Background & objectives: Nasopharyngeal and oropharyngeal swab (NPS and OPS) collection is widely accepted as the preferred method for obtaining respiratory samples. However, it has certain disadvantages which may be overcome by gargling. The primary objective of this study was to assess agreement between gargle lavage and swab as an appropriate respiratory sample for the detection of SARS-CoV-2. The secondary objective was to assess the patient acceptability of the two sampling methods. Methods: It was a cross-sectional study done at a tertiary care hospital in New Delhi, India, on 50 confirmed COVID-19 patients. Paired swab (NPS and OPS) and gargle samples were taken within 72 h of their diagnosis. Samples were processed by reverse transcription-polymerase chain reaction (RT-PCR) for detection of SARS-CoV-2. Post-sample collection, a 10-point scale was administered to assess the level of discomfort with either of the collection methods. Results: All gargle samples were positive and comparable to their corresponding swab samples irrespective of the symptoms and duration of illness. The cycle threshold (C_t) values for gargle samples were slightly higher but comparable to those of swabs. Bland-Altman plot showed good agreement between the two methods. Majority (72%) of the patients reported moderate-to-severe discomfort with swab collection in comparison to 24 per cent reporting only mild discomfort with gargle collection. Interpretation & conclusions: Our preliminary results show that the gargle lavage may be a viable alternative to swabs for sample collection for the detection of SARS-CoV-2. Adoption of gargle lavage for sample collection will have a significant impact as it will enable easy self-collection, relieve healthcare workers and also lead to substantial cost savings by reducing the need for swabs and personal protective equipment.

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Background & objectives: Population-based seroepidemiological studies measure the extent of SARS-CoV-2 infection in a country. We report the findings of the first round of a national serosurvey, conducted to estimate the seroprevalence of SARS-CoV-2 infection among adult population of India. Methods: From May 11 to June 4, 2020, a randomly sampled, community-based survey was conducted in 700 villages/wards, selected from the 70 districts of the 21 States of India, categorized into four strata based on the incidence of reported COVID-19 cases. Four hundred adults per district were enrolled from 10 clusters with one adult per household. Serum samples were tested for IgG antibodies using COVID Kavach ELISA kit. All positive serum samples were re-tested using Euroimmun SARS-CoV-2 ELISA. Adjusting for survey design and serial test performance, weighted seroprevalence, number of infections, infection to case ratio (ICR) and infection fatality ratio (IFR) were calculated. Logistic regression was used to determine the factors associated with IgG positivity. Results: Total of 30,283 households were visited and 28,000 individuals were enrolled. Population-weighted seroprevalence after adjusting for test performance was 0.73 per cent [95% confidence interval (CI): 0.34-1.13]. Males, living in urban slums and occupation with high risk of exposure to potentially infected persons were associated with seropositivity. A cumulative 6,468,388 adult infections (95% CI: 3,829,029-11,199,423) were estimated in India by the early May. The overall ICR was between 81.6 (95% CI: 48.3-141.4) and 130.1 (95% CI: 77.0-225.2) with May 11 and May 3, 2020 as plausible reference points for reported cases. The IFR in the surveyed districts from high stratum, where death reporting was more robust, was 11.72 (95% CI: 7.21-19.19) to 15.04 (9.26-24.62) per 10,000 adults, using May 24 and June 1, 2020 as plausible reference points for reported deaths. Interpretation & conclusions: Seroprevalence of SARS-CoV-2 was low among the adult population in India around the beginning of May 2020. Further national and local serosurveys are recommended to better inform the public health strategy for containment and mitigation of the epidemic in various parts of the country.

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Background & objectives: In December 2019, a novel coronavirus (SARS-CoV-2) emerged in China and rapidly spread globally including India. The characteristic clinical observations and outcomes of this disease (COVID-19) have been reported from different countries. The present study was aimed to describe the clinico-demographic characteristics and in-hospital outcomes of a group of COVID-19 patients in north India. Methods: This was a prospective, single-centre collection of data regarding epidemiological, demographic, clinical and laboratory parameters, management and outcome of COVID-19 patients admitted in a tertiary care facility in north India. Patient outcomes were recorded as death, discharge and still admitted. Results: Data of 144 patients with COVID-19 were recorded and analyzed. The mean age of the patients was 40.1±13.1 yr, with 93.1 per cent males, and included 10 (6.9%) foreign nationals. Domestic travel to or from affected States (77.1%) and close contact with COVID-19 patients in congregations (82.6%) constituted the most commonly documented exposure. Nine (6.3%) patients were smokers, with a median smoking index of 200. Comorbidities were present in 23 (15.9%) patients, of which diabetes mellitus (n=16; 11.1%) was the most common. A significant proportion of patients had no symptoms (n=64; 44.4%); among the symptomatic, cough (34.7%) was the most common symptom followed by fever (17.4%) and nasal symptoms (2.15%). Majority of the patients were managed with supportive treatment with hydroxychloroquine and azithromycin given on a case-to-case basis. Only five (3.5%) patients required oxygen supplementation, four (2.8%) patients had severe disease requiring intensive care, one required mechanical ventilation and mortality occurred in two (1.4%) patients. The time to reverse transcription-polymerase chain reaction (RT-PCR) negativity was 16-18 days. Interpretation & conclusions: In this single-centre study of 144 hospitalized patients with confirmed COVID-19 in north India, the characteristic findings included younger age, high proportion of asymptomatic patients, long time to PCR negativity and low need for intensive care unit care.

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Despite the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there are limited data emerging in children. This review provides an update on clinical features, diagnosis, epidemiology, management and prevention of coronavirus disease 2019 (COVID-19) in children. Specific characteristics noted in children and their implications in disease management as well as transmission control are highlighted. Besides respiratory symptoms, gastrointestinal and atypical features such as chilblains, neurological symptoms and multisystem inflammation are also reported. Younger infants and those with comorbidity were found to be at risk of severe illness. Infected pregnant women and neonates were reported to have good prognosis. It is possible to manage the children with mild disease at home, with strict infection prevention control measures; severely affected require respiratory support and intensive care management. There are anecdotal reports of using antiviral and immunomodulatory drugs, benefit of which needs to be confirmed in clinical trials. A significant percentage of asymptomatic infection in children has epidemiological implication as these may act as links in transmission chain in the community. There is a need for systematic data on extra-pulmonary manifestations and atypical features, risk factors of severity, role of imaging and biomarkers, testing and management strategies and trials with antivirals and immunomodulatory drugs in children. The psychosocial effects of quarantine, closure of schools, lack of play activities and impact of lockdown need to be addressed. Understanding the biological basis for the profound age-dependent differential outcome of COVID-19 infection is important. Elucidating the protective mechanisms in children may aid in developing novel treatment strategies.

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Background & objectives: In this study we describe the epidemiological data, comorbidities, clinical symptoms, severity of illness and early outcome of patients with coronavirus disease 2019 (COVID-19) from a tertiary care teaching hospital in New Delhi, India. Methods: In this preliminary analysis of a prospective observational study, all adult patients admitted to the screening intensive care unit (ICU) of the institute who fulfilled the WHO case definition of COVID-19 and confirmed to have SARS-CoV-2 infection by reverse transcription-polymerase chain reaction were included. Demographics, clinical data and 24 h outcome were assessed. Results: The preliminary analysis of 235 patients revealed that the mean age was 50.7±15.1 yr and 68.1 per cent were male. Fever (68.1%), cough (59.6%) and shortness of breath (71.9%) were the most common presenting symptoms. Hypertension (28.1%) and diabetes mellitus (23.3%) were the most common associated comorbid illnesses. Patients with mild, moderate, severe and critical illness were 18.3, 32.3, 31.1 and 18.3 per cent, respectively, at the time of ICU admission. The proportions (95% confidence interval) of patients requiring any form of oxygen therapy, oxygen therapy by high-flow nasal cannula and invasive mechanical ventilation were 77, 21.7 and 25.5 per cent, respectively, within 24 h of hospital admission. The 24 h ICU mortality was 8.5 per cent, and non-survivors had higher respiratory rate (P <0.01, n=198) and lower baseline oxyhaemoglobin saturation (P <0.001, n=198) at presentation and higher baseline serum lactate (P <0.01, n=122), total leucocyte count (P <0.001, n=186), absolute neutrophil count (P <0.001, n=132), prothrombin time (P <0.05, n=54) and INR (P <0.05, n=54) compared to survivors. Interpretation & conclusions: Nearly half of the patients presented with severe and critical disease and required high-flow nasal oxygen or invasive mechanical ventilation at admission. Severity of the presenting respiratory illness, haematological parameters and lactate rather than age or presence of comorbidity predicted early death within 24 h.

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The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been predominantly a respiratory manifestation. Currently, with evolving literature, neurological signs are being increasingly recognized. Studies have reported that SARS-CoV-2 affects all aspects of the nervous system including the central nervous system (CNS), peripheral nervous system (PNS) and the muscular system as well. Not all patients have reverse transcription-polymerase chain reaction positive for the virus in the cerebrospinal fluid, and diagnosing the association of the virus with the myriad of neurological manifestations can be a challenge. It is important that clinicians have a high-index of suspicion for COVID-19 in patients presenting with new-onset neurological symptoms. This will lead to early diagnosis and specific management. Further studies are desired to unravel the varied neurological manifestations, treatment, outcome and long-term sequel in COVID-19 patients.

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Background & objectives: Public health and diagnostic laboratories are facing huge sample loads for COVID-19 diagnosis by real-time reverse transcription-polymerase chain reaction (RT-PCR). High sensitivity of optimized real-time RT-PCR assays makes pooled testing a potentially efficient strategy for resource utilization when positivity rates for particular regions or groups of individuals are low. We report here a comparative analysis of pooled testing for 5- and 10-sample pools by real-time RT-PCR across 10 COVID-19 testing laboratories in India. Methods: Ten virus research and diagnostic laboratories (VRDLs) testing for COVID-19 by real-time RT-PCR participated in this evaluation. At each laboratory, 100 nasopharyngeal swab samples including 10 positive samples were used to create 5- and 10-sample pools with one positive sample in each pool. RNA extraction and real-time RT-PCR for SARS-CoV-2-specific E gene target were performed for individual positive samples as well as pooled samples. Concordance between individual sample testing and testing in the 5- or 10-sample pools was calculated, and the variation across sites and by sample cycle threshold (C_t) values was analyzed. Results: A total of 110 each of 5- and 10-sample pools were evaluated. Concordance between the 5-sample pool and individual sample testing was 100 per cent in the C_t value ≤30 cycles and 95.5 per cent for C_tvalues ≤33 cycles. Overall concordance between the 5-sample pooled and individual sample testing was 88 per cent while that between 10-sample pool and individual sample testing was 66 per cent. Although the concordance rates for both the 5- and 10-sample pooled testing varied across laboratories, yet for samples with C_t values ≤33 cycles, the concordance was ≥90 per cent across all laboratories for the 5-sample pools. Interpretation & conclusions: Results from this multi-site assessment suggest that pooling five samples for SARS-CoV-2 detection by real-time RT-PCR may be an acceptable strategy without much loss of sensitivity even for low viral loads, while with 10-sample pools, there may be considerably higher numbers of false negatives. However, testing laboratories should perform validations with the specific RNA extraction and RT-PCR kits in use at their centres before initiating pooled testing.

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Background & objectives: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has spread rapidly, causing unprecedented case fatalities across the world. The first laboratory-confirmed case of COVID-19 and also the first death associated with the disease in the eastern part of Uttar Pradesh (UP) was reported from Basti on March 31, 2020. The present study describes a cluster of 17 cases including one death of confirmed COVID-19 in Basti, UP, India. Methods: A 25 year old male from Basti, UP, India, who died of respiratory failure was diagnosed post-mortem as SARS-CoV-2 positive. Contact tracing carried out by the district administration found 16 cases positive among tested contacts. A detailed retrospective investigation in the form of one-to-one interview was carried out with 16 recovered individuals to understand the transmission dynamics and clinical characteristics. Results: The findings showed that the cluster transmission occurred at three levels: first was direct contact with the index case which resulted in two secondary cases. Second, at a household level where four of the seven susceptible contacts got infected, and the third was an event (funeral) where 50 individuals participated and this resulted in seven SARS-CoV-2-infected individuals in whom infection could be directly linked to a funeral gathering. The index case had associated comorbidities and succumbed to death. Most of the cases were asymptomatic except two individuals, who developed mild symptoms. The mean duration of quarantine facility was 21.6±7.3 days, and the average time taken for the first negative test after testing positive to COVID-19 was 12±4.1 days. Interpretation & conclusions: The funeral acted as a super-spreader event for the transmission of infection among family members, relatives and others. Active contact tracing and confirmation of infection among the contacts led to the isolation of 16 SARS-CoV-2 positive cases and hence the limited spread of the disease. Asymptomatic carriers and super-spreader events are among the major challenges in the control and prevention of SARS-CoV-2 transmission. Early testing, quarantine and social distancing may play key role in breaking the chain of transmission.

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Background & objectives: The global pandemic caused by SARS-CoV-2 virus has challenged public health system worldwide due to the unavailability of approved preventive and therapeutic options. Identification of neutralizing antibodies (NAb) and understanding their role is important. However, the data on kinetics of NAb response among COVID-19 patients are unclear. To understand the NAb response in COVID-19 patients, we compared the findings of microneutralization test (MNT) and plaque reduction neutralization test (PRNT) for the SARS-CoV-2. Further, the kinetics of NAb response among COVID-19 patients was assessed. Methods: A total of 343 blood samples (89 positive, 58 negative for SARS-CoV-2 and 17 cross-reactive and 179 serum from healthy individuals) were collected and tested by MNT and PRNT. SARS-CoV-2 virus was prepared by propagating the virus in Vero CCL-81 cells. The intra-class correlation was calculated to assess the correlation between MNT and PRNT. The neutralizing endpoint as the reduction in the number of plaque count by 90 per cent (PRNT₉₀) was also calculated. Results: The analysis of MNT and PRNT quantitative results indicated that the intra-class correlation was 0.520. Of the 89 confirmed COVID-19 patients, 64 (71.9%) showed NAb response. Interpretation & conclusions: The results of MNT and PRNT were specific with no cross-reactivity. In the early stages of infection, the NAb response was observed with variable antibody kinetics. The neutralization assays can be used for titration of NAb in recovered/vaccinated or infected COVID-19 patients.

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Background & objectives: The genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belonging to the family Coronaviridae, encodes for structural, non-structural, and accessory proteins, which are required for replication of the virus. These proteins are encoded by different genes present on the SARS-CoV-2 genome. The expression pattern of these genes in the host cells needs to be assessed. This study was undertaken to understand the transcription pattern of the SARS-CoV-2 genes in the Vero CCL-81 cells during the course of infection. Methods: Vero CCL-81 cells were infected with the SARS-CoV-2 virus inoculum having a 0.1 multiplicity of infection. The supernatants and cell pellets were harvested after centrifugation at different time points, post-infection. The 50% tissue culture infective dose (TCID₅₀)and cycle threshold (C_t) values of the E and the RdRp-2 genes were calculated. Next-generation sequencing of the harvested sample was carried out to observe the expression pattern of the virus by mapping to the SARS-CoV-2 Wuhan HU-1 reference sequence. The expressions were in terms of the reads per kilobase million (RPKM) values. Results: In the inital six hours post-infection, the copy numbers of E and RdRp-2 genes were approximately constant, which raised 10 log-fold and continued to increase till the 12 h post-infection (hpi). The TCID₅₀ was observed in the supernatant after 7 hpi, indicating the release of the viral progeny. ORF8 and ORF7a, along with the nucleocapsid transcript, were found to express at higher levels. Interpretation & conclusions: This study was a step towards understanding the growth kinetics of the SARS-CoV-2 replication cycle. The findings indicated that ORF8 and ORF7b gene transcripts were expressed in higher amounts indicating their essential role in viral replication. Future studies need to be conducted to explore their role in the SARS-CoV-2 replication.

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