A Pilot Seroprevalence Study Suggests Silent Zika virus Transmission in Bhopal Region of Central India.

BACKGROUND: Several sporadic cases and outbreaks of Zika virus disease have been reported from different states of India. OBJECTIVES: This paper explored the possibility of any ongoing transmission of Zika virus (ZIKV) in the Bhopal region of Central India, where the last outbreak of this disease was reported in 2018. MATERIALS AND METHODS: We screened a group of 75 febrile patients who had already tested negative for the locally endemic causes of fever like dengue, chikungunya, enteric fever, malaria, and scrub typhus and two groups of asymptomatic healthy individuals represented by blood donors (n = 75) and antenatal mothers (n = 75). We tested blood samples of febrile patients for ZIKV RNA using real-time polymerase chain reaction (PCR), and for the healthy individuals, we determined anti-zika immunoglobulin G (IgG) antibodies using enzyme-linked immunosorbent assay. RESULTS: ZIKV RNA was not detected in any of the 75 samples tested by real-time PCR assay. Among the voluntary blood donors and antenatal mothers, a total of 10 (15.38%) and 5 (6.66%) individuals were found to be seropositive for anti-ZIKV IgG antibodies, respectively. The seropositive group was found to have higher age 33.06 (±10.83) years as compared to seronegative individuals 26.60 (±5.12) years (P = 0.037). CONCLUSION: This study, which is the first survey of seroprevalence of anti-Zika antibodies from India, reports an overall seropositivity rate of 10% for anti-Zika antibodies among the healthy population, suggesting an ongoing, low level, silent transmission of ZIKV in the local community.

Th2-predominant immune response underlies the pathogenesis of Dengue.

BACKGROUND: Dengue is a rapidly emerging pandemic-prone disease, whose manifestations range from asymptomatic infection to life-threatening complications like Dengue Hemorrhagic Fever and Dengue Shock Syndrome. This study investigates and compares the immune response in clinically defined cohorts of Dengue with and without warning signs, with the aim of identifying immunological correlates of clinical disease and potential markers of disease severity. METHODS: Blood samples, collected from study participants fulfilling the WHO definition of Dengue with and without warning signs and healthy volunteers, were analyzed using flow cell-based fluorometric methods for cytokines and chemokines. Gene expression analysis, using RT-PCR, was conducted on T helper cell subset-specific transcription factors and cytokines. Demographic details, virological markers, serotype distribution, and hematological parameters were also investigated in all the subjects. RESULTS: The 35 participants recruited in the study, included 11 healthy volunteers and 12 patients each fulfilling the WHO criteria of Dengue with and without warning signs. While the demographic characteristics and serotype distribution was similar in Dengue with and without warning signs cohorts of the disease, platelet counts and Aspartate Aminotransferase (AST) levels changed significantly between Dengue with and without warning signs patients. Plasma cytokine analysis showed up-regulation of IL-4, IL-10, IP-10, and MCP-1 in Dengue patients compared to healthy volunteers. Disease severity was associated with elevated levels of IL-10, IP-10, IL-4, MCP-1, and MIP-1α. IL-8 and MIP-1α were significantly up-regulated in Dengue with warning sign compared to Dengue without warning signs cases. Transcription factor analysis indicated increased expression of RORα, FoxP3, and GATA3 in Dengue patients. mRNA expression of TGFß and IL-4 was also elevated in Dengue patients. A positive correlation between mRNA expression of IL-4 and plasma IL-4 was observed. CONCLUSION: The study reveals a Th2-predominant immune response in all Dengue patients, regardless of disease severity, with overexpression of IL-8 and MIP-1α being observed in patients with warning signs.

Relative Consolidation of the Kappa Variant Pre-Dates the Massive Second Wave of COVID-19 in India.

India experienced a tragic second wave after the end of March 2021, which was far more massive than the first wave and was driven by the emergence of the novel delta variant (B.1.617.2) of the SARS-CoV-2 virus. In this study, we explored the local and national landscape of the viral variants in the period immediately preceding the second wave to gain insight into the mechanism of emergence of the delta variant and thus improve our understanding of the causation of the second wave. We randomly selected 20 SARS-CoV-2 positive samples diagnosed in our lab between 3 February and 8 March 2021 and subjected them to whole genome sequencing. Nine of the 20 sequenced genomes were classified as kappa variant (B.1.617.1). The phylogenetic analysis of pan-India SARS-CoV-2 genome sequences also suggested the gradual replacement of the α variant with the kappa variant during this period. This relative consolidation of the kappa variant was significant, since it shared 3 of the 4 signature mutations (L452R, E484Q and P681R) observed in the spike protein of delta variant and thus was likely to be the precursor in its evolution. This study demonstrates the predominance of the kappa variant in the period immediately prior to the second wave and underscores its role as the "bridging variant" between the α and delta variants that drove the first and second waves of COVID-19 in India, respectively.

Testing of four-sample pools offers resource optimization without compromising diagnostic performance of real time reverse transcriptase-PCR assay for COVID-19.

Quick identification and isolation of SARS-CoV-2 infected individuals is central to managing the COVID-19 pandemic. Real time reverse transcriptase PCR (rRT-PCR) is the gold standard for COVID-19 diagnosis. However, this resource-intensive and relatively lengthy technique is not ideally suited for mass testing. While pooled testing offers substantial savings in cost and time, the size of the optimum pool that offers complete concordance with results of individualized testing remains elusive. To determine the optimum pool size, we first evaluated the utility of pool testing using simulated 5-sample pools with varying proportions of positive and negative samples. We observed that 5-sample pool testing resulted in false negativity rate of 5% when the pools contained one positive sample. We then examined the diagnostic performance of 4-sample pools in the operational setting of a diagnostic laboratory using 500 consecutive samples in 125 pools. With background prevalence of 2.4%, this 4-sample pool testing showed 100% concordance with individualized testing and resulted in 66% and 59% reduction in resource and turnaround time, respectively. Since the negative predictive value of a diagnostic test varies inversely with prevalence, we re-tested the 4-sample pooling strategy using a fresh batch of 500 samples in 125 pools when the prevalence rose to 12.7% and recorded 100% concordance and reduction in cost and turnaround time by 36% and 30%, respectively. These observations led us to conclude that 4-sample pool testing offers the optimal blend of resource optimization and diagnostic performance across difference disease prevalence settings.

Molecular and phylogenetic analysis of Chikungunya virus in Central India during 2016 and 2017 outbreaks reveal high similarity with recent New Delhi and Bangladesh strains.

Central India witnessed Chikungunya virus (CHIKV) outbreaks in 2016 and 2017. The present report is a hospital based cross-sectional study on the serological and molecular epidemiology of the outbreak. Mutational and phylogenetic analysis was conducted to ascertain the genetic relatedness of the central Indian strains with other Indian and global strains. Chikungunya infection was confirmed in the clinically suspected patients by the detection of anti-CHIKV IgM antibody by ELISA and viral RNA by RT-PCR. A representative set of the RT-PCR positive samples were sequenced for E1 gene and analyzed to identify the emerging mutations and establish their phylogenetic relationship, particularly with other contemporary strains. Phylogenetic analysis revealed the present strains to be of East Central South African (ECSA) genotype. Emergence of a variant strain was observed in the year 2016, which became the predominant strain in this region in 2017. The strains showed significant identity with recent New Delhi strains of 2015 and 2016 and Bangladesh strains of 2017. The epidemic mutation A226V which emerged in 2006 outbreaks of India and Indian Ocean Islands was found to be absent in the current strains. Among the important mutations viz. K211E, M269 V, D284E, I317V & V322A observed in the recent strains. I317V is a novel mutation which has emerged very recently as it was found only in central Indian (2016, 2017), New Delhi strains (2015, 2016) and Bangladesh strains (2017). This study has identified a unique mutation E1:I317V in the Central Indian strains, which is present only in recent New Delhi and Bangladesh strains till date. This study highlights the need for continuous molecular surveillance of circulating CHIKV strains in order to facilitate the prompt identification of novel strains of this virus and enable the elucidation of their clinical correlates.

Co-circulation of dengue virus serotypes in Central India: Evidence of prolonged viremia in DENV-2.

In view of paucity of information on serotype distribution of Dengue virus (DENV) in Central India, we undertook a cross-sectional study to identify clinical and virological characteristics of DENV serotypes that circulated in this region during the 2016 outbreak. Suspected cases were screened by ELISA for NS1 antigen and anti-DENV IgM antibodies. Serologically confirmed cases were subjected to RT-PCR based detection and serotyping. The RT-PCR results were confirmed by nucleotide sequencing. Genome-wide association was undertaken with DENV sequences from ViPR database and the immune evasion potential of infecting serotypes was ascertained by computing antigenic variability in B cell and Cytotoxic T cell (CTL) epitopes of all DENV proteins. The immunological basis of more prolonged viremia in DENV2-infected patients was also addressed through sequencing of NS2a gene and comparing the CTL activity in NS2a sequences identified among patients with ≤5 days and >5 days of illness. Among 166 serologically confirmed Dengue patients, 75 were positive for DENV RNA. Serotyping revealed predominance of DENV-1 and DENV-2, followed by DENV-3. Co-infection with multiple serotypes was observed in 15.5% of cases. In ~40% cases, DENV RNA was detectable beyond 5 days, among whom majority were DENV-2 infected (p = .044). Highest prevalence of antigenic variability was observed in B cell and CTL epitopes of DENV-2. The potential association between prolonged viremia and higher ability for immune evasion in DENV-2 patients was further corroborated with the observation of poorer HLA-I binding affinity in CTL epitopes observed in NS2a sequences retrieved from patients with >5 days of illness, compared to those with ≤5 days. This is the first report from central India revealing circulation of all DENV serotypes and high prevalence of co-infection with multiple serotypes. We also observed prolonged viremia upon DENV-2 infection, which could be potentially associated with its superior immune evasion potential.