A comparative analysis of the second and third wave of the Covid-19 pandemic: an experience from a tertiary care hospital in Western India.

Introduction. As the world was still recovering from the 2020 pandemic, the devastating impact of Covid-19 driven by the Delta variant shook the world in 2021. As the second wave was declining, there was an unusual surge in Covid-19 positive cases by the end of 2021 which led to global concern about the change in virus characteristics.Hypothesis/gap statement. Whole genome sequencing is critical for understanding a rapidly progressing pandemic.Aim. To provide an insight into the major differences encountered in the changing characteristics between the second and third waves of the pandemic at a tertiary care hospital in India.Methods. A retrospective observational cohort analysis was conducted on Covid-positive patients during the second wave of the Covid-19 pandemic (from March 2021 to April 2021) and the third wave of the Covid-19 pandemic (from December 2021 to January 2022).Results. Out of 303 Covid-19 positive cases, 52 samples were tested by whole genome sequencing during the second wave and 108 during the third wave. A decline of 18.5 % was observed in the case fatality rate from the second wave to the third wave. There was a 5 % decline in the number of patients admitted with ARDS and a 16.3 % decline in the number of patients with co-morbidities.In total, 51.9 percent of cases were due to the Delta variant during the second wave and 95 percent due to the Omicron variant during the third wave. We found that 36.5 % of Covid-positive patients during the second wave had been vaccinated compared to 40 % in the third wave.Conclusion. Whole genome sequencing of clinical samples from a wide range of individuals during a viral epidemic will enable us to develop a more rapid public health response to new variants and identify the required vaccine modifications more quickly.

SARS-CoV-2 variants: Impact on biological and clinical outcome.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that was first identified in December 2019, in Wuhan, China was found to be the etiological agent for a novel respiratory infection that led to a Coronavirus Induced Disease named COVID-19. The disease spread to pandemic magnitudes within a few weeks and since then we have been dealing with several waves across the world, due to the emergence of variants and novel mutations in this RNA virus. A direct outcome of these variants apart from the spike of cases is the diverse disease presentation and difficulty in employing effective diagnostic tools apart from confusing disease outcomes. Transmissibility rates of the variants, host response, and virus evolution are some of the features found to impact COVID-19 disease management. In this review, we will discuss the emerging variants of SARS-CoV-2, notable mutations in the viral genome, the possible impact of these mutations on detection, disease presentation, and management as well as the recent findings in the mechanisms that underlie virus-host interaction. Our aim is to invigorate a scientific debate on how pathogenic potential of the new pandemic viral strains contributes toward development in the field of virology in general and COVID-19 disease in particular.

Humoral Immune Response Profile of COVID-19 Reveals Severity and Variant-Specific Epitopes: Lessons from SARS-CoV-2 Peptide Microarray.

The amaranthine scale of the COVID-19 pandemic and unpredictable disease severity is of grave concern. Serological diagnostic aids are an excellent choice for clinicians for rapid and easy prognosis of the disease. To this end, we studied the humoral immune response to SARS-CoV-2 infection to map immunogenic regions in the SARS-CoV-2 proteome at amino acid resolution using a high-density SARS-CoV-2 proteome peptide microarray. The microarray has 4932 overlapping peptides printed in duplicates spanning the entire SARS-CoV-2 proteome. We found 204 and 676 immunogenic peptides against IgA and IgG, corresponding to 137 and 412 IgA and IgG epitopes, respectively. Of these, 6 and 307 epitopes could discriminate between disease severity. The emergence of variants has added to the complexity of the disease. Using the mutation panel available, we could detect 5 and 10 immunogenic peptides against IgA and IgG with mutations belonging to SAR-CoV-2 variants. The study revealed severity-based epitopes that could be presented as potential prognostic serological markers. Further, the mutant epitope immunogenicity could indicate the putative use of these markers for diagnosing variants responsible for the infection.

SARS-CoV-2 variants: Impact on biological and clinical outcome

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that was first identified in December 2019, in Wuhan, China was found to be the etiological agent for a novel respiratory infection that led to a Coronavirus Induced Disease named COVID-19. The disease spread to pandemic magnitudes within a few weeks and since then we have been dealing with several waves across the world, due to the emergence of variants and novel mutations in this RNA virus. A direct outcome of these variants apart from the spike of cases is the diverse disease presentation and difficulty in employing effective diagnostic tools apart from confusing disease outcomes. Transmissibility rates of the variants, host response, and virus evolution are some of the features found to impact COVID-19 disease management. In this review, we will discuss the emerging variants of SARS-CoV-2, notable mutations in the viral genome, the possible impact of these mutations on detection, disease presentation, and management as well as the recent findings in the mechanisms that underlie virus-host interaction. Our aim is to invigorate a scientific debate on how pathogenic potential of the new pandemic viral strains contributes toward development in the field of virology in general and COVID-19 disease in particular.

Development of a PNA-DiSc2 based portable absorbance platform for the detection of pathogen nucleic acids.

We report the development of a portable absorption (PortAbs)-based pathogen nucleic acid detection system using peptide nucleic acid (PNA) and a cyanine dye, DiSc2(5). When the dye binds to the PNA-DNA hybrid, it results in a characteristic ∼110 nm shift in the dye absorbance, which we measure using PortAbs. The protocol involves amplification of the target DNA, PNA-DNA hybridization and dye complexing steps followed by absorption measurement. The system is built using a broad-spectrum photodiode whose output is amplified and then measured by a high resolution (24 or 32 bit) analog-to-digital converter. The excitation pulses of light are delivered by a color-changing LED. The sequence of excitation, measurement and display of results are all controlled by an embedded Raspberry-Pi board (or alternatively a laptop). At higher concentrations of the target amplicon (∼200 ng), the color change can be detected visually. At lower concentrations, PortAbs outperforms a plate reader and can detect target DNA as low as 30 ng or approximately 10 nM which is at least 10 fold better than previously reported studies. We validate the methodology using SARS-CoV-2 clinical samples containing about 1000 copies of the viral RNA and show that the entire workflow takes about 90 min. The cost of the complete standalone system is less than INR 40 000 (approx. 500 USD).

SARS-CoV-2 severity classification from plasma sample using confocal Raman spectroscopy.

The world is on the brink of facing coronavirus's (COVID-19) fourth wave as the mutant forms of viruses are escaping neutralizing antibodies in spite of being vaccinated. As we have already witnessed that it has encumbered our health system, with hospitals swamped with infected patients observed during the viral outbreak. Rapid triage of patients infected with SARS-CoV-2 is required during hospitalization to prioritize and provide the best point of care. Traditional diagnostics techniques such as RT-PCR and clinical parameters such as symptoms, comorbidities, sex and age are not enough to identify the severity of patients. Here, we investigated the potential of confocal Raman microspectroscopy as a powerful tool to generate an expeditious blood-based test for the classification of COVID-19 disease severity using 65 patients plasma samples from cohorts infected with SARS-CoV-2. We designed an easy manageable blood test where we used a small volume (8 µl) of inactivated whole plasma samples from infected patients without any extra solvent usage in plasma processing. Raman spectra of plasma samples were acquired and multivariate exploratory analysis PC-LDA (principal component based linear discriminant analysis) was used to build a model, which segregated the severe from the non-severe COVID-19 group with a sensitivity of 83.87%, specificity of 70.60% and classification efficiency of 76.92%. Among the bands expressed in both the cohorts, the study led to the identification of Raman fingerprint regions corresponding to lipids (1661, 1742), proteins amide I and amide III (1555, 1247), proteins (Phe) (1006, 1034), and nucleic acids (760) to be differentially expressed in severe COVID-19 patient's samples. In summary, the current study exhibits the potential of confocal Raman to generate simple, rapid, and less expensive blood tests to triage the severity of patients infected with SARS-CoV-2.

Delta variant SARS-CoV-2 infections in pediatric cases during the second wave in India.

BACKGROUND: During October 2020, Delta variant was detected for the first time in India and rampantly spread across the globe. It also led to second wave of pandemic in India which affected millions of people. However, there is limited information pertaining to the SARS-CoV-2 strain infecting the children in India. METHODS: Here, we assessed the SARS-CoV-2 lineages circulating in the pediatric population of India during the second wave of the pandemic. Clinical and demographic details linked with the nasopharyngeal/oropharyngeal swabs (NPS/OPS) collected from SARS-CoV-2 cases (n = 583) aged 0-18 year and tested positive by real-time RT-PCR were retrieved from March to June 2021. RESULTS: Symptoms were reported among 37.2% of patients and 14.8% reported to be hospitalized. The E gene CT value had significant statistical difference at the point of sample collection when compared to that observed in the sequencing laboratory. Out of these 512 sequences 372 were VOCs, 51 were VOIs. Most common lineages observed were Delta, followed by Kappa, Alpha and B.1.36, seen in 65.82%, 9.96%, 6.83% and 4.68%, respectively in the study population. CONCLUSION: Overall, it was observed that Delta strain was the leading cause of SARS-CoV-2 infection in Indian children during the second wave of the pandemic. We emphasize on the need of continuous genomic surveillance in SARS-CoV-2 infection even amongst children.

COVID-19 Serological Survey-3 Prior to Second Wave in Mumbai, India.

Background: Subsequent to serosurveys 1 and 2 for COVID-19 carried out in three wards of Mumbai in July and August 2020, Municipal Corporation of Greater Mumbai conducted serosurvey 3 in March 2021. This was to identify the extent of exposure by testing specific IgG antibodies against COVID-19. Material and Methods: A cross-sectional study was conducted to find the prevalence of seropositivity in Mumbai, which included 10,197 samples belonging to patients visiting public dispensaries (slum population, 6006) and private (nonslum population, 4191) laboratories of Aapli Chikitsa network for blood investigations for non-COVID illnesses. The ward-wise number of unlinked anonymous samples from 24 wards was predecided by using probability proportionate sampling. The samples were collected using quota sampling technique as per predecided sample for each ward. These samples collected from nonimmunized individuals were tested for IgG antibodies at the Molecular Biology Laboratory of Kasturba Hospital for Infectious Diseases by chemiluminescence assay (CLIA) method. Results: The overall seropositivity was found to be 36.3% (41.6% in slum and 28.59% in nonslum population). It was more in city wards (38.28%) followed by western suburb (36.47%) and then eastern suburb wards (34.86%), matching with the proportion of cases in these wards during the study period. There was no significant difference in seropositivity among males and females and in different age groups. Conclusions: Seropositivity is higher in slum areas than nonslum areas. It has reduced in slum areas and increased in nonslum areas as compared to findings of serosurveys 1 and 2. This explains the detection of a greater number of cases from nonslum areas in the second wave. The average seropositivity of 36.3% justifies the necessity of immunization on a wider scale in the city. Periodic serosurveys are required at fixed intervals to monitor the trend of infection and level of herd immunity.

Isolation and Genomic Characterization of SARS-CoV-2 Omicron Variant Obtained from Human Clinical Specimens.

Due to the failure of virus isolation of the Omicron variant in Vero CCL-81 from the clinical specimens of COVID-19 cases, an initial in vivo and subsequent in vitro approach was utilized for the isolation of the virus. A total of 74 oropharyngeal/nasopharyngeal specimens were collected from SARS-CoV-2 positive international travellers and a contact case at Delhi and Mumbai, India. All the specimens were sequenced using next-generation sequencing and simultaneously inoculated onto Vero CCL-81 cells for virus isolation. Subsequently, two omicron positive specimens were inoculated into Syrian hamsters for two passages. The initial passage of the positive hamster specimens was inoculated onto Vero CCL-81 cells. The clinical specimens, hamster specimens, and Vero CCL-81 passages were sequenced to assess the mutational changes in different host species. The replication of the Omicron variant in hamsters was confirmed with the presence of a high viral load in nasal turbinate and lung specimens of both passages. The successful isolation of the virus from hamster specimens with Vero CCL-81 was observed with cytopathic effect in infected cells and high viral load in the cell suspension. The genome analysis revealed the presence of L212C mutation, Tyrosine 69 deletion, and C25000T nucleotide change in spike gene of hamster passage sequences and an absence of V17I mutation in E gene in hamster passage sequences, unlike human clinical specimen and Vero CCL-81 passages. No change was observed in the furin cleavage site in any of the specimen sequences, suggesting intact pathogenicity of the virus isolate. Our data demonstrated successful isolation of the Omicron variant with the in vivo method first followed by in vitro method. The virus isolate could be used in the future to explore different aspects of the Omicron variant.

Detection and isolation of SARS-CoV-2 Eta variant from the international travelers and local residents of India.

International travel has been the major source for the rapid spread of new SARS-CoV-2 variants across the globe. During SARS-CoV-2 genomic surveillance, a total of 212 SARS-CoV-2 positive clinical specimens were sequenced using next-generation sequencing. A complete SARS-CoV-2 genome could be retrieved from 90 clinical specimens. Of them, 14 sequences belonged to the Eta variant from clinical specimens of international travelers (n = 12) and local residents (n = 2) of India, and 76 belonged to other SARS-CoV-2 variants. Of all the Eta-positive specimens, the virus isolates were obtained from the clinical specimens of six international travelers. Many variants of interest have been found to cause substantial community transmission or cluster infections. The detection of this variant with lethal E484K mutation across the globe and India necessitates persistent genomic surveillance of the SARS-CoV-2 variants, which would aid in taking preventive action.

Opposing roles for sMAdCAM and IL-15 in COVID-19 associated cellular immune pathology.

Immune cell dysregulation and lymphopenia characterize COVID-19 pathology in moderate to severe disease. While underlying inflammatory factors have been extensively studied, homeostatic and mucosal migratory signatures remain largely unexplored as causative factors. In this study, we evaluated the association of circulating IL-6, soluble mucosal addressin cell adhesion molecule (sMAdCAM), and IL-15 with cellular dysfunction characterizing mild and hypoxemic stages of COVID-19. A cohort of SARS-CoV-2 infected individuals (n = 130) at various stages of disease progression together with healthy controls (n = 16) were recruited from COVID Care Centres (CCCs) across Mumbai, India. Multiparametric flow cytometry was used to perform in-depth immune subset characterization and to measure plasma IL-6 levels. sMAdCAM, IL-15 levels were quantified using ELISA. Distinct depletion profiles, with relative sparing of CD8 effector memory and CD4+ regulatory T cells, were observed in hypoxemic disease within the lymphocyte compartment. An apparent increase in the frequency of intermediate monocytes characterized both mild as well as hypoxemic disease. IL-6 levels inversely correlated with those of sMAdCAM and both markers showed converse associations with observed lympho-depletion suggesting opposing roles in pathogenesis. Interestingly, IL-15, a key cytokine involved in lymphocyte activation and homeostasis, was detected in symptomatic individuals but not in healthy controls or asymptomatic cases. Further, plasma IL-15 levels negatively correlated with T, B, and NK count suggesting a compensatory production of this cytokine in response to the profound lymphopenia. Finally, higher levels of plasma IL-15 and IL-6, but not sMAdCAM, were associated with a longer duration of hospitalization.

Immune profile and responses of a novel dengue DNA vaccine encoding an EDIII-NS1 consensus design based on Indo-African sequences.

The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens, and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1-4 and a dengue virus (DENV)-2 non-structural protein 1 (NS1) protein-coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on the whole genome sequencing of clinical isolates in India and complemented with data from Africa. This sequence was further optimized for protein expression. In silico structural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. The vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Assaying intracellular interferon (IFN)-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios, and immune gene profiling suggests a strong Th1-dominant immune response. Finally, the passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse-adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design by offering a novel vaccine candidate with a possible broad-spectrum protection and a successful clinical translation either as a stand alone or in a mix and match strategy.

Community transmission of SARS-CoV-2 with B.1.1.7 lineage in Mumbai, India.

The B.1.1.7 (Alpha) variant has been detected in Mumbai, India during February 2021. Subsequently, we retrieved 43 sequences from specimens of 51 COVID-19 cases from Mumbai. The sequence analysis revealed that the cases were mainly affected with Alpha variant which suggests its role in community transmission of SARS-CoV-2 in Mumbai, India.

Clinical Characterization and Genomic Analysis of Samples from COVID-19 Breakthrough Infections during the Second Wave among the Various States of India.

From March to June 2021, India experienced a deadly second wave of COVID-19, with an increased number of post-vaccination breakthrough infections reported across the country. To understand the possible reason for these breakthroughs, we collected 677 clinical samples (throat swab/nasal swabs) of individuals from 17 states/Union Territories of the country who had received two doses (n = 592) and one dose (n = 85) of vaccines and tested positive for COVID-19. These cases were telephonically interviewed and clinical data were analyzed. A total of 511 SARS-CoV-2 genomes were recovered with genome coverage of higher than 98% from both groups. Analysis of both groups determined that 86.69% (n = 443) of them belonged to the Delta variant, along with Alpha, Kappa, Delta AY.1, and Delta AY.2. The Delta variant clustered into four distinct sub-lineages. Sub-lineage I had mutations in ORF1ab A1306S, P2046L, P2287S, V2930L, T3255I, T3446A, G5063S, P5401L, and A6319V, and in N G215C; Sub-lineage II had mutations in ORF1ab P309L, A3209V, V3718A, G5063S, P5401L, and ORF7a L116F; Sub-lineage III had mutations in ORF1ab A3209V, V3718A, T3750I, G5063S, and P5401L and in spike A222V; Sub-lineage IV had mutations in ORF1ab P309L, D2980N, and F3138S and spike K77T. This study indicates that majority of the breakthrough COVID-19 clinical cases were infected with the Delta variant, and only 9.8% cases required hospitalization, while fatality was observed in only 0.4% cases. This clearly suggests that the vaccination does provide reduction in hospital admission and mortality.

Severe SARS-CoV-2 Breakthrough Reinfection With Delta Variant After Recovery From Breakthrough Infection by Alpha Variant in a Fully Vaccinated Health Worker.

Background: Post infection immunity and post vaccination immunity both confer protection against COVID-19. However, there have been many whole genome sequencing proven reinfections and breakthrough infections. Both are most often mild and caused by Variants of Concern (VOC). Methods: The patient in our study underwent serial COVID-19 RT-PCR, blood tests for serology, acute phase reactants, and chest imaging as part of clinical care. We interviewed the patient for clinical history and retrieved reports and case papers. We retrieved stored RT-PCR positive samples for whole genome sequencing (WGS) of SARS-CoV-2 from the patient's breakthrough infections and the presumed index case. Findings: The patient had three RT-PCR confirmed SARS-CoV-2 infections. Two breakthrough infections occurred in quick succession with the first over 3 weeks after complete vaccination with COVISHIELD and despite post-vaccination seroconversion. The first breakthrough infection was due to the Alpha variant and the second due to the Delta variant. The Delta variant infection resulted in hypoxia, hospitalization, and illness lasting seven weeks. Serial serology, acute phase reactants, and chest imaging supported WGS in establishing distinct episodes of infection. WGS established a fully vaccinated family member as the index case. Interpretation: The patient had an Alpha variant breakthrough infection despite past infection, complete vaccination, and seroconversion. Despite boosting after this infection, the patient subsequently had a severe Delta variant breakthrough infection. This was also a WGS proven reinfection and, therefore, a case of breakthrough reinfection. The patient acquired the infection from a fully vaccinated family member.

Clinical, Serological, Whole Genome Sequence Analyses to Confirm SARS-CoV-2 Reinfection in Patients From Mumbai, India.

Background: SARS-CoV-2 infection may not provide long lasting post-infection immunity. While hundreds of reinfections have reported only a few have been confirmed. Whole genome sequencing (WGS) of the viral isolates from the different episodes is mandatory to establish reinfection. Methods: Nasopharyngeal (NP), oropharyngeal (OP) and whole blood (WB) samples were collected from paired samples of four individuals who were suspected of SARS-CoV-2 reinfection based on distinct clinical episodes and RT-PCR tests. Details from their case record files and investigations were documented. RNA was extracted from the NP and OP samples and subjected to WGS, and the nucleotide and amino acid sequences were subjected to genome and protein-based functional annotation analyses. Serial serology was performed for Anti-N IgG, Anti- S1 RBD IgG, and sVNT (surrogate virus neutralizing test). Findings: Three patients were more symptomatic with lower Ct values and longer duration of illness. Seroconversion was detected soon after the second episode in three patients. WGS generated a genome coverage ranging from 80.07 to 99.7%. Phylogenetic analysis revealed sequences belonged to G, GR and "Other" clades. A total of 42mutations were identified in all the samples, consisting of 22 non-synonymous, 17 synonymous, two in upstream, and one in downstream regions of the SARS-CoV-2 genome. Comparative genomic and protein-based annotation analyses revealed differences in the presence and absence of specific mutations in the virus sequences from the two episodes in all four paired samples. Interpretation: Based on the criteria of genome variations identified by whole genome sequencing and supported by clinical presentation, molecular and serological tests, we were able to confirm reinfections in two patients, provide weak evidence of reinfection in the third patient and unable to rule out a prolonged infection in the fourth. This study emphasizes the importance of detailed analyses of clinical and serological information as well as the virus's genomic variations while assessing cases of SARS-CoV-2 reinfection.

A Multi-omics Longitudinal Study Reveals Alteration of the Leukocyte Activation Pathway in COVID-19 Patients.

Severe coronavirus disease 2019 (COVID-19) infection may lead to lung injury, multi-organ failure, and eventually death. Cytokine storm due to excess cytokine production has been associated with fatality in severe infections. However, the specific molecular signatures associated with the elevated immune response are yet to be elucidated. We performed a mass-spectrometry-based proteomic and metabolomic analysis of COVID-19 plasma samples collected at two time points. Using Orbitrap Fusion LC-MS/MS-based label-free proteomic analysis, we identified around 10 significant proteins, 32 significant peptides, and 5 metabolites that were dysregulated at the severe time points. Few of these proteins identified by quantitative proteomics were validated using the multiple reaction monitoring (MRM) assay. Integrated pathway analysis using distinct proteomic and metabolomic signatures revealed alterations in complement and coagulation cascade, platelet aggregation, myeloid leukocyte activation pathway, and arginine metabolism. Further, we highlight the role of leukocyte activation and arginine metabolism in COVID-19 pathogenesis and targeting these pathways for COVID-19 therapeutics.