INVESTIGATING LONG COVID MARKERS IN MYOCARDIAL INFARCTION PATIENTS: A CROSS-SECTIONAL STUDY

Covid 19 caused by SARS COV-2 is a global health concern that has led to the death of nearly 70 lakh people worldwide as of 2024. The incidence as well as severity of the disease have reduced considerably following global efforts to combat the virus. It usually results in acute infection in symptomatic patients that resolves in four weeks in majority of the cases but in almost 30% of the cases it may lead to prolonged symptoms for months or even years post infection. This results in development of what is termed as long covid. Due to the sheer number of infections caused by SARS COV-2 during the pandemic it is very important to understand post acute sequelae in this case termed long covid. According to WHO, long covid infection is the continuation or development of new symptoms three months after initial SARS COV 2 infection, which last for at least 2 months without any other explanation. Although at present there are no definitive gold standard investigations for diagnosing long covid many biomarkers have been proposed. Recently Soraya Maria Menezes et. al. have proposed a two gene biomarker of FYN and SARS COV 2 Antisense RNA with a high sensitivity and specificity. Although the exact mechanistic pathway for the expression of FYN is not known, it has been shown by multiple studies to have been upregulated in COVID infections. We believe that upregulation of CD55/DAF leading to dysregulation of the complement system caused by covid infection might be further leading to an increased FYN expression in these patients. Although further studies are required to determine the exact nature of this interaction, we believe that proper investigation of the host kinome might lead to therapeutic breakthroughs in treatment of viral infections caused by covid.

Long covid may result in the development of many systemic disorders like immune dysregulation, neurodegenerative diseases and most importantly increased cardiovascular events among others. COVID 19 pandemic lately has been shown to be linked to increased incidence of myocardial infarction (MI) and stroke especially in young adults, a populations previously considered as relatively less at risk for such events. A meta analysis by Zuin M et. al. found that history of covid was significantly associated with MI. Covid 19 infection has been hypothesised to interfere with the cardiovascular system through three principal mechanisms. First being direct myocardial damage due to entry of virus into the myocardial cells. This has been supported by certain studies showing cardiac tissue tropism of the virus due to expression of ACE 2 receptors in these cells  as seen in certain studies. Secondly, the interaction of the virus with its principal receptor ACE 2 leading to the dysregulation of the RAS system thus leading to indirect myocardial damage and finally the effect of the virus on promoting a systemic inflammatory immune status long after the resolution of the disease as shown by various studies. Given this scenario, it is important to investigate any potential relationship between the development of long covid and subsequent systemic manifestations playing a foul role in development of MI in young adults with a history of SARS COV 2 infection. Persistent viral load in the bloodstream or covid viral residual protein related inflammation might play a role in precipitating such outcomes.Thus using the biomarkers FYN and SARS COV 2 Antisense RNA we propose to investigate the prevalence of long covid in this population to ascertain its role in the development of MI in young adults.

Objective

To determine the persistent presence of long covid markers, antisense SARS-CoV-2 RNA and FYN RNA in young patients with a previous history of hospitalization due to acute covid infection suffering from MI.

To find a correlation between angiographic severity using GENSINI scores and long covid marker expression.

Methodology

Type of study : Cross Sectional study

Study Design : In this study 40 patients will be included following selection criteria after obtaining informed consent.

Duration of the study : 18 months

Sample Size: 40 young myocardial infarction patients

Inclusion & Exclusion Criteria :

Inclusion Criteria:

Participants must be between 18 to 45 years old.

Participants must have a confirmed diagnosis of acute MI documented  in their medical records based on the diagnosis of STEMI defined by the European Society of Cardiology (ESC)/American  College of Cardiology Foundation (ACCF).

NSTEMI is diagnosed in patients determined to have symptoms consistent with ACS and troponin elevation but without ECG changes consistent with STEMI.

Exclusion Criteria:

Individuals younger than 18 years or older than 45 years.

Congenital heart disease

Cardiomyopathies

Pericardial diseases

Patients on pacemakers

Individuals who are unable to provide informed consent, such as those with cognitive impairment  or legal incapacity.

Ethical consideration: Ethical clearance obtained from the Institutional Ethical Committee.

Written Consent: Written informed consent will be obtained from all the participants before enrolment in the study.

Study Protocol:

Sample collection :

Needle aspiration of venous blood samples from the excised patient into two 3 ml K2 EDTA tubes will be collected.

Gene Expression Studies:

Extracting RNA: RNA will be extracted using the Nucleospin RNA extraction kit from Takara. Analysis of the purity and concentration of RNA will be performed using a nanodrop spectrophotometer. cDNA will be synthesized and primers for antisense SARS-CoV-2 ( SARS-CoV-2_orf1ab_REV-mRNA ) and FYN RNA primers will be designed to perform RT-qPCR.

RT qPCR: Will be performed using Tb green reagents on Quantstudio 6 and relative expression of will be calculated by using Delta Delta Ct method.

Statistical Analysis

The data obtained will be  entered in a Microsoft Excel sheet, and statistical analysis will be  performed using statistical package for the social sciences ( Version 20 ). In gene expression plots, fold change is plotted, but statistical analysis will be made on threshold cycle (ACt) values. The p-value <0.05 will be considered significant. Correlation will be performed between various variables.

Implications

The results obtained through this study will help us better understand the effect that covid might have on development of long term systemic disorders such as MI . By investigating the presence of long covid markers, we can better understand its link to MI. This study provides novel insights into MI and will further lead to identification of future therapeutic targets for prevention and treatment. The results will inform public health policy in regard to MI in young adults and guide future research in this area.