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 Table of Contents  
Year : 2021  |  Volume : 10  |  Issue : 2  |  Page : 108-114

Diagnostically fighting the coronavirus disease 2019 pandemic: A general perspective

1 Department of Biochemistry, Centre of Excellence in Molecular Biology and Regenerative Medicine, JSS Medical College, JSS Academy of Higher Education and Research, Mysore, Karnataka, India
2 Department of Biochemistry, JSS Medical College, JSS Academy of Higher Education and Research, Mysore, Karnataka, India

Date of Submission05-Sep-2020
Date of Decision15-Jan-2021
Date of Acceptance19-Jan-2021
Date of Web Publication18-May-2021

Correspondence Address:
Dr. Devananda Devegowda
Department of Biochemistry, Centre of Excellence in Molecular Biology and Regenerative Medicine, JSS Medical College, JSS Academy of Higher Education and Research, Mysore - 570 015, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijhas.IJHAS_217_20

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The recent outbreak of severe acute respiratory syndrome-coronavirus 2 has brought human survival and existence to an all-time low. The infection seems to be uncontrollable, and the scientific community has no answer yet. The only means of keeping the infection under the check is the effective usage of both serological and molecular diagnostic testing. The article provides the readers with the basic understanding of the current pandemic situation and educates them regarding the same. It is essential that members of all the scientific background are aware of the current pandemic at its basic level. This would direct the scientific community to work toward warding off the pandemic which is the need of the hour.

Keywords: Coronavirus, coronavirus disease 2019, molecular diagnostics, severe acute respiratory syndrome-coronavirus 2, serology, vaccine

How to cite this article:
Ramesh PS, Arun B, Devegowda D. Diagnostically fighting the coronavirus disease 2019 pandemic: A general perspective. Int J Health Allied Sci 2021;10:108-14

How to cite this URL:
Ramesh PS, Arun B, Devegowda D. Diagnostically fighting the coronavirus disease 2019 pandemic: A general perspective. Int J Health Allied Sci [serial online] 2021 [cited 2023 Nov 29];10:108-14. Available from: https://www.ijhas.in/text.asp?2021/10/2/108/316283

  Introduction Top

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is globally considered as pandemic, causing over 23 million outbreaks worldwide. It emerged from Wuhan, China in December.[1],[2] The virus was initially termed as Wuhan coronavirus or 2019 novel coronavirus (2019-nCov) by the Chinese researchers, the World Health Organization (WHO) finally named it as SARS-CoV-2, and the disease as coronavirus disease 2019 (COVID-19).[3] As of September 3, 2020, a total of 25,884,895 confirmed cases globally, with 859,130 deaths had been reported by the WHO. The coronavirus belongs to a family coronoviridae known to cause various symptoms such as pneumonia, fever, breathing difficulty, and lung infection.[4],[5] Tyrrell and Bynoe were the first ones to isolate the coronavirus from the respiratory track of an adult with common cold dating back to the year 1965.[6] Four decades after the discovery, coronaviruses have caused three pandemics, namely, SARS in 2003, middle-east respiratory syndrome (MERS) in 2012, and COVID-19 which is ongoing.[6],[7] Initial case was classified as pneumonia of unknown etiology, later Chinese Center for Disease Control and Prevention (CDC) and local CDCs attributed it to the novel coronavirus family.[8]

Coronaviruses contain a single-stranded RNA and are divided into α, β, γ, and δ-CoV.[9] The alpha and beta coronaviruses originate from mammals, specifically from the bats, gamma, and delta coronaviruses originate from birds and swine.[10] The genome size varies between 26 kb and 32 kb. The alpha-coronaviruses comprise HCoVNL63 and HCoV-229E, and the beta-coronaviruses comprise HCoV-OC43, HCoV-HKU1, SARS-CoV, MERS, and SARS-CoV-2.[11] Previously, six human coronaviruses had been discovered, and the novel coronavirus is the seventh known coronavirus, which belongs to beta group of viruses.[10],[12] Like all coronaviruses, COVID-19 consists of three viral proteins: a spike protein, a membrane protein, and an envelope protein. The novel coronavirus has a genome similar to that of the SARS-CoV[4] with 80% nucleotide identity[12] and is believed to be transmitted from animals[10] and requires the angiotensin-converting enzyme 2 (ACE2) as a receptor to enter the cell.[7] The spike protein of the virus binds to the cell receptor and cause membrane fusion.[13] The common symptoms at the onset include fever, cough, and myalgia, and few patients showed symptoms of rhinorrhea, sneezing, or sore throat. The complications include acute respiratory distress syndrome, acute cardiac injury, and secondary infection.[14] The virus is transmitted human to human[15] through aerosols, by sneeze or cough droplets, or by direct person-to-person contact or indirect contact of the contaminated object.[16] Wind speed, humidity, and temperature play a major role in the survival of the virus.[17] The human coronavirus can survive up to 9 days in normal temperature, causing infection.[18] With the widespread of this virus like wildfire, development of vaccine against it is the current challenge. Diagnostic testing plays a major role in the identification and containment of any infection. This enables the rapid implementation of control measures which has the potential to limit the spread to a certain extent.[15] As more COVID-19 cases appear in and around the world, there is an unmet need for the development of rapid, easy-to-use, and cost-effective diagnostic tests. In the ongoing coronavirus pandemic, the diagnostic tests have become an essential tool to track the spread of the COVID-19 disease. This is a concern which has caught the eye of the scientific community. The research labs have to step up in to developing their own diagnostics to meet the crushing need.

In this mini review, we examine how viral diagnostic tests work, the ones which are available commercially and are Food and Drug Administration (FDA) approved. A special emphasis is given on India's scenario which seems to be struggling to get the statistics of COVID-19 right, as the number of infected cases shows large signs of fluctuation every day. The country's lockdown and health policy looked like working out very well initially and but later on started failing miserably in majority of the places due to the poor awareness and lack of access to facilities with the cases of COVID-19 being dangerously underestimated.

  The Pandemic Scare Top

From being unknown to most of the population to being the most talked and feared disease, the specter of COVID-19 infection has come into the limelight over the past couple of months. What has made most people worry is the fact that there is no definitive cure or vaccine yet for this disease and that it carries a very high mortality rate.[19],[20] Thanks to the strict government policies which advocated the preventive measures by locking down the whole country in stepwise phases which helped curb the spreading of viral infection. However, unlocking has proved to be a disaster as the country's overview of COVID-19 infection does not show any signs of slowing down. As of August 23, 2020, India is the 3rd worst affected country in the world with over 3 million confirmed cases and 56,883 deaths with the infection rate still accelerating (https://www.worldometers.info/coronavirus/country/india).

The symptoms of COVID-19 are similar to many other diseases, such as influenza, making diagnostic tests quintessential to pinpoint people who actually have COVID-19. In addition to identifying the infected people, the diagnostic tests can also help determine who has recovered from COVID-19, as well as improve our understanding of how the virus spreads and help monitor the effectiveness of control measures. The two principal tests used in the diagnostics currently worldwide are the molecular testing detecting the viral genetic material and the serological testing detecting antigens/antibodies against the virus.

  Molecular Testing of Coronavirus Disease 2019 Top

Testing is the most ideal approach to be certain that someone is positive for the COVID-19 infection. Alongside measures such as social distancing and self-isolation, quality testing is an essential part of efforts to contain and control the COVID-19 pandemic, which currently has no approved vaccine or disease-specific treatment. Typically, in any pandemic circumstance, the assays should be high throughput, implying that laboratories can test high number of samples in a brief time frame and accuracy, which means a reliable test result. This can be accomplished by analyzing patient samples using real-time reverse transcription polymerase chain reaction (real-time RT-PCR) technology. Basically, the technology works by detecting the RNA component of the virus that causes COVID-19. The specimens are collected in a solution containing the lysis buffer of the specified nucleic acid extraction kit. This extracted RNA will be a mix of the patient's own genetic material and the virus's RNA, if present. The single-strand RNA is reverse transcribed to double-strand DNA using a reverse transcriptase enzyme. Additional short fragments known as primers (for the specific targets) that are complementary to specific parts of the transcribed viral DNA will be added. If the viral genetic material is present in a sample, these fragments attach themselves to target sections of the viral DNA and get amplified in the process. The dye used in the assay will tag the amplifying segment which can be then used to detect the virus in real time.

  The Paradigm in Assay Selection for Molecular Detection of Coronavirus Disease 2019 Top

During the initial days of identification of COVID-19, random-amplification and deep-sequencing methods contributed very well.[21],[22] Such molecular methods including next-generation sequencing will continue to play its role to determine the mutations of COVID-19 in future but are impractical to be used as a diagnostic tool. Most of the molecular methods developed for the diagnosis of COVID-19 including those from the U.S. Centers for Disease Control and Prevention involve real-time RT-PCR assays. Other molecular methods that are being developed and evaluated worldwide include loop-mediated isothermal amplification (PCR), multiplex isothermal amplification, and microarray-based assays.[23],[24],[25]

Even though real-time RT-PCR is a boon to the molecular testing world, target selection for the assay is a definite challenge. A major advantage of real-time RT-PCR assays is that amplification and analysis are done simultaneously, and the results can be made available in real time. Furthermore, since the assay is performed within a closed system, it minimizes false-positive results which are usually associated with amplification product contamination. Within their positive-sense, single-stranded RNA genome, coronaviruses have a number of molecular targets that can be used for PCR assays [Figure 1]b. The genes encoding structural proteins which are usually targeted include spike glycoproteins (S), envelope (E), transmembrane (M), and nucleocapsid (N). In addition, some researchers have also explored the possibility of targeting species-specific accessory genes that are required for viral replication, namely, RdRp (RNA-dependent RNA polymerase), HE (hemagglutinin-esterase), and open reading frame 1a (ORF1a) and ORF1b.[26],[27]
Figure 1: Schematic representation of coronavirus disease 2019 (a) structure and (b) genome organization. The genome organization of coronavirus disease 2019 viral RNA (~30kb) was adapted from GenBank accession number MN908947

Click here to view

It is important to include at least two molecular targets in the assay to avoid potential cross-reactivity with the other endemic coronaviruses and also to negate the potential genetic drift of COVID-19. The CDC recommends two nucleocapsid protein targets (N1 and N2), while the WHO recommends first-line screening with an E gene assay followed by a confirmatory assay using the RdRp gene.[28],[29] Investigators from different countries have used various molecular targets and one such study utilized two sequence regions (ORF1b and Nucleo-capsid protein [N protein]) that are highly conserved among coronavirus subtypes for the inceptive RT-PCR testing.[30] Currently, there are no evidences suggesting that any one of the sequence regions offers a unique advantage for clinical testing. However, CDC came up with a RT-PCR diagnostic panel and listed few primers and probes for the detection of novel coronaviruses 2019 and can be found in their official website (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html). The ideal design would be to include one conserved region and one specific region as the viruses are known to evolve within the newer populations owing to the effect of genetic drift. In a recent study, Chan et al. compared the assay performance of three novel real-time RT-PCR assays targeting the RdRp/Hel, S, and N genes of COVID-19. Among them, the COVID-19-RdRp/Hel assay had not only higher sensitivity and specificity but also the lowest limit of detection.[27]

  Serological Testing of Coronavirus Disease 2019 Top

Serology is used to measure the host immune response to an infection and is best utilized retrospectively. Various kit manufacturing companies and research institutions are striving to develop rapid serological methods have proven to be useful in confirming the COVID-19 infections. The technique previously has had an important role in assessing the epidemiology of other infectious disease outbreaks previously.[31],[32] Lateral flow assays (LFAs) for both IgM and IgG antibodies undeniably have played an important role allowing the epidemiologists to determine the burden of infection and the overall mortality during the COVID-19 outbreak. However, serology detection is not likely to play a role in active case management of COVID-19 infection due to the nonspecific responses of IgM and longer duration required to develop specific IgG responses. However, as the outbreak progresses, serological testing can aid in diagnosis or confirmation of late COVID-19 cases and also to determine the immunity of frontline workers.

COVID-19 serology testing mainly relies on targeted antibodies binding to virus-specific antigens. Blood serum from suspected patient is collected and applied to a testing platform such as microtiter plate or a chip containing the viral antigen. The blood gets drawn through the capillary action inside the device where it mixes with the antigens. If the patient has been exposed to the viral infection, then the body would have developed corresponding antibodies against COVID-19 which will be recognized. Currently, on the market, the available platforms for COVID-19 serology tests include LFAs, ELISA, and chemiluminescent immunoassays. Each of these assays differs in the way they detect antibody–antigen binding.

The antigen specificity dictates the accurate interpretation of serology tests. If antigens are not pathogen specific, then the chances of cross-reactivity increases and the reliability of test results decrease. Commercially available serological testing kits target the surface and the nucleocapsid proteins as antigens to develop their kits [Figure 1]a. The following viral antigens to detect antibodies for COVID-19 infection: (i) Spike proteins which are monomeric surface proteins that bind to host cells and aids virus entry. Each monomer contains two subunits: S1 that binds to host cell receptors and S2 facilitates fusion of viral and cellular membranes. (ii) N protein is a basic RNA-binding protein that plays an important role in replication and transcription within the host cells. (iii) Receptor Binding Domain (RBD) – RBD is a portion of the S1 subunit that binds ACE2, the human receptor for the virus.

  How is India Handling Coronavirus Disease 2019 Diagnostics? Top

In India, The Indian Council of Medical Research (ICMR), the apex body for formulation, coordination, and promotion of biomedical research has been monitoring the coronavirus situation since the pandemic breakout. Currently, RT-PCR probes for diagnosis of COVID-19 are procured from USA by ICMR-National Institute of Virology (NIV) and are distributed for evaluation to the various testing laboratories across the country. These US FDA EUA/CE IVD-approved kits can be only used after the intimation to ICMR and the approval from DCGI (Drugs Controller General of India). ICMR has established a fast-track mechanism for validation of non-US FDA EUA/CE IVD-approved kits at ICMR NIV and the information gets updated frequently on https://www.icmr.gov.in/.

Although RT-PCR is the standard test carried out in testing the COVID-19, ICMR has widened the range of testing by implementing rapid antibody, ELISA, and TrueNat testing. While RT-PCR takes around 3–5 h for obtaining the result, the latter requires around 30–60 min to obtain results. Until the end of May, RT-PCR had capped a cost of Rs. 4500 per test. However, later on the Union Health Ministry and ICMR interfered and has asked the private laboratories to step down the pricing. A total of 199 kits of various manufacturers have been evaluated by ICMR validation centers, out of which 96 were found to be satisfactory kits and were approved for testing (https://www.icmr.gov.in/pdf/covid/kits/RT_PCR_Tests_Kits_Evaluation_Summ_11082020.pdf). Seegene and SD Biosensor are South Korean RT-PCR-based diagnostic kits in India which has received government approval and are widely used. SD Biosensor's Standard M nCoV RT Detection kit detects the E gene and the RdRP gene strains,[33] whereas Seegene's Allplex 2019-nCOV Assay detects the E gene, RdRP gene, and N gene[34] and delivers the diagnosis in <4 h.

Rapid antigen test is another technique implemented for testing, the advantage of this test is that it takes hardly 20–30 min to obtain results and also it is cost effective, costing Rs. 600 per test. In India, 19 diagnostic kits have been validated, of which only three kits have been approved by the NIV, Pune, and 15 kits do not have approvals, although they have obtained licenses and certificates in other countries (https://www.icmr.gov.in/pdf/covid/kits/List_of_rapid_antigen_kits_07082020_v.pdf). The government has also approved 12 rapid antibody test kits such as BioMednomics (USA), Getein Biotech (China), Sensing Self Ltd (Singapore), Hangzhou Biotest Biotech (China), AmonMed Biotechnology Co (China), Beijing Tigsun Diagnostics Co Ltd (China), Biomaxima (Poland), CTK Biotech (USA), Hunan Lituo Biotechnology Co (China), Vivacheck Lab (China), and Wondfo (China). Recently, two private companies – Indian company MyLab and German firm Altona Diagnostics got government approval to supply COVID-19 test kits.

On exposure to the COVID-19, IgG and IgM antibodies are produced to fight against the pathogen. The IgM antibodies are produced in 4–7 days after pathogenic challenge to the body while the IgG antibodies are usually produced between 10 and 14 days.[35],[36] The ICMR had earlier approved Indian biopharma company Transasia's ELISA antibody IgG testing kit, which is the first Indian company to get the approval in the country. However, recently, the marketing license of the same had been suspended by the central drugs standard control organization as the kit was removed by USFDA (https://indianexpress.com/article/india/kit-import-licence-of-3-firms-cancelled-15-suspended-order-based-on-ban-by-usfda-6523542/). ICMR has also designed a Covid Kavach ELISA IgG test through the NIV in Pune. The other manufacturers of ELISA kits in India are Zydus Cadila, Meril Diagnostics, Voxtur Bio, Trivitron Healthcare, J Mitra and Co, Karwa Enterprise, and Avecon Healthcare (https://www.icmr.gov.in/pdf/covid/kits/ELISA_CLIA_Kits_List_07082020.pdf).

  Current Issues and Challenges in the Future of Clinical Testing Top

As already discussed, COVID-19 serology assays are designed to be specific for the virus, but how can it be assured that they will not cross react with other coronavirus subtypes? Cross-reactivity with the common coronaviruses would be a major issue since 50%–70% of children have antibodies to one or more coronaviruses, and 80%–90% of the adults have antibodies to all four coronaviruses.[37] Theoretically, the rapid antigen LFAs would provide the advantage of a fast turnaround time and affordable pricing for the detection of COVID-19, but they are bound to suffer poor sensitivity, especially in the early stages of infection. Given the variability of the viral loads in patients, there is concern that antigen detection may miss true positive cases due to low infectious burden or sampling discrepancy.

A key question that still remains unsolved is that if a person was exposed to COVID-19, develops antibodies, and in future, if the same person comes in contact with the virus again, will they be protected? How long will the immunity last? To answer such debatable questions, it is essential to develop serological tests that target not the less specific IgM isotype but the longer lasting, highly specific IgG antibodies. In addition, one should also remember that just the presence of IgG antibodies does not guarantee protection from future infection. However, a recent study reported that rhesus macaques that were re-challenged to SARS-CoV-2 strain nearly a month after the initial infection, did not show any clinical signs of viral dissemination.[38] Even though the data are limited, it offers hope that the development of neutralizing antibodies may provide immunity against reinfection of SARS-CoV-2. Furthermore, there is no have definitive proof yet and it is hard to tell how long the immunity will last if it exists.

COVID-19 diagnostic testing has been rapid and has scaled up globally. This increase in testing has been valuable in making public health decisions as well as monitoring patient care. However, many low- and middle-income countries have faced numerous difficulties. The demand to supply exceeds despite the rapid scale-up in diagnostics, and the distribution of the available supply has been far from the expectations of the WHO. Many manufacturers have entered the diagnostics market, and it has been nothing but a challenge to ensure adequate test quality and reliability to evaluate which ones work best. Due to the pandemic, the shipping of supplies has been disrupted the national and international transportation and has further added to the slowing down of testing in some countries. Some diagnostic laboratories have been overburdened with the volume of testing, sometimes causing testing errors or delay in reporting the results.

  The Vaccines and the Path Forward Top

The much awaited vaccines for the novel SARS-CoV-2 are already here, and it required an intense global R and D activity for the development of the vaccine with the fast-tracked human clinical testing. The world saw a surge in the vaccine development with diverse platforms of technology coming into play. In the middle of March 2020, the pandemic forced numerous nonprofit organizations, private/industry sectors, and academic research institutes to invest their time and money in developing vaccines for the viral infection spreading like a wildfire. Multiple technology platforms were evaluated including the DNA and RNA, viral peptides, recombinant proteins, live attenuated, and inactive virus approaches. Of all the various approaches, the novel platforms based on the nucleic acids offered great flexibility in terms of antigen manipulation and potential for rapid development of vaccines.

The global effort of the R and D in response to the unexpected pandemic was unprecedented in terms of scale and speed, and the vaccines were expected to be made available by early 2021. The worldwide endeavor to create a safe and effective COVID-19 vaccine is bearing fruit and rightly so a handful of vaccines now have been authorized around the globe; many more remain in development. When all hopes were lost with the pandemic affecting uncontrollably, India looked toward becoming self-reliant in the development and production of COVID-19 vaccine and initially at least five major pharmaceutical industries of India engaged in the vaccine development collaborating with various research institutes. Finally, Covishield vaccine developed by Oxford-Astra Zeneca in association with Serum Institute of India, Pune and Covaxin developed by Bharat Biotech, Hyderabad, in association with ICMR won the race to develop and mass produce the vaccines.

As of January 16, 2021, there are eight vaccines that are approved on a full scale to be administered, some in multiple countries and some only in the country of the vaccine origin. [Table 1] summarizes currently authorized vaccines, their origin, and the platform on which the vaccine is developed.
Table 1: Details of the currently authorized vaccines for COVID-19

Click here to view

  Concluding Remarks Top

The current pandemic has been the biggest public health crisis; the world has seen since the great World War II. The ongoing, freakish outbreak of COVID-19 worldwide has once again highlighted the importance of the laboratory diagnosis in order to limit the spread of coronavirus as well as to appropriately monitor those patients who have a serious infection. This mini review has opined about the significance of diagnostic testing, challenges regarding such testing for SARS-CoV-2, current scenario in India and the unresolved questions.

The restrictions issued on various activities in relation to COVID-19 have slowly started to be lifted off. Diagnostic testing will continue to play an important role in the identification of people infected with SARS-CoV-2, aid in guiding their treatment, indicate who needs to be isolated to curb the further spread of the infection. There are a lot of speculations on the vaccines which are on the verge of development and diagnostics will still be essential to make sure such vaccines are used efficiently worldwide. Diagnostically made available surveillance data can help identify which parts of the population needs vaccine the most, the populations still at risk of new infections, and the ones which are infected and recovering well. Without a doubt, there is an urgent need for the development of affordable point of care diagnostic testing with short turnaround time which will help in patient management in real time and also make effective clinical decisions. In order to defeat this COVID-19 pandemic, the only solution is a multifaceted approach and diagnostic testing is a key factor.


Pushkal Sinduvadi Ramesh acknowledges the Senior Research Fellowship from Council of Scientific and Industrial Research, Government of India (Sanction no. 08/721[0001]/2018-EMR-I)

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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