has recovered. This expands our understanding of post-acute COVID-19 sequelae by providing a better concept of why certain patients have post-acute COVID-19 mortality risk.

Previous studies have shown that patients who are hospitalized with COVID-19 have an increased risk of mortality 12 months after recovery (5). Those findings suggest that prevention of COVID-19 hospitalizations is of paramount importance. However, some patients will be hospitalized. The finding that elevated inflammation during the initial hospitalization episode is associated with mortality risk after recovery suggests that it may be worthwhile treating the viral episode but also consider treating the hyperinflammation. The NIH recommendations for care of COVID-19 hospitalized patients recommend steroids only for patients who need supplemental oxygen (16). The finding that the use of steroids prescribed upon discharge from the hospital and the corresponding reduced risk of mortality indicate that treating inflammation after the acute COVID-19 episode may act as a buffer to the downstream mortality risk from the initial COVID-19 episode (1415). Perhaps this requires a reconceptualization of COVID-19 as both an acute disease and potentially a chronic disease because of the lingering risks. Future research is needed to see if ongoing treatment for inflammation in a clinical trial has positive benefits.

There are several strengths and limitations to this study. The strengths of this study include the PCR validated COVID-19 tests at baseline for the cohort. Further, the linked electronic health record allows us to look not only at health care utilization like hospitalizations and both inpatient and outpatient medication but also laboratory tests like CRP levels. The cohort also allows us to have a substantial follow-up time.

In terms of limitations, the first that needs to be considered is that the analysis was based on hospitalized patients seen in one health system with a regional catchment area. Although more than 1200 hospitalized patients with PCR validated COVID-19 diagnoses were included in the analysis, and the cohort was followed for 12 months, the primary independent variable was systemic inflammation which should not be substantially affected by region of the country. Second, the data are observational. Thus, the analyses related to steroids and downstream mortality require a clinical trial to confirm these suggestive findings. Third, we did not have death certificates available to us to compute cause of death. The Social Security Death Index in partnership with the EHR allows us to be confident that the patient died and so we have a strong measure of all-cause mortality but we were unable to determine specific causes of death within this database. Fourth, although there are a variety of other markers of inflammation (e.g., D dimer, IL 6), CRP is one of the most robust measures of systemic inflammation. Moreover, it is much more widely used and was the most prevalent marker among the patients in the study.

In conclusion, hyperinflammation present with severe COVID-19 is associated with an increased mortality risk after hospital discharge. Although suggestive, treatment with anti-inflammatory medications like steroids upon hospital discharge is associated with a decreased post-acute COVID-19 mortality risk. This suggests that treating inflammation may also benefit other post-acute sequelae like long COVID. A reconceptualization of COVID-19 as both an acute and chronic condition may be useful.

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Life-threatening inflammation is turning COVID-19 into a chronic disease

Authors: Chris Melore MAY 13, 2022 Study Finds

Long COVID continues to be a lingering problem for more and more coronavirus patients in the months following their infection. Now, a new study contends that the life-threatening inflammation many patients experience — causing long-term damage to their health — is turning COVID-19 into a chronic condition.

“When someone has a cold or even pneumonia, we usually think of the illness being over once the patient recovers. This is different from a chronic disease, like congestive heart failure or diabetes, which continue to affect patients after an acute episode. We may similarly need to start thinking of COVID-19 as having ongoing effects in many parts of the body after patients have recovered from the initial episode,” says first author Professor Arch G. Mainous III, vice chair for research in the Department of Community Health and Family Medicine at the University of Florida Gainesville, in a media release.

“Once we recognize the importance of ‘long COVID’ after seeming ‘recovery’, we need to focus on treatments to prevent later problems, such as strokes, brain dysfunction, and especially premature death.”

COVID inflammation increases risk of death one year later

The study finds COVID patients experiencing severe inflammation while in the hospital saw their risk of death skyrocket by 61 percent over the next year post-recovery.

Inflammation raising the risk of death after an illness is a seemingly confusing concept. Typically, inflammation is a natural part of the body’s immune response and healing process. However, some illnesses including COVID-19 cause this infection-fighting response to overshoot. Previous studies call this the “cytokine storm,” an event where the immune system starts attacking healthy tissue.

“COVID-19 is known to create inflammation, particularly during the first, acute episode. Our study is the first to examine the relationship between inflammation during hospitalization for COVID-19 and mortality after the patient has ‘recovered’,” Prof. Mainous says.

“Here we show that the stronger the inflammation during the initial hospitalization, the greater the probability that the patient will die within 12 months after seemingly ‘recovering’ from COVID-19.”

There is a way to stop harmful inflammation

The study examined the health records of 1,207 adults hospitalized for COVID-19 in the University of Florida health system between 2020 and 2021. Researchers followed them for at least one year after discharge — keeping track of their C-reactive protein (CRP) levels. This protein is secreted by the liver and is a common measure of systemic inflammation.

Results show patients with a more severe case of the virus and those needing oxygen or ventilation had higher CRP levels during their hospitalization. The patients with the highest CRP concentrations had a 61-percent increased risk of death over the next year after their release from the hospital.

However, the team did find that prescribing anti-inflammatory steroids after hospitalization lowered the risk of death by 51 percent. Study authors say their findings show that the current recommendations for care after a coronavirus infection need to change. Researchers recommend more widespread use of orally taken steroids following a severe case of COVID.

Warning to anyone who’s had Covid as scientists discover symptoms that can last for TWO YEARS

Authors: Vanessa Chalmers, Digital Health Reporter May 11 2022  May 12 2022

DOCTORS have discovered the symptoms of Covid that can last for two years or more. 

Their research has shown that half of patients admitted to hospital are still likely to have at least one persistent problem two years later.

The study, published in The Lancet Respiratory Medicine, has the longest follow-up period of patients to date. 

Researchers are only able to analyse what symptoms exist after two years given the coronavirus emerged in late 2019.

So it’s possible problems like fatigue and anxiety could stick around even longer.

Lead author Professor Bin Cao, of the China-Japan Friendship Hospital, China, said: “Our findings indicate that for a certain proportion of hospitalised Covid-19 survivors, while they may have cleared the initial infection, more than two years is needed to recover fully from Covid-19.”

The study involved almost 1,200 patients, aged 57 on average, who were infected with the bug in the early phase of the pandemic.

They had all been treated in Wuhan, China, then assessed six months, 12 months and two years after discharge.

Researchers looked at their walking abilities, mental health, quality of life and more.

Covid patients were generally found to be in poorer health than those in the general population two years after infection.

They reported:

  • Fatigue or muscle weakness (31 per cent of Covid patients compared to five per cent in the general population)
  • Sleep difficulties (51 per cent compared with 14 per cent)
  • Pain or discomfort (23 per cent compared with five per cent)
  • Anxiety or depression (12 per cent compared with five per cent)

Joint pain, palpitations, dizziness, and headaches were also more common among previously hospitalised Covid patients.

Not all of those hospitalised were affected, however.

More than half (55 per cent) of participants had at least one symptom of Covid at two years, and were therefore considered “long Covid” patients.

The researchers then compared the long Covid group with the group of participants who had endured Covid, but gotten better. 

Those with long Covid had more pain (35 per cent vs 10 per cent), and mobility issues (five per cent vs one per cent) than their fully recovered counterparts.

Some 13 per cent showed symptoms of anxiety and 11 per cent depression, compared with three per cent and one per cent in non-long Covid patients, respectively. 

The researchers said it’s not possible to say whether problems like these are specific to Covid, or whether other hospital patients experience them.

Long Covid is defined as someone who still battles symptoms beyond four weeks in the UK.

It may be defined as ongoing Covid (four to 12 weeks), or post-Covid syndrome (more than 12 weeks) by medics.

The symptoms may include fatigue, a cough, breathlessness, muscle or joint pain, loss of taste of smell and brain fog.

Factors Associated with Post-Acute Sequelae of SARS-CoV-2 (PASC) After Diagnosis of Symptomatic COVID-19 in the Inpatient and Outpatient Setting in a Diverse Cohort

Authors: Sun M. Yoo MD, MPHTeresa C. Liu MD, MPHYash Motwani BSMyung S. Sim DrPH

Nisha Viswanathan MDNathan Samras MDFelicia Hsu MD & Neil S. Wenger MD, MPH 

Journal of General Internal Medicine (2022)

ABSTRACT

Background

The incidence of persistent clinical symptoms and risk factors in Post-Acute Sequelae of SARS-CoV-2 (PASC) in diverse US cohorts is unclear. While there are a disproportionate share of COVID-19 deaths in older patients, ethnic minorities, and socially disadvantaged populations in the USA, little information is available on the association of these factors and PASC.

Objective

To evaluate the association of demographic and clinical characteristics with development of PASC.

Design

Prospective observational cohort of hospitalized and high-risk outpatients, April 2020 to February 2021.

Participants

One thousand thirty-eight adults with laboratory-confirmed symptomatic COVID-19 infection.

Main Measures

Development of PASC determined by patient report of persistent symptoms on questionnaires conducted 60 or 90 days after COVID-19 infection or hospital discharge. Demographic and clinical factors associated with PASC.

Key Results

Of 1,038 patients with longitudinal follow-up, 309 patients (29.8%) developed PASC. The most common persistent symptom was fatigue (31.4%) followed by shortness of breath (15.4%) in hospitalized patients and anosmia (15.9%) in outpatients. Hospitalization for COVID-19 (odds ratio [OR] 1.49, 95% [CI] 1.04–2.14), having diabetes (OR, 1.39; 95% CI 1.02–1.88), and higher BMI (OR, 1.02; 95% CI 1–1.04) were independently associated with PASC. Medicaid compared to commercial insurance (OR, 0.49; 95% CI 0.31–0.77) and having had an organ transplant (OR 0.44, 95% CI, 0.26–0.76) were inversely associated with PASC. Age, race/ethnicity, Social Vulnerability Index, and baseline functional status were not associated with developing PASC.

Conclusions

Three in ten survivors with COVID-19 developed a subset of symptoms associated with PASC in our cohort. While ethnic minorities, older age, and social disadvantage are associated with worse acute COVID-19 infection and greater risk of death, our study found no association between these factors and PASC.

INTRODUCTION

As millions of people recover from COVID-19 amidst the global pandemic, many continue to report an array of persistent symptoms after infection, termed post-acute sequelae of SARS-CoV-2 (PASC). Commonly reported PASC symptoms range from fatigue and dyspnea to “brain fog” and anosmia, with ongoing disability and disruption of work, social, and home lives.1,2,3,4 Centers for Disease Control and Prevention classify the array of symptoms lasting 4 or more weeks after COVID-19 infection as a Post-COVID Condition, which may include long-haul COVID, long COVID, or PASC. Most efforts to describe PASC and factors associated with PASC have focused on hospitalized adult patients1,2,4 and more recently on patient with mild COVID-19 infection treated in the outpatient setting up to 9 months after infection.5,6 Although there are studies that have looked at factors associated with PASC,7 there are no prospective cohort studies to our knowledge that have evaluated the association of ethnicity, social vulnerability, and insurance status with developing PASC. Although COVID-19 has disproportionately impacted racial and ethnic minority groups, previously studied PASC cohorts in the USA have small proportions of Latinx, Black or African American, and Asian American and Pacific Islander (AAPI) patients.5

In April 2020, University of California Los Angeles (UCLA) Health created a COVID Ambulatory Monitoring Program to clinically care for the diverse group of COVID-19 patients discharged from the hospital as well as high-risk COVID-19 patients cared for in the outpatient setting. Patients were eligible from all parts of the UCLA Health system, which includes more than 40 primary care clinics and 20 UCLA Hospitalist services in hospitals across southern California. This COVID-19 program collected standardized clinical data to guide the approach to monitoring and care. Using these patient-reported data combined with clinical information from the electronic health record (EHR), we describe a population-based cohort of patients with symptomatic COVID-19, characterize the timeline of persistent clinical symptoms, and identify factors associated with PASC.

PASC terminology is in evolution and can include a wide range of clinical manifestations including psychiatric manifestations and evidence of organ dysfunction as a result of SARS-CoV-2, including new symptoms or clinical findings that were not evident at the time of acute COVID-19 infection. For our evaluation, we focused on a subset of symptoms associated with PASC as described in the Clinical and Functional Survey (available in Appendix), which we will reference as PASC in our paper.

METHODS

A longitudinal, prospective cohort of adults with laboratory confirmed SARS-CoV-2 infection was enrolled in the UCLA COVID Ambulatory Program starting April 2020. Standardized follow-up was performed to monitor patients with COVID-19 discharged from the two UCLA hospitals, UCLA patients discharged from 20 other local hospitals, and UCLA outpatients who were referred by their primary care providers. COVID-19 patients discharged from the general ward services were approached post-discharge to enroll them in the program. Outpatients with new infections deemed clinically high risk by their primary care providers were referred for enrollment. A questionnaire administered by nurses over the telephone collected information on functional status prior to COVID-19 infection and post-COVID clinical symptoms. Follow-up monitoring continued at 30, 60, and 90 days after hospital discharge for post-discharge patients or date of positive COVID-19 test for outpatients to evaluate for persistent symptoms. A multidisciplinary team of primary care physicians and specialists followed patients to address persistent symptoms or clinical deterioration. The study was approved by the UCLA institutional review board (IRB#20-001358).

A monitoring questionnaire (available in the Appendix) assessed whether the patient felt that his or her health was back to normal. The survey queried baseline function by asking about maximal exertion level prior to COVID-19 infection: vigorous activities such as running, lifting heavy objects, and participating in strenuous sports; moderate activities, such as moving a table, pushing a vacuum cleaner, bowling, or playing golf; climbing one flight of stairs; walking one block; lifting or carrying groceries; bathing or dressing yourself.8 Functional limitation over the past 4 weeks was assessed using this item during each survey. Perceived cognitive deficits were evaluated with three questions modified from the Perceived Deficits Questionnaire-59 that ask whether patients in the last 4 weeks had trouble getting things organized, had trouble concentrating on things, or forgetting what the patient talked about after a telephone conversation. Lastly, patients were asked about the following symptoms over the past 4 weeks: fever, chills or night sweats; loss of smell or taste; fatigue; shortness of breath; chest pain; numbness or tingling; nausea, vomiting, or diarrhea; muscle aches; and rash.

Demographic characteristics (age, gender, race, ethnicity) were obtained from the EHR, as were a history of diabetes mellitus or organ transplant, body mass index (BMI), Elixhauser comorbidity index,10 and level of medical care required for the initial COVID-19 illness (ambulatory care, emergency room, hospital, and intensive care unit [ICU]). Insurance was collapsed into commercial, Medicare, Medicaid, and none/other. Social Vulnerability Index (SVI) was calculated and split into quartiles.11

Baseline demographic characteristics, functional status, and clinical characteristics were evaluated in the full cohort and compared among patients treated initially in the outpatient setting, in the inpatient setting, and in the ICU. Patients were characterized as having PASC if they noted persistent COVID-19 symptoms on the 90-day post-discharge survey (or the 60-day survey if the 90-day survey was incomplete). Survey attrition flow diagram is included in the Appendix (Supplemental Figure 1). Baseline characteristics of PASC patients in the inpatient and outpatient settings were compared to patients without persisting post-acute symptoms. Among patients with PASC, symptoms were compared at the time of acute infection, 30 days, 60 days, and 90 days, presented separately for patients with PASC who received their initial COVID-19 treatment in the inpatient versus outpatient setting.

Statistical Analysis

Summary statistics (i.e., mean, standard deviation SD, and percentage) for demographics and clinical characteristics are reported for the full cohort. We performed t-tests for a difference in means between groups for age, BMI, and Elixhauser Comorbidity Index and used chi-square tests to test differences in proportions between groups for sex, race/ethnicity, diabetes, transplant, payor status, and baseline functional status. We used a multivariable logistic regression model to evaluate the factors associated with the development of PASC. The pre-specified factors included in the model were demographics (age, sex, race), clinical characteristics (diabetes, BMI, transplant status), insurance type, SVI, COVID-19 care venue, and baseline function. We performed 5 multiple imputations (MI) for Elixhauser (6.0% missingness, and 10.3% unknown) and performed logistic regression analysis for each imputed set. We did not include Elixhauser in the final logistic regression model after finding that it was not statistically significant in those MI sets and the inclusion of imputed Elixhauser in the model did not affect the estimates of odds ratios (ORs) of other factors. In order to further characterize clinical factors associated with PASC, we disaggregated the components of the Elixhauser index and evaluated their individual relationships with PASC using chi-square and Fisher’s exact tests. Factors included in the final model were selected regardless of their statistical significance considering the clinical importance and to examine the effect of socioeconomic status on the PASC outcome. Two-sided P value <0.05 was considered statistically significant and analyses were done using SAS 9.4 (Cary, NC).

RESULTS

Participant Characteristics

Of the 1,296 enrolled in the program between April 2020 to February 2021, 1,038 patients (80.1%) completed follow-up surveys at 30, 60, or 90 days after hospital discharge or outpatient diagnosis and were included in the study. Of the 800 patients treated for COVID-19 in the hospital, 152 (19%) received treatment in the ICU. Of the 238 patients treated as outpatients, 36 (15.1%) received care in the emergency department. The mean age of the cohort was 60 years (interquartile range, 37 to 83) with an even split by gender overall, but more women receiving outpatient care. Thirty percent were White and 42% Latinx. More than one-third of the cohort had diabetes, over 10% had received a solid organ transplant, and mean BMI was nearly 30. Forty-two percent of patients had commercial insurance and mean SVI was 0.46 (interquartile range 0.16 to 0.76). At baseline, most patients could complete vigorous or moderate activities. Men, patients with diabetes, and Latinx patients were more likely to have been admitted to the hospital or the ICU (Table 1).Table 1 Demographics and Clinical Characteristics of COVID-19 PatientsFull size table

Persistent Symptoms

Of the 1038 patients, 309 (29.8%) reported persistent symptoms on the follow-up survey at least 60 days after the acute illness, defined as PASC in this cohort. Of the 800 patients who received treatment for COVID-19 in the hospital, 246 (30.8%) developed PASC whereas 63 (26.5%) of the 238 treated as high-risk outpatients developed PASC.

Symptoms during the acute period of COVID-19 illness were reported on the 30-day survey. Of the PASC patients who completed the 30-day survey (n=231), the most commonly reported symptoms were fatigue in 169 patients (73.2%) followed by shortness of breath in 147 patients (63.6%), fevers and chills in 119 (51.5%), and muscle aches in 117 patients (50.6%). In terms of persistent symptoms at least 60 days after infection, fatigue was the most commonly reported symptom (31.4%), followed by shortness of breath (13.9%), and loss of taste or smell (9.8%). Persistent fever (1.9%) and rash (< 1%) were rare. When comparing hospitalized to outpatient PASC patients, fatigue was the most common persistent symptom in both groups. The next most common symptom in hospitalized patients was shortness of breath (15.4%), whereas it was loss of taste or smell (15.9%) in outpatients (Figure 1A, B).

figure 1
Figure 1

Factors Associated with PASC

In bivariate analyses, factors associated with PASC were different among patients treated for COVID-19 in the inpatient versus outpatient setting. In the outpatient setting, PASC patients were younger, more likely to be White, women, and commercially insured. In the inpatient setting, age and race were unrelated to PASC, and women were more likely to report symptoms consistent with PASC. In both settings, transplant patients were less likely to develop PASC and baseline functional status prior to COVID-19 infection was not related (Table 2).Table 2 Demographics and Characteristics of COVID-19 PASC Cohort and Non-PASC Cohort by Highest Level of CareFull size table

In adjusted analyses, hospitalization for COVID-19 (OR 1.49, 95% confidence interval [CI] 1.04–2.14), having diabetes (OR, 1.39; 95% CI 1.02–1.88), and higher BMI (OR, 1.02; 95% CI 1.0002–1.04) were independently associated with developing PASC. Having Medicaid compared to commercial insurance (OR, 0.49; 95% CI 0.31–0.77) or a history of organ transplant (OR 0.44, 95% CI, 0.26–0.76) was inversely associated with developing PASC. Age, race/ethnicity, SVI, and baseline functional status were not associated with developing PASC (Fig. 2).

figure 2
Figure 2

DISCUSSION

In this prospective cohort of individuals with COVID-19 infection, 30% of patients developed a subset of symptoms associated with PASC. This large, diverse cohort achieved a longitudinal follow-up of 80% of patients after the acute COVID-19 illness. Studying a diverse population treated in a single health system, we are able to control for factors such as access and quality of care often lacking in existing studies.5,12 This health system-based COVID-19 population reveals the startling findings that age, race, and economic disadvantage appear unassociated with development of PASC. This contrasts with COVID-19 infection rates, hospitalizations, and deaths that are disproportionately higher in racial and ethnic minority communities and older people.13,14

Existing prospective studies have not evaluated race and ethnicity and its association with PASC. Sudre et al. found increasing age, female gender, hospitalization, and more than five symptoms in the first week of illness to be associated with PASC, but were unable to analyze the impact of ethnicity due to incomplete data.7 Other studies have described hospitalized patients as more likely to be older, not White, and with lower household incomes compared to outpatients,12 but evaluation of the association between race/ethnicity, SVI, insurance, and PASC were not done.

Current data indicate that ethnic minorities represent a disproportionate number of COVID-19 cases, hospitalizations, and deaths in the USA.13,14,15 Ethnic minorities in the USA diagnosed with COVID-19 were likely to have a greater number of underlying clinical comorbidities,16 lower socioeconomic status,17 loss of health insurance during the pandemic,18,19 and poor access to healthcare. In cohort studies of patients hospitalized for COVID-19, race and ethnicity were not independently associated with in-hospital mortality after adjusting for factors such as age, sex, comorbidities, insurance, and neighborhood deprivation.16,20 This suggests that access to care is an important factor in COVID-19 outcomes. In our study, race and ethnicity was not associated with developing PASC. One possible explanation is that patients had access to the same health system with standardized follow-up. Another possible explanation is that factors that contribute to risks of contracting COVID-19 are not as important in the COVID-19 recovery process and development of PASC. Another factor to consider is whether report of symptoms and expectations for recovery differ across socioeconomic, ethnic, and racial groups and whether the tools used to detect PASC equitably capture these reports.

Existing literature suggests that women were more likely than men to develop PASC,7 whereas our data found a non-statistically significant trend in this direction. It has been suggested this may be due to differences in immunity with a higher prevalence of autoimmune disease in women.21 Diabetes and elevated BMI were associated with developing PASC, which may be due to increased inflammation seen in SARS-CoV-222 potentially leading to microvascular and macrovascular complications.23,24 Interestingly, surviving transplant patients were less likely to develop PASC. The dampened host inflammatory response to COVID-19 for those on immunosuppression agents may play a role, as suggested by a prior study showing solid organ transplant recipients had a faster decline in disease severity over time.25

Surprisingly, patients with commercial insurance had double the likelihood of developing PASC compared to patients with Medicaid. This association will be important to explore further to understand if insurance status in this group is representing unmeasured demographic factors or exposures.

In the acute period of illness, the most commonly reported clinical symptoms among patients who fit our PASC definition were fatigue then shortness of breath, in line with existing studies.1,2 Although fatigue was the most common persistent symptom in hospitalized and non-hospitalized patients, this was followed by loss of sense of smell in outpatients and shortness of breath in inpatients, which is in line with previous studies.3,5 This variation in symptoms at presentation and over time suggests differences in the clinical phenotypes of those with mild to moderate COVID-19 treated in the outpatient setting compared to those with severe COVID-19 requiring hospitalization.

Strengths of our study include a large diverse cohort of COVID-19 patients prospectively followed in a single health system with a large number of organ transplant patients, and standardized longitudinal data to assess symptom evolution over time. Study limitations include potential bias from subjective rating of symptoms and functional status, evaluation of a limited subset of symptoms encapsulated by PASC, not having a comparator group of patients with persistent symptoms after non-COVID hospital admissions, and limited information about pre-existing conditions in our patient population. In addition, survivorship bias may exist where the analysis was limited to individuals that survived to at least 30 days after COVID-19 diagnosis, and referral bias in the outpatient cohort, as only patients deemed clinically high risk were referred to the program, which may affect the generalizability in the outpatient cohort.

CONCLUSION

In this diverse prospective cohort of symptomatic COVID-19 patients treated in the inpatient and outpatient setting, nearly one-third developed a subset of symptoms associated with PASC. The most common symptoms that persisted were different in outpatients compared to inpatients, with fatigue and anosmia in outpatients and fatigue and dyspnea in inpatients. Finally, the lack of relationship between factors related to more serious COVID-19 illness (age, ethnicity, baseline function, and socioeconomic vulnerability) suggests that the long-term effects of COVID-19 may vary from those producing acute illness. These findings, along with variation by insurance status and the protective nature of transplantation, should stimulate additional study to understand the pathophysiologic factors underlying PASC, as well as the tools and methods used to detect PASC. Understanding the effects of long COVID will allow for more effective education among patients and providers, and allow for appropriate healthcare resource utilization in the evaluation and treatment of PASC.

References

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Only 29% of hospitalized COVID-19 patients fully well one year on: Study

Authors: CNA 24 Apr 2022 08:02AM

PARIS: Not even one in three people have completely recovered from COVID-19 a full year after being hospitalized with the disease, a United Kingdom study indicated on Sunday (Apr 24), warning that long COVID could become a common condition.

The study involving more than 2,300 people also found that women were 33 per cent less likely to fully recover than men.

It also found that obese people were half as likely to fully recover, while those who needed mechanical ventilation were 58 per cent less likely.

The study looked at the health of people who were discharged from 39 British hospitals with COVID-19 between March 2020 and April 2021, then assessed the recovery of 807 of them five months and one year later.

Just 26 per cent reported a full recovery after five months, and that number rose only slightly to 28.9 per cent after a year, according to the study published in the Lancet Respiratory Medicine journal.

“The limited recovery from five months to one year after hospitalisation in our study across symptoms, mental health, exercise capacity, organ impairment and quality of life is striking,” said study co-leader Rachel Evans of the National Institute for Health and Care Research.

The most common long COVID symptoms were fatigue, muscle pain, poor sleep, slowing down physically and breathlessness.

“Without effective treatments, long COVID could become a highly prevalent new long-term condition,” said study co-lead Christopher Brightling of the University of Leicester.

The study, which will be presented at the European Congress of Clinical Microbiology and Infectious Diseases, is ongoing, and will continue to monitor the patients’ health.

Longest Covid infection lasted more than 16 months, tests show

Authors: Michelle Roberts

UK doctors believe they have documented the longest Covid infection on record – a patient they treated who had detectable levels of the virus for more than 16 months, or 505 days, in total.

The unnamed individual had other underlying medical conditions and died in hospital in 2021.

Persistent infections such as this are still rare, say the London medics.

Most people naturally clear the virus, but the patient in question had a severely weakened immune system.

Chronic infections like these need studying to improve our understanding of Covid and the risks it can pose, say experts.

The patient first caught Covid in early 2020. They had symptoms and the infection was confirmed with a PCR test.

They were in and out of hospital many times over the next 72 weeks, for both routine checks and care.

On each occasion – about 50 in all – they tested positive, meaning they still had Covid.

The doctors, from King’s College London and Guy’s and St Thomas’ NHS Foundation Trust, say detailed lab analysis revealed it was the same, persistent infection, rather than repeated bouts.

The patient could not shake the infection, even after being given antiviral drugs.

This is different to “long Covid”, where symptoms persist after the infection has gone.

One of the medics who will be presenting the findings at a medical conference – the European Congress of Clinical Microbiology and Infectious Diseases – is Dr Luke Blagdon Snell.

He told the BBC: “These were throat swab tests that were positive each time. The patient never had a negative test. And we can tell it was one continuous infection because the genetic signature of it – the information we got from sequencing the viral genome – was unique and constant in that patient.”

Prolonged infections are rare but important, say the researchers, because they might give rise to new variants of Covid – although that did not happen in this case, or other ones that they studied.

Dr Snell said: “The virus is still adapting to the human host when people are infected for a long time. It might provide an opportunity for Covid to accrue new mutations.

“Some of these patients that we have studied have mutations that have been seen in some of the variants of concern.”

He stressed that none of the nine patients they checked had spawned a new dangerous variant.

Someone with a chronic infection might not be contagious to others, he added.

Dr David Strain from the University of Exeter Medical School, said: “We know that every time the virus replicates, it must reproduce its RNA – equivalent to manually copying a text book. We know if we were to transcribe an entire book we would make mistakes, so too does the virus. Every copy will produce mutations.

“Although Omicron did not arise in these particular individuals, this demonstrates a very clear pathway by which vaccine resistant variants may arise. Whereas with BA.2 we have got lucky, that the mutation is associated with a less severe illness, there is no guarantee that the next iteration will be the same.”

About 30% of COVID-19 patients suffer from ‘long COVID’ – study

Surprisingly, ethnicity, older age, and socioeconomic status, all linked with severe illness, were not associated with the long COVID syndrome.

Authors JERUSALEM POST STAFF Published: APRIL 19, 2022 

Two years into the COVID-19 pandemic, researchers are still learning about the effects of the virus, including how many people suffer from “long COVID” – When symptoms of the disease linger long past infection. 

New University of California Los Angeles (UCLA) research finds that 30% of people treated for COVID-19 developed Post Acute Sequelae of COVID-19 (PASC), most commonly known as “long COVID.”

According to the study, which was recently published in Springer, patients with a history of hospitalization, diabetes, and higher body mass index were most likely to develop the condition, while those covered by Medicaid, as opposed to commercial health insurance, or had undergone an organ transplant were less likely to acquire it.

 SARS-CoV-2, the novel coronavirus that causes COVID-19 (illustrative). (credit: PIXABAY)

Surprisingly, demographics that are linked with severe illness and greater risk of death from COVID-19, such as ethnicity, older age and socioeconomic status, were not associated with long COVID syndrome. 

The UCLA researchers studied 1,038 people who were enrolled in the UCLA COVID Ambulatory Program between April 2020 and February 2021. Of those, 309 people were living with long COVID. In hospitalized patients, the most persistent symptoms were fatigue and shortness of breath (31% and 15%, respectively). Loss of sense of smell (16%) was the most reported symptom in outpatients.

“This study illustrates the need to follow diverse patient populations longitudinally to understand the Long COVID disease trajectory and evaluate how individual factors such as pre-existing co-morbidities, sociodemographic factors, vaccination status and virus variant type affect type and persistence of Long COVID symptoms,” said Dr. Sun Yoo, health sciences assistant clinical professor at David Geffen School of Medicine at UCLA and medical director of the Extensivist Program.  “Studying outcomes in a single health system can minimize variation in quality of medical care. Our study also raises questions such as: Why were patients with commercial insurance twice as likely to develop Long COVID than patients insured through Medicaid? Because persistent symptoms can be subjective in nature, we need better tools to accurately diagnose Long COVID and to differentiate it from exacerbations of other emerging or chronic conditions. Finally, we need to ensure equitable access to outpatient Long COVID care.” 

Alzheimer’s-like signaling in brains of COVID-19 patients

Authors: Steve Reiken,Leah Sittenfeld,Haikel Dridi,Yang Liu,Xiaoping Liu,Andrew R. Marks First published: 03 February 2022  https://doi.org/10.1002/alz.12558

Abstract

Introduction

The mechanisms that lead to cognitive impairment associated with COVID-19 are not well understood.

Methods

Brain lysates from control and COVID-19 patients were analyzed for oxidative stress and inflammatory signaling pathway markers, and measurements of Alzheimer’s disease (AD)-linked signaling biochemistry. Post-translational modifications of the ryanodine receptor/calcium (Ca2+) release channels (RyR) on the endoplasmic reticuli (ER), known to be linked to AD, were also measured by co-immunoprecipitation/immunoblotting of the brain lysates.

Results

We provide evidence linking SARS-CoV-2 infection to activation of TGF-β signaling and oxidative overload. The neuropathological pathways causing tau hyperphosphorylation typically associated with AD were also shown to be activated in COVID-19 patients. RyR2 in COVID-19 brains demonstrated a “leaky” phenotype, which can promote cognitive and behavioral defects.

Discussion

COVID-19 neuropathology includes AD-like features and leaky RyR2 channels could be a therapeutic target for amelioration of some cognitive defects associated with SARS-CoV-2 infection and long COVID.

1 NARRATIVE

1.1 Contextual background

Patients suffering from COVID-19 exhibit multi-system organ failure involving not only pulmonary1 but also cardiovascular,2 neural,3 and other systems. The pleiotropy and complexity of the organ system failures both complicate the care of COVID-19 patients and contribute, to a great extent, to the morbidity and mortality of the pandemic.4 Severe COVID-19 most commonly manifests as viral pneumonia-induced acute respiratory distress syndrome (ARDS).5 Respiratory failure results from severe inflammation in the lungs, which arises when SARS-CoV-2 infects lung cells. Cardiac manifestations are multifactorial and include hypoxia, hypotension, enhanced inflammatory status, angiotensin-converting enzyme 2 (ACE2) receptor downregulation, endogenous catecholamine adrenergic activation, and direct viral-induced myocardial damage.67 Moreover, patients with underlying cardiovascular disease or comorbidities, including congestive heart failure, hypertension, diabetes, and pulmonary diseases, are more susceptible to infection by SARS-CoV-2, with higher mortality.67

In addition to respiratory and cardiac manifestations, it has been reported that approximately one-third of patients with COVID-19 develop neurological symptoms, including headache, disturbed consciousness, and paresthesias.8 Brain tissue edema, stroke, neuronal degeneration, and neuronal encephalitis have also been reported.2810 In a recent study, diffuse neural inflammatory markers were found in >80% of COVID-19 patient brains, processes which could contribute to the observed neurological symptoms.11 Furthermore, another pair of frequent symptoms of infection by SARS-CoV-2 are hyposmia and hypogeusia, the loss of the ability to smell and taste, respectively.3 Interestingly, hyposmia has been reported in early-stage Alzheimer’s disease (AD),3 and AD type II astrocytosis has been observed in neuropathology studies of COVID-19 patients.10

Systemic failure in COVID-19 patients is likely due to SARS-CoV-2 invasion via the ACE2 receptor,9 which is highly expressed in pericytes of human heart8 and epithelial cells of the respiratory tract,12 kidney, intestine, and blood vessels. ACE2 is also expressed in the brain, especially in the respiratory center and hypothalamus in the brain stem, the thermal center, and cortex,13 which renders these tissues more vulnerable to viral invasion, although it remains uncertain whether SARS-CoV-2 virus directly infects neurons in the brain.14 The primary consequences of SARS-CoV-2 infection are inflammatory responses and oxidative stress in multiple organs and tissues.1517 Recently it has been shown that the high neutrophil-to-lymphocyte ratio observed in critically ill patients with COVID-19 is associated with excessive levels of reactive oxygen species (ROS) and ROS-induced tissue damage, contributing to COVID-19 disease severity.15

Recent studies have reported an inverse relationship between ACE2 and transforming growth factor-β (TGF-β). In cancer models, decreased levels of ACE2 correlated with increased levels of TGF-β.18 In the context of SARS-CoV-2 infection, downregulation of ACE2 has been observed, leading to increased fibrosis formation, as well as upregulation of TGF-β and other inflammatory pathways.19 Moreover, patients with severe COVID-19 symptoms had higher blood serum TGF-β concentrations than those with mild symptoms,20 thus further implicating the role of TGF-β and warranting further investigation.

Interestingly, reduced angiotensin/ACE2 activity has been associated with tau hyperphosphorylation and increased amyloid beta (Aβ) pathology in animal models of AD.2122 The link between reduced ACE2 activity and increased TGF-β and tau signaling in the context of SARS-CoV-2 infection needs further exploration.

Our laboratory has shown that stress-induced ryanodine receptor (RyR)/intracellular calcium release channel post-translational modifications, including oxidation and protein kinase A (PKA) hyperphosphorylation related to activation of the sympathetic nervous system and the resulting hyper-adrenergic state, deplete the channel stabilizing protein (calstabin) from the channel complex, destabilizing the closed state of the channel and causing RyR channels to leak Ca2+ out of the endoplasmic/sarcoplasmic reticulum (ER/SR) in multiple diseases.2329 Increased TGF-β activity can lead to RyR modification and leaky channels,30 and SR Ca2+ leak can cause mitochondrial Ca2+ overload and dysfunction.29 Increased TGF-β activity31 and mitochondrial dysfunction32 are also associated with SARS-CoV-2 infection.

Here we show that SARS-CoV-2 infection is associated with adrenergic and oxidative stress and activation of the TGF-β signaling pathway in the brains of patients who have succumbed to COVID-19. One consequence of this hyper-adrenergic and oxidative state is the development of tau pathology normally associated with AD. In this article, we investigate potential biochemical pathways linked to tau hyperphosphorylation. Based on recent evidence that has linked tau pathology to Ca2+ dysregulation associated with leaky RyR channels in the brain,333 we investigated RyR2 biochemistry and function in COVID-19 patient brains.

RESEARCH-IN-CONTEXT

  1. Systematic review: The authors reviewed the literature using PubMed. While the mechanisms that lead to cognitive impairment associated with COVID-19 are not well understood, there have been recent reports studying SARS-CoV-2 infection and brain biochemistry and neuropathology. These relevant citations are appropriately cited.
  2. Interpretation: Our findings link the inflammatory response to SARS-CoV-2 infection with the neuropathological pathways causing tau hyperphosphorylation typically associated with Alzheimer’s disease (AD). Furthermore, our data indicate a role for leaky ryanodine receptor 2 (RyR2) in the pathophysiology of SARS-CoV-2 infection.
  3. Future directions: The article proposes that the alteration of cellular calcium dynamics due to leaky RyR2 in COVID-19 brains is associated with the activation of neuropathological pathways that are also found in the brains of AD patients. Both the cortex and cerebellum of SARS-CoV-2–infected patients exhibited a reduced expression of the Ca2+ buffering protein calbindin. Decreased calbindin could render these tissues more vulnerable to cytosolic Ca2+ overload. Ex vivo treatment of the COVID-19 brain using a Rycal drug (ARM210) that targets RyR2 channels prevented intracellular Ca2+ leak in patient samples. Future experiments will explore calcium channels as a potential therapeutic target for the neurological complications associated with COVID-19.

1.2 Study conclusions and disease implications

Our results indicate that SARS-CoV-2 infection activates inflammatory signaling and oxidative stress pathways resulting in hyperphosphorylation of tau, but normal amyloid precursor protein (APP) processing in COVID-19 patient cortex and cerebellum. There was reduced calbindin expression in both cortex and cerebellum rendering both tissues vulnerable to Ca2+-mediated pathology. Moreover, COVID-19 cortex and cerebellum exhibited RyR Ca2+ release channels with the biochemical signature of ‘‘leaky’’ channels and increased activity consistent with pathological intracellular Ca2+ leak. RyR2 were oxidized, associated with increased NADPH oxidase 2 (NOX2), and were PKA hyperphosphorylated on serine 2808, both of which cause loss of the stabilizing subunit calstabin2 from the channel complex promoting leaky RyR2 channels in COVID-19 patient brains. Furthermore, ex vivo treatment of COVID-19 patient brain samples with the Rycal drug ARM210, which is currently undergoing clinical testing at the National Institutes of Health for RyR1-myopathy (ClinicalTrials.gov Identifier: NCT04141670), fixed the channel leak. Thus, our experiments demonstrate that SARS-CoV-2 infection activates biochemical pathways linked to the tau pathology associated with AD and that leaky RyR Ca2+ channels may be a potential therapeutic target for the neurological complications associated with COVID-19.

The molecular basis of how SARS-CoV-2 infection results in ‘‘long COVID’’ is not well understood, and questions regarding the role of defective Ca2+ signaling in the brain in COVID-19 remain unanswered. A recent comprehensive molecular investigation revealed extensive inflammation and degeneration in the brains of patients that died from COVID-19,34 including in patients with no reported neurological symptoms. These authors also reported overlap between marker genes of AD and genes that are upregulated in COVID-19 infection, consistent with the findings of increased tau pathophysiology reported in the present study. We propose a potential mechanism that may contribute to the neurological complications caused by SARS-CoV-2: defective intracellular Ca2+ regulation and activation of AD-like neuropathology.

TGF-β belongs to a family of cytokines involved in the formation of cellular fibrosis by promoting epithelial-to-mesenchymal transition, fibroblast proliferation, and differentiation.35 TGF-β activation has been shown to induce fibrosis in the lungs and other organs by activation of the SMAD-dependent pathway. We have previously reported that TGF-β/SMAD3 activation leads to NOX2/4 translocation to the cytosol and its association with RyR channels, promoting oxidization of the channels and depletion of the stabilizing subunit calstabin in skeletal muscle and in heart.2830 Alteration of Ca2+ signaling may be particularly crucial in COVID-19-infected patients with cardiovascular/neurological diseases due, in part, to the multifactorial RyR2 remodeling after the cytokine storm, increased TGF-β activation, and increased oxidative stress. Moreover, SARS-CoV-2–infected patients exhibited a hyperadrenergic state. The elevated expression of glutamate carboxypeptidase 2 (GCPII) in COVID-19 brains reported in the present study could also contribute directly to increased PKA signaling of RyR2 by reducing PKA inhibition via metabotropic glutamate receptor 3 (mGluR3).36 Hyperphosphorylation of RyR2 channels can promote pathological remodeling of the channel and exacerbate defective Ca2+ regulation in these tissues. The increased Ca2+/cAMP/PKA signaling could also open nearby K+ channels which could potentially weaken synaptic connectivity, reduce neuronal firing,36 and could activate Ca2+ dependent enzymes.

Interestingly, both the cortex and cerebellum of SARS-CoV–2-infected patients exhibited a reduced expression of the Ca2+ buffering protein calbindin. Decreased calbindin could render these tissues more vulnerable to the cytosolic Ca2+ overload. This finding is in accordance with previous studies showing reduced calbindin expression levels in Purkinje cells and the CA2 hippocampal region of AD patients3739 and in cortical pyramidal cells of aged individuals with tau pathology.3340 In contrast to the findings in the brains of COVID-19 patients in the present study, calbindin was not reduced in the cerebellum of AD patients, possibly protecting these cells from AD pathology.3941

Leaky RyR channels, leading to increased mitochondrial Ca2+ overload and ROS production and oxidative stress, have been shown to contribute to the development of tau pathology associated with AD.3232933 Recent studies of the effects of COVID-19 on the central nervous system have found memory deficits and biological markers similar to those seen in AD patients.4243 Our data demonstrate increased activity of enzymes responsible for phosphorylating tau (pAMPK, pGSK3β), as well as increased phosphorylation at multiple sites on tau in COVID-19 patient brains. The tau phosphorylation observed in these samples exhibited some differences from what is typically observed in AD, occurring in younger patients and in areas of the brain, specifically the cerebellum, that usually do not demonstrate tau pathology in AD patients. Taken together, these data suggest a potential contributing mechanism to the development of tau pathology in COVID-19 patients involving oxidative overload-driven RyR2 channel dysfunction. Furthermore, we propose that these pathological changes could be a significant contributing factor to the neurological manifestations of COVID-19 and in particular the “brain fog” associated with long COVID, and represent a potential therapeutic target for ameliorating these symptoms. For example, tau pathology in the cerebellum could explain the recent finding that 74% of hospitalized COVID-19 patients experienced coordination deficits.44 The data presented also raise the possibility that prior COVID-19 infection could be a potential risk factor for developing AD in the future.

The present study was limited to the use of existing autopsy brain tissues at the Columbia University Biobank from SARS-CoV-2–infected patients. The number of subjects is small and information on their cognitive function as well as their brain histopathology and levels of Aβ in cerebrospinal fluid and plasma are lacking. Furthermore, we did not have access to a suitable animal model of SARS-CoV-2 infection in which to test whether the observed biochemical changes in COVID-19 brains and potential cognitive and behavioral deficits associated with the brain fog of long COVID could be reversed or attenuated by therapeutic interventions. The design of future studies should include larger numbers of subjects that are age- and sex-matched. The cognitive function of SARS-CoV-2–infected patients who presented cognitive symptoms should be assessed and regularly monitored. Moreover, it is important to know whether the observed neuropathological signaling is unique to SARS-CoV-2 infection or are common to all other viral infections. Previous studies have reported cognitive impairment in Middle East respiratory syndrome45 as well as Ebola4647 patients. Retrospective studies comparing the incidence and the magnitude of cognitive impairments caused by these different viral infections would improve our understanding of these neurological complications of viral infections.

2 CONSOLIDATED RESULTS AND STUDY DESIGN

There were increased markers of oxidative stress (glutathione disulfide [GSSG]/ glutathione [GSH]) in the cortex (mesial temporal lobe) and cerebellum (cerebellar cortex, lateral hemisphere) of COVID-19 tissue. Kynurenic acid, a marker of inflammation, was increased in COVID-19 cortex and cerebellum brain lysates compared to controls, is in accordance with recent studies showing a positive correlation between kynurenic acid and cytokines and chemokine levels in COVID-19 patients.4850

To determine whether SARS-CoV-2 infection also increases tissue TGF-β activity, we measured SMAD3 phosphorylation, a downstream signal of TGF-β, in control and COVID-19 tissue lysates. Phosphorylated SMAD3 (pSMAD3) levels were increased in COVID-19 cortex and cerebellum brain lysates compared to controls, indicating that SARS-CoV-2 infection increased TGF-β signaling in these tissues. Interestingly, brain tissues from COVID-19 patients exhibited activation of the TGF-β pathway, despite the absence of the detectable (by immunohistochemistry and polymerase chain reaction, data not shown) virus in these tissues. These results suggest that the TGF-β pathway is activated systemically by SARS-CoV-2, resulting in its upregulation in the brain, as well as other organs. In addition to oxidative stress, COVID-19 brain tissues also demonstrated increased PKA and calmodulin-dependent protein kinase II association domain (CaMKII) activity, most likely associated with increased adrenergic stimulation. Both PKA and CaMKII phosphorylation of tau have been reported in tauopathies.5152

The hallmarks of AD brain neuropathology are the formation of Aβ plaques from abnormal APP processing by BACE1, as well as tau ‘‘tangles’’ caused by tau hyperphosphorylation.53 Brain lysates from COVID-19 patients’ autopsies demonstrated normal BACE1 and APP levels compared to controls. The patients analyzed in the present study were grouped by age (young ≤ 58 years old, old ≥ 66 years old) to account for normal, age-dependent changes in APP and tau pathology. Abnormal APP processing was only observed in brain lysates from patients diagnosed with AD. However, AMPK and GSK3β phosphorylation were increased in both the cortex and cerebellum in COVID-19 brains. Activation of these kinases in SARS-CoV-2–infected brains leads to a hyperphosphorylation of tau consistent with AD tau pathology in the cortex. COVID-19 brain lysates from older patients showed increased tau phosphorylation at S199, S202, S214, S262, and S356. Lysates from younger COVID-19 patients showed increased tau phosphorylation at S214, S262, and S356, but not at S199 and S202, demonstrating increased tau phosphorylation in both young and old individuals and suggesting a tau pathology similar to AD in COVID-19–affected patients. Interestingly, both young and old patient brains demonstrated increased tau phosphorylation in the cerebellum, which is not typical of AD.

RyR channels may be oxidized due to the activation of the TGF-β signaling pathway.30 NOX2 binding to RyR2 causes oxidation of the channel, which activates the channel, manifested as an increased open probability that can be assayed using 3[H]ryanodine binding.54 When the oxidization of the channel is at pathological levels, there is destabilization of the closed state of the channel, resulting in spontaneous Ca2+ release or leak.2730 To determine the effect of the increased TGF-β signaling associated with SARS-CoV-2 infection on NOX2/RyR2 interaction, RyR2 and NOX2 were co-immunoprecipitated from brain lysates of COVID-19 patients and controls. NOX2 associated with RyR2 in brain tissues from SARS-CoV-2–infected individuals were increased compared to controls.

Given the increased oxidative stress and increased NOX2 binding to RyR2 seen in COVID-19 brains, RyR2 post-translational modifications were investigated. Immunoprecipitated RyR2 from brain lysates demonstrated increased oxidation, PKA phosphorylation on serine 2808, and depletion of the stabilizing protein subunit calstabin2 in SARS-CoV-2–infected tissues compared to controls. This biochemical remodeling of the channel is known as the ‘‘biochemical signature’’ of leaky RyR2235556 that is associated with destabilization of the closed state of the channel. This leads to SR/ER Ca2+ leak, which contributes to the pathophysiology of a number of diseases including AD.232426305557 RyR channel activity was determined by binding of 3[H]ryanodine, which binds only to the open state of the channel. RyR2 was immunoprecipitated from tissue lysates and ryanodine binding was measured at both 150 nM and 20 μM free Ca2+. RyR2 channels from SARS-CoV-2–infected brain tissue demonstrated abnormally high activity (increased ryanodine binding) compared to channels from control tissues at physiologically resting conditions (150 nM free Ca2+), when channels should be closed. Interestingly, cortex and cerebellum of SARS-CoV-2–infected patients also exhibited a reduced expression of the Ca2+ binding protein calbindin. Calbindin is typically not reduced in the cerebellum of AD patients, possibly providing some protection against AD pathology. The low calbindin levels in the cerebellum of COVID-19 brains could contribute to the observed tau pathology in this brain region. An additional atypical finding in the COVID-19 brains studied in this investigation is an increased level of GCPII. This could contribute to the observed RyR PKA phosphorylation by increasing cAMP and inhibiting the metabotropic glutamate receptor type 3.36

3 DETAILED METHODS AND RESULTS

3.1 Methods

3.1.1 Human samples

De-identified human heart, lung, and brain tissue were obtained from the COVID BioBank at Columbia University. The cortex samples were from the mesial temporal lobe and the cerebellum samples were from the cerebellar cortex, lateral hemisphere. The Columbia University BioBank functions under standard operating procedures, quality assurance, and quality control for sample collection and maintenance. Age- and sex-matched controls exhibited absence of neurological disorders and cardiovascular or pulmonary diseases. Sex, age, and pathology of patients are listed in Table 1.TABLE 1. Sex, age, and pathology of COVID-19 patients

Patient NumberSexAgePathology
1Male57Acute hypoxic-ischemic injury in the hippocampus, pons, and cerebellum.
2Female38Hypoxic ischemic encephalopathy, severe, global.
3Male58Hypoxic/ischemic injury, global, widespread astrogliosis/microgliosis.
4Male84Dementia. Beta-amyloid plaques are noted in cortex and cerebellum.
5Female80Severe hypoxic ischemic encephalopathy, severe. Global astrogliosis and microgliosis. Mild Alzheimer-type pathology.
6Female74Acute hypoxic-ischemic encephalopathy, global, moderate to severe. Arteriolosclerosis, mild. Metabolic gliosis, moderate
7Male66Left frontal subacute hemorrhagic infarct. Multifocal subacute infarcts in pons and left cerebral peduncle. Global astrogliosis and microgliosis (see microscopic description). Alzheimer’s pathology.
8Female76Hypoxic ischemic encephalopathy, moderate. Alzheimer’s pathology. Atherosclerosis, moderate. Arteriolosclerosis, moderate
9Male72Hypoxic/ischemic injury, acute to subacute, involving hippocampus, medulla and cerebellum. Mild atherosclerosis. Mild arteriolosclerosis
10Male71Hypoxic-ischemic encephalopathy, acute, global, mild to moderate. Diffuse Lewy body disease, neocortical type, consistent with Parkinson disease dementia. Atherosclerosis, severe. Arteriolosclerosis, mild.

Lysate preparation and Western blots

Tissues (50 mg) were isotonically lysed using a Dounce homogenizer in 0.25 ml of 10 mM Tris maleate (pH 7.0) buffer with protease inhibitors (Complete inhibitors from Roche). Samples were centrifuged at 8000 × g for 20 minutes and the protein concentrations of the supernatants were determined by Bradford assay. To determine protein levels in tissue lysates, tissue proteins (20 μg) were separated by 4% to 20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots were developed using the following antibodies: pSMAD3 (Abcam, 1:1000), SMAD3 (Abcam, 1:1000), AMPK (Abcam, 1:1000), tau (Thermo Fisher, 1:1000), pTauS199 (Thermo Fisher, 1:1000), pTauS202/T205 (Abcam, 1:1000), pTauS262 (Abcam, 1:1000), GSK3β (Abcam, 1:1000), pGSK3βS9 (Abcam, 1:1000), pGSK3βT216 (Abcam, 1:1000), APP (Abcam, 1:1000), BACE1 (Abcam, 1:1000), GAPDH (Santa Cruz Biotech, 1:1000), CTF-β (Santa Cruz Biotechnology, Inc., 1:1000), Calbindin (Abcam, 1:1000), and GCPII (Thermo Fisher, 1:4000).

Analyses of ryanodine receptor complex

Tissue lysates (0.1 mg) were treated with buffer or 10 μM Rycal (ARM210) at 4°C. RyR2 was immunoprecipitated from 0.1 mg lung, heart, and brain using an anti-RyR2 specific antibody (2 μg) in 0.5 ml of a modified radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.2, 0.9% NaCl, 5.0 mm NaF, 1.0 mm Na3VO4, 1% Triton X-100, and protease inhibitors; RIPA) overnight at 4°C. RyR2-specific antibody was an affinity-purified polyclonal rabbit antibody using the peptide CKPEFNNHKDYAQEK corresponding to amino acids 1367–1380 of mouse RyR2 with a cysteine residue added to the amino terminus. The immune complexes were incubated with protein A-Sepharose beads (Sigma) at 4°C for 1 hour, and the beads were washed three times with RIPA. The immunoprecipitates were size-fractionated on SDS-PAGE gels (4%–20% for RyR2, calstabin2, and NOX2) and transferred onto nitrocellulose membranes for 1 hour at 200 mA. Immunoblots were developed using the following primary antibodies: anti-RyR2 (Affinity BioReagents, 1:2500), anti-phospho-RyR-Ser(pS)-2808 (Affinity BioReagents 1:1000), anti- calstabin2 (FKBP12 C-19, Santa Cruz Biotechnology, Inc., 1:2500), and anti-NOX2 (Abcam, 1:1000). To determine channel oxidation, the carbonyl groups in the protein side chains were derivatized to DNP by reaction with 2,4-dinitrophenylhydrazine. The DNP signal associated with RyR2 was determined using a specific anti-DNP antibody according to the manufacturer using an Odyssey system (LI-COR Biosciences) with infrared-labeled anti-mouse and anti-rabbit immunoglobulin G (IgG; 1:5000) secondary antibodies.

Ryanodine binding

RyR2 was immunoprecipitated from 1.5 mg of tissue lysate using an anti-RyR2 specific antibody (25 μg) in 1.0 ml of a modified RIPA buffer overnight at 4°C. The immune complexes were incubated with protein A-Sepharose beads (Sigma) at 4°C for 1 hour, and the beads were washed three times with RIPA buffer, followed by two washes with ryanodine binding buffer (10 mM Tris-HCl, pH 6.8, 1 M NaCl, 1% CHAPS, 5 mg/ml phosphatidylcholine, and protease inhibitors). Immunoprecipitates were incubated in 0.2 ml of binding buffer containing 20 nM [3H] ryanodine and either of 150 nM and 20 μm free Ca2+ for 1 hour at 37°C. Samples were diluted with 1 ml of ice-cold washing buffer (25 mm Hepes, pH 7.1, 0.25 m KCl) and filtered through Whatman GF/B membrane filters pre-soaked with 1% polyethyleneimine in washing buffer. Filters were washed three times with 5 ml of washing buffer. The radioactivity remaining on the filters is determined by liquid scintillation counting to obtain bound [3H] ryanodine. Nonspecific binding was determined in the presence of 1000-fold excess of non-labeled ryanodine.

GSSG/GSH ratio measurement and SMAD3 phosphorylation

Approximately 20 mg of tissue suspended in 200 μL of ice-cold phosphate-buffered saline/0.5% NP-40, pH6.0 was used for lysis. Tissue was homogenized with a Dounce homogenizer with 10 to 15 passes. Samples were centrifuged at 8000 × g for 15 minutes at 4°C to remove any insoluble material. Supernatant was transferred to a clean tube. Deproteinizing of the samples was accomplished by adding 1 volume ice-cold 100% (w/v) trichloroacetic acid (TCA) into five volumes of sample and vortexing briefly to mix well. After incubating for 5 minutes on ice, samples were centrifuged at 12,000 × g for 5 minutes at 4°C and the supernatant was transferred to a fresh tube. The samples were neutralized by adding NaHCO3 to the supernatant and vortexing briefly. Samples were centrifuged at 13,000 × g for 15 minutes at 4°C and supernatant was collected. Samples were then deproteinized, neutralized, TCA was removed, and they were ready to use in the assay. The GSSG/GSH was determined using a ratio detection assay kit (Abcam, ab138881). Briefly, in two separate assay reactions, GSH (reduced) was measured directly with a GSH standard and Total GSH (GSH + GSSG) was measured by using a GSSG standard. A 96-well plate was set up with 50 μL duplicate samples and standards with known concentrations of GSH and GSSG. A Thiol green indicator was added, and the plate was incubated for 60 minutes at room temperature (RT). Fluorescence at Ex/Em = 490/520 nm was measured with a fluorescence microplate reader and the GSSG/GSH for samples were determined comparing fluorescence signal of samples with known standards.

Kynurenic acid assay

Kynurenic acid (KYNA) concentration in brain lysates was determined using an enzyme-linked immunosorbent assay (ELISA) kit for KYNA (ImmuSmol). Briefly, samples (50 μl) were added to a microtiter plate designed to extract the KCNA from the samples. An acylation reagent was added for 90 minutes at 37°C to derivatize the samples. After derivatization, 50 μl of the prepared standards and 100 μl samples were pipetted into the appropriate wells of the KYNA microtiter plate. KYNA Antiserum was added to all wells and the plate was incubated overnight at 4°C. After washing the plate four times, the enzyme conjugate was added to each well. The plate was incubated for 30 minutes at RT on a shaker at 500 rpm. The enzyme substrate was added to all wells and the plate was incubated for 20 minutes at RT. Stop solution was added to each well. A plate reader was used to determine the absorbance at 450 nm. The sample signals were compared to a standard curve.

PKA activity assay

PKA activity in brain lysates was determined using a PKA activity kit (Thermo Fisher, EIAPKA). Briefly, samples were added to a microtiter plate containing an immobilized PKA substrate that is phosphorylated by PKA in the presence of ATP. After incubating the samples with ATP at RT for 2 hours, the plate was incubated with the phospho-PKA substrate antibody for 60 minutes. After washing the plate with wash buffer, goat anti-rabbit IgG horseradish peroxidase (HRP) conjugate was added to each well. The plate was aspirated, washed, and TMB substrate was added to each well, which was then incubated for 30 minutes at RT. A plate reader was used to determine the absorbance at 450 nm. The sample signals were compared to a standard curve.

CaMKII activity assay

CaMKII activity in brain lysates was determined using the CycLex CaM kinase II Assay Kit (MBL International). Briefly, samples were added to a microtiter plate containing an immobilized CaMKII substrate that is phosphorylated by CaMKII in the presence of Mg2+ and ATP. After incubating the samples in kinase buffer containing Mg2+ and ATP at RT for 1 hour, the plate was washed and incubated with the HRP conjugated anti-phospho-CaMKII substrate antibody for 60 minutes. The plate was aspirated, washed, and TMB substrate was added to each well, which was then incubated for 30 minutes at RT. A plate reader was used to determine the absorbance at 450 nm. The sample signals were compared to a standard curve.

Statistics

Group data are presented as mean ± standard deviation. Statistical comparisons between the two groups were determined using an unpaired t-test. Values of P < .05 were considered statistically significant. All statistical analyses were performed with GraphPad Prism 8.0.

3.2 Results

3.2.1 Oxidative stress and TGF-β, PKA, and CaMKII activation

Oxidative stress levels were determined in brain tissues (cortex, cerebellum) from COVID-19 patient autopsy tissues and controls by measuring the ratio of GSSG to GSH by an ELISA kit. COVID-19 patients exhibited significant oxidative stress with a 3.8- and 3.2-fold increase in GSSG/GSH ratios in cortex (Ctx) and cerebellum (CB) compared to controls, respectively (Figure 1A). High circulating levels of kynurenine have been reported in COVID-19.4850 However, the expression of KYNA in COVID-19 brain tissue has not been examined. Levels in the Ctx and CB were measured using an ELISA kit. COVID-19 brains had a significant increase in the Ctx and CB compared to controls (Figure 1A). An additional marker of tissue inflammation is increased cytokine expression. SMAD3 phosphorylation, a downstream signal of TGF-β, was increased in COVID-19 Ctx and CB tissue lysates compared to controls (Figure 1B and 1C). Increased adrenergic activation in the brain of patients infected with SARS-CoV-2 was also demonstrated by measuring PKA activity in the Ctx and CB and CaMKII activity was increased as well (Figure 1D).

Details are in the caption following the image
FIGURE 1Open in figure viewerIncreased oxidative stress, inflammatory and adrenergic signaling in brains of COVID-19 patients. A, Bar graph depicting the glutathione disulfide (GSSG)/ glutathione (GSH) ratio and kynurenic acid (KYNA) enzyme-linked immunsorbent assay signal from control (n = 6) and COVID-19 (n = 6) tissue lysates. CB, cerebellum; Ctx, cortex. Data are mean ± standard deviation (SD). *P < .05 control versus COVID-19. B, Western blots showing phospho-SMAD3 and total SMAD3 from control (n = 4) and COVID-19 (n = 7) brain lysates. C, Bar graphs depicting quantification of pSMAD3/SMAD3 from Western blot signals in B. D, Calmodulin-dependent protein kinase II association domain (CaMKII) and protein kinase A (PKA) activity of brain tissue lysates. Data are mean ± SD. *P < .05 control versus COVID-19

Activation of AD-linked signaling

Both PKA and CaMKII have been directly implicated in the increased phosphorylation of tau associated with AD.5152 Because COVID-19 brain lysates had increased PKA and CaMKII activity, AD-linked biochemistry was evaluated in the COVID-19 brain lysates. Normal APP processing was observed in COVID-19 brain lysates as demonstrated by normal BACE1 and APP levels compared to controls (Figure 2A and B). Abnormal APP processing was only observed in brain lysates from patients diagnosed with AD (see Table 1 for patient details). However, phosphorylation/activation of AMPK and GSK3β was observed in SARS-CoV-2–infected patient brain lysates. Activation of these kinases along with the activation of PKA and CaMKII (Figure 1) leads to a hyperphosphorylation of tau at multiple residues (Figure 2C and D). Tau hyperphosphorylation in the cerebellum is not typical of AD pathology. The CB tau pathology demonstrated in COVID-19 warrants further investigation.

Details are in the caption following the image
FIGURE 2Open in figure viewerHyperphosphorylation of tau but normal amyloid precursor protein (APP) processing in COVID-19 brains. A, Brain (CB, cerebellum; Ctx, cortex) lysates were separated by 4% to 20% polyacrylamide gel electrophoresis. Immunoblots were developed for pAMPK, AMPK, GSK3β, pGSK3β (T216), APP, BACE1, and GAPDH loading control. The numbers (1–10) above immunoblots refer to patient numbers listed in Table 1. B, Bar graphs showing quantification of pAMPK, pGSK3β, APP/GAPDH, and BACE1/GAPDH from Western blots in (A). Data are mean ± standard deviation (SD). *P < .05 control versus COVID-19; **P < .05 CB versus Ctx; #P < .05 COVID (Young) versus COVID (Old). C, Immunoblots of brain lysates showing total tau and tau phosphorylation on residues S199, S202/T205, S214, S262, and S356. D, Bar graphs showing quantification phosphorylated tau at the residues shown on Western blots in (C). Data are mean ± SD. *P < .05 control versus COVID-19; **P < .05 CB versus Ctx; #P < .05 COVID (Young) versus COVID (Old)

RyR2 channel oxidation and leak

RyR2 biochemistry was investigated to determine whether RyR2 in COVID-19 brain tissues demonstrated a “leaky” phenotype. Increased NOX2/RyR2 binding was shown in Ctx and CB lysates from SARS-CoV-2–infected individuals compared to controls using co-immunoprecipitation (Figure 3A and B). In addition, RyR2 from SARS-CoV-2–infected brains had increased oxidation, increased serine 2808 PKA phosphorylation, and depletion of the stabilizing protein subunit calstabin2 compared to controls (Figure 3A and B). RyR channels exhibiting these characteristics can be inappropriately activated at low cytosolic Ca2+ concentrations resulting in a pathological ER/SR Ca2+ leak. 3[H]Ryanodine binding to immunoprecipitated RyR2 was measured at both 150 nM and 20 μM free Ca2+. Because ryanodine binds only to the open state of the channel under these conditions, 3[H]Ryanodine binding may be used as a surrogate measure of channel open probability. The total amount of RyR immunoprecipitated was the same for control and COVID-19 samples (data not shown). Increased RyR2 channel activity at resting conditions (150 nM free Ca2+) was observed in COVID-19 channels compared to controls (Figure 3C). Under these conditions, RyR channels should be closed. Rebinding of calstabin2 to RyR2, using a Rycal, has been shown to reduce SR/ER Ca2+ leak, despite the persistence of the channel remodeling. Indeed, calstabin2 binding to RyR2 was increased when COVID-19 patient brain tissue lysates were treated ex vivo with the Rycal drug ARM210 (Figure 3A and B). Abnormal RyR2 activity observed at resting Ca2+ concentration was also decreased by Rycal treatment (Figure 3C).

Details are in the caption following the image
FIGURE 3Open in figure viewerDysregulation of calcium-handling proteins in COVID-19 brains. A, Western blots depicting ryanodine receptor 2 (RyR2) oxidation, protein kinase A (PKA) phosphorylation, and calstabin2 or NADPH oxidase 2 (NOX2) bound to the channel from brain (CB, cerebellum; Ctx, cortex) lysates. B, Bar graphs quantifying DNP/RyR2, pS2808/RyR2, and calstabin2 and NOX2 bound to the channel from the Western blots. Data are mean ± standard deviation (SD). *P < .05 control versus COVID-19; # P < .05 COVID-19 versus COVID-19+ARM210. C, 3[H]ryanodine binding from immunoprecipitated RyR2. Bar graphs show ryanodine binding at 150 nM Ca2+ as a percent of maximum binding (Ca2+ = 20 μM). Data are mean ± SD. *P < .05 control versus COVID-19; #P < .05 COVID-19 versus COVID-19+ARM210. D, Western blots showing the levels of glutamate carboxypeptidase 2 (GCPII), calbindin, and GAPDH loading control in brain (Ctx, CB). E, Bar graphs quantifying GCPII/GAPDH and calbindin/GAPDH from the western blots. Data are mean ± SD. *P < .05 control versus COVID-19

An interesting finding concerning the tau phosphorylation in brain lysates from SARS-CoV-2 patients was the increase of phosphorylation at multiple sites in the cerebellum. This is atypical of AD. One potential mechanism to explain this finding is the significantly decreased levels of calbindin expressed in COVID-19 cerebellum (Figure 3D3E). The decreased cerebellar calbindin levels could make this area of the brain more susceptible to Ca2+-induced activation of enzymes upstream of tau phosphorylation. Moreover, increased GCPII expression was observed in COVID-19 cortex and cerebellar lysates (Figure 3D3E), which would reduce mGluR3 inhibition of PKA signaling and could contribute to the PKA hyperphosphorylation of RyR2.

Model for the role for leaky RyR2 in the pathophysiology of SARS-CoV-2 infection

Our data indicate a role for leaky RyR2 in the pathophysiology of SARS-CoV-2 infection (Figure 4). In addition to the brain of COVID-19 patients, we observed increased systemic oxidative stress and activation of the TGF-β signaling pathway in lung, and heart, which correlates with oxidation-driven biochemical remodeling of RyR2 (Figure 3 and S1 in supporting inormation). This RyR2 remodeling results in intracellular Ca2+ leak, which can play a role in heart failure progression, pulmonary insufficiency, as well as cognitive dysfunction.232628 The alteration of cellular Ca2+ dynamics has also been implicated in COVID-19 pathology.5859 Taken together, the present data suggest that leaky RyR2 may play a role in the long-term sequelae of COVID-19, including the “brain fog” associated with SARS-CoV-2 infection which could be a forme fruste of AD,60 and could predispose long COVID patients to developing AD later in life. Leaky RyR2 channels may be a therapeutic target for amelioration of some of the persistent cognitive deficits associated with long COVID.

Details are in the caption following the image
FIGURE 4Open in figure viewerSARS-CoV-2 infection results in leaky ryanodine receptor 2 (RyR2) that may contribute to cardiac, pulmonary, and cognitive dysfunction. SARS-CoV-2 infection targets cells via the angiotensin-converting enzyme 2 (ACE2) receptor, inducing inflammasome stress response/activation of stress signaling pathways. This results in increased transforming growth factor-β (TGF-β) signaling, which activates SMAD3 (pSMAD) and increases NADPH oxidase 2 (NOX2) expression and the amount of NOX2 associated with RyR2. Increased NOX2 activity at RyR2 oxidizes the channel, causing calstabin2 depletion from the channel macromolecular complex, destabilization of the closed state, and ER/SR calcium leak that is known to contribute to cardiac dysfunction,55 arrhythmias,61 pulmonary insufficiency,2325 and cognitive and behavioral abnormalities associated with neurodegenreation.2426 Decreased calbindin in COVID-19 may render brain more susceptible to tau pathology. Rycal drugs fix the RyR2 channel leak by restoring calstabin2 binding and stabilizing the channel closed state. Fixing leaky RyR2 may improve cardiac, pulmonary, and cognitive function in COVID-19.

Acute Mesenteric Ischemia in COVID-19 Patients

Authors: Dragos Serban 1,2,*† , Laura Carina Tribus 3,4,†, Geta Vancea 1,5,† , Anca Pantea Stoian, Ana Maria Dascalu 1,* Andra Iulia Suceveanu 6Ciprian Tanasescu 7,8, Andreea Cristina Costea 9 Mihail Silviu Tudosie 1, Corneliu Tudor 2, Gabriel Andrei Gangura 1,10, Lucian Duta 2 and Daniel Ovidiu Costea 6,11,

Abstract:

Acute mesenteric ischemia is a rare but extremely severe complication of SARS-CoV-2 infection. The present review aims to document the clinical, laboratory, and imaging findings, management, and outcomes of acute intestinal ischemia in COVID-19 patients. A comprehensive search was performed on PubMed and Web of Science with the terms “COVID-19” and “bowel ischemia” OR “intestinal ischemia” OR “mesenteric ischemia” OR “mesenteric thrombosis”. After duplication removal, a total of 36 articles were included, reporting data on a total of 89 patients, 63 being hospitalized at the moment of onset. Elevated D-dimers, leukocytosis, and C reactive protein (CRP) were present in most reported cases, and a contrast-enhanced CT exam confirms the vascular thromboembolism and offers important information about the bowel viability. There are distinct features of bowel ischemia in non-hospitalized vs. hospitalized COVID-19 patients, suggesting different pathological pathways. In ICU patients, the most frequently affected was the large bowel alone (56%) or in association with the small bowel (24%), with microvascular thrombosis. Surgery was necessary in 95.4% of cases. In the non-hospitalized group, the small bowel was involved in 80%, with splanchnic veins or arteries thromboembolism, and a favorable response to conservative anticoagulant therapy was reported in 38.4%. Mortality was 54.4% in the hospitalized group and 21.7% in the non-hospitalized group (p < 0.0001). Age over 60 years (p = 0.043) and the need for surgery (p = 0.019) were associated with the worst outcome. Understanding the mechanisms involved and risk factors may help adjust the thromboprophylaxis and fluid management in COVID-19 patients.

1. Introduction Acute mesenteric ischemia (AMI) is a major abdominal emergency, characterized by a sudden decrease in the blood flow to the small bowel, resulting in ischemic lesions of the intestinal loops, necrosis, and if left untreated, death by peritonitis and septic shock. In nonCOVID patients, the etiology may be mesenteric arterial embolism (in 50%), mesenteric arterial thrombosis (15–25%), venous thrombosis (5–15%), or less frequent, from nonocclusive causes associated with low blood flow [1]. Several systemic conditions, such as arterial hypertension, atrial fibrillation, atherosclerosis, heart failure, or valve disease are risk factors for AMI. Portal vein thrombosis and mesenteric vein thrombosis can be seen with celiac disease [2], appendicitis [3], pancreatitis [4], and, in particular, liver cirrhosis and hepatocellular cancer [5]. Acute intestinal ischemia is a rare manifestation during COVID-19 disease, but a correct estimation of its incidence is challenging due to sporadic reports, differences in patients’ selection among previously published studies, and also limitations in diagnosis related to the strict COVID-19 regulations for disease control and difficulties in performing imagistic investigations in the patients in intensive care units. COVID-19 is known to cause significant alteration of coagulation, causing thromboembolic acute events, of which the most documented were pulmonary embolism, acute myocardial infarction, and lower limb ischemia [6]. Gastrointestinal features in COVID-19 disease are relatively frequently reported, varying from less than 10% in early studies from China [7,8] to 30–60%, in other reports [9,10]. In an extensive study on 1992 hospitalized patients for COVID-19 pneumonia from 36 centers, Elmunzer et al. [7] found that the most frequent clinical signs reported were mild and self-limited in up to 74% of cases, consisting of diarrhea (34%), nausea (27%), vomiting (16%), and abdominal pain (11%). However, severe cases were also reported, requiring emergency surgery for acute bowel ischemia or perforation [5,8]. The pathophysiology of the digestive features in COVID-19 patients involves both ischemic and non-ischemic mechanisms. ACE2 receptors are present at the level of the intestinal wall, and enterocytes may be directly infected by SARS-CoV-2. The virus was evidenced in feces and enteral walls in infected subjects [4,11–13]. In a study by Xu et al., rectal swabs were positive in 8 of 10 pediatric patients, even after the nasopharyngeal swabs became negative [14]. However, the significance of fecal elimination of viral ARN is still not fully understood in the transmission chain of the SARS-CoV-2 infection. On the other hand, disturbance of lung-gut axis, prolonged hospitalization in ICU, and the pro coagulation state induced by SARS-CoV-2 endothelial damage was incriminated for bowel ischemia, resulting in intestinal necrosis and perforation [8,9,15]. Early recognition and treatment of gastrointestinal ischemia are extremely important, but it is often challenging in hospitalized COVID-19 patients with severe illness. The present review aims to document the risk factors, clinical, imagistic, and laboratory findings, management, and outcomes of acute intestinal ischemic complications in COVID-19 patients. 2. Materials and Methods A comprehensive search was performed on PubMed and Web of Science with the terms “COVID-19” AND (“bowel ischemia” OR “intestinal ischemia” OR “mesenteric ischemia” OR “mesenteric thrombosis”). All original papers and case reports, in the English language, for which full text could be obtained, published until November 2021, were included in the review. Meeting abstracts, commentaries, and book chapters were excluded. A hand search was performed in the references of the relevant reviews on the topic. 2.1. Data Extraction and Analysis The review is not registered in PROSPERO. A PRISMA flowchart was employed to screen papers for eligibility (Figure 1) and a PRISMA checklist is presented as a Supple- J. Clin. Med. 2022, 11, 200 3 of 22 mentary File S1. A data extraction sheet was independently completed by two researchers, with strict adherence to PRISMA guidelines. J. Clin. Med. 2022, 11, 200 3 2.1. Data Extraction and Analysis The review is not registered in PROSPERO. A PRISMA flowchart was employedscreen papers for eligibility (Figure 1) and a PRISMA checklist is presented as a Supmentary File S1. A data extraction sheet was independently completed by two researchwith strict adherence to PRISMA guidelines. Figure 1. PRISMA 2020 flowchart for the studies included in the review. The relevant data abstracted from these studies are presented in Tables 1–3. COV19 diagnosis was made by PCR assay in all cases. All patients reported with COVIDdisease and mesenteric ischemia were documented in terms of age, sex, comorbidittime from SARS-CoV-2 infection diagnosis, presentation, investigations, treatment, outcome. A statistical analysis of the differences between acute intestinal ischemia in pviously non-hospitalized vs. previously hospitalized patients was performed. The pottial risk factors for an adverse vital prognosis were analyzed using SciStat® softw(www.scistat.com (accessed on 25 November 2021)). Papers that did not provide sufficient data regarding evaluation at admission, domentation of SARS-CoV-2 infection, or treatment were excluded. Patients suffering frother conditions that could potentially complicate intestinal ischemia, such as liver cirrsis, hepatocellular carcinoma, intraabdominal infection (appendicitis, diverticulitis), pcreatitis, and celiac disease were excluded. Any disagreement was solved by discussioFigure 1. PRISMA 2020 flowchart for the studies included in the review. The relevant data abstracted from these studies are presented in Tables 1–3. COVID-19 diagnosis was made by PCR assay in all cases. All patients reported with COVID-19 disease and mesenteric ischemia were documented in terms of age, sex, comorbidities, time from SARS-CoV-2 infection diagnosis, presentation, investigations, treatment, and outcome. A statistical analysis of the differences between acute intestinal ischemia in previously nonhospitalized vs. previously hospitalized patients was performed. The potential risk factors for an adverse vital prognosis were analyzed using SciStat® software (www.scistat.com (accessed on 25 November 2021)). Papers that did not provide sufficient data regarding evaluation at admission, documentation of SARS-CoV-2 infection, or treatment were excluded. Patients suffering from other conditions that could potentially complicate intestinal ischemia, such as liver cirrhosis, hepatocellular carcinoma, intraabdominal infection (appendicitis, diverticulitis), pancreatitis, and celiac disease were excluded. Any disagreement was solved by discussion. J. Clin. Med. 2022, 11, 200 4 of 22 Table 1. Patients with intestinal ischemia in retrospective studies on hospitalized COVID-19 patients. Study No of Patients with Gastrointestinal Ischemia (Total No of COVID-19 Patients in ICU) Sex (M; F) Age (Mean) BMI Time from Admission to Onset (Days) Abdominal CT Signs Intraoperative/Endoscopic Findings Treatment Outcomes Kaafarani HMA [16] 5 (141); 3.8% 1;3 62.5 32.1 51.5 (18–104) days NA Cecum-1—patchy necrosis Cecum_ileon-1 Small bowel-3; yellow discoloration on the antimesenteric side of the small bowel; 1 case + liver necrosis Surgical resection NA Kraft M [17] 4 (190); 2.1% NA NA NA NA NA Bowel ischemia + perforation (2) Bowel ischemia + perforation (1) MAT+massive bowel ischemia (1) Right hemicolectomy (2) Transverse colectomy (1) Conservative, not fit for surgery Recovery (3) Death (1) Yang C [18] 20 (190 in ICU; 582 in total); 10.5% 15:5 69 31.2 26.5 (17–42) Distension Wall thickness Pneumatosis intestinalis Perforation SMA or celiac thrombosis no info Right hemicolectomy 7(35%) Sub/total colectomy12 (60%) Ileocecal resection 1(5%) Recovery (11) Death (9) Hwabejire J [19] 20 13:7 58.7 32.5 13 (1–31) Pneumatosis intestinalis 42% Portal venous gas (33%) Mesenteric vessel patency 92% large bowel ischemia (8) small bowel ischemia (4) both (8) yellow discoloration of the ischemic bowel resection of the ischemic segment abdomen left open + second look (14) Recovery (10) Death (10) O’Shea A [20] 4 (142); 2.8% NA NA NA NA bowel ischemia, portal vein gas, colic pneumatosis NA NA NA Qayed E [21] 2 (878); 0.22% NA NA NA NA NA diffuse colonic ischemia (1) Small + large bowel ischemia and pneumatosis (1) Total colectomy (1) Extensive resection (1) Recovery (1) Death (1) NA: not acknowledged; MAT: mesenteric artery thrombosis; SMA: superior mesenteric artery. J. Clin. Med. 2022, 11, 200 5 of 22 Table 2. Case reports and case series presenting gastrointestinal ischemia in hospitalized COVID-19 patients under anticoagulant medication. Article Sex Age Comorbidities Time from COVID-19 Diagnosis; Time from Admission (Days) ICU; Type of Ventilation Clinical Signs at Presentation Leukocytes (/mm3 ) CRP (mg/L) Lactat mmol/L Ferritin (ng/mL) LDH (U/L) Thrombocytes (/mm3) D-Dimers (ng/mL) Abdominal CT Signs Treatment Outcome Azouz E [22] M 56 none 1; 2 (hospitalized for acute ischemic stroke) No info abdominal pain and vomiting No info – – – – – – Multiple arterial thromboembolic complications: AMS, right middle cerebral artery, a free-floating clot in the aortic arch Anticoagulation (no details), endovascular thrombectomy Laparotomy + resection of necrotic small bowel loops No info Al Mahruqi G [23] M 51 none 26; 24 yes, intubated Fever, metabolic acidosis, required inotropes 30,000 – 7 687 – – 2.5 Non-occlusive AMI Hypoperfused small bowel, permeable aorta, SMA, IMA + deep lower limb thrombosis enoxaparin 40 mg/day from admission; surgery refused by family death Ucpinar BA [24] F 82 Atrial fibrillation, hypertension, chronic kidney disease 3; 3 no – 14,800 196 5.1 – – – 1600 SMA thrombosis; distended small bowel, with diffuse submucosal pneumatosis portomesenteric gas fluid resuscitation; continued ceftriaxone, enoxaparin 0.4cc twice daily; not operable due to fulminant evolution Death Karna ST [25] F 61 DM, hypertension 4; 4 Yes, HFNO diffuse abdominal pain with distention 21,400 421.6 1.4 – – 464,000 No thrombosis of the distal SMA with dilated jejunoileal loops and normal enhancing bowel wall. Iv heparin 5000 ui, followed by 1000 ui, Ecospin and clopidogrel Laparotomy after 10 days with segmental enterectomy of the necrotic bowel Death by septic shock and acute renal failure Singh B [26] F 82 Hypertension, T2DM 32; 18 Yes, Ventilator support severe diffuse abdominal distension and tenderness 22,800 308 2.5 136 333 146,000 1.3 SMA—colic arteries thrombosis pneumatosis intestinalis affecting the ascending colon and cecum laparotomy, ischemic colon resection, ileostomy; heparin in therapeutic doses preand post-surgery slow recovery J. Clin. Med. 2022, 11, 200 6 of 22 Table 2. Cont. Article Sex Age Comorbidities Time from COVID-19 Diagnosis; Time from Admission (Days) ICU; Type of Ventilation Clinical Signs at Presentation Leukocytes (/mm3 ) CRP (mg/L) Lactat mmol/L Ferritin (ng/mL) LDH (U/L) Thrombocytes (/mm3) D-Dimers (ng/mL) Abdominal CT Signs Treatment Outcome Nakatsutmi K [27] F 67 DM, diabetic nephropathy requiring dialysis, angina, postresection gastric cancer 16; 12 ICU, intubation hemodynamic deterioration, abdominal distension 15,100 32.14 – – – – 26.51 edematous transverse colon; abdominal vessels with sclerotic changes laparotomy, which revealed vascular micro thrombosis of transverse colon—right segment resection of the ischemic colonic segment, ABTHERA management, second look, and closure of the abdomen after 24 h death Dinoto E [28] F 84 DM, hypertension, renal failure 2; 2 no Acute abdominal pain and distension; 18,000 32.47 – – 431 – 6937 SMA origin stenosis and occlusion at 2 cm from the origin, absence of bowel enhancement Endovascular thrombectomy of SMA; surgical transfemoral thrombectomy and distal superficial femoral artery stenting Death due to respiratory failure Kiwango F [29] F 60 DM, hypertension 12; 3 no Sudden onset abdominal pain 7700 – – – – – 23.8 Not performed Not performed due to rapid oxygen desaturation Massive bowel acute ischemia death J. Clin. Med. 2022, 11, 200 7 of 22 Table 3. Case reports and case series presenting gastrointestinal ischemia in non-hospitalized COVID-19 patients. Article Sex Age Comorbidities Time from COVID-19 Diagnosis (Days) Clinical Signs at Presentation Leukocyte Count (/mm3 ) CRP (mg/L) Lactate mmol/L Ferritin (ng/mL) LDH (U/L) Thrombocytes (/mm3 ) D-Dimers (ng/mL) Abdominal CT Signs Treatment Outcome Sevella, P [30] M 44 none 10 Acute abdominal pain constipation, vomiting 23,400 – – – 1097 360,000 1590 Viable jejunum, ischemic bowel, peritoneal thickening with fat stranding; free fluid in the peritoneal cavity LMWH 60 mg daily Piperacillin 4g/day Tazobactam 500 mg/day Extensive small bowel + right colon resection death Nasseh S [31] M 68 no info First diagnosis epigastric pain and diarrhea for 4 days 17,660 125 – – – – 6876 terminal segment of the ileocolic artery thrombosis; thickening of the right colon wall and the last 30 cm of the small bowl unfractionated heparin laparoscopy -no bowel resection needed recovery Aleman W [32] M 44 none 20 severe abdominopelvic pain 36,870 – – 456.23 – 574,000 263.87 absence of flow at SMV, splenic, portal vein; Small bowel loop dilatation and mesenteric fat edema enoxaparin and pain control medication 6 days, then switched to warfarin 6 months recovery Jeilani M [33] M 68 Alzheimer disease, COPD 9 Sharp abdominal pain +distension 12,440 307 – – – 318,000 897 a central venous filling defect within the portal vein extending to SMV; no bowel wall changes LMWH, 3 months recovery Randhawa J [34] F 62 none First diagnosis right upper quadrant pain and loss of appetite for 14 days Normal limits – – – 346 – – large thrombus involving the SMV, the main portal vein with extension into its branches Fondaparinux 2.5. mg 5 days, then warfarin 4 mg (adjusted by INR), 6 months recovery Cheung S [35] M 55 none 12 (discharged for 7 days) Nausea, vomiting and worsening generalized abdominal pain with guarding 12,446 – 0.68 – – – – low-density clot, 1.6 cm in length, causing high-grade narrowing of the proximal SMA continuous heparin infusion continued 8 h postoperative, Laparotomy with SMA thromboembolectomy and enterectomy (small bowel) recovery J. Clin. Med. 2022, 11, 200 8 of 22 Table 3. Cont. Article Sex Age Comorbidities Time from COVID-19 Diagnosis (Days) Clinical Signs at Presentation Leukocyte Count (/mm3 ) CRP (mg/L) Lactate mmol/L Ferritin (ng/mL) LDH (U/L) Thrombocytes (/mm3 ) D-Dimers (ng/mL) Abdominal CT Signs Treatment Outcome Beccara L [36] M 52 none 22 (5 days after discharge and cessation prophylactic LWMH) vomiting and abdominal pain, tenderness in epigastrium and mesogastrium 30,000 222 – – – – – arterial thrombosis of vessels efferent of the SMA with bowel distension Enterectomy (small bowel) LMWH plus aspirin 100 mg/day at discharge recovery Vulliamy P [37] M 75 none 14 abdominal pain and vomiting for 2 days 18,100 3.2 – – – 497,000 320 intraluminal thrombus was present in the descending thoracic aorta with embolic occlusion of SMA Catheter-directed thrombolysis, enterectomy (small bowel) recovery De Barry O [38] F 79 none First diagnosis Epigastric pain, diarrhea, fever for 8 days, acute dyspnea 12600 125 5.36 – – – – SMV, portal vein, SMA, and jejunal artery thrombosis Distended loops, free fluid anticoagulation Resection of affected colon+ ileum, SMA thrombolysis, thrombectomy death Romero MCV [39] M 73 smoker, DM, hypertension 14 severe abdominal pain, nausea. fecal emesis, peritoneal irritation 18,000 – – – – 120,000 >5000 RX: distention of intestinal loops, inter-loop edema, intestinal pneumatosis enoxaparin (60 mg/0.6 mL), antibiotics (no info) enterectomy, anastomotic fistula, reintervention death Posada Arango [40] M F F 62 22 65 None Appendectomy 7 days before left nephrectomy, 5 3 15 colicative abdominal pain at food intake; unsystematized gastrointestinal symptoms; abdominal pain in the upper hemiabdomen 20,100 – – – – – – – – 1536 – – 534 – – – – – – – – Case 1: thrombus in distal SMA and its branches, intestinal loops dilatation, hydroaerical levels, free fluid thrombosis of SMV Case 2: SMV thrombosis and adiacent fat edema Case 3: thrombi in the left jejunal artery branch with infarction of the corresponding jejunal loops Case 1: Laparotomy: extensive jejunum + ileum ischemia; surgery could not be performed Case 2: Anticoagulation analgesic and antibiotics Case 3: segmental enterectomy Case 1: death Case 2: recovery Case 3: recovery J. Clin. Med. 2022, 11, 200 9 of 22 Table 3. Cont. Article Sex Age Comorbidities Time from COVID-19 Diagnosis (Days) Clinical Signs at Presentation Leukocyte Count (/mm3 ) CRP (mg/L) Lactate mmol/L Ferritin (ng/mL) LDH (U/L) Thrombocytes (/mm3 ) D-Dimers (ng/mL) Abdominal CT Signs Treatment Outcome Pang JHQ [41] M 30 none First diagnosis colicky abdominal pain, vomiting – – – – – – 20 SMV thrombosis with diffuse mural thickening and fat stranding of multiple jejunal loops conservative, anticoagulation with LMWH 1mg/kc, twice daily, 3 months; readmitted and operated for congenital adherence causing small bowel obstruction recovery Lari E [42] M 38 none First diagnosis abdominal pain, nausea, intractable vomiting, and shortness of breath Mild leukocytosis – 2.2 – – – 2100 extensive thrombosis of the portal, splenic, superior, and inferior mesenteric veins + mild bowel ischemia Anticoagulation, resection of the affected bowel loop No info Carmo Filho A [43] M 33 Obesity (BMI: 33), other not reported 7 severe low back pain radiating to the hypogastric region – 58.2 – 1570 – – 879 enlarged inferior mesenteric vein not filled by contrast associated with infiltration of the adjacent adipose planes enoxaparin 5 days, followed by long term oral warfarin recovery Hanif M [44] F 20 none 8 abdominal pain and abdominal distension 15,900 62 – 1435.3 825 633,000 2340 not performed evidence of SMA thrombosis; enterectomy with exteriorization of both ends recovery Amaravathi U [45] M 45 none 5 Acute epigastric and periumbilical pain – Normal value 1.3 324.3 – – 5.3 SMA and SMV thrombus i.v. heparin; Laparotomy with SMA thrombectomy; 48 h Second look: resection of the gangrenous bowel segment No info Al Mahruqi G [23] M 51 none 4 generalized abdominal pain, nausea, vomiting 16,000 – – 619 – – 10 SMA thrombosis and non-enhancing proximal ileal loops consistent with small bowel ischemia unfractionated heparin, thrombectomy + repeated resections of the ischemic bowel at relook (jejunum+ileon+cecum) Case 2: recovery J. Clin. Med. 2022, 11, 200 10 of 22 Table 3. Cont. Article Sex Age Comorbidities Time from COVID-19 Diagnosis (Days) Clinical Signs at Presentation Leukocyte Count (/mm3 ) CRP (mg/L) Lactate mmol/L Ferritin (ng/mL) LDH (U/L) Thrombocytes (/mm3 ) D-Dimers (ng/mL) Abdominal CT Signs Treatment Outcome Goodfellow M [46] F 36 RYGB, depression, asthma 6 epigastric pain, irradiating back, nausea 9650 1.2 0.7 – – – – abrupt cut-off of the SMV in the proximal portion; diffuse infiltration of the mesentery, wall thickening of small bowel IV heparin infusion, followed by 18,000 UI delteparin after 72 h recovery Abeysekera KW [26] M 42 Hepatitis B 14 right hypochondrial pain, progressively increasing for 9 days – – – – – – – enhancement of the entire length of the portal vein and a smaller thrombus in the mid-superior mesenteric vein, mural edema of the distal duodenum, distal small bowel, and descending colon factor Xa inhibitor apixaban 5 mg ×2/day, 6 months – recovery RodriguezNakamura RM [27] M F 45 42 -vitiligo -obesity 14 severe mesogastric pain, nausea, diaphoresis 16,400 18,800 367 239 – – 970 – – – 685,000 – 1450 14,407 Case 1: SMI of thrombotic etiology with partial rechanneling through the middle colic artery, and hypoxic-ischemic changes in the distal ileum and the cecum Case 2: thrombosis of the portal and mesenteric veins and an abdominopelvic collection in the mesentery with gas Case 1: resection with entero-enteral anastomosis; rivaroxaban 10 mg/day, 6 months Case 2: Loop resection, entero-enteral manual anastomosis, partial omentectomy, and cavity wash (fecal peritonitis) Case 1: Recovery Case 2: death Plotz B [47] F 27 SLE with ITP First diagnosis acute onset nausea, vomiting, and non-bloody diarrhea – – – – – – 5446 diffuse small bowel edema enoxaparin, long term apixaban at discharge recovery J. Clin. Med. 2022, 11, 200 11 of 22 Table 3. Cont. Article Sex Age Comorbidities Time from COVID-19 Diagnosis (Days) Clinical Signs at Presentation Leukocyte Count (/mm3 ) CRP (mg/L) Lactate mmol/L Ferritin (ng/mL) LDH (U/L) Thrombocytes (/mm3 ) D-Dimers (ng/mL) Abdominal CT Signs Treatment Outcome Chiu CY [48] F 49 Hypertension, DM, chronic kidney disease 28 diffuse abdominal pain melena and hematemesis – – – – – – 12,444 distended proximal jejunum with mural thickening laparotomy, proximal jejunum resection no info Farina D [49] M 70 no info 3 abdominal pain, nausea 15,300 149 – – – – – acute small bowel hypoperfusion, SMA thromboembolism not operable due to general condition Death SMA: superior mesenteric artery; SMV: superior mesenteric vein; DM: diabetes mellitus; T2DM: type 2 diabetes mellitus; AMI: acute mesenteric ischemia; IMV: inferior mesenteric vein; RYGB: Roux-en-Y gastric bypass (bariatric surgery). J. Clin. Med. 2022, 11, 200 12 of 22 2.2. Risk of Bias The studies analyzed in the present review were comparable in terms of patient selection, methodology, therapeutic approach, and the report of final outcome. However, there were differences in the reported clinical and laboratory data. The sample size was small, most of them being case reports or case series, which may be a significant source of bias. Therefore, studies were compared only qualitatively. 3. Results After duplication removal, a total of 36 articles were included in the review, reporting data on a total of 89 patients. Among these, we identified 6 retrospective studies [16–21], documenting intestinal ischemia in 55 patients admitted to intensive care units (ICU) with COVID-19 pneumonia for whom surgical consult was necessary (Table 1). We also identified 30 case reports or case series [22–51] presenting 34 cases of acute bowel ischemia in patients positive for SARS-CoV-2 infection in different clinical settings. 8 cases were previously hospitalized for COVID-19 pneumonia and under anticoagulant medication (Table 2). In 26 cases, the acute ischemic event appeared as the first symptom of COVID-19 disease, or in mild forms treated at home, or after discharge for COVID -19 pneumonia and cessation of the anticoagulant medication (Table 3). 3.1. Risk Factors of Intestinal Ischemia in COVID-19 Patients Out of a total of 89 patients included in the review, 63 (70.7%) were hospitalized for severe forms of COVID-19 pneumonia at the moment of onset. These patients were receiving anticoagulant medication when reported, consisting of low molecular weight heparin (LMWH) at prophylactic doses. The incidence of acute intestinal ischemia in ICU patients with COVID-19 varied widely between 0.22–10.5% (Table 1). In a study by O’Shea et al. [20], 26% of hospitalized patients for COVID-19 pneumonia who underwent imagistic examination, presented results positive for coagulopathy, and in 22% of these cases, the thromboembolic events were with multiple locations. The mean age was 56.9 years. We observed a significantly lower age in non-hospitalized COVID-19 patients presenting with acute intestinal ischemia when compared to the previously hospitalized group (p < 0.0001). There is a slight male to female predominance (M:F = 1:68). Obesity might be considered a possible risk factor, with a reported mean BMI of 31.2–32.5 in hospitalized patients [16,18,19]. However, this association should be regarded with caution, since obesity is also a risk factor for severe forms of COVID-19. Prolonged stay in intensive care, intubation, and the need for vasopressor medication was associated with increased risk of acute bowel ischemia [8,18,19]. Diabetes mellitus and hypertension were the most frequent comorbidities encountered in case reports (8 in 34 patients, 23%), and 7 out of 8 patients presented both (Table 4). There was no information regarding the comorbidities in the retrospective studies included in the review. 3.2. Clinical Features in COVID-19 Patients with Acute Mesenteric Ischemia Abdominal pain, out of proportion to physical findings, is a hallmark of portomesenteric thrombosis, typically associated with fever and leukocytosis [4]. Abdominal pain was encountered in all cases, either generalized from the beginning, of high intensity, or firstly localized in the epigastrium or the mezogastric area. In cases of portal vein thrombosis, the initial location may be in the right hypochondrium, mimicking biliary colic [26,34]. Fever is less useful in COVID-19 infected patients, taking into consideration that fever is a general sign of infection, and on the other hand, these patients might be already under antipyretic medication. J. Clin. Med. 2022, 11, 200 13 of 22 Table 4. Demographic data of the patients included in the review. Nr. of Patients 89 M 48 (61.5% *) F 30 (38.5% *) NA 11 The first sign of COVID-19 6 (6.7%) Home treated 17 (19.1%) Hospitalized • ICU 63 (70.7%) 58 (92% of hospitalized patients) Discharged 3 (3.3%) Time from diagnosis of COVID-19 infection • Non-Hospitalized • Hospitalized (*when mentioned) 8.7 ± 7.4 (1–28 days) 9.6 ± 8.3 (1–26 days) Time from admission in hospitalized patients 1–104 days Age (mean) • Hospitalized • Non-hospitalized 59.3 ± 12.7 years 62 ± 9.6 years. (p < 0.0001) 52.8 ± 16.4 years. BMI 31.2–32.5 Comorbidities • Hypertension • DM • smokers • Atrial fibrillation • COPD • Cirrhosis • RYGB • Vitiligo • Recent appendicitis • Operated gastric cancer • Alzheimer disease • SLE 8 7 2 1 2 1 1 1 1 1 1 1 *: percentage calculated in known information group; BMI: body mass index; COPD: chronic obstructive pulmonary disease; SLE: systemic lupus erythematosus. Other clinical signs reported were nausea, anorexia, vomiting, and food intolerance [23,31,38,45]. However, these gastrointestinal signs are encountered in 30–40% of patients with SARS-CoV-2 infection. In a study by Kaafarani et al., up to half of the patients with gastrointestinal features presented some degrees of intestinal hypomotility, possibly due to direct viral invasion of the enterocytes and neuro-enteral disturbances [16]. Physical exam evidenced abdominal distension, reduced bowel sounds, and tenderness at palpation. Guarding may be evocative for peritonitis due to compromised vascularization of bowel loops and bacterial translocation or franc perforation [35,39]. A challenging case was presented by Goodfellow et al. [25] in a patient with a recent history of bariatric surgery with Roux en Y gastric bypass, presenting with acute abdominal pain which imposed the differential diagnosis with an internal hernia. Upcinar et al. [24] reported a case of an 82-years female that also associated atrial fibrillation. The patient was anticoagulated with enoxaparin 0.4 cc twice daily before admission and continued the anticoagulant therapy during hospitalization for COVID-19 pneumonia. Bedside echocardiography was performed to exclude atrial thrombus. Although SMA was reported related to COVID-19 pneumonia, atrial fibrillation is a strong risk factor for SMA of non-COVID-19 etiology. J. Clin. Med. 2022, 11, 200 14 of 22 In ICU patients, acute bowel ischemia should be suspected in cases that present acute onset of digestive intolerance and stasis, abdominal distension, and require an increase of vasopressor medication [19]. 3.3. Imagistic and Lab Test Findings D-dimer is a highly sensitive investigation for the prothrombotic state caused by COVID-19 [45] and, when reported, was found to be above the normal values. Leukocytosis and acute phase biomarkers, such as fibrinogen and CRP were elevated, mirroring the intensity of inflammation and sepsis caused by the ischemic bowel. However, there was no significant statistical correlation between either the leukocyte count (p = 0.803) or D-dimers (p = 0.08) and the outcome. Leucocyte count may be within normal values in case of early presentation [34]. Thrombocytosis and thrombocytopenia have been reported in published cases with mesenteric ischemia [30,35,42,46,50]. Lactate levels were reported in 9 cases, with values higher than 2 mmol/L in 5 cases (55%). LDH was determined in 6 cases, and it was found to be elevated in all cases, with a mean value of 594+/−305 U/L. Ferritin is another biomarker of potential value in mesenteric ischemia, that increases due to ischemia-reperfusion cellular damage. In the reviewed studies, serum ferritin was raised in 7 out of 9 reported cases, with values ranging from 456 to 1570 ng/mL. However, ferritin levels were found to be correlated also with the severity of pulmonary lesions in COVID-19 patients [52]. Due to the low number of cases in which lactate, LDH, and ferritin were reported, no statistical association could be performed with the severity of lesions or with adverse outcomes. The location and extent of venous or arterial thrombosis were determined by contrastenhanced abdominal CT, which also provided important information on the viability of the intestinal segment whose vascularity was affected. Radiological findings in the early stages included dilated intestinal loops, thickening of the intestinal wall, mesenteric fat edema, and air-fluid levels. Once the viability of the affected intestinal segment is compromised, a CT exam may evidence pneumatosis as a sign of bacterial proliferation and translocation in the intestinal wall, pneumoperitoneum due to perforation, and free fluid in the abdominal cavity. In cases with an unconfirmed diagnosis of COVID-19, examination of the pulmonary basis during abdominal CT exam can add consistent findings to establish the diagnosis. Venous thrombosis affecting the superior mesenteric vein and or portal vein was encountered in 40.9% of reported cases of non-hospitalized COVID-19 patients, and in only one case in the hospitalized group (Table 5). One explanation may be the beneficial role of thrombotic prophylaxis in preventing venous thrombosis in COVID-19 patients, which is routinely administrated in hospitalized cases, but not reported in cases treated at home with COVID-19 pneumonia. In ICU patients, CT exam showed in most cases permeable mesenteric vessels and diffuse intestinal ischemia affecting the large bowel alone (56%) or in association with the small bowel (24%), suggesting pathogenic mechanisms, direct viral infection, small vessel thrombosis, or “nonocclusive mesenteric ischemia” [16]. 3.4. Management and Outcomes The management of mesenteric ischemia includes gastrointestinal decompression, fluid resuscitation, hemodynamic support, anticoagulation, and broad antibiotics. Once the thromboembolic event was diagnosed, heparin, 5000IU iv, or enoxaparin or LMWH in therapeutic doses was initiated, followed by long-term oral anticoagulation and/or anti-aggregating therapy. Favorable results were obtained in 7 out of 9 cases (77%) of splanchnic veins thrombosis and in 2 of 7 cases (28.5%) with superior mesenteric artery thrombosis. At discharge, anticoagulation therapy was continued either with LMWH, for a period up to 3 months [33,36,41], either, long term warfarin, with INR control [32,34,41] or apixaban 5 mg/day, up to 6 months [26,47]. No readmissions were reported. J. Clin. Med. 2022, 11, 200 15 of 22 Table 5. Comparative features in acute intestinal ischemia encountered in previously hospitalized and previously non-hospitalized COVID-19 patients. Parameter Hospitalized (63) NonHospitalized (26) p * Value Type of mesenteric ischemia: • Arterial • Venous • Mixt (A + V) • Diffuse microthrombosis • Multiple thromboembolic locations • NA 5 (14.7% *) 1 (2.9%) 0 30 (88.2%) 2 (5.8%) 29 10 (38.4%) 11 (42.3%) 2 (7.6%) 3 (11.5%) 1 (3.8%) 0 p < 0.0001 Management: • Anticoagulation therapy only • Endovascular thrombectomy • Laparotomy with ischemic bowel resection • None (fulminant evolution) 0 2 (1 + surgery) (3%) 60 (95.4%) 2 (3%) 10 (38.4%) 2 (+surgery) 15 (57.6%) 1 (3.8%) p < 0.0001 Location of the resected segment: • Colon • Small bowel • Colon+small bowel • NA 35 (56%) 10 (16%) 15 (24%) 6 0 12 (80%) 3 (20%) 0 p < 0.0001 Outcomes: • Recovery • Death • NA 26 (46.4%) 30 (54.4%) 7 17 (79.3%) 5 (21.7%) 3 p = 0.013 * calculated for Chi-squared test. Antibiotic classes should cover anaerobes including F. necrophorum and include a combination of beta-lactam and beta-lactamase inhibitor (e.g., piperacillin-tazobactam), metronidazole, ceftriaxone, clindamycin, and carbapenems [4]. In early diagnosis, during the first 12 h from the onset, vascular surgery may be tempted, avoiding the enteral resection [25,53]. Endovascular management is a minimally invasive approach, allowing quick restoration of blood flow in affected vessels using techniques such as aspiration, thrombectomy, thrombolysis, and angioplasty with or without stenting [40]. Laparotomy with resection of the necrotic bowel should be performed as quickly as possible to avoid perforation and septic shock. In cases in which intestinal viability cannot be established with certainty, a second look laparotomy was performed after 24–48 h [43] or the abdominal cavity was left open, using negative pressure systems such as ABTHERA [51], and successive segmentary enterectomy was performed. Several authors described in acute bowel ischemia encountered in ICU patients with COVID-19, a distinct yellowish color, rather than the typical purple or black color of ischemic bowel, predominantly located at the antimesenteric side or circumferentially with affected areas well delineated from the adjacent healthy areas [18,19]. In these cases, patency of large mesenteric vessels was confirmed, and the histopathological reports J. Clin. Med. 2022, 11, 200 16 of 22 showed endothelitis, inflammation, and microvascular thrombosis in the submucosa or transmural. Despite early surgery, the outcome is severe in these cases, with an overall mortality of 45–50% in reported studies and up to 100% in patients over 65 years of age according to Hwabejira et al. [19]. In COVID-19 patients non hospitalized at the onset of an acute ischemic event, with mild and moderate forms of the disease, the outcome was less severe, with recovery in 77% of cases. We found that age over 60 years and the necessity of surgical treatment are statistically correlated with a poor outcome in the reviewed studies (Table 6). According to the type of mesenteric ischemia, the venous thrombosis was more likely to have a favorable outcome (recovery in 80% of cases), while vascular micro thombosis lead to death in 66% of cases. Table 6. Risk factors for severe outcome. Parameters Outcome: Death p-Value Age • Age < 60 • Age > 60 27.2% 60% 0.0384 * 0.043 ** Surgery • No surgery • surgery 0% 60% 0.019 ** Type of mesenteric ischemia • Arterial • Venous • Micro thrombosis 47% 20% 66% 0.23 ** D dimers Wide variation 0.085 * 0.394 ** Leucocytes Wide variation (9650–37,000/mmc) 0.803 0.385 ** * One-way ANOVA test; ** Chi-squared test (SciStat® software, www.scistat.com (accessed on 25 November 2021)). 4. Discussions Classically, acute mesenteric ischemia is a rare surgical emergency encountered in the elderly with cardiovascular or portal-associated pathology, such as arterial hypertension, atrial fibrillation, atherosclerosis, heart failure, valve disease, and portal hypertension. However, in the current context of the COVID-19 pandemic, mesenteric ischemia should be suspected in any patient presenting in an emergency with acute abdominal pain, regardless of age and associated diseases. Several biomarkers were investigated for the potential diagnostic and prognostic value in acute mesenteric ischemia. Serum lactate is a non-specific biomarker of tissue hypoperfusion and undergoes significant elevation only after advanced mesenteric damage. Several clinical trials found a value higher than 2 mmol/L was significantly associated with increased mortality in non-COVID-patients. However, its diagnostic value is still a subject of debate. There are two detectable isomers, L-lactate, which is a nonspecific biomarker of anaerobic metabolism, and hypoxia and D-lactate, which is produced by the activity of intestinal bacteria. Higher D-lactate levels could be more specific for mesenteric ischemia due to increased bacterial proliferation at the level of the ischemic bowel, but the results obtained in different studies are mostly inconsistent [53,54]. Several clinical studies found that LDH is a useful biomarker for acute mesenteric ischemia, [55,56]. However, interpretation of the results may be difficult in COVID-19 patients, as both lactate and LDH were also found to be independent risk factors of severe forms of COVID-19 [57,58]. The diagnosis of an ischemic bowel should be one of the top differentials in critically ill patients with acute onset of abdominal pain and distension [50,59]. If diagnosed early, the J. Clin. Med. 2022, 11, 200 17 of 22 intestinal ischemia is potentially reversible and can be treated conservatively. Heparin has an anticoagulant, anti-inflammatory, endothelial protective role in COVID-19, which can improve microcirculation and decrease possible ischemic events [25]. The appropriate dose, however, is still a subject of debate with some authors recommending the prophylactic, others the intermediate or therapeutic daily amount [25,60]. We found that surgery is associated with a severe outcome in the reviewed studies. Mucosal ischemia may induce massive viremia from bowel epithelium causing vasoplegic shock after surgery [25]. Moreover, many studies reported poor outcomes in COVID-19 patients that underwent abdominal surgery [61,62]. 4.1. Pathogenic Pathways of Mesenteric Ischemia in COVID-19 Patients The intestinal manifestations encountered in SARS-CoV-2 infection are represented by inflammatory changes (gastroenteritis, colitis), occlusions, ileus, invaginations, and ischemic manifestations. Severe inflammation in the intestine can cause damage to the submucosal vessels, resulting in hypercoagulability in the intestine. Cases of acute cholecystitis, splenic infarction, or acute pancreatitis have also been reported in patients infected with SARS-CoV-2, with microvascular lesions as a pathophysiological mechanism [63]. In the study of O’Shea et al., on 146 COVID-19 hospitalized patients that underwent CT-scan, vascular thrombosis was identified in 26% of cases, the most frequent location being in lungs [20]. Gastrointestinal ischemic lesions were identified in 4 cases, in multiple locations (pulmonary, hepatic, cerebellar parenchymal infarction) in 3 patients. The authors raised awareness about the possibility of underestimation of the incidence of thrombotic events in COVID-19 patients [20]. Several pathophysiological mechanisms have been considered, and they can be grouped into occlusive and non-occlusive causes [64]. The site of the ischemic process, embolism or thrombosis, may be in the micro vascularization, veins, or mesenteric arteries. Acute arterial obstruction of the small intestinal vessels and mesenteric ischemia may appear due to hypercoagulability associated with SARS-CoV-2 infection, mucosal ischemia, viral dissemination, and endothelial cell invasion vis ACE-2 receptors [65,66]. Viral binding to ACE2Receptors leads to significant changes in fluid-coagulation balance: reduction in Ang 2 degradation leads to increased Il6 levels, and the onset of storm cytokines, such as IL-2, IL-7, IL-10, granulocyte colony-stimulating factor, IgG -induced protein 10, monocyte chemoattractant protein-1, macrophage inflammatory protein 1-alpha, and tumor necrosis factor α [67], but also in the expression of the tissue inhibitor of plasminogen -1, and a tissue factor, and subsequently triggering the coagulation system through binding to the clotting factor VIIa [68]. Acute embolism in small vessels may be caused by the direct viral invasion, via ACE-2 Receptors, resulting in endothelitis and inflammation, recruiting immune cells, and expressing high levels of pro-inflammatory cytokines, such as Il-6 and TNF-alfa, with consequently apoptosis of the endothelial cells [69]. Capillary viscometry showed hyperviscosity in critically ill COVID-19 patients [70,71]. Platelet activation, platelet–monocyte aggregation formation, and Neutrophil external traps (NETs) released from activated neutrophils, constitute a mixture of nucleic DNA, histones, and nucleosomes [59,72] were documented in severe COVID-19 patients by several studies [70,71,73]. Plotz et al. found a thrombotic vasculopathy with histological evidence for lectin pathway complement activation mirroring viral protein deposition in a patient with COVID19 and SLE, suggesting this might be a potential mechanism in SARS-CoV-2 associated thrombotic disorders [47]. Numerous alterations in fluid-coagulation balance have been reported in patients hospitalized for COVID-19 pneumonia. Increases in fibrinogen, D-dimers, but also coagulation factors V and VIII. The mechanisms of coagulation disorders in COVID-19 are not yet fully elucidated. In a clinical study by Stefely et al. [68] in a group of 102 patients with severe disease, an increase in factor V > 200 IU was identified in 48% of cases, the levels determined being statistically significantly higher than in non-COVID mechanically J. Clin. Med. 2022, 11, 200 18 of 22 ventilated or unventilated patients hospitalized in intensive care. This showed that the increased activity of Factor V cannot be attributed to disease severity or mechanical ventilation. Additionally, an increase in factor X activity was shown, but not correlated with an increase in factor V activity, but with an increase in acute phase reactants, suggesting distinct pathophysiological mechanisms [74]. Giuffre et al. suggest that fecal calcoprotein (FC) may be a biomarker for the severity of gastrointestinal complications, by both ischemic and inflammatory mechanisms [75]. They found particularly elevated levels of FC to be well correlated with D-dimers levels in patients with bowel perforations, and hypothesized that the mechanism may be related to a thrombosis localized to the gut and that FC increase is related to virus-related inflammation and thrombosis-induced ischemia, as shown by gross pathology [76]. Non-occlusive mesenteric ischemia in patients hospitalized in intensive care units for SARS-CoV-2 pneumonia requiring vasopressor medication may be caused vasospastic constriction [19,64,65]. Thrombosis of the mesenteric vessels could be favored by hypercoagulability, relative dehydration, and side effects of corticosteroids. 4.2. Question Still to Be Answered Current recommendations for in-hospital patients with COVID-19 requiring anticoagulation suggest LMWH as first-line treatment has advantages, with higher stability compared to heparin during cytokine storms, and a reduced risk of interaction with antiviral therapy compared to oral anticoagulant medication [77]. Choosing the adequate doses of LMWH in specific cases—prophylactic, intermediate, or therapeutic—is still in debate. Thromboprophylaxis is highly recommended in the absence of contraindications, due to the increased risk of venous thrombosis and arterial thromboembolism associated with SARS-CoV-2 infection, with dose adjustment based on weight and associated risk factors. Besides the anticoagulant role, some authors also reported an anti-inflammatory role of heparin in severe COVID-19 infection [66,78,79]. Heparin is known to decrease inflammation by inhibiting neutrophil activity, expression of inflammatory mediators, and the proliferation of vascular smooth muscle cells [78]. Thromboprophylaxis with enoxaparin could be also recommended to ambulatory patients with mild to moderate forms of COVID-19 if the results of prospective studies show statistically relevant benefits [80]. In addition to anticoagulants, other therapies, such as anti-complement and interleukin (IL)-1 receptor antagonists, need to be explored, and other new agents should be discovered as they emerge from our better understanding of the pathogenetic mechanisms [81]. Several studies showed the important role of Il-1 in endothelial dysfunction, inflammation, and thrombi formation in COVID-19 patients by stimulating the production of Thromboxane A2 (TxA2) and thromboxane B2 (TxB2). These findings may justify the recommendation for an IL-1 receptor antagonist (IL-1Ra) which can prevent hemodynamic changes, septic shock, organ inflammation, and vascular thrombosis in severe forms of COVID-19 patients [80–82]. 5. Conclusions Understanding the pathological pathways and risk factors could help adjust the thromboprophylaxis and fluid management in COVID-19 patients. The superior mesenteric vein thrombosis is the most frequent cause of acute intestinal ischemia in COVID-19 nonhospitalized patients that are not under anticoagulant medication, while non-occlusive mesenteric ischemia and microvascular thrombosis are most frequent in severe cases, hospitalized in intensive care units. COVID-19 patients should be carefully monitored for acute onset of abdominal symptoms. High-intensity pain and abdominal distension, associated with leukocytosis, raised inflammatory biomarkers and elevated D-dimers and are highly suggestive for mesenteric ischemia. The contrast-enhanced CT exam, repeated, if necessary, offers valuable information regarding the location and extent of the acute ischemic event. Early diagnosis and treatment are essential for survival.

J. Clin. Med. 2022, 11, 200 19 of 22 Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jcm11010200/s1, File S1: The PRISMA 2020 statement. Author Contributions: Conceptualization, D.S., L.C.T. and A.M.D.; methodology, A.P.S., C.T. (Corneliu Tudor); software, G.V.; validation, A.I.S., M.S.T., D.S. and L.D.; formal analysis, A.C.C., C.T. (Ciprian Tanasescu); investigation, G.A.G.; data curation, D.O.C.; writing—original draft preparation, L.C.T., A.M.D., G.V., D.O.C., G.A.G., C.T. (Corneliu Tudor); writing—review and editing, L.D., C.T. (Ciprian Tanasescu), A.C.C., D.S., A.P.S., A.I.S., M.S.T.; visualization, G.V. and L.C.T.; supervision, D.S., A.M.D. and D.S. have conducted the screening and selection of studies included in the review All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. 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COVID-19 Can Infect and Harm Digestive Organs

Authors: E.J. Mundell

 The coronavirus isn’t just attacking the lungs: New research shows it’s causing harm to the gastrointestinal tract, especially in more advanced cases of COVID-19.

A variety of imaging scans performed on hospitalized COVID-19 patients showed bowel abnormalities, according to a study published online May 11 in Radiology. Many of the effects were severe and linked with clots and impairment of blood flow.

“Some findings were typical of bowel ischemia, or dying bowel, and in those who had surgery we saw small vessel clots beside areas of dead bowel,” said study lead author Dr. Rajesh Bhayana, who works in the department of radiology at Massachusetts General Hospital in Boston.

“Patients in the ICU can have bowel ischemia for other reasons, but we know COVID-19 can lead to clotting and small vessel injury, so bowel might also be affected by this,” Bhayana explained in a journal news release.

One expert unconnected to the new study said the findings aren’t surprising.

“Our emerging understanding of COVID-19 has found the disease to have multisystem involvement including the nervous, cardiac, vascular [excess clotting] and finally the digestive systems, among others,” said Dr. Sherif Andrawes. He directs endoscopy in the division of gastroenterology and hematology at Staten Island University in New York City.

“It seems that this disease is intricate, in the sense that it can involve multiorgan systems, rather than being a disease of the respiratory system solely,” Andrawes said.

In fact, a study published online May 13 in the journal Science Immunology has found evidence that SARS-CoV-2, the virus behind COVID-19, can infect the human digestive system.

Researchers led by Siyuan Ding of Washington University School of Medicine in St. Louis, said their findings “highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression.”

That seems to be borne out by the Boston study.

That research included 412 COVID-19 patients who were hospitalized between March 27 and April 10. They averaged 57 years of age, and 134 of them underwent abdominal imaging, including 137 radiographs, 44 ultrasounds, 42 CT scans, and one MRI.