Seroprevalence of anti-SARS-CoV-2 IgG antibodies in admitted patients at a tertiary referral centre in North India (preprint)

Background: Seroprevalence of IgG antibodies against SARS-CoV-2 is an important tool to estimate true burden of infection in a given population. Serosurveys, though being conducted in different parts of India, are not readily published in entirety and often do not report on the different characteristics of the population studied. In this present study, we aimed to serially estimate the seroprevalence of anti-SARS-CoV-2 IgG antibody over 11 months at one of the largest government hospital in India. Method In this cross-sectional study which was conducted between 9th June 2020 and 27th April 2021, consecutive patients admitted to medicine wards or intensive care units, who were negative for SARS-CoV-2 by RT-PCR or CBNAAT were included. The clinic-demographic features of the subjects were recorded in pre-formed questionnaires. Anti-SARS-CoV2 antibody levels targeting recombinant spike receptor-binding domain (RBD) protein of SARS CoV-2 were estimated in serum sample by the ELISA method. Results A total of 916 patients were recruited over 11 months with mean age(plus/minus SD) 39.79 plus/minus 14.9 of years and 55% of population being males. In total 264(28.8%) patients were found to be seropositive. Residency in Delhi and non-smoking status conferred a higher risk for seropositivity. The adjusted odds ratio for seropositivity with regards to no smoking and residence out of Delhi were .31 plus/minus .09 (Odds ratio plus/minus S.E) and .65 plus/minus .1 (Odds ratio plus/minus S.E) respectively. No other factors like age, socio-economic status, contact history etc. showed significant relationship with seropositivity. Conclusion The seropositivity rate among hospitalized patients was found to increase with time (from 8.45% to 38%) over a period of 9 months. Residence in Delhi and non-smokers had higher risk for seropositivity on multivariate analysis.

Selection of appropriate reference genes for normalization of qRT-PCR based gene expression analysis in SARS-CoV-2, and Covid-associated Mucormycosis infection (preprint)

Selection of reference genes in quantitative PCR is critical to determine accurate and reliable mRNA expression. Despite this knowledge, not a single study has investigated the expression stability of housekeeping genes to determine their suitability to act as reference gene in SARS-CoV-2 or Covid19 associated mucormycosis (CAM) infections. Herein, we address these gaps by investigating the expression stability of nine most commonly used housekeeping genes including TBP, CypA, B2M, 18S, PGC-1α, GUSB, HPRT-1, β-ACTIN and GAPDH in the patients of varying severity (asymptomatic, mild, moderate and severe). We observed significant differences in the expression of candidate genes across the disease spectrum. Next, using various statistical algorithms (delta Ct, Normfinder, Bestkeeper, RefFinder and GeNorm), we observed that CypA demonstrated the most consistent expression across the patients of varying severity and emerged as the most suitable gene in Covid19, and CAM infections. Incidentally, the most commonly used reference gene GAPDH showed maximum variations and found to be the least suitable. Lastly, comparative evaluation of expression of NRF2 is performed following normalization with GAPDH and CypA to highlight the relevance of an appropriate reference gene. Our results reinforce the idea of a selection of housekeeping genes only after validation especially during infections.

Molecular signature of postmortem lung tissue from COVID-19 patients suggests distinct trajectories driving mortality (preprint)

The precise molecular mechanisms behind severe life-threatening lung abnormalities during severe SARS-CoV-2 infections are still unclear. To address this challenge, we performed whole transcriptome sequencing of lung autopsies from 31 patients suffering from severe COVID-19 related complications and 10 uninfected controls. Using a metatranscriptome analysis of lung tissue samples we identified the existence of two distinct molecular signatures of lethal COVID-19. The dominant "classical" signature (n=23) showed upregulation of unfolded protein response, steroid biosynthesis and complement activation supported by massive metabolic reprogramming leading to characteristic lung damage. The rarer signature (n=8) potentially representing "Cytokine Release Syndrome" (CRS) showed upregulation of IL1 cytokines such CCL19 but absence of complement activation and muted inflammation. Further, dissecting expression of individual genes within enriched pathways for patient signature suggests heterogeneity in host response to the primary infection. We found that the majority of patients cleared the SARS-CoV-2 infection, but all suffered from acute dysbiosis with characteristic enrichment of opportunistic pathogens such as Gordonia bronchialis in "classical" patients and Staphylococcus warneri in CRS patients. Our results suggest two distinct models of lung pathology in severe COVID-19 patients that can be identified through the status of the complement activation, presence of specific cytokines and characteristic microbiome. This information can be used to design personalized therapy to treat COVID-19 related complications corresponding to patient signature such as using the identified drug molecules or mitigating specific secondary infections.

ACE2 Expression in Lungs of Severe COVID-19 Infection: A Study on Minimally Invasive Post- mortem Tissue Samples (preprint)

Angiotensin-converting enzyme 2 (ACE2) is a key host protein by which severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) enters and multiplies within cells. The level of ACE2 expression in the lung is hypothesised to correlate with an increased risk of severe infection and complications in COVID-19 (COrona VIrus Disease 2019). To test this hypothesis, we compared the protein expression status of ACE2 by immunohistochemistry (IHC) in post-mortem lung samples of patients who died of severe COVID-19 and lung samples obtained from non-COVID-9 patients for other indications. IHC for CD61 and CD163 were performed for assessment of platelet-rich microthrombi and macrophages, respectively. IHC for SARS-CoV-2 viral antigen was also performed. Quantification of immunostaining, random sampling, and correlation analysis was used to substantiate the morphologic findings. Our results show that among a total of 44 COVID-19 post-mortem lung tissues and 15 lung biopsies in non-COVID-19 patients included, ACE2 protein expression was significantly higher in COVID-19 patients than in controls, regardless of sample size. Histomorphology in COVID-19 lungs showed diffuse alveolar damage (DAD), acute bronchopneumonia, and acute lung injury with SARS-CoV-2 viral protein detected in a subset of cases. ACE2 expression levels positively correlated with increased expression levels of CD61 and CD163. In conclusion, our results show significantly higher ACE2 protein expression in severe COVID-19 disease, correlating with increased macrophage infiltration and microthrombi, suggesting a pathobiological role in disease severity.

Novel Risk Factors for Coronavirus Disease-Associated Mucormycosis (CAM): A Case Control Study During the Outbreak in India (preprint)

Background: The epidemiology of the Coronavirus-disease associated mucormycosis (CAM) syndemic is poorly elucidated. In this study, we aimed to analyse the risk factors for CAM, in addition to those established for mucormycosis prior to COVID-19 pandemic.Methods: We performed a case-control study comparing patients diagnosed with CAM with those who had recovered from COVID-19 without developing mucormycosis. Information on comorbidities, glycaemic control, and practices related to COVID-19 prevention and management was recorded.Findings: One hundred fifty-two cases of CAM and 200 controls diagnosed with COVID-19 during April–May 2021 were included. In the CAM group, symptoms of mucormycosis began a mean 18·9 ± 9·1 days after the symptom onset of COVID-19. All, but one patient, carried either of the conventional risk factors: diabetes and steroid use. On multivariate regression analysis, risk factors associated with increased odds of CAM included presence of diabetes (aOR 3·5, 95%CI 1·1–11), poor glycaemic control (p<0.05 for blood glucose levels >200 mg/dL), use of systemic steroids (aOR 7·7,95% CI 2·4–24·7), prolonged use of cloth and surgical masks (vs N95 masks, aOR 17·5, 95% CI 4·6-66·7), and repeated nasopharyngeal swab testing (aOR 1·6,95% CI 1·2–2·2).Interpretation: CAM is strongly associated with diabetes, poor glycaemic control, and systemic steroid use. Novel risk factors identified in our study include prolonged use of cloth and surgical masks vis-a-vis N95 masks, and repeated nasopharyngeal swab testing. Oxygen therapy and need for hospitalization for COVID-19 did not affect the risk of CAM.Funding Information: This was an investigator-initiated non-funded study.Declaration of Interests: The authors declare that they have no competing interests.Ethics Approval Statement: The study was approved by the Institute Ethics Committee, AIIMS, Delhi, India. [IECPG-353/28.05.21].

Novel risk factors for Coronavirus disease-associated mucormycosis (CAM): a case control study during the outbreak in India (preprint)

BackgroundThe epidemiology of the Coronavirus-disease associated mucormycosis (CAM) syndemic is poorly elucidated. We aimed to identify risk factors that may explain the burden of cases and help develop preventive strategies. MethodsWe performed a case-control study comparing cases diagnosed with CAM and those who had recovered from COVID-19 without developing mucormycosis (controls). Information on comorbidities, glycemic control, and practices related to COVID-19 prevention and treatment was recorded. Results352 patients (152 cases and 200 controls) diagnosed with COVID-19 during April-May 2021 were included. In the CAM group, symptoms of mucormycosis began a mean 18.9 (SD 9.1) days after onset of COVID-19, and predominantly rhino-sinus and orbital involvement was present. All, but one, CAM cases carried conventional risk factors of diabetes and steroid use. On multivariable regression, increased odds of CAM were associated with the presence of diabetes (adjusted OR 3.5, 95%CI 1.1-11), use of systemic steroids (aOR 7.7,95% CI 2.4-24.7), prolonged use of cloth and surgical masks (vs no mask, aOR 6.9, 95%CI 1.5-33.1), and repeated nasopharyngeal swab testing during the COVID-19 illness (aOR 1.6,95% CI 1.2-2.2). Zinc therapy, probably due to its utility in immune function, was found to be protective (aOR 0.05, 95%CI 0.01-0.19). Notably, the requirement of oxygen supplementation or hospitalization did not affect the risk of CAM. ConclusionJudicious use of steroids and stringent glycemic control are vital to preventing mucormycosis. Use of clean masks, preference for N95 masks if available, and minimizing swab testing after the diagnosis of COVID-19 may further reduce the incidence of CAM.

Use of HFNC in COVID-19 patients in non-ICU setting: Experience from a tertiary referral centre of north India and a systematic review of literature (preprint)

Introduction The rapid surge of cases and insufficient numbers of intensive care unit (ICU) beds have forced hospitals to utilise their general wards for administration of non-invasive respiratory support including HFNC(High Flow Nasal Cannula) in severe COVID-19. However, there is a dearth of data on the success of such advanced levels of care outside the ICU setting. Therefore, we conducted an observational study at our centre, and systematically reviewed the literature, to assess the success of HFNC in managing severe COVID-19 cases outside the ICU. Methods A retrospective cohort study was conducted in a tertiary referral centre where records of all adult COVID-19 patients (over 18 years) requiring HFNC support were between September and December 2020 were analysed. HFNC support was adjusted to target SpO2 over 90% and respiratory rate less than 30 per min. The clinical, demographic, laboratory, and treatment details of these patients were retrieved from the medical records and entered in predesigned proforma. Outcome parameters included duration of oxygen during hospital stay, duration of HFNC therapy, length of hospital stay and death or discharge. HFNC success was denoted when a patient did not require escalation of therapy to NIV or invasive mechanical ventilation, or shifting to the ICU, and was eventually discharged from the hospital without oxygen therapy; otherwise, the outcome was denoted as HFNC failure. Systematic review was also performed on the available literature on the experience with HFNC in COVID-19 patients outside of ICU settings using the MEDLINE, Web of Science and Embase databases. Statistical analyses were performed with the use of STATA software, version 12, OpenMeta[Analyst], and visualization of the risk of bias plot using robvis. Results Thirty-one patients receiving HFNC in the ward setting, had a median age of 62 (50 - 69) years including 24 (77%) males. Twenty-one (68%) patients successfully tolerated HFNC and were subsequently discharged from the wards, while 10 (32%) patients had to be shifted to ICU for non-invasive or invasive ventilation, implying HFNC failure. Patients with HFNC failure had higher median D-dimer values at baseline (2.2 mcg/ml vs 0.6 mcg/ml, p=0.001) and lower initial SpO2 on room air at admission (70% vs 80%, p=0.026) as compared to those in whom HFNC was successful .A cut-off value of 1.7 mg/L carried a high specificity (90.5%) and moderate sensitivity (80%) for the occurrence of HFNC failure. Radiographic severity scoring as per the BRIXIA score was comparable in both the groups(11 vs 10.5 out of 18, p=0.78 ). After screening 98 articles, total of seven studies were included for synthesis in the systematic review with a total of 820 patients, with mean age of the studies ranging from 44 to 83 years and including 62% males. After excluding 2 studies from the analysis, the pooled rates of HFNC failure were 36.3% (95% CI 31.1% - 41.5%) with no significant heterogeneity (I2 =0%, p=0.55). Conclusions Our study demonstrated successful outcomes with use of HFNC in an outside of ICU setting among two-thirds of patients with severe COVID-19 pneumonia. Lower room air SpO2 and higher D-dimer levels at presentation were associated with failure of HFNC therapy leading to ICU transfer for endotracheal intubation or death. Also, the results from the systematic review demonstrated similar rates of successful outcomes concluding that HFNC is a viable option with failure rates similar to those of ICU settings in such patients.

Microscopic imaging of bronchoalveolar fluids of COVID-19 positive intubated patients reveals the different level of SARS-CoV-2 infection on oral squamosal epithelial cells

COVID-19 pandemic has been a global health emergency due to its association with severe pneumonia and high rate of mortality. In the current study, we reported the direct evidence for the variable level of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infection on several types of oral epithelial cells isolated from the aspirated oropharyngeal and bronchoalveolar secretions of severely infected and intubated patients using cytology, confocal based immunofluorescence imaging (IF), scanning electron microscope (SEM) and transmission electron microscopic (TEM) studies. Cytological analysis showed the presence of keratinised and non-keratinised oral epithelial cells with viral inclusion bodies in stratum granulosum and intermedium cells. IF imaging using SARS-CoV-2 spike protein specific antibody confirmed the presence of virus inside the stratum spinosum/granulosum (keratinised) and stratum intermedium/superficial cells (non-keratinised). No SARS-CoV-2 viruses were seen in stratum corneum cells. SEM analysis also confirms the absence of virus like structure on stratum corneum surface while viruses like structure were seen on the stratum spinosum/granulosum/stratum/intermedium and stratum superficial cells. This advanced microscopic study confirms directly that the virus tends to infect and multiply in the metabolically active or sub-basal cells of oral epithelium and absent in the inactive keratinised stratum corneum from shedding zones of oral mucosa.

ReCOVer study: A Cross-sectional Observational Study to Identify the Rehabilitation Need in Post-discharge COVID-19 Survivors (preprint)

IntroductionWith the increasing number of Coronavirus disease-2019 (COVID-19) cases there is simultaneous increase in recovered cases too. There are many post-covid complications where need for rehabilitation intervention is very conspicuous especially pulmonary, neurological complications. Hence data are of utmost importance to find out those rehabilitation needs among post-covid survivors. Methods and analysisReCOVer (Rehabilitation Need in Post-discharge COVID-19 Survivors), a cross-sectional observational study protocol has been planned to find out rehab-need by assessing International Classification of Functioning, Disability and Health (ICF) core data set, COVID-19 Yorkshire Rehab Screen (C19-YRS) tool, The Post-COVID-19 Functional Status (PCFS) scale, barriers to functional independence and rehab services (affordability & availability). Post-discharge (minimum 1 weeks) Covid patients (required hospitalisation) will be included in the study. Study will be conducted through Telerehabilitation facility. Study will conform to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines. Ethics and disseminationStudy received ethical approval from Institute Ethics Committee, All India Institute of Medical Sciences (AIIMS), New Delhi, India. Findings will be disseminated at scientific conferences/meetings, peer-reviewed journals, and to relevant stakeholders including the ministry of health (if required).

Clinical correlation of lung ultrasound profiles in patients with COVID-19 infection (preprint)

BackgroundLung ultrasound is a popular point of care test that correlates well with computed tomography for lung pathologies. While previous studies have shown its ability to detect COVID-19 related lung pathology, we aimed to evaluate the utility of lung ultrasound in the triage and prognostication of COVID-19 patients by determining its ability to predict clinical severity and outcomes. MethodsThis was a prospective, cross-sectional, observational, single centre study done at JPNATC and AIIMS, New Delhi, India. Consenting eligible patients aged 18 years or more were included if hospitalised with microbiologically confirmed COVID-19 and classified as mild, moderate (respiratory rate >24/min OR SpO2<94% on room air) and severe COVID-19 (respiratory rate >30/min OR SpO2<90% on room air) at the time of enrolment. The lungs were systematically assessed with ultrasound after division into 14 zones (4 anteriorly, 4 axillary and 6 posteriorly). Clinical and laboratory parameters including arterial blood gas analysis at the time of evaluation were recorded. Patients were followed till death or discharge. The primary objective was to determine the correlation between clinical severity and lung ultrasound profiles (no. of A, B and C profiles, and the total number of areas involved). Secondary objectives included assessment of the correlation between lung ultrasound profiles and clinical outcomes and development of a statistical model incorporating ultrasound and clinical parameters to allow prediction of COVID-19 related severity and outcomes. FindingsBetween October 1, 2020, and January 31,2021, patients were screened for inclusion and total n=60 patients were evaluated and included in the final analysis. The most common abnormality seen were B lines, seen in at least one zone in n=53 (88.33%) of cases. A median of 9 (IQR: 5-12) zones of the 14 assessed had a B-profile. The total number of abnormal areas (zones with a B or C profile) correlated significantly with the PaO2/FiO2 ratio ({rho}= -0.7232, p<0.0001) and SpO2/FiO2 ratio ({rho}= -0.6866, p<0.0001), and differed significantly between mild and moderate vs severe cases (p=0.0026 mild vs moderate, p<0.0001 mild vs severe, p=0.0175 moderate vs severe). The total number of B lines were predictors of mortality (p=0.0188, OR 1.03, 95% CI 1.003-1.060). Statistical models that incorporated total number of B-lines, CRP and anticoagulation use could predict mortality (p=0.0124, pseudo R2=0.1740) with an AUC= 0.7682 (95% CI=0.6176-0.9188), and the total number of involved areas and LDH levels could distinguish severe disease from mild/moderate disease (p<0.0001, Pseudo R2=0.3822), AUC = 0.8743 (95% CI=0.7752-0.9733). A simplified cut off of [≥]6 involved areas (of the 14 assessed) was 100% sensitive and 52% specific for differentiating severe disease from mild and moderate ones. InterpretationIn patients with COVID-19, increasing involvement of the lungs as assessed by ultrasonography correlates significantly with clinical severity and outcomes. These findings may be utilized in future prospective studies to validate the use of lung ultrasound to triage and prognosticate patients with COVID-19 infection. Author ApprovalAll authors have seen and approved the manuscript Competing interestsThere are no potential competing interests Data availability StatementAll data referred to in the manuscript shall be provided when asked for. DisclaimersThe authors have nothing to disclose Funding statementNo funding source was involved.

Clinical Correlation of Lung Ultrasound Profiles in Patients with COVID-19 Infection (preprint)

Background: Lung ultrasound is a popular point of care test that correlates well with computed tomography for lung pathologies. While previous studies have shown its ability to detect COVID-19 related lung pathology, we aimed to evaluate the utility of lung ultrasound in the triage and prognostication of COVID-19 patients by determining its ability to predict clinical severity and outcomes.Methods: This was a prospective, cross-sectional, observational, single centre study done at JPNATC and AIIMS, New Delhi, India. Consenting eligible patients aged 18 years or more were included if hospitalised with microbiologically confirmed COVID-19 and classified as mild, moderate (respiratory rate >24/min OR SpO2<94% on room air) and severe COVID-19 (respiratory rate >30/min OR SpO2 <90% on room air) at the time of enrolment. The lungs were systematically assessed with ultrasound after division into 14 zones (4 anteriorly, 4 axillary and 6 posteriorly). Clinical and laboratory parameters including arterial blood gas analysis at the time of evaluation were recorded. Patients were followed till death or discharge. The primary objective was to determine the correlation between clinical severity and lung ultrasound profiles (no. of A, B and C profiles, and the total number of areas involved). Secondary objectives included assessment of the correlation between lung ultrasound profiles and clinical outcomes and development of a statistical model incorporating ultrasound and clinical parameters to allow prediction of COVID-19 related severity and outcomes.Findings: Between October 1, 2020, and January 31,2021, patients were screened for inclusion and total n=60 patients were evaluated and included in the final analysis. The most common abnormality seen were B lines, seen in at least one zone in n=53 (88.33%) of cases. A median of 9 (IQR: 5-12) zones of the 14 assessed had a B-profile. The total number of abnormal areas (zones with a B or C profile) correlated significantly with the PaO2/FiO2 ratio (ρ= -0.7232, p<0.0001) and SpO2/FiO2 ratio (ρ= -0.6866, p<0.0001), and differed significantly between mild and moderate vs severe cases (p=0.0026 mild vs moderate, p<0.0001 mild vs severe, p=0.0175 moderate vs severe). The total number of B lines were predictors of mortality (p=0.0188, OR 1.03, 95% CI 1.003-1.060). Statistical models that incorporated total number of B-lines, CRP and anticoagulation use could predict mortality (p=0.0124, pseudo R2=0.1740) with an AUC= 0.7682 (95% CI=0.6176-0.9188), and the total number of involved areas and LDH levels could distinguish severe disease from mild/moderate disease (p<0.0001, Pseudo R2=0.3822), AUC = 0.8743 (95% CI=0.7752-0.9733). A simplified cut off of ≥6 involved areas (of the 14 assessed) was 100% sensitive and 52% specific for differentiating severe disease from mild and moderate ones.Interpretation: In patients with COVID-19, increasing involvement of the lungs as assessed by ultrasonography correlates significantly with clinical severity and outcomes. These findings may be utilized in future prospective studies to validate the use of lung ultrasound to triage and prognosticate patients with COVID-19 infection.Funding Statement: No funding source was involved.Declaration of Interests: The authors have nothing to discloseEthics Approval Statement: The study was reviewed and cleared by the Institute Ethics Committee (IEC).

The Association Between Hypocalcemia and Outcome in COVID-19 Patients: a Retrospective Study (preprint)

Background: Calcium has been shown to have a vital role in the pathophysiology of SARS-CoV and MERS-CoV diseases but less is known about hypocalcemia in COVID-19 patients and its association with the disease severity and the final outcome. Therefore, this study was conducted with an aim to assess the clinical features in the COVID-19 patients having hypocalcemia and to observe its impact on COVID-19 disease severity and final outcome.Method: In this retrospective study, consecutive COVID-19 patients of all age groups were enrolled. Demographical, clinical and laboratory details were collected and analysed. On the basis of albumin-corrected calcium level patients were classified into normocalcemic (n=51) and hypocalcemic (n=110). Death was the primary outcome. Results: The mean age of hypocalcemic were significantly lower (p<0.05). A significantly higher number of normocalcemic patients had severe COVID-19 disease(92.73%, p<0.01), had comorbidities (82.73%, p<0.05) and required ventilator support(39.09%, p<0.01)compared to the hypocalcemic patients. The mortality rate was significantly higher (33.63%, p<0.05) in the hypocalcemic patients when compared with the normocalcemic patients (15.69%). Haemoglobin (p<0.01), hematocrit (p<0.01) and red cell count (p<0.01) were significantly lower with higher levels of absolute neutrophil count (<0.05) and neutrophil to lymphocyte ratio (p<0.01) in the hypocalcemic patients. Albumin-corrected calcium level had a significant positive correlation with haemoglobin level, haematocrit, red cell count, total protein, albumin and albumin to globulin ratio and a significant negative correlation with absolute neutrophil count and neutrophil to lymphocyte ratio.Conclusion: The disease severity, ventilator requirement and mortality were considerably higher in hypocalcemic COVID-19 patients. 

A two-arm, randomized, controlled, multi-centric, open-label Phase-2 study to evaluate the efficacy and safety of Itolizumab in moderate to severe ARDS patients due to COVID-19 (preprint)

An uncontrolled increase in cytokine production may lead to systemic hyperinflammation, vascular hypo-responsiveness, increased endothelial permeability, hypercoagulation, multi-organ dysfunction and eventually death in moderate to severely ill COVID-19 patients. Targeting T-cells, an important driver of the hyperinflammatory response, in the treatment of COVID-19, could potentially reduce mortality and improve survival rates. Itolizumab is an anti-CD6 humanized monoclonal antibody with an immunomodulating action on Teffector cells that downregulates T-cell activation, proliferation and subsequent production of various chemokines and cytokines. The efficacy and safety of Itolizumab for the treatment of cytokine release syndrome in patients with moderate to severe acute respiratory distress syndrome (ARDS) due to COVID-19 was evaluated in a multi-centric, open-label, two-arm, controlled, randomized, phase 2 study. Eligible patients were randomized (2:1) to arm A (best supportive care + Itolizumab) and arm B (best supportive care). The primary outcome of interest was reduction in all-cause mortality 30 days after enrolment. Thirty-six patients were screened, 5 were treated as first dose sentinels and the rest were randomized, whilst 4 patients were considered screen failures. Two patients in the Itolizumab treatment arm discontinued prior to receiving the first dose and were replaced. At the end of 1 month, there were 3 deaths in arm B, and none in arm A (p= 0.0296). At the end of the follow-up period, more patients in Arm A had improved SpO2 without increasing FiO2 (p=0.0296), improved PaO2 (p=0.0296), and reduction in IL-6 (43 pg/ml vs 212 pg/ml; p=0.0296) and tumor necrotic factor- (9 pg/ml vs 39 pg/ml; p=0.0253) levels. Itolizumab was generally safe and well tolerated, and transient lymphopenia (11 patients in Arm A) and infusion reactions (7 patients) were the commonly reported treatment related safety events. These encouraging results indicate that larger clinical trials are warranted to establish the role of Itolizumab in controlling immune hyperactivation in COVID-19.

Clinico-pathological features in fatal Covid-19 Infection: A Preliminary Experience of a Tertiary Care Centre in North India using Post-Mortem Minimally Invasive Tissue Biopsies (preprint)

Background: The Covid-19 pandemic began in China in December 2019. India is the second most affected country, as of November 2020 with more than a 8.5million cases. Covid-19 infection primarily involves the lung with the severity of illness varying from influenza-like illness to acute respiratory distress syndrome. Other organs have also found to be variably affected. Studies evaluating the histopathological changes of Covid-19 are critical in providing a better understanding of the disease pathophysiology and guiding treatment. Minimally invasive biopsy techniques (MITS/B) provide an easy and suitable alternative to complete autopsies. In this prospective single-center study we present the histopathological examination of 37 patients who died with complications of Covid-19. Methods: This was an observational study conducted in the Intensive Care Unit of JPN Trauma Centre AIIMS. A total of 37 patients who died of Covid-19 were enrolled in the study. Post-mortem percutaneous biopsies were taken with the help of surface landmarking/ultrasonography guidance from lung, heart, liver, and kidneys; after obtaining ethical consent. The biopsy samples were then stained with haematoxylin and eosin stain. Immunohistochemistry (IHC) was performed using CD61 and CD163 in all lung cores. SARS-CoV-2 virus was detected using IHC with primary antibodies in selected samples. Details regarding demographics, clinical parameters, hospital course, treatment details, and laboratory investigations were also collected for clinical correlation. Results: A total of 37 patients underwent post-mortem minimally invasive tissue sampling. Mean age of the patients was 48.7years and 59.5% of them were males. Respiratory failure was the most common complication seen in 97.3%. Lung histopathology showed acute lung injury and diffuse alveolar damage in 78% of patients. Associated bronchopneumonia was seen in 37.5% of patients and scattered microthrombi were visualized in 21% of patients. Immunostaining with CD61 and CD163 highlighted megakaryocytes and increased macrophages in all samples. Immunopositivity for SARS-CoV-2 was observed in Type II pneumocytes. Acute tubular injury with epithelial vacuolization was seen in 46% of the renal biopsies but none of them showed evidence of microvascular thrombosis. 71% of the liver tissue cores showed evidence of Kupfer cell hyperplasia. 27.5% had evidence of submassive hepatic necrosis and 14% had features of acute on chronic liver failure. All the heart biopsies showed non-specific features such as hypertrophy with nucleomegaly with no evidence of myocardial necrosis in any of the samples. Conclusions The most common finding in this cohort is the diffuse alveolar damage with demonstration of SARS-CoV-2 protein in the acute phase of DAD. Microvascular thrombi were rarely identified in the lung, liver and kidney. Substantial hepatocyte necrosis, hepatocyte degeneration, Kupffer cell hypertrophy, micro, and macrovesicular steatosis unrelated to microvascular thrombi suggests that liver might be a primary target of Covid-19. This study highlights the importance of MITS/B in better understanding the pathological changes associated with Covid-19.

Outcome of Conservative Therapy in COVID-19 Patients Presenting with Gastrointestinal Bleeding (preprint)

Background/ObjectiveThere is a paucity of data on the management of gastrointestinal (GI) bleeding in patients with COVID-19 amid concerns about the risk of transmission during endoscopic procedures. We aimed to study the outcomes of conservative treatment for GI bleeding in patients with COVID-19. MethodsIn this retrospective analysis, 24 of 1342 (1.8%) patients with COVID-19, presenting with GI bleeding from 22 April to 22 July 2020, were included. ResultsThe mean age of patients was 45.8{+/-}12.7 years; 17 (70.8%) were males; upper GI (UGI) bleeding: lower GI (LGI) 23:1. Twenty-two (91.6%) patients had evidence of cirrhosis-21 presented with UGI bleeding while one had bleeding from hemorrhoids. Two patients without cirrhosis were presumed to have non-variceal bleeding. The medical therapy for UGI bleeding included vasoconstrictors-somatostatin in 17 (73.9%) and terlipressin in 4 (17.4%) patients. All patients with UGI bleeding received proton pump inhibitors and antibiotics. Packed red blood cells (PRBCs), fresh frozen plasma and platelets were transfused in 14 (60.9%), 3 (13.0%) and 3 (13.0%), respectively. The median PRBCs transfused was 1 (0-3) unit(s). The initial control of UGI bleeding was achieved in all 23 patients and none required an emergency endoscopy. At 5-day follow-up, none rebled or died. Two patients later rebled, one had intermittent bleed due to gastric antral vascular ectasia, while another had rebleed 19 days after discharge. Three (12.5%) cirrhosis patients succumbed to acute hypoxemic respiratory failure during hospital stay. ConclusionConservative management strategies including pharmacotherapy, restrictive transfusion strategy, and close hemodynamic monitoring can successfully manage GI bleeding in COVID-19 patients and reduce need for urgent endoscopy. The decision for proceeding with endoscopy should be taken by a multidisciplinary team after consideration of the patients condition, response to treatment, resources and the risks involved, on a case to case basis.

Poor Outcomes in Patients with Cirrhosis and COVID-19 (preprint)

Background and AimThere is a paucity of data on the clinical presentations and outcomes of Coronavirus disease 2019(COVID-19) in patients with underlying liver disease. We aimed to summarize the presentations and outcomes of COVID-19 positive patients and compare with historical controls.MethodsPatients with known chronic liver disease who presented with superimposed COVID- 19(n=28) between 22nd April and 22nd June 2020 were studied. Seventy-eight cirrhotic patients from historical controls were taken as comparison group.ResultsA total of 28 COVID patients- two without cirrhosis, one with compensated cirrhosis, sixteen with acute decompensation (AD), and nine with acute-on-chronic liver failure(ACLF) were included. The etiology of cirrhosis was alcohol(n=9), non-alcoholic fatty liver disease(n=2), viral(n=5), autoimmune hepatitis(n=4), and cryptogenic cirrhosis(n=6). The clinical presentations included complications of cirrhosis in 12(46.2%), respiratory symptoms in 3(11.5%) and combined complications of cirrhosis and respiratory symptoms in 11(42.3%) patients. The median hospital stay was 8(7-12) days. The mortality rate in COVID-19 patients was 42.3%(11/26), as compared to 23.1%(18/78) in the historical controls(p=0.077). All COVID-19 patients with ACLF(9/9) died compared to 53.3%(16/30) in ACLF of historical controls(p=0.015). Mortality rate was higher in COVID patients with compensated cirrhosis and AD as compared to historical controls 2/17(11.8%) vs 2/48(4.2%), though not statistically significant (p=0.278). Requirement of mechanical ventilation independently predicted mortality (hazard ratio, 13.68). Both non-cirrhotic patients presented with respiratory symptoms and recovered uneventfully.ConclusionCOVID-19 is associated with poor outcomes in patients with cirrhosis, with worst survival rates in ACLF. Mechanical ventilation is associated with a poor outcome.