Development of robust, indigenous ELISA for detection of IgG antibodies against CoV-2 N and S proteins: mass screening.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has adversely affected humankind and caused millions of deaths globally since January 2020. Robust and quick serological tests such as antibody detection assays for SARS-CoV-2 provide relevant information and aid in the process of vaccine development and diagnostics, as well as in sero-epidemiological monitoring of antibody response to the virus. The receptor-binding domain (RBD) of spike and nucleocapsid protein are specific targets for detecting SARS-CoV-2 antibodies. Here, we present the development of a stable spike (S) and nucleocapsid (N) protein-based ELISA antibody detection test "CoroSuchak," with 99% sensitivity, 98% specificity, cost-effective, and detection in a minimum time for serodiagnosis and mass screening of the population for antibodies against SARS-CoV-2. Blood samples were analyzed from 374 SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) positive, 772 negative and asymptomatic, and 874 random groups of subjects. We found that the antibody titer was significantly higher (p < 0.0001) in infected and vaccinated group compared to the only vaccinated and only infected group. Using enzyme-linked immunosorbent assay (ELISA), we detected SARS-CoV-2 immunoglobulin G (IgG) antibodies in 118/123 (96%) infected individuals, 570/653 (87%) non-infected but vaccinated individuals, 231/237 (97%) individuals who were both infected and vaccinated, and 499/874 (57%) from randomly selected individuals from the first and second waves of the pandemic. Similarly in the third wave, 14/14 (100%) infected and 16/20 (80%) RT-PCR-negative but symptomatic subjects were detected. Thus, the highly sensitive and specific in-house developed ELISA antibody detection kit "CoroSuchak" is extremely useful to determine the seroprevalence of SARS-CoV-2 antibodies in the coronavirus-exposed population. KEY POINTS: •Indigenous kit using a combination of spike and nucleocapsid proteins and peptide sequences. •High sensitivity and specificity to detect variants. •Highly sensitive for mass screening.

Impact of high altitude on composition and functional profiling of oral microbiome in Indian male population.

The oral cavity of human contains bacteria that are critical for maintaining the homeostasis of the body. External stressors such as high altitude (HA) and low oxygen affect the human gut, skin and oral microbiome. However, compared to the human gut and skin microbiome, studies demonstrating the impact of altitude on human oral microbiota are currently scarce. Alterations in the oral microbiome have been reported to be associated with various periodontal diseases. In light of the increased occurrence of HA oral health related problems, the effect of HA on the oral salivary microbiome was investigated. We conducted a pilot study in 16 male subjects at two different heights i.e., H1 (210 m) and H2 (4420 m). Total of 31 saliva samples,16 at H1 and 15 at H2 were analyzed by utilizing the 16S rRNA high-throughput sequencing, to explore the relationship between the HA environment and salivary microbiota. The preliminary results suggesting that, the most abundant microbiome at the phylum level are: Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Interestingly, 11 genera were identified at the both heights with different relative abundances. In addition, the salivary microbiome was more diverse at H1 compared to H2 as demonstrated by decreased alpha diversity. Further, predicted functional results indicate that microbial metabolic profiles significantly decreased at H2 as compared to H1, including two major metabolic pathways involving carbohydrates, and amino acids. Our findings show that HA induces shifts in the composition and structure of human oral microbiota which can affect host health homeostasis.

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.

Probiotics maintain the gut microbiome homeostasis during Indian Antarctic expedition by ship.

Ship voyage to Antarctica is a stressful journey for expedition members. The response of human gut microbiota to ship voyage and a feasible approach to maintain gut health, is still unexplored. The present findings describe a 24-day long longitudinal study involving 19 members from 38th Indian Antarctic Expedition, to investigate the impact of ship voyage and effect of probiotic intervention on gut microbiota. Fecal samples collected on day 0 as baseline and at the end of ship voyage (day 24), were analyzed using whole genome shotgun sequencing. Probiotic intervention reduced the sea sickness by 10% compared to 44% in placebo group. The gut microbiome in placebo group members on day 0 and day 24, indicated significant alteration compared to a marginal change in the microbial composition in probiotic group. Functional analysis revealed significant alterations in carbohydrate and amino acid metabolism. Carbohydrate-active enzymes analysis represented functional genes involved in glycoside hydrolases, glycosyltransferases and carbohydrate binding modules, for maintaining gut microbiome homeostasis. Suggesting thereby the possible mechanism of probiotic in stabilizing and restoring gut microflora during stressful ship journey. The present study is first of its kind, providing a feasible approach for protecting gut health during Antarctic expedition involving ship voyage.

Effects of Acute Exposure to Hypobaric Hypoxia on Mucosal Barrier Injury and the Gastrointestinal Immune Axis in Rats.

High altitude-induced gastrointestinal (GI) problems are potentially life-threatening. GI tract bleeding and inflammation are the major problems induced by hypobaric hypoxia (HH). In this study, effects of acute exposure to HH up to 14 days at 7620 m on GI immune function have been studied. To fulfill these objectives, Sprague-Dawley (SD) rats were divided into five groups namely Control and HH exposed (1, 3, 7, and 14 days). All groups except control were exposed to 7620 m of HH in an animal decompression chamber for the respective time intervals. Different degrees of intestinal mucosal damage in terms of increased mucosal permeability and disruption of intestinal villi were observed for different time intervals. HH exposure also upregulated secretory immunoglobulin A (sIgA) and proinflammatory cytokines in GI lavage along with proinflammatory markers such as toll-like receptor 4 (TLR4) and inducible nitric oxide synthase (iNOS). HH exposure of rats for 7 days significantly increased interleukin-17 (IL-17) and natural killer (NK) cell and dendritic cell populations compared with unexposed control rats. However, the number of naive T cells was significantly decreased in Peyer's patches. Our results connect HH to GI immune axis and highlight Th17 cells and proinflammatory molecules as potential therapeutic targets to counteract HH-induced GI dysfunction.