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ORIGINAL ARTICLE Table of Contents  
Ahead of print publication
SARS-CoV-2 Infection after Effects: Multi-Organ Damage through Oxygen Radicals


1 Department of Natural Products, National Institute of Pharmaceutical Education and Research; Department of Lab Services, Apollo Multi-Speciality Hospital; Department of Biochemistry, Jagannath Gupta Institute of Medical Sciences and Hospital, Budge-Budge, Kolkata, West Bengal, India
2 Department of Natural Products, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India
3 Department of Lab Services, Apollo Multi-Speciality Hospital, Kolkata, West Bengal, India
4 Department of Biochemistry, Jagannath Gupta Institute of Medical Sciences and Hospital, Budge-Budge, Kolkata, West Bengal, India
5 Department of Pathology, Apollo Hospitals Educational and Research Foundation, New Delhi, India

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Date of Submission23-Aug-2022
Date of Decision21-Sep-2022
Date of Acceptance30-Sep-2022
Date of Web Publication17-Nov-2022
 

  Abstract 


Introduction: SARS-CoV-2 respiratory infection leads to two-layered pathology in time (a) immediate pathology and recovery or fatal ending and (b) long periods of remission followed by unexplained clinical expressions involving one or more systems with various clinical presentations, even leading to loss of lives. Among the common causative factors affecting nearly all organs and systems, oxygen radicals and oxygen-derived species (ROS) should rank conclusively on top. Objective: This clinical report, part of a community screening of unexplained clinical outcomes in post-COVID presentation, carries findings focusing on ROS production and possible ROS-induced damages. Materials and Methods: Flow cytometry was used to quantify the samples' total ROS, superoxide production, and apoptosis quantification. Results: Observations indicate unexplained nearly uniform enhanced ROS production in all these long COVID subjects, although clinical presentations varied from no complaints to the requirement of advanced interventions. Conclusion: Causative factors leading to raised oxygen-derived toxic intermediaries (ROS) in initiating these variable long COVID presentations are ill-understood yet possibly merit mass screenings and possible intense anti-oxidative therapy given that such antioxidant therapy through oral medications led to rapid lowering of ROS production and improvement of clinical presentations.

Keywords: COVID-19, multi-organ affection, post-COVID cardiac affection, radicals and oxygen-derived species, SARS-CoV-2, superoxide


How to cite this URL:
Datta D, Singh R, Velayutham R, Bhattacharya A, Ray U, Dasgupta S, Dutta S, Saha A, Roy D, Ghosh S, Arumugam S, Datta P, Ganguly N K. SARS-CoV-2 Infection after Effects: Multi-Organ Damage through Oxygen Radicals. Apollo Med [Epub ahead of print] [cited 2022 Nov 28]. Available from: https://apollomedicine.org/preprintarticle.asp?id=361434





  Introduction Top


The SARS-CoV-2 pandemic has been the cause of unseen instances of global death and debility.[1] With the primary variant (Delta) passage between 2020 and 2021, the death toll had an unseen peak in each country, particularly in India, South-East Asia, Europe, and North America.[2] Since the second half of 2021, although viral variants have been in circulation significantly, clinical incidences have remained reasonable and under the control of collective health-care facilities in each country, while death counts have gone down significantly around the baseline.

SARS-CoV-2, a member of the corona family, shares viral antigens with other members of the family, including molecular details of binding and internalization on the respiratory epithelium.[3],[4] It is interesting, in this context, that nearly 25%–30% of respiratory tract infections round the year are caused by the corona family,[5] and there is a strong likelihood that cross-protection against SARS-CoV-2 from such shared antigen[s] may have played a significant role in the protection of the unvaccinated/part-vaccinated population– particularly young children– in a country like India where resource limitation, nonavailability of required mass-level vaccine infrastructure, lack of awareness, and religious restrictions are abundant.[6]

This is a report from an ongoing mass screening program where the postvaccine cell-mediated immune status of the population at large is being studied along with probing of possible causative factor(s) in postcorona multisystem affections.

Current clinical assessment of postcorona or postvaccine immune status considers humoral immunity (IgG) only, whereas it is the peripheral-most parameter resulting from innumerable cell–cell, cell–protein, and protein - protein interactions in Cell-mediated immunity (CMI). Therefore, a clearer in-depth molecular assessment of such immune status needs extensive screenings of the CMI network, which is an assured clinical improbability at a mass scale. Therefore, this study tries to squeeze scientific necessity into clinical affordability and resource availability, particularly in debilitated health-care systems (like India), so that clinical assessment may finally be grounded on minimum parameter scrutiny without undermining the quality of the decision process.


  Materials and Methods Top


Study groups

Clinical samples were collected from Apollo Multispeciality. Patients were grouped as.

Normal subjects

Healthy individuals with no previous medical history, aged 20–24 years, were selected as control subjects for the study.

Long SARS-CoV-2 infection with cardiac-coronary presentation

A 56 year old otherwise healthy male present complained of chest discomfort in April 2022. ECG showed signs of ischaemic heart disease. Echocardiography revealed septalhyperkinesia with left ventricular dysfunction (Left ventricular Ejection Fraction was 48%).CT angio was done which showed 70-80% occlusion of the left circumflex artery. Past history of the patient was significant for an episode of COVID 19 in June 2020 which was managed with hydroxychloroquine,vitamins and anti –pyretic. He did not require oxygensupplementation during the covid 19 episode. For the current episode he was prescribed beta blockers vasodilators, anticoagulants and high dose antioxidants (150 mg levocarnitine +200mg Vitamin E). Despite ongoing medical therapy, the patient expired 4 months later due to an episode of massive AMI [Supplementary Figure 1].



Active SARS-CoV-2 infection with cardiac–coronary presentation

A 68 years old diabetic, hypertensive female patient presented with shortness of breath for the last 3 Days and mild fever for the last 7 days. Significant findings include left ventricular dilatation with severe systolic dysfunction (Left ventricular ejection fraction was 30% and there was grade 2 Mitral regurgitation) on ECHO. The ECG showed atrial fibrillation with rapid ventricular response. The COVID RT PCR was positive (CT of N gene 25 and RdRP 26). The results of the laboratory parameters are summarized. She was treated with anti-arrhythmic drugs, oxygen supplementation and other supportive medications. She improved and was discharged 2 weeks later in hemodynamically stable condition [Supplementary Figure 2].



No SARS-CoV-2 infection with cardiac-coronary presentation

Three patients aged 60-69 years were included in this category. The history of these patients detailed below:

  • Patient 1: A 62 years hepatitis B positive male presented with chest pain for past 2 days. The ECG revealed a non-ST elevated AMI. Critical double-vessel coronary artery disease was discovered during coronary angiography. Percutaneous transluminal angioplasty with stenting was performed. The post procedural stage was uneventful [Supplementary Figure 3]a.
  • Patient 2: For last 6 months, he had been experiencing occasional chest pain with exertional dyspnea that was getting worse. Percutaneous transluminal coronary angioplasty and stenting were performed after a coronary angiography revealed double-vessel coronary artery disease. The patient recovered well [Supplementary Figure 3]b
  • A 70 years male presented with intermittent chest pain associated with exertional dyspnoea since last 6 months which was progressive in nature. Coronary angiogram revealed double vessel coronary artery disease for which percutaneous transluminal coronary angioplasty and stenting was done. Post procedural period was uneventful. [Supplementary Figure 3]c.



Recent SARS-CoV-2 infection with Cardiac–Coronary presentation-Two patients with different age groups were selected

  • Patient 1: In April 2022, a diabetic and hypertensive female (70–74 years) was admitted with complaints of dizziness and presyncope attacks. In January 2022, she was diagnosed with COVID-19, for which she was treated with oxygen supplementation and other supportive drugs. During the current stay, the ECG revealed bradycardia with bifascicular heart block, and the angiography revealed minimal coronary abnormalities. She had a permanent pacemaker implanted and has improved since then [Supplementary Figure 4]a
  • Patient 2: At the beginning of April 2022, a 54 years male complained of chest pain. Septal hypokinesia was detected on echocardiography, along with left ventricular failure (left ventricular ejection fraction was 48%). ECG revealed a block in the left bundle branch. A CT angiogram revealed a 70%–80% blockage of the left circumflex artery. Past history revealed that the patient had COVID 19, for which he was placed in home isolation and treated with favipiravir, Hydroxychloroquine sulfate (HCQS), ivermectin, and vitamins. During his COVID sickness, his saturation was effectively maintained, and he did not require oxygen supplementation. His cardiac issues are currently treated with beta-blockers and Angiotensin-converting enzyme (ACE) medications [Supplementary Figure 4]b.



Long SARS-CoV-2 infection with renal presentation

In September 2020, a 50 years female patient with borderline renal parenchymal disease was admitted to the hospital with fever, anorexia, weakness, body ache, and dizziness. She was found to have a COVID infection. The CT value for COVID RT-PCR was N 25, and the RdRP value was 26. She was given conservative treatment and did not require the use of steroids or oxygen. Subsequently over the months her renal parameters deteriorated (creatinine 2.9 mg/dl and urea 61 mg/dl) [Supplementary Figure 5].



Long SARS-CoV-2 infection with no clinical presentation

Both patients aged 35 and 39 years were asymptomatic and placed into home isolation. No specific treatment was initiated [Supplementary Figure 6].



Reagents and material

Annexin/PI kit (640914) purchased from Biolegend, 2'-7'-Dichlorodihydrofluorescein diacetate (DCFDA) (D6883) from Sigma-Aldrich, and MitoSOX (M36008) purchased from Thermo scientific.

Quantification of radicals and oxygen-derived species by DCFDA

One hundred microliters of blood sample was added to the FACS tube, 3 ml of ×1 lysis buffer was added, and it was incubated for 10 min. The sample was centrifuged at 1500 rpm speed for 5 min. The supernatant was discarded, and the pellet was washed with ×1 phosphate buffered saline (PBS). The supernatant was again discarded, and 10 μM of DCFDA reagent was added. Quantification of radicals and oxygen-derived species (ROS) was done by flow cytometry.

Quantification of mitochondrial-induced superoxide radical by MitoSOX

One hundred milliliters of blood sample was added to the FACS tube, 3 ml of 1X lysis buffer was added, and it was incubated for 10 min. The sample was centrifuged at 1500 rpm speed for 5 min. The supernatant was discarded, the pellet was washed with 1X PBS, and 30 μM of MitoSOX reagent was added. Flow cytometry was used for the quantification of superoxide production.

Apoptotic cell quantification by Annexin V and PI

One hundred milliliters of blood sample was added to the FACS tube, 3 ml of 1X lysis buffer was added, and it was incubated for 10 min. The sample was centrifuged at 1500 rpm speed for 5 min. The supernatant was discarded, the pellet was washed with 1X PBS, and 5 μl of Annexin V FITC and 10 μl of PI were added. Quantification of apoptosis was done by flow cytometry.

Flow cytometry

Flow cytometry measurements were performed with BD LSRFortessa Cell Analyzer (BD Biosciences, San Jose, USA). FITC channel was utilized to capture the DCFDA, Annexin V, and other FITC-tagged markers. PE-CF594 was used to capture the MitoSOX and other PE-tagged markers. Twenty thousand were acquired for the sample.


  Results and Discussion Top


SARS-CoV-2 infection had a long passage of transformations over the last two years--from being a respiratory pathogen in the beginning to an inducer of uncontrolled systemic inflammation and finally to an initiator of multi-system affection and shutdown leading to fatal endings. While reports are abundant in the immediate events of the pathogen causing severe respiratory affections,[7] lung fibrosis leading to failure,[8] near uncontrolled systemic inflammatory responses,[9] and even parallel clinical presentations of cardiac affection,[10] reports of delayed secondary organ affections – cardiac, neural, and renal are not very infrequent.

Individuals with cardiac complications in an apparent “long SARS-CoV-2” infection showed a significant increase in total ROS production compared to other groups without comorbidities, an overall increase of 56% (3.16% increment from mitochondrial fraction) [Supplementary Figure 1]b,[Supplementary Figure 1]c. This shows a predominance of unexplained ROS without any obvious inflammatory response (~53%), which is being heightened in this report as a possible candidate to be under scrutiny in all active, immediate past, and long-past cases of SARS-CoV-2 infection. Post-COVID renal infection cases presented with enhanced ROS production to the extent of 13.8% [Supplementary Figure 5]. Apoptotic cell population in patients with cardiac coronary disease with long SARS-CoV-2 infection showed a remarkable increase (13.1%) versus normal subjects (1.19%) [Supplementary Figure 1]a.


  Discussion Top


ROS, oxygen-derived molecules which oxidize nearly any biomolecules, are of two types;

  1. Generated by NOX-2, an NADPH oxidase in scavenging and competent immune cells such as macrophages and PMN cells. This is physiological and constitutes a primary step in the body's defense mechanism
  2. An unexplained ROS generated in SARS-CoV-2 and other infections, causing uncontrolled oxidative stress and immune cell dysregulation, also known as a cytokine storm.[11],[12]


Cytokine dysregulation and immune cell dysfunction constitute a usual clinical presentation in advanced active SARS-CoV-2 infection, while unexplained ROS without any obvious inflammatory clinical presentation in old SARS-COV-2 cases warrants closer examination. Observed cases being part of an ongoing community screening for assessment of CMI in post-SARS-CoV-2 episodes drew immediate attention in a couple of past covid patients who were long past the currently agreed *post covid* window of 3-6 months after primary infection was long crossed. One of the patients presented with cardiac symptoms after 22 months of primary infection needing institutional interventions, while the other patient showed renal complaints about 7 months after primary infection. While the other patient showed renal complaints about 7 months after primary infection, In both, in between periods when completely disease-free and symptom-free.

Enhanced total ROS beyond the stipulated postcorona time window with ~95% of enhanced amount coming from nonmitochondrial unexplained fraction and the remaining ~5% from mitochondrial fraction merits a closer examination of ROS in all SARS-CoV-2 cases, particularly beyond the primary infection and up to a very liberally extended time window as possible diagnostic and prognostic clinical markers coming days. Interestingly, oral antioxidant therapy with Vitamin E and other institutional interventions gave a nearly dramatic resolution of the “long COVID case” with the cardiac presentation. Unfortunately, the patient, discontinued aggressive antioxidant therapy and succumbed to Myocardial infarction (MI) 3 weeks postdischarge.

Expectedly, active and recent COVID cases also gave reasonable increments in combined ROS titers. Considering all the presented categories, it seems there is a need for active antioxidant aggressive therapy in all SARS-CoV-2 cases, unlike the present casual approach. Along with this aggressive antioxidant therapy, clinical assessment of gross ROS and MitoSOX fraction can be considered active dynamic diagnostic and prognostic parameters in all SARS-CoV-2 cases, both during the infection and for reasonable periods after the resolution of primary infection.

Closer examination of possible unexplained ROS long after primary infection possibly hints at the presence of non-structural protein(s) like Coronavirus 3a, in-part remaining *molecularly hidden* in system in SARS-COV-1 and SARS-COV-2 post-infections; nonstructural 3a proteins activate the NLRP3 inflammasome in macrophages and thereby increase MitoSOX component.[13] Furthermore, there is a possibility of the formation of an abnormal complex of IgG with SARS-CoV-2 S-protein, resulting in hyper-inflammatory responses in macrophages associated with altered glycosylation of the Fc fraction of IgG.[14] Of course circulating IgG gets extensively glycosylated in the Fc portion through S-protein interactions and hence perhaps needs continuous experimental clinical probing for glycosylated IgG or free-S-protein as possible diagnostic and prognostic marker(s) through subsequent broad-based community screenings. In comparison, mitROS and overall titer enhancement of free radicals can be explained in several ways in acute cases, but explaining hyper-ROS reactions following long delays in clinical events need a required alert.

None of the patients in the presented study showed signs of intravascular coagulation. However, the endothelium is a definite target of both whole viral particles and inflammatory cytokines during the active phase or in recent COVID-19 infections.[3] ROS-mediated endothelial damage and activation have–in turn–quite a few routes, such as tumor necrosis factor-dependent, enhanced endothelial permeability, or an imbalanced NO-ROS synthesis in post-COVID hypertensive cases.[12],[15],[16]


  Conclusion Top


Recent SARS-CoV-2 or active SARS-CoV-2 have explainable enhanced ROS titers, a part of which is contributed by MitoSOX logically as part of the inflammatory process. However, the molecular event in unexplained ROS, including a parallel surge in MitoSOX, remains obscure, particularly when the primary SARS-CoV-2 infection and the subsequent organ or multi-organ presentation have a longer time gap than covered under the current definition of postcorona syndrome or long COVID-19 conditions and need scrutiny both for early diagnostic parameter and for continued prognostic markers. Deciphering a possible preventive and therapeutic approach through neutralizing ROS and its MitoSOX fraction through more vigorous routine antioxidant therapy is also of immediate importance.

Declaration of patient consent

The authors certify that he has obtained all appropriate patient consent forms. In the form, the patient has given his consent for his images and other clinical information to be reported in the journal. The patient understands that his name and initials will not be published, and due efforts will be made to conceal his identity, but anonymity cannot be guaranteed.

Acknowledgment

We would like to acknowledge NIPER Kolkata and Apollo Multispeciality Hospitals for providing the facility for the experimental work.

Conflicts of interest

There are no conflicts of interest.

Ethical statement

Ethical clearance was done by the ethical committee at Apollo Multispeciality Hospital, Kolkata, following the ICMR guidelines. The study has been conducted in accordance with the ethical principles mentioned in the Declaration of Helsinski (2013).

Funding

Funding is provided by NIPER Kolkata.

Author contribution

  • RS and AB carried out all the experiments
  • UR, SD, SD, AS, DR, and SG collects the samples and maintain case history and patient consent
  • AS, PD, NK, and VR-Gives a critical review of the manuscript
  • DD-Was the originator of the work, guided, analyzed data analysis, and presented the manuscript.




 
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Correspondence Address:
Debatosh Datta,
National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/am.am_122_22





 

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    -  Datta D
    -  Singh R
    -  Velayutham R
    -  Bhattacharya A
    -  Ray U
    -  Dasgupta S
    -  Dutta S
    -  Saha A
    -  Roy D
    -  Ghosh S
    -  Arumugam S
    -  Datta P
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