Probable cases are tested. It is an immunoassay test that detects the viral antigen. In India, the ICMR has proposed to use the antigen detection kits called as Standard Q COVID-19 Ag detection kit (developed by the South Korean company S D Biosensor). The kit consists of covid antigen test device, viral extraction tube with viral lysis buffer and sterile swab for sample collection. 11 Samples are collected from the respiratory tract using the sterile swab provided in the kit. If the antigen is present in the sample, it will bind to the paper strip containing specific antibodies covered with plastic casing and produces visually detectable signals within 15 minutes. If the result is negative, then further confirmation to be done by RT-PCR (Reverse Transcription Polymerase Chain Reaction). The rapid antigen detection testing is useful in early detection of covid19, as it shows positivity only when the antigen is replicating. False positive results are reported with this test, as the antibodies on the strip can detect other antigens too. Hence this test is not preferred commonly and the results from this testing needs further validation. 12

In this test the antibodies produced against corona virus infection is detected. But this test is not specific to covid19 and detects antibodies produced against any corona virus. In addition if the test is carried out too early in the disease process after the exposure to the virus, the antibodies would not have formed and hence not detected. Therefore, it is not used nowadays. 13 (Figure 3)

The confirmatory test for covid 19 is by antigen detection tests, which could be Polymerase Chain Reaction (PCR takes 2-3 hours), Reverse Transcription Polymerase Chain Reaction (RT-PCR takes 3-4 hours), Reverse transcription Loop-Mediated Isothermal Amplification (RT- LAMP takes), Recombinase Polymerase Amplification (RPA takes) or CRISPR-based diagnostics that takes 15-60 minutes. 9

PCR was introduced by Kary Mullis in the 1980, where in billions of copies of a specific DNA sample of interest can be produced and used for analysis. The major limitation of this test is that the target sequence should be known before adding the primer. 14

The basic components and reagents for PCR are - DNA template (DNA target region to amplify), DNA polymerase (enzyme that polymerizes new DNA strands by binding and elongation to form double-stranded DNA), heat-resistant Taq polymerase (remains intact during the high-temperature DNA denaturation process), DNA primers (each of the sense and anti-sense strands of the DNA), Deoxynucleoside triphosphates (the building blocks from which the DNA polymerase synthesizes a new DNA strand), buffer solution and the bivalent cations, typically magnesium and potassium ions. 14(Figure 4)

PCR consists of a 20–40 repeated thermal cycles involving temperature changes.

Initialization: It is the first step and is required for activation of DNA polymerases by hot-start PCR.12 It consists of heating the reaction chamber for 1–10 minutes, to a temperature of 94–96℃ (201–205 F), or 98℃ (208 F), if extremely thermo stable polymerases are used.

Denaturation: It is the process of DNA melting or denaturation, by breaking the hydrogen bonds of the double-stranded DNA between complementary bases into two single-stranded DNA strands. This is carried out at 94–98℃ (201–208 F) for 20–30 seconds is the first cycle. Annealing: The next step is annealing at 50–65℃ (122–149 F) for 20–40 seconds where primers get annealed to each of the single-stranded DNA templates at the 3' end of each strand.

Extension/Elongation: 75^o–80℃ (167–176 F), wherein the temperature depends on the DNA polymerase. New DNA strand is formed by adding free dNTPs to the DNA template in the 5'-to 3' direction. Millions of DNA copies can be obtained by following these steps.

To calculate the number of DNA copies per cycle is 2ⁿ (where n is the number of cycles). Thus, a reaction set for 30 cycles results in 2³⁰, or 1,073,741,824, copies of the original double-stranded DNA target region. Then chamber is cooled to 4–15°C (39–59 F).

Gene target for SARS-CoV2 are RdRP- RNA dependent RNA polymerase, ORF (open reading frames ORF1a/b) and N2 nucleocapsid. It takes 5-6 hours to get the result. 15

In this procedure, first RNA primers and reverse transcriptase enzyme are added for DNA formation. Then the PCR procedure is continued. Nowadays automatic machines are used for the entire laboratory test, so is the case for molecular confirmation test for Covid19. Only the test sample need to be added and the results will be out in 80 minutes. 15

Recently researchers have developed CRISPR gene-editing technology which helps in detection of coronavirus in just 5 minutes. It is inexpensive lab equipment. The principal of this test identifies the sequence of coronavirus RNA by creating a “guide” RNA (complementary to the target RNA sequence) which binds to it in solution. When this happens CRISPR tool’s Cas13 “scissors” enzyme cuts single-stranded RNA apart. These release fluorescent particles into the solution. When the laser light is passed it releases fluorescent light which identifies the presence of corona virus. However it is not yet commercially available for use. 16

Chest CT is an important and fast imaging tool used in the diagnosis of Covid-19-infected patients. The CT severity score index is used to assess the lung changes in patients involved by Covid-19. It can also predict the disease severity by showing the percentage of lung involvement and hence can also be useful in predicting the disease prognosis. It is based on approximate estimation of pulmonary involved areas. 17, 18

Each of the five lung lobes are visually scored and given a score from 1 to 5:

Then, the final score will be the summation of individual lobar scores and will be out of 25 (total score); the total lung involvement is then obtained by multiplying the total score times 4. 17, 18

Saliva is one of the non invasive tool that is been used as a diagnostic test in many diseases. RTPCR of the throat and nasopharyngeal swab (NPS) is the most commonly used test for the diagnosis of covid -19. However, NPS necessitates the availability of skilled technical staff, causes significant patient discomfort during test sample collection, and is associated with high risk of infection to healthcare workers involved. Several studies have investigated the presence of SARS-CoV-2 RNA in saliva. Several of those studies confirmed reliable detection of SARS-CoV-2 in the saliva of patients with COVID-19. Saliva offered sensitivity and specificity for SARS-CoV-2 detection comparable to that of the current standard of nasopharyngeal and throat swabs. However, the utility of saliva in diagnosing COVID-19 infection remains understudied. Clinical studies with larger patient populations that measure recordings at different stages during the disease are still necessary to confirm the accuracy of COVID-19 diagnosis with saliva. Nevertheless, the utility of saliva as a diagnostic tool opens the possibility of using rapid and less invasive diagnostic strategies by targeting bioanalytes rather than the pathogen. The literature supports that saliva offers a simple sample collection method compared to technique sensitive NPS and has the advantage of point-of-care testing for initial screening in community or hospital-based set-up. 19, 20

Viruses undergo constant change over time with most changes having no impact , however, sometimes there may be severe impact on the property of the virus, the disease process, its severity, or the performance of vaccines, therapeutic medicines, diagnostic tools, or other public health and social measures. SARS CoV-2 too has undergone various genomic changes since its identification for the first time. These variants include : Variant being manitered (VBM)- Alpha (B.1.1.7 and Q lineages), Beta (B.1.351 and descendent lineages), Gamma (P.1 and descendent lineages), Epsilon (B.1.427 and B.1.429), Eta (B.1.525), Iota (B.1.526), Kappa (B.1.617.1), 1.617.3, Mu (B.1.621, B.1.621.1) andZeta (P.2); Variants of concern (VOC) - Delta (B.1.617.2 and AY lineages) and Omicron (B.1.1.529). The variant currently posing serious threat to the world is Omicron. 21, 22 Due to the constant genomic evolution of the virus, the current diagnostics and even the efficiency of vaccines stands questionable.