**3. Available diagnostic assays**

The severity of the disease varies in diagnosed patients ranging from asymptomatic or mild cases to severe cases. The former can be cured by supportive care but the latter depends upon extracorporeal membrane oxygenation. Once patients reached to symptomatic stage, they become contagious and start to shed and spread the virus. Mass screening and accessible diagnostics always play a vital role to constrain the transmission and spread of the virus thus in reduction of mortality rate. Proper infection tracing is needed in the assessment of overall health impacts and statistics. Till now, there are various strategies for diagnosing COVID-19, which are mainly based on viral nucleic acid or antigen detection, and detection of the host's immunological responses. Description about the available test is below:

#### **3.1 Molecular test**

Till now, confirmatory diagnosis for COVID-19 is based on molecular approaches only. These are considered to be first-line methods. Nucleic acid testing based on real-time reverse transcription-polymerase chain reaction (RT-PCR) is the main technique for laboratory diagnosis. It involves nucleic acid amplification test to detect unique sequences within SARS-CoV-2 genome. RT-PCR is a two step process. In the first step, viral RNA is converted to cDNA using a reverse transcriptase enzyme, and the second step involves the amplification of only the selected region using gene-specific primers and further quantification is carried out as fluorescently labeled hydrolysis probe produces fluorescent signals [29]. Since the release of entire genome sequence of the virus from scientists of China, many countries, such as England, Germany, South Korea, Turkey, Russia, the USA, India, and China, launched their clinical-grade RT-PCR kits for SARS-CoV-2 detection. For RT-PCR kits, samples are taken from various infected parts of the body, including nasopharyngeal, oropharyngeal, or nasal swabs, upper and lower respiratory tract aspirates, bronchoalveolar lavage, and the sputum [30]. Main components in RT-PCR-based Kits are the reverse transcription and amplification enzymes, specific primers and probes for amplification of the selected viral genome regions, and authorized reagents for negative, positive, and internal controls, target genes, corresponding primer, and probe sequences used in RT-PCR kits so far for SARS-CoV-2 detection. Various research groups have been proposed the use of different set of target genes, corresponding primer, and probe sequences. Generally, the commercial kits based on the RT-PCR are only operated in well-equipped laboratory conditions and require skilled persons [31]. Pixel by

#### *Current Status of COVID-19 Diagnostics DOI: http://dx.doi.org/10.5772/intechopen.96955*

LabCorp COVID-19 Test Home Collection Kit made home collection possible. It contains a specimen biohazard bag, pre-labeled return FedEx envelope, saline tube, insulated specimen pouch, nasal specimen collection, swab gel pack (for sample cooling), shipping box, and the user guideline [32].

Other molecular-based technologies like LAMP, RT-LAMP, and rRT-LAMP amplify nucleic acid isothermally without any use of a thermocycler. DNA polymerase along with multiple primers, six or four as inner and outer primers, is used to amplify the target sequence. In RT-LAMP, analysis of results is done by the change of color, fluorescence, or turbidity in the PCR tubes, which makes it a simple and practical technique. Use of multiple primers gives specificity in results. RT-LAMP is a fast and specific method which completes detection of SARS-CoV-2 in 1–2 h without any need of a trained molecular biologist [33].

Another isothermal nucleic acid amplification based assay is SHERLOCK assay, which also employs the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) enzymology for the detection of the target nucleic acid. Upon the binding of CRISPR RNA to the target sequence, the nonspecific endonuclease activity of Cas13 or Cas12 starts, leading to the cleavage of nearby reporter RNAs which generates signal for detection. SHERLOCK is a very specific and sensitive assay for diagnosis as Cas13 does not get activated if two or more mismatches are present in the target RNA, and it can easily discriminate between SARS-CoV-2 and other similar viruses [34].

Advantages:


#### Disadvantages:


#### **3.2 Antibody test**

In an antibody test, existence and concentration of IgG and IgM antibodies is measured in the blood/serum/plasma samples of infected patients. It can determine if the body encounters with a pathogen like SARS-CoV-2 virus. Common antibody tests are based on lateral flow type assays (LFA) and enzyme-linked immunosorbent type assays (ELISA) [35]. An antibody LFA test detects the presence of the specific antibodies in the patient's blood sample. A SARS-CoV-2 antigen(s) is already immobilized on a sample pad. The sample is loaded onto the sample pad at end port

where Colloidal gold (CG) or quantum dot (QD)-labeled detection antibodies are present. Sample along with labeled antibodies moves through the strip by capillary action to the test line and control line [36]. If SARS-CoV-2 antibodies are present in the sample, they will be captured by the labeled detection antibody and will bind to the immobilized antigen at test line. Even if the SARS-CoV-2 antibodies are absent in the sample, the gold-labeled antibodies will still be captured at the control line and a band on the strip will appear due to the accumulation of CG or QD.

ELISA tests are performed in multi-well plates coated with the recombinant viral antigen. If antibodies (IgG or IgM) against the SARS-CoV-2 antigen are present in the sample, a binding between coated antigen and SARS-CoV-2 antibodies occurs. Then, secondary anti-human antibodies are added to bind with SARS-CoV-2 Ag-Ab complex. Secondary antibodies are enzyme labeled (usually horseradish peroxidase). Upon addition of an enzyme substrate, a color-changing reaction happens. In the absence of the antibody of interest, no color is generated. In a modified version of ELISA, that is, chemiluminescent immunoassays (CLIA), the binding of the secondary antibody is confirmed by a chemiluminescent substrate [37]. ELISA test is a multistep process that demands a well-equipped lab, but LFA can be used at home without any training [38, 39].

#### **3.3 Antigen test**

An antigen is a non-self particle/fragment/molecule that can induce the immune system to produce antibody against pathogens, hence protects the body. The antigen test is also an immunoassay which detects viral components (i.e., S glycoprotein, M protein, or released N protein) or directly virus. Unlike the antibody-based methods, antigen tests detect the active viral infection, not the recovery situation. Because antigens precede antibodies, antigen test could be more reliable than antibody tests. Antigen tests can also be operated on LFA strips for rapid detection or in ELISA plates for increased sensitivity, and high throughput uses [29, 40].

Advantages:


Disadvantages:

• Low sensitivity as it requires high level of viral load for testing.

#### **3.4 Other methods**

Several other methods for diagnosis have been proposed. Some of them are novel and at research stage and some are based on conventional technology, for example, computed tomography (CT) scans. Aptamers functionalized with quantum dots (Qds), paper-based assays, semiconductor-based binding assays, surface plasmon resonance-based assays, piezoelectric immune sensors, and electrochemical sensors have been developed. A CT scan reveals about the possible abnormalities due to the viral infection in the chest [41]. Other detection


### **Table 1.**

*Developed kits for COVID-19 diagnosis.*

technologies developed for the identification of SARS-CoV-2 are based on the presence of different biomarkers in bio-fluids. Increased concentrations of C-reactive protein, D-dimer, lymphocytes, leukocytes, and blood platelets and elevated levels of serum urea, creatinine, and cystatin C can be utilized for diagnosis. Biosensors based on plasmonic sensing and field effect transistor (FET) have been developed for mass screening. Localized surface plasmon resonance (LSPR) sensor detects the SARS-CoV-2 nucleic acid with combined use of photo-thermal effect and plasmon sensing [42]. In FET-based biosensors, biological molecules modify the charge distribution of the surface, or they generate a surface potential by binding to the surface, which is further measured as a conductance value [43]. For mass screening of such pandemic, we need a global e-platform to control the spread of the virus which is now possible with emergence of data science and advancement in mobile telecommunications. In this view, mHealth is a useful development, which is an application of mobile devices like smarphones, onboard optics/sensor based patient monitoring devices, and wireless/Bluetooth technology. Such advancements lead to easy collection of large epidemiological data based on contact tracing, automation of inventory mangement, digital, and fast reporting of the new cases. It can also help further in supply chain management of limited resources for affected areas and also help government in policy making [44]. Indian government has also launched "Aarogya setu" app during COVID-19 pandemic [45]. Information about some of the COVID-19 diagnostic kits based on above discussed methods are listed in **Table 1**.
