Severe Acute Respiratory Syndrome Coronavirus-2 - A Surge of CoronaVirus Disease-2019: An Epidemiological Study in Coimbatore District.

Context: Coronavirus disease-2019 (COVID-19) is an ongoing pneumonia-like cluster syndrome which originated in Wuhan city of China and is still now on escalation, causing severe outbreaks all over the world. Being a ribonucleic acid (RNA) virus which has the low proofreading RNA-dependent RNA polymerase leads to many mutations and that serves as the major cause for the progress of the disease. As per the recent research works done, 99% of COVID-19 severe acute respiratory syndrome coronavirus (SARS-CoV-2) are due to pangolin-associated coronavirus which causes the super spreading events of coronavirus. SARS-CoV-2 was identified in the nasopharyngeal swabs received in the viral transport medium at optimum temperature. Materials and
Methods: The tests were conducted for a time period of 1 year from July 2020 to June 2021. A total of 77,824 samples were tested in the laboratory as per ICMR guidelines using approved RNA extraction kits and polymerase chain reaction kits.
Results: In the total of 77,824 samples tested in our laboratory, 14174 positives were identified. In that, about seven positive cases (0.004%) were identified in the month of July 2020 which increased to the maximum in September 2020 to about 865 positive cases (6%) which is the peak of first wave COVID-19 in Coimbatore district, Tamil Nadu. Out of 77,824 samples tested, the actual cumulative laboratory-confirmed positive cases of about 14174 were identified. In that, 7731 (55%) male positive cases were identified, 6171 (43%) female positive cases were identified, and 270 (2%) children who were below 12 years of age also were tested positive. Conclusions: The findings of the study indicated a high predominance of SARS-CoV-2 infection in the male gender population when compared to females and children below 12 years of age in Coimbatore district as of June 2021. The surge of cases was high in September 2020 as well as in May 2021, indicating the first and second wave of COVID-19. Copyright:

INTRODUCTION

A global alarm of coronavirus disease-2019 (COVID-19) is still an ongoing threat in all parts of the world. This global threat had its beginning in Wuhan city of China in December 2019. COVID-19 is now currently called severe acute respiratory syndrome coronavirus-2 (SARS-COV-2) which has affected a total of 3,16,662 people across 188 countries till March 2020.[1] The severity of the disease is continuously increasing uninterruptedly. The initial origin or the “start point” of this deadly virus was a seafood market in Wuhan, Hubei district, in Mainland China, which gradually moved on to Italy, which has the maximum number of cases and the death rate is escalating day by day.[23] In India, the first case was a medical student who returned to his native Kerala from Wuhan on January 30, 2020.[4] The disease presents with fever, cough, myalgia, and fatigue leading to severe acute respiratory disease by cytokine storm and that leads to the end of life.[5]

India's leading laboratory surveillance testing for COVID-19 was the Indian Council of Medical Research which in the initial phase conducted COVID-19 testing in 78 selected national reference laboratories.[6] By January 2021, World Health Organization (WHO) has declared 10,639,684 confirmed cases and 153,184 death all over the world.[7] India has the second-highest cases of coronavirus disease-2019 (COVID-19) as of December 18, 2020, with a total positive cases of 782,915 and deaths at 11,954 in Tamil Nadu.[8] The cases are identified by the laboratory real-time quantitative polymerase chain reaction (RT-qPCR) testing methods using ICMR approved kits, and based on this testing, the number of cases is reported. However, the case identification is not true to the extent as many remain asymptomatic which goes unidentified.[9] To understand the spread of infection among the population, certain seroepidemiological cross-sectional surveys are recommended by WHO to measure the rate of spread of infection and then to take necessary steps for containment accordingly.[10]

Severe acute respiratory syndrome coronavirus-2 – its structure and pathogenesis:

The agent which causes the COVID-19 is a single-stranded positive-sense irregular club-shaped ribonucleic acid (RNA) virus with spike-like projections on their surfaces. They belong to the novel member of 2b beta CoV of the family Coronaviridae. The main reservoir for this SARS-COV-2 was found to be Bats for which exception is HCoV-OC43 and HCoV-HKU1 type virus, where rodents have been identified as the reservoirs.[11]

Since SARS-CoV-2 is an RNA virus, it has the capability to mutate because of the presence of RNA-dependent RNA polymerase enzyme (RdRp/RP) which leads to rapid transmission among humans. As per the Victorian Infectious Disease Research Laboratory, Australia, the cultivation of SARS-CoV-2 in human epithelial cell lines is better when compared to earlier SARS and Middle East respiratory syndrome CoV.[12] The growth of the virus is also successfully done in other countries such as China, the USA, Singapore, Japan, and India. Other cell lines such as VeroCCL81 and Vero6 are also under trial.[1314]

The coronavirus genome encodes both the nonstructural and structural proteins. The structural proteins include spike surface glycoprotein (S), small envelope proteins (E), matrix protein (M), nucleocapsid protein (N), and eight accessory proteins which encode the 3' terminus, whereas the open reading frame encodes the 5' terminus. The nonstructural proteins code for viral replication inside the host cells and structural protein-like spike glycoprotein attaches to the host cell surface through angiotensin-converting enzyme-2 receptor (ACE-2) main receptor.[15] ACE-2 receptors are found in lung, heart, ileum, kidney, and bladder. In lungs, ACE-2 receptors are highly expressed in lung epithelial cells.[16] Due to this injury to the lung, it starts producing interleukin (IL)-6 in large numbers in addition to IL-8. This serves as the responsibility for major inflammatory reactions leading to severe symptoms such as acute respiratory distress syndrome.[17181920]

MATERIALS AND METHODS

RT-PCR was done to identify the SARS-CoV-2 in nasopharyngeal swabs received in viral transport medium at optimum temperature in Pathogenix Labs, Coimbatore. All the samples received in the laboratory are treated as potentially infectious and received with proper precautions of wearing gloves and a laboratory coat. A total of 77,824 SARS-CoV-2 cases were tested in the laboratory for a period of 1 year from July 2020 to June 2021. As per the ICMR guidelines, RNA extraction was done using Zybio Nucleic Acid Extraction kit and RT-PCR detection using CoviDx Assure SARS-CoV-2 kit.

RNA extraction is done by the magnetic bead method, where all the reagents are brought to room temperature before starting the extraction. Working solution is prepared using the extraction reagent I (guanidinium isothiocyanate, tris buffer, and dimethyl carbinol), magnetic beads solution, and Proteinase K provided in the kit. All reagents and solutions prepared are used within 30 min of preparation. This working solution is then added to the sample in a 1.5-ml centrifuge tube. It is then heated for 4 min on a dry bath at 550°C. After centrifuge, the mixtures are placed on the magnetic separator for 1 min, and then discard the supernatant. Then, the extraction reagent II (tris buffer and NaCl) is added on and the same procedure is repeated. At the end, the elution buffer (deionized water) is added and heated for 2 min at 800°C. The magnetic beads have specific polymeric nucleic acids (DNA/RNA) adsorbed on their surface. These nucleic acids are separated from the liquid phase when the magnetic separator is used by washing steps in between will remove the residual impurities and inhibitors that are present. At the end, nucleic acids are eluded from the magnetic beads by changing the liquid phase conditions.

Once the RNA is extracted, 17 ul of master mix preparation of COVIDX Assure SARS-CoV-2 kit is added on to 8 ul of the RNA extracted and using ROCHE Diagnostics RT-PCR detection machine which is represented in Figure 1 with thermal cycling conditions adjusted according to the Master Mix kit used. On completion, analysis of the data is done as per the manufacturer setup.

Figure 1

Polymerase chain reaction machine

RESULTS

This study was conducted for a period of 1 year in Pathogenix Labs, Coimbatore, from July 2020 to June 2021. A total of 77,824 samples were tested in the laboratory as per ICMR guidelines and using ICMR approved RNA extraction kits and PCR kits. In the total of 77,824 samples tested in our laboratory, 14174 positives were identified. In that, about seven positive cases (0.004%) were identified in the month of July 2020 which increased to the maximum in September 2020 to about 865 positive cases (6%) which is the peak of first wave COVID-19 in Coimbatore district, Tamil Nadu.

There was a drastic decrease in the number of cases in January, February, and March of 2021 to about 0.3%, 0.5%, and 1%, respectively. There is a surge in the total number of cases in April 2021 to about 988 positive cases (7%) which is followed by a dramatic increase in the month of May 2021 to about 7717 cases (54%), and in June 2021, 3103 positive cases (22%), respectively. This scenario strongly suggests the rise in the number of cases in Coimbatore district as per our laboratory data suggesting the second wave in Coimbatore district which is illustrated in Table 1 as well as in Figure 2.

Table 1

Data for the year 2020-2021

Month	Total number of samples tested	Total number of positive cases (%)
July 2020	20	7 (0.04)
Auguest 2020	1663	223 (2)
September 2020	7154	865 (6)
Octobar 2020	6669	550 (4)
November 2020	3085	235 (2)
December-2020	6907	205 (1)
January 2021	1346	54 (0.3)
February-2021	1397	66 (0.5)
March 2021	1938	161 (1)
April 2021	5404	988 (7)
May 2021	23,101	7717 (54)
June 2021	19,140	3103 (22)

Figure 2

Prevalence of coronavirus disease 2019 positivity in Pathogenix Lab for the year 2020–2021

Out of 77,824 samples tested, the actual cumulative laboratory-confirmed positive cases of about 14174 were identified. In that, 7731 (55%) male positive cases were identified, 6171 (43%) female positive cases were identified, and 270 (2%) children who were below 12 years of age also were tested positive. This shows the higher predominance of the COVID-19 male population when compared to the females, and in children , it was very low to about 2% in both the waves of COVID-19 surge. This is illustrated in Figure 3.

Figure 3

Positivity in the gender population

CONCLUSIONS

The findings of the study indicated a high predominance of SARS-CoV-2 infection in the male gender population when compared to females and children below 12 years of age in Coimbatore district as of June 2021. The surge of cases was higher in September 2020 as well as in May 2021, indicting the first and second wave of COVID-19. Increasing the testing of more number of samples in future will give us information on the rapidity of transmission and also help us to evaluate the impact of containment strategies that are followed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.