Pressured cytotoxic T cell epitope strength among SARS-CoV-2 variants correlates with COVID-19 severity.

Heterogeneity in susceptibility among individuals to COVID-19 has been evident through the pandemic worldwide. Cytotoxic T lymphocyte (CTL) responses generated against pathogens in certain individuals are known to impose selection pressure on the pathogen, thus driving emergence of new variants. In this study, we probe the role played by host genetic heterogeneity in terms of HLA-genotypes in determining differential COVID-19 severity in patients. We use bioinformatic tools for CTL epitope prediction to identify epitopes under immune pressure. Using HLA-genotype data of COVID-19 patients from a local cohort, we observe that the recognition of pressured epitopes from the parent strain Wuhan-Hu-1 correlates with COVID-19 severity. We also identify and rank list HLA-alleles and epitopes that offer protectivity against severe disease in infected individuals. Finally, we shortlist a set of 6 pressured and protective epitopes that represent regions in the viral proteome that are under high immune pressure across SARS-CoV-2 variants. Identification of such epitopes, defined by the distribution of HLA-genotypes among members of a population, could potentially aid in prediction of indigenous variants of SARS-CoV-2 and other pathogens.

Simple and low-cost nucleic acid extraction methods for detection of SARS-CoV2 in self-collected saliva and dry oral swabs.

Background: Ongoing need of alternative strategies for SARS-CoV-2 detection is undeniable. Self-collected samples without viral transport media (VTM), coupled with simple nucleic acid extraction methods for SARS-CoV-2 PCR are beneficial. Objectives: To evaluate results of SARS-CoV-2 PCR using simple nucleic acid extraction methods from self -collected saliva and oral swabs without VTM. Methods: A cross-sectional single-centre study was conducted on 125 participants (101 SARS-CoV-2 positive cases and 24 controls). PCR was performed following five simple nucleic acid extraction methods on self -collect saliva and oral swabs without VTM and results were compared with gold standard PCR. For saliva, kit-based extraction (SKE), Proteinase K and Heat extraction (SPHE), only Heat extraction (SHE) methods and for dry oral swabs, Proteinase K and Heat extraction (DPHE) and only Heat extraction (DHE) was performed. Results: SARS-CoV-2 was detected in self-collected saliva and oral swabs. 93.07% were correctly classified as positive by SKE, 69.31% by SHE, 67.33% by SPHE, 67.33% by DPHE and 55.45% by DHE. Discriminant power of SKE was significantly higher than other methods (p-value < 0.001) with good- fair agreement of alternate extraction methods against gold standard. Conclusion: Combination of self-collected saliva/ oral-swab without VTM and alternative RNA extraction methods offer a simplified, economical substitute strategy for SARS-CoV-2 detection.

A 9-gene biomarker panel identifies bacterial coinfections in culture-negative COVID-19 cases.

Confirmatory diagnosis of bacterial coinfections with COVID-19 is challenging due to the limited specificity of the widely used gold-standard culture sensitivity test despite clinical presentations. A misdiagnosis can either lead to increased health complications or overuse of antibiotics in COVID-19 patients. With a multi-step systems biology pipeline, we have identified a 9-gene biomarker panel from host blood that can identify bacterial coinfection in COVID-19 patients, even in culture-negative cases. We have also formulated a qPCR-based score that diagnoses bacterial coinfection with COVID-19 with the accuracy, specificity, and sensitivity of 0.93, 0.96, and 0.89, respectively. This gene signature and score can assist in the clinical decision-making process of necessary and timely prescription of antibiotics in suspected bacterial coinfection cases with COVID-19 and thereby help to reduce the associated morbidity and mortality.

Evaluation of hospital environment for presence of Mucorales during COVID-19-associated mucormycosis outbreak in India - a multi-centre study.

BACKGROUND: An unprecedented rise in the number of COVID-19-associated mucormycosis (CAM) cases has been reported in India. Myriad hypotheses are proposed for the outbreak. We recently reported uncontrolled diabetes and inappropriate steroid therapy as significant risk factors for the outbreak. However, Mucorales contamination of hospital environment was not studied. AIM: To perform a multi-centre study across India to determine possible Mucorales contamination of hospital environment during the outbreak. METHODS: Eleven hospitals from four zones of India representing high to low incidence for mucormycosis cases were included in the study. Samples from a variety of equipment used by the patients and ambient air were collected during May 19th, 2021 through August 25th, 2021. FINDINGS: None of the hospital equipment sampled was contaminated with Mucorales. However, Mucorales were isolated from 11.1% air-conditioning vents and 1.7% of patients' used masks. Other fungi were isolated from 18% of hospital equipment and surfaces, and 8.1% of used masks. Mucorales grew from 21.7% indoor and 53.8% outdoor air samples. Spore counts of Mucorales in air were significantly higher in the hospitals of North and South zones compared to West and East zones (P < 0.0001). Among Mucorales isolated from the environment, Rhizopus spp. were the most frequent genus. CONCLUSION: Contamination of air-conditioning vents and hospital air by Mucorales was found. Presence of Mucorales in these areas demands regular surveillance and improvement of hospital environment, as contamination may contribute to healthcare-associated mucormycosis outbreaks, especially among immunocompromised patients.

Predicted CTL responses from pressured epitopes in SARS-CoV-2 correlate with COVID-19 severity (preprint)

Heterogeneity in susceptibility among individuals to COVID-19 has been evident through the pandemic worldwide. Protective cytotoxic T lymphocyte (CTL) responses generated against pathogens in certain individuals are known to impose selection pressure on the pathogen, thus driving emergence of new variants. In this study, we focus on the role played by host genetic heterogeneity in terms of HLA-genotypes in determining differential COVID-19 severity in patients and dictating mechanisms of immune evasion adopted by SARS-CoV-2 due to the imposed immune pressure at global and cohort levels. We use bioinformatic tools for CTL epitope prediction to identify epitopes under immune pressure. Using HLA-genotype data of COVID-19 patients from a local cohort, we observe that asymptomatic individuals recognize a larger number of pressured epitopes which could facilitate emergence of mutations at these epitopic regions to overcome the protectivity they offer to the host. Based on the severity of COVID-19, we also identify HLA-alleles and epitopes that offer higher protectivity against severe disease in infected individuals. Finally, we shortlist a set of pressured and protective epitopes that represent regions in the viral proteome that are under higher immune pressure across SARS-CoV-2 variants due to the protectivity they offer. Identification of such epitopes could potentially aid in prediction of indigenous variants of SARS-CoV-2 and other pathogens, defined by the distribution of HLA-genotypes among members of a population.

Laboratory preparedness for SARS-CoV-2 testing in India: Harnessing a network of Virus Research & Diagnostic Laboratories.

Background & objectives: An outbreak of respiratory illness of unknown aetiology was reported from Hubei province of Wuhan, People's Republic of China, in December 2019. The outbreak was attributed to a novel coronavirus (CoV), named as severe acute respiratory syndrome (SARS)-CoV-2 and the disease as COVID-19. Within one month, cases were reported from 25 countries. In view of the novel viral strain with reported high morbidity, establishing early countrywide diagnosis to detect imported cases became critical. Here we describe the role of a countrywide network of VRDLs in early diagnosis of COVID-19. Methods: The Indian Council of Medical Research (ICMR)-National Institute of Virology (NIV), Pune, established screening as well as confirmatory assays for SARS-CoV-2. A total of 13 VRDLs were provided with the E gene screening real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay. VRDLs were selected on the basis of their presence near an international airport/seaport and their past performance. The case definition for testing included all individuals with travel history to Wuhan and symptomatic individuals with travel history to other parts of China. This was later expanded to include symptomatic individuals returning from Singapore, Japan, Hong Kong, Thailand and South Korea. Results: Within a week of standardization of the test at NIV, all VRDLs could initiate testing for SARS-CoV-2. Till February 29, 2020, a total of 2,913 samples were tested. This included both 654 individuals quarantined in the two camps and others fitting within the case definition. The quarantined individuals were tested twice - at days 0 and 14. All tested negative on both occasions. Only three individuals belonging to different districts in Kerala were found to be positive. Interpretation & conclusions: Sudden emergence of SARS-CoV-2 and its potential to cause a pandemic posed an unsurmountable challenge to the public health system of India. However, concerted efforts of various arms of the Government of India resulted in a well-coordinated action at each level. India has successfully demonstrated its ability to establish quick diagnosis of SARS-CoV-2 at NIV, Pune, and the testing VRDLs.