This descriptive study was carried out at Department of Microbiology of a tertiary care teaching hospital over a four month period between 21^st March 2020 and 8^th July 2020. Nasopharyngeal and/or oropharyngeal (NP/OP) swabs were collected in VTM and were received in a triple layer packaging and maintaining the cold chain from dependent collection centre as per COVID-19 sample collection guidelines given by ICMR [7,8]. As per current ICMR strategy everyone was grouped in to one of nine patient categories based on symptoms and clinical details [8]. Based on symptoms, patients were grouped as asymptomatic, Influenza Like Illness (ILI) and Severe Acute Respiratory Infection (SARI). Patient profile was taken into consideration as Healthcare Worker (HCW) or pregnant women or traveller. History of contact to a confirmed case was also noted. The testing was performed by ICMR approved RT-PCR kit, Super Script (SS) III Invitrogen, for qualitative detection of SARS-CoV-2 genetic targets; Envelope (E gene), Hong Kong University open reading frame 1b (HKU) and Ribonucleic Acid (RNA) dependent RNA Polymerase (RdRP) gene [9]. To begin with, RNA extraction from the NP swab in VTM was performed using spin column based method. Extracted RNA was subjected to RT-PCR as per standard operating procedures of ICMR-NIV (National Institute of Virology) [9]. The patient level data was captured from the specimen referral form submitted by each hospital and from the online ICMR portal (https://cvstatus.icmr.gov.in/). Confidentiality of patients was maintained, and all individuals were identified by unique sample identification number.

The patient level variables were tabulated in Microsoft Excel spreadsheet for further analysis. The variables studied included age, gender, place of residence, symptom status, hospitalisation status. The demographic and clinical data of samples tested were summarised by medians and IQR for continuous variables and by proportions for categorical variables.

A total of 12,187 samples were received between 21^st March to 8^th July 2020. However, the data from 11,196 individuals were complete and were included in the analysis. Overall, 2053 samples were tested positive for SARS-CoV-2 indicating positivity rate of 18.33%. A steady increase in the monthly average positivity rate was noted during the study period. The COVID-19 positivity in the month of March, April, May, June, and July were 3.84% (n=26), 2.27% (n=2422), 7.83% (n=4222), 28.50% (n=4070) and 34.96% (n=1447), respectively (date-wise data is given in supplementary file). The daily sample received and the cumulative sample tested along with positivity trend is shown in [Table/Fig-1,2].

[Table/Fig-1]:

SARS-CoV-2 infection trend of respiratory samples tested between 21^st March 2020 to 8^th July 2020.

[Table/Fig-2]:

Cumulative sample tested for SARS-CoV-2 infection and COVID-19 positivity-trend from March to July 2020.

The study population was male predominant (n=6871; 61.37%) with median (IQR) age of 33 (24-46) years. SARS-CoV-2 positivity was highest (27.62%) in individuals above 61 years of age. The positivity rate in each age group is shown in [Table/Fig-3]. Of these 11,196 individuals, 74.58% were found to be asymptomatic. The SARS-CoV-2 positivity rate in individuals with SARI was 14.17%. The SARS-CoV-2 positivity rate among asymptomatic and symptomatic individuals were 17.02% and 22.17%, respectively, and this difference was not significant [Table/Fig-4]. The common symptoms at the time of testing are shown in [Table/Fig-5]. Distribution of patients in various ‘ICMR patient category’ (Category 1 to 9) is shown in [Table/Fig-6].

[Table/Fig-4]:

COVID-19 detection in symptomatic versus asymptomatic individuals (n=11,196).

[Table/Fig-5]:

Frequency of symptoms in individuals at the time of SARS-CoV-2 diagnostic testing.

Highest number of SARS-CoV-2 infected cases were seen in asymptomatic direct and high risk contacts of lab confirmed cases, i.e., ICMR category 5a (63.91%). Health Care Workers (HCW) belonging to category 3 (symptomatic HCW) and 5b (asymptomatic HCW) constituted 11.35% (n=1271) of the sample tested. The overall SARS-CoV-2 positivity rate in symptomatic HCW and asymptomatic HCW were 0.78% and 2.48%, respectively.

Globally, COVID-19 pandemic has affected over 232 countries with over 25 million cases till the end of August [10]. India is currently ranked third with over 3.5 million cases [10]. Elevated infectivity of the virus coupled with high population density, sparse resources and reckless human behaviour have fuelled the spread of pandemic in India. However, in a short span of four months, the all round efforts of Government of India were successful in expanding COVID-19 diagnostic testing capacity. By 11^th June 2020, there were 877 ICMR approved labs testing for COVID-19 [11]. Working on “Test, Track and Treat” strategy, for early detection and containment of cases, presently there are 1400 ICMR approved laboratories operational across India [11,12]. Currently, testing data across all diagnostic laboratories is entered into unified ICMR data entry portal, as per the ICMR guidelines [5]. This will help in guiding the National strategies appropriately. In this descriptive study, authors have analysed data from over 12,000 individuals tested at the laboratory from March to July 2020. Maharashtra is leading in number of COVID-19 cases in India. In this study, the overall SARS-CoV-2 sample positivity rate was 18.33%. Present study data indicate increase in SARS-CoV-2 positivity from 3.03-28.42% from March to July 2020.

The highest number of samples was received from age group of 21-40 years with an average positivity rate of 15.33%. Data from other cities of Maharashtra and rest of India also indicate maximum burden of COVID-19 in younger population [5,12]. People of 21-40 years age groups constitute working class and are less compliant to interventions like quarantine, public use of mask and social distancing. Overcrowding in urban cities of Pune and Nashik at home, workplace or during commuting, could explain higher positivity in younger population. Younger people being less compliant had increased risk of getting exposed to infection. Besides many young people being asymptomatic carriers were the main drivers of the pandemic. Also, the data may be skewed towards younger age group as younger people are more approachable to COVID-19 testing. Whereas, older people are restricted to home and are subjected to testing only when they get sick. This study indicated highest (27.62%) SARS-CoV-2 positivity in individuals above 61 years of age. Prior studies have also indicated higher affection and mortality in elderly individuals [13,14]. This age group have additional co-morbidity in making them prone for increased severity of disease and hospitalisation. Existing COVID-19 statistics also indicate higher mortality rate in Maharashtra (3.5%) as compared to the rest of India (2.05%) [15].

Results indicate male predominance which corroborates with other studies [16,17]. Males are more susceptible to SARS-CoV-2 infection due to higher expression of ACE-2 receptors in males as compared to females [16]. Besides females are found to have higher macrophage and neutrophil activity with higher antibody production [16]. Moreover, in Indian setting, males contribute to a higher percentage of outdoor working population thereby increasing the risk of exposure.

Although the number of asymptomatic people tested was higher, there was no difference in the COVID-19 positivity rate between symptomatic and asymptomatic people. The asymptomatic individuals are main source of spread of infection in susceptible Indian population. The higher asymptomatic population is indicative of active contact tracing efforts. The commonest symptoms reported were fever, cough and breathlessness. The study results indicated considerable affection of HCW. These HCW are the real COVID-19 warriors, involved in day-to-day management of infected patients and their personal health needs to be safe guarded. The HCW should be on priority list of immunisation, whenever a protective vaccine is made available.

The study results should be seen in the light of certain limitations. The study analysed the samples received only during the initial four months of pandemic. Non-pharmacological intervention and government policy will influence the future course of pandemic in Indian setting. Also, authors could not ascertain the outcome of the patient following detection of SARS-CoV-2 infection.

This descriptive study is preliminary assessment of 11,196 samples tested for SARS-CoV-2 by RT-PCR at our laboratory from March to July 2020. The study showed rising positivity trend of COVID-19 requiring strengthening and capacity building of laboratory network for early diagnosis. The disease containment strategy needs to be focussed on younger population. Integration of laboratory would provide nationwide data for generating meaningful strategies.