Pharma Focus Asia

Standard Operating Protocol (SOP) for The Clinical Diagnosis of Novel Coronavirus(COVID-19) Infection

Mohanarangan Sundararam, Research Scholar, Crystal Growth Centre, Anna University

Jaison Jeevanandam, Faculty, Engineering and Science, Curtin University

The outbreak of novel Coronavirus Infection (COVID-19) has dramatically affected the lives of humans throughout the world with increasing number of asymptomatic and symptomatic cases every day, where laboratory testing is essential to identify, categorise and quarantine the COVID-19 infected patients via proper diagnosis. The serological findings, molecular testing, and Computer Tomography scan (CT scan) are followed as the Standard Operating Protocol(SOP) for the confirmation of COVID-19 infection. These combinatorial diagnosis approach possess several advantages and certain limitations based on the cost, sensitivity, accuracy and rapid detection of novel coronavirus infection.

The outbreak of a novel Coronavirus (COVID-19) in 2020 has led to an unexpected alteration in human lives and economic activities. There is a global increase in the number of COVID-19 infected people, every day and the ability of this virus to spread rapidly has been a major concern for governments, which has led to lockdown measures for several months to mitigate its spreading rate. COVID-19 has been identified to cause Severe Acute Respiratory Syndrome (SARS) by novel Coronavirus 2 (CoV2), which has affected almost every category of humans in the world, irrespective of their age, race or geographical regions. Several reports showed that this viral infection is lethal to elders and patients with comorbid as well as immune-compromised condition and are included as high-risk groups. In addition, youngsters with immunity will be an asymptomatic carrier of this virus towards high risk patients. Thus, proper clinical diagnosis of COVID-19 is highly essential to provide specific treatment, isolation and monitor virus infected patients during this pandemic situation. It can be noted that increasing the number of laboratory tests, assessments and evaluations are the crucial diagnostic strategy, that are followed by the governments to successfully control this viral infection. In particular, diagnostic methods, such as serological evaluations, molecular methods, and Computer Tomography scan (CT scan) are currently followed as a Standard Operating Protocol (SOP) for the confirmation of COVID-19 in patients. However, these methods possess certain advantages as well as limitations, depending on their cost, sensitivity, accuracy and detection in real-time.

Serological Evaluation

In general, the serological tests are used to determine the antibodies, that are produced against a specific pathogen in a person after 7 days of symptomatic infection. The incubation period of novel coronavirus or SARS-CoV-2 is 14 days and hence the test will be performed after the incubation period to identify the infection of COVID-19 in patients. There are three distinct types of antibodies, such as Immunoglobulin G (IgG), Immunoglobulin M (IgM), and Immunoglobulin A (IgA) will be secreted in COVID-19 infected patients, which will be beneficial in diagnosing the exact stage of infection/disease. In the early stage of infection, IgM levels will be higher in the serum of infected person, whereas IgG levels will be higher after the reduction of the infection. Recently, a rapid detection kit has been developed by Udugumaet al. (2020) with a paper-like membrane strip, which is coated with two lines, where gold nanoparticle as well as antibody conjugates are present in one line and captured antibodies on the other line. In this unique diagnostic tool, the blood or urine of the patient will be collected in the membrane to detect the presence of antibodies in COVID-19 infected patients via alteration in the paper strip colour from red to blue. Further, Enzyme Linked Immunosorbant Assay (ELISA) is another significant method for the detection of target antibodies. Recently, ELISA assay was used to evaluate the presence of COVID-19 infection in 167 samples collected from 94 patients. The study showed that the IgG antibodies can be detected via later flow ELISA approach after 14-25 days of viral infection with 92.1 per cent of sensitivity, compared to IgG ELISA (89.5 per cent). In addition, aptamers were also proposed to be beneficial in the identification and evaluation of antibody after COVID-19 infection. Moreover, serological evaluation has a sensitivity, specificity, and accuracy of 57 per cent, 100 per cent, & 69 per cent for IgM and 81 per cent, 100 per cent and 86 per cent for IgG, respectively. However, serological assay can be used as a preliminary diagnostic method to identify the presence of COVID-19 infection and can be used to identify the percentage of ‘herd immunity’ in a community. Even though, this method is rapid and sensitivity, its specificity is low and molecular methods, such as RT-PCR is required to confirm the viral infection.

Molecular Methods

Molecular methods to identify the presence of specific viral protein, such as real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR), are widely used throughout the world as a confirmatory test for COVID-19 infection. The specimen from upper respiratory tract at the right anatomic position is essential for a prompt and accurate molecular diagnosis of COVID-19. The single-stranded Ribonucleic Acid (RNA) genome and genes encoding structural proteins, including envelope glycoproteins Spike (S), Envelope (E), Transmembrane(M), Helicase (Hel), and Nucleocapsid(N) can be used as molecular targets of COVID-19 in PCR assays. Further, a cycle threshold value of less than 40 is defined as a COVID-19 positive in real-time RT-PCR assay with 100 per cent of accuracy. However, this method can provide false negative results, during early stage of infection (viral loads may not be present at the site of sampling) or improper sampling procedure. Furthermore, this molecular method is not applicable (IgG detection) for determining COVID-19 after the recovery as in serological evaluations and the high cost as well as sample preparation involved in the RT-PCR are the major limitations of this molecular method.

Computer Tomography

The term ‘Computed Tomography’(CT) refers to a computerised x-ray imaging procedure in which a narrow beam of x-ray is applied towards a patient to produce signals that are processed by a computer to generate cross-sectional images of the body. Moreover, CT computer uses sophisticated mathematical techniques to construct a 2D image of the patient’s body part. The thickness of the tissue represented in each image can vary depending on the CT machine, which usually ranges from 1-10 mm. If the patient with severe symptoms of COVID-19 is tested negative via RT-PCR, CT scans of the lungs are currently used to confirm the presence or absence of the SARS-CoV-2. It is worthy to note that the sensitivity (97.2 per cent) of CT scan is higher, compared to the RT-PCR method (83.3 per cent). Additionally, the cost of CT scans is expensive, however it is necessary to perform both CT and RT-PCR test for the person with mild symptoms of COVID-19 to monitor the disease and mitigate its spread. Recently, we have been working on a rapid test kit for the detection of SARS-CoV-2 infection using the saliva of the patient. The test kit is fabricated using novel green synthesised gold nanorods coated with specific biomolecule, which can change its colour after binding to the spike protein. We believe that this rapid test kit will be beneficial in the swift identification of the infected patients and to control the spread of this novel coronavirus.

Mohanarangan Sundararam

Mohanarangan Sundararam is a research scholar, pursing his Ph.D. in Crystal Growth Centre, Anna University, Chennai, India. His Ph.D. project is about ‘Instrumentation development for the biochemical assays with enzymatic reaction’, which involves several biochemical, biotechnology, clinical lab and bio-analytic aspects. He has several years of experience as a biochemist in various hospitals and laboratories. Further, he has authored a book chapter, few research articles and conference proceedings related to biosensors for biochemical assays. His current research focuses on enzymatic assays and biosensor fabrication. Currently, he is also working as a lecturer in Academy of Competitive Examinations and Research Training (ACERT), Chennai, India.

Jaison Jeevanandam

Jaison Jeevanandam obtained his Ph.D. in the Department of chemical engineering, Faculty of Engineering and Science from Curtin University, Malaysia. His Ph.D. project is about ‘Novel nanomedicine for reversing insulin resistance in Type 2 diabetes’ which involved cross-disciplinary research between physics, chemistry, biology, medicine and engineering. He has experience in nanoparticle synthesis, especially in green synthesis using plant extracts, characterisation, cytotoxic analysis of nanoparticles,and also in vitro diabetic models. His current research focuses on the application of bionanotechnology in the fabrication of nanoformulation to deliver drugs for the treatment of several diseases, especially diabetes. Till date, Dr. Jaison has authored about 25 book chapters, 33 journal articles along with some conference papers and H-index of 11. Currently, Dr. Jaison is working as a lecturer in Academy of Competitive Examinations and Research Training (ACERT), Chennai, India.

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