Association of Angiotensin Converting Enzyme 2 (ACE2) Gene Polymorphisms with Disease Severity in Indian Covid-19 Patients: A Next-gen Sequencing Approach

The clinical picture of COVID-19 ranges from asymptomatic to mild, moderate or severe disease sometimes leading to death. Differences in the interaction between SARS-CoV-2 Spike (S) protein and angiotensin converting enzyme 2 (ACE2) protein may lead to differences in disease severity. We studied whether ACE2 polymorphisms are associated with disease severity and outcome. We recruited 114 patients between July 2020 - March 2022 confirmed positive by RTPCR for COVID-19 with different degrees of severity (21 mild, 29 moderate, 34 severe, 30 death) and 30 controls (10 non-vaccinated+ 20 vaccinated) who were RT-PCR negative inspite of high-risk contact. Next-gen sequencing was done on MiSeq (Illumina) using amplicon-based targeted sequencing approach using a custom-designed panel to sequence all the exons of ACE2 gene. SPSS ver.26 was used for analysis. The following ACE2 variants were identified on the Local Run Manager (LRM) software from Illumina: (i) rs2285666 (c.439+4G>A) splice region variant, in controls (60%) and Patients (45.8%), (ii) rs4646140 (c.802+24G>A) intronic variant in 4/114 patients and 1/30 controls, (iii) rs41303171 ( c.2158A>G) missense variant in 2/114 patients, (iv) rs536749578 (c.2114+9T>C) intron variant, (v) rs763994205 (c.868A>C) missense variant and (vi) rs7595907 (c.656G>A) missense variant in 1/114 patient each only. rs2285666 was observed in equal frequency ( 60%) in vaccinated and non-vaccinated controls. rs2285666 was observed amongst different severity groups: Mild (80.95%), Moderate (37.93%), Severe (44.11%), and Death (56.67%) revealing association with disease severity, probably having a protective effect. However, these results need to be confirmed on larger sample sizes.

A study on the morbid histopathological changes in COVID-19 patients with or without comorbidities using minimally invasive tissue sampling

Introduction: COVID-19 causes morbid pathological changes in different organs including lungs, kidney, liver,etc especially in those who succumb. Though clinical outcomes in those with comorbidities are known to be different from those without - not much is known about the differences at histopathological level. Aim(s): It was to compare the morbid histopathological changes in COVID-19 patients between those who were immunocompromised(Gr 1), malignancy(Gr 2) or had cardiometabolic conditions (hypertension, diabetes or coronary artery disease)(Gr 3). Method(s): Post-mortem tissue sampling (MITS) was done from the lungs, kidney, heart, and liver using biopsy gun within two hours of death. Routine (H & E stain) and special stains (AFB, SM, PAS) were done besides immunohistochemistry. Result(s): A total of 100 patients underwent MITS and data of 92 were included (immunocompromised: 27, maligancy:18, cardiometabolic conditions:71). Within lung histopathology, capillary congestion was more in those with malignancy while others like diffuse alveolar damage, microthrombi, pneumocyte hyperplasia etc was equally distributed. Within liver, architecture distortion was significantly different in immunocompromised while steatosis, portal inflammation, Kupffer cell hypertrophy, confluent necrosis were equally distributed. There was a trend towards higher acute tubular injury in those with cardiometabolic conditions as compared to the other groups. No significant histopathological differences in heart was discerned. Conclusion(s): Certain histopathological features are markedly different in different groups (Gr 1,2 and3)of COVID-19 patients with fatal outcome.

An inexpensive and rapid diagnostic method for detection of SARS-CoV-2 RNA by loop-mediated isothermal amplification (LAMP).

SARS-CoV-2 is a public pandemic health concern globally. Nasopharyngeal and oropharyngeal swab samples are used for Covid-19 viral detection. Sample collection procedure was tedious and uncomfortable and unsuitable for biochemical and CBC analysis in swab samples. Biochemistry and CBC tests are key determinant in management of Covid-19 patients. We developed a LAMP test to detect viral RNA in blood samples. LAMP is required four specific primers targeting the internal transcribed S-region and loop primers for viral RNA amplification. RNA was extracted from blood samples by TRIzol method. LAMP reaction was performed at 60 °C for 1 hour and amplicons were visualized in HNB dye. No cross-reactivity was seen with HBV, HCV, and HIV infected sample. Out of 40 blood samples, 33 samples were positive for LAMP and Q-PCR analysis, one sample was positive for LAMP and negative for Q-PCR, two samples were negative for LAMP but positive for Q-PCR, and four blood samples were negative for LAMP and Q-PCR. LAMP method has an accuracy of 92.50%, with sensitivity and specificity of 94.28% and 80%, respectively. Thus, LAMP diagnostic test has proved reliable, fast, inexpensive and can be useful for detection where the limited resources available.•LAMP method is a potential tool for detection of SARS-CoV-2.•Blood samples are the key determinant for routine diagnostics as well as molecular diagnostics.•LAMP assay is an appropriate diagnostics method which offers greater simplicity, low cost, sensitivity, and specificity than other methods in molecular diagnostics.

ACE2 protein expression in lung tissues of severe COVID-19 infection.

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 COrona VIrus Disease 2019 (COVID-19). 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-19 patients for other indications. IHC for CD61 and CD163 was performed for the assessment of platelet-rich microthrombi and macrophages, respectively. IHC for SARS-CoV-2 viral antigen was also performed. In a total of 55, 44 COVID-19 post-mortem lung samples were tested for ACE2, 36 for CD163, and 26 for CD61, compared to 15 non-covid 19 control lung sections. Quantification of immunostaining, random sampling, and correlation analysis were used to substantiate the morphologic findings. Our results show that ACE2 protein expression was significantly higher in COVID-19 post-mortem lung tissues 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 were 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.

Clinico-pathological features in fatal COVID-19 infection: a preliminary experience of a tertiary care center in North India using postmortem minimally invasive tissue sampling.

OBJECTIVES: To study the histopathology of patients dying of COVID-19 using post-mortem minimally invasive sampling techniques. METHODS: This was a single-center observational study conducted at JPNATC, AIIMS. Thirty-seven patients who died of COVID-19 were enrolled. Post-mortem percutaneous biopsies were taken from lung, heart, liver, kidney and stained with hematoxylin and eosin. Immunohistochemistry was performed using CD61 and CD163. SARS-CoV-2 virus was detected using IHC with primary antibodies. RESULTS: The mean age was 48.7 years and 59.5% were males. Lung histopathology showed diffuse alveolar damage in 78% patients. Associated bronchopneumonia was seen in 37.5% and scattered microthrombi in 21% patients. Immunopositivity for SARS-CoV-2 was observed in Type II pneumocytes. Acute tubular injury with epithelial vacuolization was seen in 46% of renal biopsies. Seventy-one percent of liver biopsies showed Kupffer cell hyperplasia and 27.5% showed submassive hepatic necrosis. CONCLUSIONS: Predominant finding was diffuse alveolar damage with demonstration of SARS-CoV-2 protein in the acute phase. Microvascular thrombi were rarely identified in any organ. Substantial hepatocyte necrosis, Kupffer cell hypertrophy, microvesicular, and macrovesicular steatosis unrelated to microvascular thrombi suggested that liver might be a primary target of COVID-19.