External quality assessment for laboratories in pan-India ILI/SARI surveillance for simultaneous detection of influenza virus and SARS-CoV-2.

Introduction: The Indian Council of Medical Research has set up a nationwide network of 28 laboratories for simultaneous surveillance of influenza virus and SARS-CoV-2 in ILI/SARI patients, using an in-house developed and validated multiplex real-time RTPCR assay. The aim of this study was to ensure the quality of testing by these laboratories by implementing an external quality assessment program (EQAP). Methods: For this EQAP, a proficiency test (PT) panel comprising tissue-culture or egg-grown influenza virus and SARS-CoV-2 was developed. The PT panel was distributed to all the participant laboratories, which tested the panel and submitted the qualitative results online to the EQAP provider. The performance of the laboratories was evaluated on qualitative criteria but cycle threshold (Ct) values were also gathered for each sample. Results: On a qualitative basis, all the laboratories achieved the criteria of 90% concordance with the results of the PT panel provider. Ct values of different samples across the laboratories were within ≤ ±3 cycles of the corresponding mean values of the respective sample. The results of this EQAP affirmed the quality and reliability of testing being done for simultaneous surveillance of influenza virus and SARS-CoV-2 in India.

Mapping the Landscape of Health Research Priorities for Effective Pandemic Preparedness in Human Mpox Virus Disease.

The global re-emergence of monkeypox (Mpox) in non-endemic regions in 2022 has highlighted the critical importance of timely virus detection and robust public health surveillance in assessing outbreaks and their impact. Despite significant Mpox research being conducted worldwide, there is an urgent need to identify knowledge gaps and prioritize key research areas in order to create a roadmap that maximizes the utilization of available resources. The present research article provides a comprehensive mapping of health research priorities aimed at advancing our understanding of Mpox and developing effective interventions for managing its outbreaks, and, as evidenced by the fact that achieving this objective requires close interdisciplinary collaboration. The key research priorities observed were identifying variants responsible for outbreaks; discovering novel biomarkers for diagnostics; establishing suitable animal models; investigating reservoirs and transmission routes; promoting the One Health approach; identifying targets for vaccination; gaining insight into the attitudes, experiences, and practices of key communities, including stigma; and ensuring equity during public health emergencies. The findings of this study hold significant implications for decision making by multilateral partners, including research funders, public health practitioners, policy makers, clinicians, and civil society, which will facilitate the development of a comprehensive plan not only for Mpox but also for other similar life-threatening viral infections.

Pan-India influenza-like illness (ILI) and Severe acute respiratory infection (SARI) surveillance: epidemiological, clinical and genomic analysis.

Background: Over time, COVID-19 testing has significantly declined across the world. However, it is critical to monitor the virus through surveillance. In late 2020, WHO released interim guidance advising the use of the existing Global Influenza Surveillance and Response System (GISRS) for the integrated surveillance of influenza and SARS-CoV-2. Methods: In July 2021, we initiated a pan-India integrated surveillance for influenza and SARS-CoV-2 through the geographically representative network of Virus Research and Diagnostic Laboratories (VRDLs) across 26 hospital and laboratory sites and 70 community sites. A total of 34,260 cases of influenza-like illness (ILI) and Severe acute respiratory infection (SARI) were enrolled from 4 July 2021 to 31 October 2022. Findings: Influenza A(H3) and B/Victoria dominated during 2021 monsoon season while A(H1N1)pdm09 dominated during 2022 monsoon season. The SARS-CoV-2 "variants of concern" (VoC) Delta and Omicron predominated in 2021 and 2022, respectively. Increased proportion of SARI was seen in extremes of age: 90% cases in < 1 year; 68% in 1 to 5 years and 61% in ≥ 8 years age group. Approximately 40.7% of enrolled cases only partially fulfilled WHO ILI and SARI case definitions. Influenza- and SARS-CoV-2-infected comorbid patients had higher risks of hospitalization, ICU admission, and oxygen requirement. Interpretation: The results depicted the varying strains and transmission dynamics of influenza and SARS-CoV-2 viruses over time, thus emphasizing the need to continue and expand surveillance across countries for improved decision making. The study also describes important information related to clinical outcomes of ILI and SARI patients and highlights the need to review existing WHO ILI and SARI case definitions.

Protocol for establishing a model for integrated influenza surveillance in Tamil Nadu, India.

The potential for influenza viruses to cause public health emergencies is great. The World Health Organisation (WHO) in 2005 concluded that the world was unprepared to respond to an influenza pandemic. Available surveillance guidelines for pandemic influenza lack the specificity that would enable many countries to establish operational surveillance plans. A well-designed epidemiological and virological surveillance is required to strengthen a country's capacity for seasonal, novel, and pandemic influenza detection and prevention. Here, we describe the protocol to establish a novel mechanism for influenza and SARS-CoV-2 surveillance in the four identified districts of Tamil Nadu, India. This project will be carried out as an implementation research. Each district will identify one medical college and two primary health centres (PHCs) as sentinel sites for collecting severe acute respiratory infections (SARI) and influenza like illness (ILI) related information, respectively. For virological testing, 15 ILI and 10 SARI cases will be sampled and tested for influenza A, influenza B, and SARS-CoV-2 every week. Situation analysis using the WHO situation analysis tool will be done to identify the gaps and needs in the existing surveillance systems. Training for staff involved in disease surveillance will be given periodically. To enhance the reporting of ILI/SARI for sentinel surveillance, trained project staff will collect information from all ILI/SARI patients attending the sentinel sites using pre-tested tools. Using time, place, and person analysis, alerts for abnormal increases in cases will be generated and communicated to health authorities to initiate response activities. Advanced epidemiological analysis will be used to model influenza trends over time. Integrating virological and epidemiological surveillance data with advanced analysis and timely communication can enhance local preparedness for public health emergencies. Good quality surveillance data will facilitate an understanding outbreak severity and disease seasonality. Real-time data will help provide early warning signals for prevention and control of influenza and COVID-19 outbreaks. The implementation strategies found to be effective in this project can be scaled up to other parts of the country for replication and integration.

Determination of a cut-off value for the serological diagnosis of scrub typhus by detecting anti-Orientia tsutsugamushi immunoglobulin M.

Background & objectives: The diagnosis of scrub typhus (ST) is usually done using enzyme-linked immunosorbent assay (ELISA) due to its ease of performance and reading objectivity. The cut-off value for ELISA needs to be calculated for each geographical location as it depends on zonal endemicity of the disease. This study was, therefore, undertaken to calculate the pan-India cut-off for anti-Orientia tsutsugamushi (OT) immunoglobulin M (IgM) by ELISA. Methods: Samples from cases (cases of ST) and controls (voluntary, consenting, healthy adults) were collected by a network of 29 laboratories across India and tested for anti-OT IgM by immunofluorescence assay (IFA), the considered gold standard test. These samples were retested by ELISA for anti-OT IgM and their optical densities (ODs) were used for cut-off estimation by receiver operating characteristic (ROC) curve. Results: Anti-OT IgM ELISA ODs from 273 controls and 136 cases were used for the cut-off estimation. The ODs of the anti-OT IgM ELISA on healthy individuals and those of confirmed ST cases ranged from 0.1 to 0.75 and 0.5 to 4.718, respectively. ROC curve-based cut-off for ELISA was calculated as 0.554 at a sensitivity of 95.2 per cent and specificity of 95.1 per cent. A value of >1 was noted to have a specificity of 100 per cent in diagnosing ST. Interpretation & conclusions: The cut-off calculated for India was similar to the previous cut-off that was used until now.

Effect of storage temperatures simulating transport conditions of nasopharyngeal swabs on the results of a chemiluminescence immunoassay (CLIA) to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen.

PURPOSE: Chemiluminescence Immunoassay (CLIA) is high throughput, rapid diagnostic test which has recently come up for the detection of SARS-CoV-2 antigen. The present study evaluated performance of CLIA antigen test in nasopharyngeal swab samples stored at different temperatures for 7 days to simulate the transport conditions and transit time across the country from remote peripheral laboratories to central facilities. MATERIALS AND METHODS: Limit of detection (LOD), sensitivity and specificity of VITROS® SARS-CoV-2 antigen assay was determined using Real-time reverse transcriptase PCR (rRT-PCR) confirmed SARS-CoV-2 positive and negative samples. To detect the effect of storage temperatures on VITROS ®SARS-CoV-2 antigen results, samples were stored at 4 â€‹°C, 25 â€‹°C & 37 â€‹°C for 7 days followed by detection of SARS-CoV-2 nucleocapsid antigen and compared with N-gene rRT-PCR. RESULTS: The VITROS® SARS-CoV-2 antigen test was found to have a sensitivity and specificity of 78.9% and 100% respectively with high sensitivity of 88.1% for samples with Ct â€‹< â€‹30. The LOD of VITROS assay was equivalent to 3800 copies of RNA per reactions as compared to 72 copies per reaction for rRT-PCR. We observed that more than 80% of samples with <30 Ct values could be detected by VITROS SARS-CoV-2 antigen assay at day 7 even when stored at 37 â€‹°C. For samples with Ct values between 26 and 30, on day 7 the positivity rate of N-antigen at 4 â€‹°C was 90.9% and 37 â€‹°C was 63.6%. CONCLUSIONS: CLIA testing can be carried out for the detection of SARS-CoV-2 N-protein in NP-swab samples transported in cold chain even with 7 days transit time, particularly for Ct â€‹< â€‹30 samples which represents cases with higher transmissibility. As drop in positivity for VITROS assay was lower as compared to rRT-PCR on day 7 in cold chain-maintained samples, the assay can be useful to screen samples received from remote peripheral areas before performing rRT-PCR.

External quality assurance of serological diagnosis of dengue, chikungunya and Japanese encephalitis virus infection.

Introduction: Dengue, chikungunya and Japanese encephalitis are the most common arthropod-borne viral diseases in India. Due to overlapping clinical symptoms, accurate, high-quality and timely laboratory-based differential diagnosis is essential for control and containment of outbreaks. This is most commonly done by detection of IgM antibodies in serum using enzyme-linked immunosorbent assays. The Resource Centre for Virus Research and Diagnostic Laboratories (VRDLs) in Pune, India organized an external quality assurance (EQA) study to check the accuracy of serological diagnostics in the VRDL network. Methods: Three panels, one each for anti-dengue virus, anti-chikungunya virus and anti-Japanese encephalitis virus IgM antibodies, comprising six human serum samples (two positive and four negative) were distributed to test the sensitivity, specificity and reproducibility of serological testing in 124 VRDLs across India in 2018-19 and 2019-20. Results: Among the 124 VRDLs, the average concordance for both 2018-19 and 2019-20 was 98%. In 2018-19, 78.33%, 13.33% and 6.66% of VRDLs reported 100% concordance, 91-99% concordance and 81-90% concordance with the reference results, respectively, and 1.66% of VRDLs had concordance <80%. In 2019-20, 79.68%, 14.06% and 4.68% of VRDLs reported 100% concordance, 91-99% concordance and 81-90% concordance with the reference results, respectively, and 1.56% of VRDLs had concordance <80%. Conclusion: The EQA programme was beneficial for assessing and understanding the performance of the VRDLs. The study data indicate good proficiency in serological diagnosis of dengue, chikungunya and Japanese encephalitis in the VRDL network laboratories. Further expansion of the EQA programme to cover other viruses of public health importance will increase confidence among the VRDL network, and generate evidence of high-quality testing.

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.

Inter-laboratory testing as a strategy for external quality assessment for qualitative detection of SARS-CoV-2 by real-time RT-PCR testing in India.

To implement the strategy of test, track and treat to tackle the ongoing COVID-19 pandemic, the number of real-time RT-PCR-based testing laboratories was increased for diagnosis of SARS-CoV-2 in the country. To ensure reliability of the laboratory results, the Indian Council of Medical Research initiated external quality assessment (EQA) by deploying inter-laboratory quality control (ILQC) activity for these laboratories by nominating 34 quality control (QC) laboratories. This report presents the results of this activity for a period of September 2020 till November 2020. A total of 597 laboratories participated in this activity and 86 per cent of these scored ≥90 per cent concordance with QC laboratories. This ILQC activity showcased India's preparedness in quality diagnosis of SARS-CoV-2.

Expansion of the measles and rubella laboratory network, India.

Objective: To expand the measles and rubella laboratory network of India by integrating new laboratories. Methods: In collaboration with the World Health Organization (WHO), the Indian government developed a 10-step scheme to systematically expand the number of laboratories performing serological and molecular testing for measles and rubella. The Indian Council of Medical Research and WHO identified suitable laboratories based on their geographical location, willingness, preparedness, past performance and adherence to national quality control and quality assurance mechanisms. The 10-step scheme was initiated with training on measles and rubella diagnostic assays followed by testing of both measles and rubella serology and molecular unknown panels, cross-verification with reference laboratories and ended with WHO on-site accreditation. Findings: After extensive training, technical support, funding and monitoring, all six selected laboratories attained passing scores of 90.0% or more in serological and molecular proficiency testing of measles and rubella. Since 2018, the laboratories are a part of the measles and rubella network of India. Within 12 months of initiation of independent reporting, the six laboratories have tested 2287 serum samples and 701 throat or nasopharyngeal swabs or urine samples. Conclusion: The process led to strengthening and expansion of the network. This proficient laboratory network has helped India in scaling up serological and molecular testing of measles and rubella while ensuring high quality testing. The collaborative model developed by the Indian government with WHO can be implemented by other countries for expanding laboratory networks for surveillance of measles and rubella as well as other infectious diseases.

SARS-CoV-2 and Influenza Virus Co-Infection Cases Identified through ILI/SARI Sentinel Surveillance: A Pan-India Report.

SARS-CoV-2/influenza virus co-infection studies have focused on hospitalized patients who usually had grave sequelae. Here, we report SARS-CoV-2/influenza virus co-infection cases from both community and hospital settings reported through integrated ILI/SARI (Influenza Like Illness/Severe Acute Respiratory Infection) sentinel surveillance established by the Indian Council of Medical Research. We describe the disease progression and outcomes in these cases. Out of 13,467 samples tested from 4 July 2021-31 January 2022, only 5 (0.04%) were of SARS-CoV-2/influenza virus co-infection from 3 different sites in distinct geographic regions. Of these, three patients with extremes of age required hospital admission, but none required ICU admission or mechanical ventilation. No mortality was reported. The other two co-infection cases from community settings were managed at home. This is the first report on SARS-CoV-2/Influenza virus co-infection from community as well as hospital settings in India and shows that influenza viruses are circulating in the community even during COVID-19. The results emphasize the need for continuous surveillance for multiple respiratory pathogens for effective public health management of ILI/SARI cases in line with the WHO (World Health Organization) recommendations.

External quality assessment of COVID-19 real time reverse transcription PCR laboratories in India.

Sudden emergence and rapid spread of COVID-19 created an inevitable need for expansion of the COVID-19 laboratory testing network across the world. The strategy to test-track-treat was advocated for quick detection and containment of the disease. Being the second most populous country in the world, India was challenged to make COVID-19 testing available and accessible in all parts of the country. The molecular laboratory testing network was augmented expeditiously, and number of laboratories was increased from one in January 2020 to 2951 till mid-September, 2021. This rapid expansion warranted the need to have inbuilt systems of quality control/ quality assurance. In addition to the ongoing inter-laboratory quality control (ILQC), India implemented an External Quality Assurance Program (EQAP) with assistance from World Health Organization (WHO) and Royal College of Pathologists, Australasia. Out of the 953 open system rRTPCR laboratories in both public and private sector who participated in the first round of EQAP, 891(93.4%) laboratories obtained a passing score of > = 80%. The satisfactory performance of Indian COVID-19 testing laboratories has boosted the confidence of the public and policy makers in the quality of testing. ILQC and EQAP need to continue to ensure adherence of the testing laboratories to the desired quality standards.

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.

Performance assessment of seven SARS-CoV-2 IgG enzyme-linked immunosorbent assays.

The pandemic of COVID-19 has caused enormous fatalities worldwide. Serological assays are important for detection of asymptomatic or mild cases of COVID-19, and sero-prevalence and vaccine efficacy studies. Here, we evaluated and compared the performance of seven commercially available enzyme-linked immunosorbent assay (ELISA)s for detection of anti-severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) immunoglobulin G (IgG). The ELISAs were evaluated with a characterized panel of 100 serum samples from qRT-PCR confirmed COVID-19 patients, collected 14 days post onset disease, 100 SARS-CoV-2 negative samples and compared the results with that of neutralization assay. Results were analysed by creating the receiver operating characteristic curve of all the assays in reference to the neutralization assay. All kits, were found to be suitable for detection of IgG against SARS-CoV-2 with high accuracy. The DiaPro COVID-19 IgG ELISA showed the highest sensitivity (98%) among the kits. The assays demonstrated high sensitivity and specificity in detecting the IgG antibodies against SARS-CoV-2. However, the presence of IgG antibodies does not always correspond to neutralizing antibodies. Due to their good accuracy indices, these assays can also aid in tracing mild infections, in cohort studies and in pre-vaccine evaluations.

An Epidemiological Analysis of SARS-CoV-2 Genomic Sequences from Different Regions of India.

The number of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) cases is increasing in India. This study looks upon the geographic distribution of the virus clades and variants circulating in different parts of India between January and August 2020. The NPS/OPS from representative positive cases from different states and union territories in India were collected every month through the VRDLs in the country and analyzed using next-generation sequencing. Epidemiological analysis of the 689 SARS-CoV-2 clinical samples revealed GH and GR to be the predominant clades circulating in different states in India. The northern part of India largely reported the 'GH' clade, whereas the southern part reported the 'GR', with a few exceptions. These sequences also revealed the presence of single independent mutations-E484Q and N440K-from Maharashtra (first observed in March 2020) and Southern Indian States (first observed in May 2020), respectively. Furthermore, this study indicates that the SARS-CoV-2 variant (VOC, VUI, variant of high consequence and double mutant) was not observed during the early phase of virus transmission (January-August). This increased number of variations observed within a short timeframe across the globe suggests virus evolution, which can be a step towards enhanced host adaptation.