Flow cytometry profiling of cellular immune response in COVID-19 infected, recovered and vaccinated individuals.

INTRODUCTION: SARS-CoV-2 has infected over 753 million individuals and caused more than 6.8 million deaths globally to date. COVID-19 disease severity has been associated with SARS-CoV-2 induced hyper inflammation and the immune correlation with its pathogenesis remains unclear. Acute viral infection is characterised by vigorous coordinated innate and adaptive activation, including an early cellular response that correlates well with the amplitude of virus specific humoral response. OBJECTIVE: The present study covers a wide spectrum of cellular immune response against COVID-19, irrespective of infection and vaccination. METHODS: We analysed immune status of (a) COVID-19 hospitalised patients including deceased and recovered patients, and compared with home isolated and non-infected healthy individuals, and (b) infected home isolated individuals with vaccinated individuals, using flow cytometry. We performed flow cytometry analysis of PBMCs to determine non-specific cell-mediated immune response. RESULTS: The immune response revealed extensive induction and activation of multiple immune lineages, including T and B cells, Th17 regulatory subsets and M1, M2 macrophages in deceased and hospitalised recovered patients, vaccinated and healthy individuals. Compromised immune cell expression was observed in deceased patients even in later stages, while expression was restored in hospitalised recovered patients and home isolated individuals. CONCLUSION: The findings associated with recovery and convalescence define a new signature of cellular immune response that persists in individuals with SARS-CoV-2 infection and vaccination. The findings will help in providing a better understanding of COVID-19 disease and will aid in developing better therapeutic strategies for treatment.

Evaluation of altered miRNA expression pattern to predict COVID-19 severity.

Outbreak of COVID-19 pandemic in December 2019 affected millions of people globally. After substantial research, several biomarkers for COVID-19 have been validated however no specific and reliable biomarker for the prognosis of patients with COVID-19 infection exists. Present study was designed to identify specific biomarkers to predict COVID-19 severity and tool for formulating treatment. A small cohort of subjects (n = 43) were enrolled and categorized in four study groups; Dead (n = 16), Severe (n = 10) and Moderate (n = 7) patients and healthy controls (n = 10). Small RNA sequencing was done on Illumina platform after isolation of microRNA from peripheral blood. Differential expression (DE) of miRNA (patients groups compared to control) revealed 118 down-regulated and 103 up-regulated known miRNAs with fold change (FC) expression ≥2 folds and p ≤ 0.05. DE miRNAs were then subjected to functional enrichment and network analysis. Bioinformatic analysis resulted in 31 miRNAs (24 Down-regulated; 7 up-regulated) significantly associated with COVID-19 having AUC>0.8 obtained from ROC curve. Seventeen out of 31 DE miRNAs have been linked to COVID-19 in previous studies. Three miRNAs, hsa-miR-147b-5p and hsa-miR-107 (down-regulated) and hsa-miR-1299 (up-regulated) showed significant unique DE in Dead patients. Another set of 4 miRNAs, hsa-miR-224-5p (down-regulated) and hsa-miR-4659b-3p, hsa-miR-495-3p and hsa-miR-335-3p were differentially up-regulated uniquely in Severe patients. Members of three miRNA families, hsa-miR-20, hsa-miR-32 and hsa-miR-548 were significantly down-regulated in all patients group in comparison to healthy controls. Thus a distinct miRNA expression profile was observed in Dead, Severe and Moderate COVID-19 patients. Present study suggests a panel of miRNAs which identified in COVID-19 patients and could be utilized as potential diagnostic biomarkers for predicting COVID-19 severity.

Gene variants in pro-coagulant and anti-coagulant genes could be prognostic genetic markers of COVID-19 susceptibility.

Background: Present study aimed to identify DNA polymorphisms (variants) which can modulate the risk of COVID-19 infection progression to severe condition. TaqMan based SNP genotyping assay was performed for 11 single nucleotide polymorphisms (SNPs) in pro-coagulant and anti-coagulant genes. Methodology: A total of 33 COVID-19 patients, including dead, severe and moderately infected individuals were compared to 35 healthy controls. Both alleles in the SNP were labelled with two different fluorescent dyes (FAM and VIC) during assay formulation. DNA of study subjects were mixed with SNP assay and TaqMan master mix on 96 well PCR plate according to manufacturer's protocol and RT-PCR was performed. Allelic discrimination assay gave clear results for presence of specific allele in each sample. Three SNPs were located in the pro-coagulant genes, another three involved in blood clot dissolution while rest five were in the genes encoding natural anti-coagulants. COVID-19 infected patients were further sub-divided into three groups, deceased (n = 16), severe (n = 10) and moderately infected (n = 7). Results: SNP genotyping showed significant differences between COVID-19 patients and controls in two SNPs, rs6133 in Selectin-P (SELP) and rs5361 in Selectin-E (SELE) gene. Also, rs2020921 and rs8176592, in clot dissolution genes, tissue Plasminogen activator (tPA) and tissue factor pathway inhibitor (TFPI) respectively showed significant genotypic and allelic difference in patients of COVID-19 compared to healthy controls. Further three SNPs rs2227589, rs757583846, and rs121918476 in natural anti-coagulant genes anti-thrombin III (ATIII), protein C (PROC), and protein S (PROS) respectively showed statistically significant difference between the study groups. Conclusion: Our findings indicate that gene variants, those involved in coagulation and anti-coagulation may play a major role in determining individual susceptibility to COVID-19.

Efficacy of convalescent plasma therapy in the patient with COVID-19: a randomised control trial (COPLA-II trial).

IMPORTANCE: No proven treatment is available for severely ill COVID-19. Therapeutic use of COVID-19 convalescent plasma (COPLA) is under investigation. OBJECTIVE: To compare the efficacy of COPLA with standard medical therapy (SMT) alone in severe COVID-19 patients. DESIGN, SETTING AND PARTICIPANTS: A multicentric, open-labelled, phase-III randomised controlled trial conducted at two treatment centres with COPLA collected at the third dedicated centre in North-India, the coordinating centre during trial from June 2020 to December 2020. The study population comprised 400 participants in the ratio of 1:1 in each treatment group. INTERVENTION: One group received COPLA with SMT (n=200), and another group received SMT only (n=200). MAIN OUTCOME MEASURES: Primary outcome was time to clinical improvement measured by a two-point reduction in the ordinal scale. Secondary outcomes included duration of O2 therapy, the proportion of patients on mechanical ventilation at day-7, mortality, SARS-CoV-2 antibody levels, cytokine levels and incidence of adverse events. RESULTS: The median time to a two-point reduction in the ordinal scale in both groups was 9 days (IQR=7-13) (p=0.328). The median duration of O2 therapy was 8 days (IQR=6-12) in COPLA and 10 days (IQR=6-12) in SMT group (p=0.64). The PaO2/FiO2 ratio showed significant improvement at 7 days in COPLA group(p=0.036). There was no difference in mortality till 28 days in both groups (p=0.62). However, if COPLA was given within 3 days of hospital admission, a significant reduction in ordinal scale was observed (p=0.04). Neutralising antibody titres in COPLA group (80 (IQR 80-80)) were higher than SMT group (0 (IQR 0-80)) at 48 hours (p=0.001). COPLA therapy led to a significant reduction in TNF-α levels at 48 hours (p=0.048) and D-dimer at 7 days (p=0.02). Mild allergic reactions were observed in 3 (1.5%) patients in COPLA group. CONCLUSION AND RELEVANCE: Convalescent plasma with adequate antibody titres should be transfused in COVID-19 patients along with SMT in the initial 3 days of hospitalisation for better clinical outcomes. TRIAL REGISTRATION NUMBER: NCT04425915.

Implications of COVID-19 on Thrombotic Profile of Severely Affected Patients.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) is a novel viral disease that spread as a global pandemic in 2020 by infecting millions of people across the world. Its clinical prognosis is dependent on various coagulatory parameters since thrombotic events are frequently associated with infection severity. METHODS: A total of 383 COVID-19 patients enrolled in Rajiv Gandhi Super Specialty Hospital, Delhi, India, were included in the present retrospective study. Patients were divided into three categories, severe (n = 141), moderate (n = 138), and mild (n = 104) based on infection severity. Various thrombotic parameters and anticoagulant levels were measured in 70 patients and further analyzed. RESULTS: Coagulopathy is seen in COVID-19 patients (n = 70) with a significant increase in fibrinogen, D-dimer levels, and prothrombin time in patients with severe and moderate disease compared to patients with a mild infection. Approximately, 70% of patients with severe and moderate disease demonstrated fibrinogen levels higher than the standard reference range. 60.41% of patients with severe disease showed significantly higher D-dimer levels. Thrombotic parameters were notably elevated in the nonsurvivors group compared to COVID-19 survivors. Nearly, 91% of patients with severe infection had anticoagulant protein S levels below the reference range. CONCLUSION: COVID-19 infection severely impacts the blood coagulation cascade, which might lead to the manifestation of severe symptoms and increased mortality in patients.

Social Stigma of COVID-19 Experienced by Frontline Healthcare Workers of Department of Anaesthesia and Critical Care of a Tertiary Healthcare Institution in Delhi.

BACKGROUND: Social stigma is associated with Coronavirus Disease-2019 (COVID-19) particularly against people who have contracted the disease or have come in contact with it. There is paucity of studies regarding the prevalence of social stigma against healthcare workers (HCWs) in COVID-19 hospitals in India. The objective of this study was to measure social stigma faced by frontline HCWs of Department of Anaesthesia and Critical Care in a COVID-19 hospital and to assess the relationship between sociodemographic characteristics and social stigma. PATIENTS AND METHODS: A cross-sectional study using a questionnaire (sociodemographic characteristics along with modified Berger HIV Stigma Scale) was conducted from October 10, 2020 to October 30, 2020, in the Department of Anaesthesia and Critical Care. The survey was distributed among frontline HCWs using Google Forms as well as Bilingual Physical Form. Total stigma and subgroups of stigma scale were measured for different sociodemographic parameters and compared. Data were presented as mean ± standard deviation. p-value <0.05 was taken as significant. RESULTS: Out of 120 frontline HCWs participated in the study, 68 (56.6%) reported severe level of COVID-19-related stigma. The mean score of COVID-19-related stigma was 41 + 7.69. Mean scores for subgroups of stigma scale, i.e., personalized stigma, disclosure concerns, negative self-image, and concerns with public attitude, were 15.60 + 4.01, 6.68 + 3.21, 5.46 + 3.22, and 13.25 + 2.44, respectively. In the univariate analysis, the overall COVID-19-related stigma scores were associated with age >30 years, male gender, lower designation (technicians and nursing orderly), lesser education, and married HCWs. In logistic regression model, only male gender was significantly associated with severity of COVID-19 stigma. CONCLUSION: This study concluded that more than half of frontline HCWs in the Department of Anaesthesia and Critical Care experienced severe social stigma during COVID-19 pandemic, with highest stigma in concerns with public attitude subgroup. Severity of stigma was associated with age, male gender, designation, education, and marital status of HCW. HIGHLIGHTS: Frontline HCWs of Department Anaesthesia and Critical Care experienced significant stigma related to COVID-19. HOW TO CITE THIS ARTICLE: Jain S, Das AK, Talwar V, Kishore J, Heena, Ganapathy U. Social Stigma of COVID-19 Experienced by Frontline Healthcare Workers of Department of Anaesthesia and Critical Care of a Tertiary Healthcare Institution in Delhi. Indian J Crit Care Med 2021;25(11):1241-1246.

Spectrum of Multisystem Inflammatory Syndrome in Children (MIS-C)-a Report of Three Cases.

We report three cases of multisystem inflammatory syndrome in children (MIS-C) during July 2020 from a tertiary care hospital with different clinical presentations and course of management. This will guide in better management of children with MIS-C. All three patients, aged 1 to 12 years old, were critically ill. They presented with common features of MIS-C, such as fever, conjunctival congestion, gastrointestinal involvement, and skin manifestations. Clinical features were suggestive of shock, coagulopathy, and multiorgan involvement. Laboratory findings revealed raised inflammatory markers, including C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and D-dimers (DD). All patients required intensive care with oxygen therapy, fluid resuscitation, inotropic agents, and broad-spectrum antibiotics. All patients received steroids, and two patients were given intravenous immunoglobulin. One patient died, and the remaining two patients were discharged. Our findings confirmed that COVID-19 may cause severe disease in children, and the presentation may vary, requiring early recognition and timely management.

Potential association of COVID-19 and ABO blood group: An Indian study.

Coronavirus disease 2019 (COVID-19) transmits from person to person mainly through respiratory droplets and coughing. Infection severity ranges from asymptomatic and mild infection to those with moderate and severe symptoms which may lead to multiple organ failure and mortality. Infection severity largely depends on individual's immune response, age and co-morbidities. Present study categorized COVID-19 infected patients based on their infection severity and linked COVID-19 severity with age, gender and ABO blood group types. Clinical details of 383 COVID-19 patients were collected from Rajiv Gandhi Super Specialty hospital (RGSSH), India; divided into three groups; mild, moderate and severe patients, based on their symptoms. Present analysis revealed that age plays major role in infection severity, as the symptoms are more severe in patients above 45 years. Infection rate was higher in males compared to females. Most patients with A(+ve) and B(+ve) blood group were severely affected compared to those of blood group type O(+ve) and AB(+ve). O(+ve) blood group was least represented in severe patients. Present findings could be helpful in generating awareness amongst the population regarding susceptibility towards the COVID-19 infection. This supportive information would help clinicians and health workers to propose new strategies and tactical solution against COVID-19 infection.