**2. Structure of SARS-CoV-2**

SARS-CoV-2 has a characteristic crown-like appearance due to the spikes formed by a major glycoprotein (Mol. Wt. ~180 kDa), i.e., SP, which has two subunits, S1 and S2 [1] (**Figure 1**). S1 has the receptor binding domain (RBD) that recognizes and binds to angiotensin converting enzyme 2 receptor (ACE2) in the lower respiratory tract of SARS-CoV-2 infected subjects [18]. In contrast, S2 has other basic elements needed for membrane fusion. The amino-terminal region of S1 subunit is the most variable immunogenic antigen. SP is the most widely studied viral protein as it is responsible for the SARS-CoV-2 infection. It is the target of all SARS-CoV-2 neutralizing antibodies and COVID-19 vaccines. On the contrary, NP (Mol. Wt. ~40 kDa) is another viral structural protein, which is the most abundant viral phosphoprotein produced and shed during the first two weeks of SARS-CoV-2 infection, with peak shedding around 10 days after infection. It exhibits high immunogenicity and can be detected in either nasal/nasopharyngeal swabs, saliva, stool, serum or urine samples [19]. The sandwich ELISA is used for the detection of NP as it is a large protein with multiple epitopes. The other structural proteins of SARS-CoV-2 are the MP and EP. MP is the most abundant protein on SARS-CoV-2, while EP is the smallest structural protein of SARS-CoV-2 that plays a role in viral assembly, release of virions, and pathogenesis [1].

## **3.** *In vitro* **diagnostics for COVID-19**

Various IVD assays have been developed for the detection of SARS-CoV-2 viral RNA, antibodies, and antigens, which encompass assays performed in certified COVID-19 diagnostic laboratories and rapid tests employed at POC settings. The various IVD assays, together with their characteristic features and bioanalytical performances, are specified in this section. Almost all the IVD assays for COVID-19 are CE IVD certified, while several are also approved by the FDA under the EUA. As shown in **Figure 2**, the RT-PCR is positive for about 3 weeks after the onset of symptoms in COVID-19 patients, while the rapid tests for viral antigen work best during the first week after the onset of symptoms [20]. On the contrary, the serology tests for detecting antibodies work best after seroconversion at the end of 3rd-week post onset of symptoms, when the COVID-19 patients enter convalescence [20, 21].

#### **3.1 Molecular diagnostics**

RT-PCR is the gold standard for the confirmatory clinical diagnosis of COVID-19 and the most used IVD assay globally. The first real-time RT-PCR assay, highly specific for SARS-CoV-2 RNA and no cross-reactivity to other coronaviruses, was developed by Tib-Molbiol, Germany, in January 2020 [22]. The assay involved the detection of SARS-CoV-2 RNA by employing envelope (E) and RNA-dependent RNA polymerase (RdRp) gene assays, where E-gene assay enabled the first-line screening and RdRp gene assay did the confirmatory

#### **Figure 2.**

*An overview of the COVID-19 biomarkers' longitudinal response and the utility of various IVD tests at different stages of COVID-19.*

testing. An alternative format, one-step RT-PCR assay, was developed to detect ORF1b and N regions of SARS-CoV-2 in less than 1.5 h [23]. It employed the N gene assay for screening and Orf1b gene assay for confirmatory analysis. But the assay could also detect SARS-CoV and other closely-related viruses as ORF1b and N regions are highly conserved among sarbecoviruses. The authors distinguished SARS-CoV-2 from SARS-CoV via sequence analysis of positive amplicons if the RT-PCR results are positive. A prospective development was the real-time RT-PCR assay to detect RdRp/helicase (H) genes of SARS-CoV-2 [24]. The assay has high sensitivity and detects COVID-19 in low viral load samples, saliva, plasma, and upper respiratory tract samples. Moreover, it showed no crossreactivity with other human coronaviruses and respiratory viruses. Subsequently, many innovative RT-PCR assays were developed by many IVD companies and research groups.

A prominent test is the CE certified and FDA EUA approved rapid, real-time RT-PCR test, i.e., the Xpert® Xpress SARS-CoV-2 test, by Cepheid, USA [25]. The assay, requiring GeneXpert Dx or GeneXpert Infinity Systems, enables the qualitative detection of SARS-CoV-2 ribonucleic acid (RNA) in specimens collected from the upper respiratory tract [25]. These include nasopharyngeal, oropharyngeal, nasal, or mid-turbinate swab or nasal wash/aspirate specimens. The rapid RT-PCR test is run on GeneXpert Instrument Systems that have automated and fully integrated process steps, i.e., sample preparation, extraction of RNA, amplification of RNA, and detection of the target sequences. The systems employ single-use disposable cartridges, which have all the RT-PCR reagents together with a sample processing control (SPC) and a probe check control (PCC). SPC controls the sample processing and monitors the presence of potential inhibitors in the RT-PCR reaction. It ensures the presence of adequate RT-PCR reaction conditions for the amplification reaction and the proper working of RT-PCR reagents. On the other hand, PCC ensures reagent rehydration, filling of PCR tube, and the presence of all reaction components in the cartridge. It also monitors the integrity of the probe and the stability of the dye. The procedure involves the sample collection and its placement into a viral transport tube that contains 3 mL transport medium or 3 mL of saline. The specimen in the tube is mixed by rapidly inverting it 5 times, followed by transferring the sample to the sample chamber of the Xpert Xpress SARS-CoV-2

#### In Vitro *Diagnostics for COVID-19: State-of-the-Art, Future Directions and Role in Pandemic… DOI: http://dx.doi.org/10.5772/intechopen.97775*

cartridge. The cartridge is then loaded onto the GeneXpert Instrument System for the automated sample processing and real-time RT-PCR. The kit comprises freezedried beads, lysis reagent, binding reagent, elution reagent, and disposable transfer pipettes, which are sufficient to process 10 specimens or quality control samples. The positive predictive agreement (PPA) and negative predictive agreement (NPA) of the test relative to the expected results were 97.8% and 95.6%, respectively. Cepheid has developed the same test on GeneXpert Xpress System, a POC system, with similar bioanalytical performance. The SARS-CoV-2 diagnosis is positive if the signal for the N2 nucleic acid target or signals from both nucleic acid targets (N2 and E) have a cycle threshold (Ct) within the valid range. But the diagnosis is presumptive positive if the signal for only the E nucleic acid target has a Ct within the valid range. It may require additional confirmatory testing. The overall assay duration is less than 45 min.

Cepheid has further developed another FDA EUA approved real-time multiplex RT-PCR test, Xpert® Xpress SARS-CoV-2/Flu/RSV, which is performed on GeneXpert Dx or GeneXpert Infinity Systems. It qualitatively detects and differentiates SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus (RSV) viral RNA in nasopharyngeal swab, nasal swab, or nasal wash/ aspirate specimens. The principle of the assay is very similar to that of Cepheid's SARS-CoV-2 test. Cepheid recommends the use of external controls in the form of inactivated viruses that are provided by ZeptoMetrix, USA. They should be used to perform external quality control with each new lot and shipment of reagents. The PPA and NPA of the test relative to the predicate RT-PCR tests (FDA EUA approved) for SARS-CoV-2, Flu A, Flu B, and RSV were 97.9% and 100%; 100% and 100%; 100% and 99%; and, 100% and 100%, respectively. The company has developed the same test on GeneXpert Xpress System (a POC system) with similar bioanalytical performance.

The Simplexa™ COVID-19 real-time RT-PCR assay from DiaSorin Molecular, Italy, is another prospective assay for the qualitative detection of SARS-CoV-2 RNA in nasopharyngeal swabs, nasal swabs, nasal wash/aspirate, and bronchoalveolar lavage specimens from COVID-19 suspects. It contains reagents that are sufficient for 24 reactions. The assay targets the ORF1ab and S gene regions of the SARS-CoV-2 genome and is run on the LIAISON® MDX instrument using the Direct Amplification Disc and other accessories. It employs fluorescent probes together with corresponding forward and reverse primers for the amplification of SARS-CoV-2 RNA and internal control RNA. The company provides Simplexa™ COVID-19 Positive Control Pack, which may be used as an external control for quality control testing. The PPA and NPA in various sample matrices were both 100% w.r.t. an established comparator.

The cobas® SARS-CoV-2 is a real-time RT-PCR assay from Roche, which qualitatively detects SARS-CoV-2 RNA in clinically-collected nasal, nasopharyngeal, and oropharyngeal swabs specimens from COVID-19 suspects. The assay is also approved for clinically-instructed self-collected nasal swab specimens. It is performed on cobas® 6800/8800 Systems, which comprise a sample supply module, a transfer module, a processing module, and an analytical module. The cobas® SARS-CoV-2 employs fully automated sample preparation involving RNA extraction and purification, which is followed by PCR amplification and detection. It targets the ORF1 a/b and E gene regions of the SARS-CoV-2 genome. The company provides the assay controls, i.e., cobas® SARS-CoV-2 Control Kit and cobas® Buffer Negative Control Kit. The PPA and NPA determined in the clinical evaluation with nasopharyngeal swab samples were both 100%.

The Vivalytic COVID-19 test developed by Bosch, Germany, in collaboration with Randox Laboratories, UK is another prospective assay [26]. The

fully-automated POC test can detect SARS-CoV-2 and nine respiratory viruses, including influenza A and B, within 2.5 h. The procedure involves sequentially taking the swab sample from the nose or throat of COVID-19 suspects, placing the swab inside a Vivalytic cartridge containing all the COVID-19 assay reagents, and plugging the cartridge into the Vivalytic analyzer.

The most prominent and rapid POC molecular test is the Abbott ID Now™ COVID-19 test [27], which qualitatively detects the viral RNA from SARS-CoV-2 specimens, i.e., throat, nasal, nasopharyngeal, or oropharyngeal swab samples, in just 5 min [27, 28]. It is a POC molecular test for the RdRp gene, which requires just a portable, touchscreen-operated, lightweight (6.6 pounds) and compact (the size of a small toaster) instrument called ID Now. It enables COVID-19 testing in hospitals, clinics, physicians' offices, or other POC settings. The test kit comprises 24 tests, positive and negative controls, pipettes, and swabs for sample collection.

#### **3.2 Antigen detection**

The LFIA-based rapid Ag tests for SARS-CoV-2 have played a phenomenal role in the COVID-19 pandemic response. They have been extensively used to screen a large population at POC settings as physician office laboratories, offices, schools, businesses, and homes. They have been approved for professional IVD use and selftesting by the FDA and several countries such as Germany. Germany has allowed the use of tens of rapid Ag tests for self-testing via approval provided by the Federal Institute for Drugs and Medical Devices (BfArM) [6]. The US FDA has granted EUA to several rapid Ag tests for SARS-CoV-2, which can be used for professional IVD use, home use, or both. Almost all the approved rapid Ag tests detect the NP of SARS-CoV-2 and have got good analytical performance. Although several reports have shown the contradictory analytical performance of rapid Ag tests, there is no doubt that such tests are of extreme importance as they have extended the outreach of SARS-CoV-2 testing enormously. The most widely used rapid Ag tests are those from Abbott, Becton Dickinson (BD), and Quidel, which have been further approved by FDA recently under EUA for the serial screening of COVID-19 suspects by testing them twice over 3 days with 24–48 h between tests.

The BinaxNOW™ COVID-19 Ag Card from Abbott Diagnostics is an LFIA that enables the qualitative detection of NP antigen from SARS-CoV-2 in direct anterior nasal (nares) swabs without viral transport media [29]. It is an immunochromatography membrane assay that employs highly sensitive and specific antibodies to detect NP from SARS-CoV-2. A test strip is constructed by immobilizing SARS-CoV-2 specific antibodies and a control Ab onto a membrane as two distinct lines and combining it with other reagents/pads. The COVID-19 Ag card is cardboard, book-shaped hinged card that has a well to hold nasal swab and the test strip mounted on opposite sides. The assay procedure involves taking the nasal swab specimen from the COVID-19 suspects and mixing it with the extraction reagent. The extraction agent disrupts the virus particles and exposes internal viral NP. It is followed by closing the card, which brings the extracted sample in contact with the test strip that starts the LFIA assay. The test results are detected visually by naked eyes after 15 min, where the presence of a pink/purple sample line shows the presence of NP in the sample. The test is intended for use in COVID-19 suspects who are within 7 days of symptoms onset. The PPA and NPA of the test were 97.1% and 98.5%, respectively, against the comparator method. FDA also approves it under EUA for the serial screening of COVID-19, where the individuals are tested twice over 3 days with at least 36 h between tests. However, the test does not differentiate between SARS-CoV-2 and SARS-CoV, and the positive results do not rule out bacterial infection or co-infection with other viruses. The negative

In Vitro *Diagnostics for COVID-19: State-of-the-Art, Future Directions and Role in Pandemic… DOI: http://dx.doi.org/10.5772/intechopen.97775*

test results should be treated as presumptive in patients beyond 7 days post onset of symptoms, where further confirmation with a molecular assay is required. It is essential that the results of the test should be read within 30 minutes, and the nasal swab specimens are used immediately after collection. Apart from the test cards, extraction reagent, and nasal swabs, the BinaxNOW™ COVID-19 Ag Card provides positive and negative control swabs. The positive control swab is a dried swab containing non-infectious recombinant SARS-CoV-2 NP, while the negative control swab has the sample matrix without any NP. It is recommended to test the positive and negative control swabs after each shipment of Ag tests and at least once for each untrained operator.

The BD Veritor™ System [30] for Rapid Detection of SARS-CoV-2 is another prospective rapid LFIA-based test that detects qualitatively the presence of SARS-CoV-2 NP in direct anterior nasal swabs from COVID-19 suspects within the first five days of the onset of symptoms. FDA also authorizes it under EUA for the serial screening of COVID-19 where the subjects are tested twice over 2 or 3 days with 24–48 h between tests. The swab specimens are placed in the extraction reagent tube for sample processing, and the processed sample is then added to the BD Veritor System test device. The SARS-CoV-2 NP in the sample form Ag-conjugate complexes by binding to antibodies conjugated to detector particles in the test strip. The complexes are then captured by the specific antibodies bound to the membrane at the test line. The BD Veritor™ System test device's test results are read after the completion of test in 15 min using the BD Veritor™ Plus Analyzer Instrument. The SARS-CoV-2 test kit includes BD Veritor™ System test devices, extraction reagent, nasal swabs, SARS-CoV-2 (+) Control Swab, and SARS-CoV-2 (−) Control Swab. Most of the assay characteristics in terms of interferences, specificity, and analysis are similar to that of Abbott's BinaxNOW™ COVID-19 Ag Card test. The specimens should be tested immediately after collection. The PPA and NPA of the test were 84% and 100%, respectively, against the RT-PCR method.

The QuickVue At-Home OTC COVID-19 Test from Quidel Corporation is another rapid test to detect SARS-CoV-2 NP qualitatively in direct anterior nasal swabs from COVID-19 suspects within the first six days of the onset of symptoms. FDA also authorizes it under EUA for the serial screening of COVID-19 suspects where they are tested twice over 2 or 3 days with 24–36 h between tests. The test procedure is very similar to that of the BD Veritor™ System for Rapid Detection of SARS-CoV-2 test except that the test strip is read visually by naked eyes after 10 min. The presence of a pink/purple-colored test line indicates the presence of SARS-CoV-2 NP in the specimen. Most assay characteristics are like that of Abbott's BinaxNOW™ COVID-19 Ag Card test. The PPA and NPA of the test were 83.5% and 99.2%, respectively, against the EUA molecular comparator assay.

All other SARS-CoV-2 rapid Ag tests detect the NP antigen qualitatively and have demonstrated good analytical performance. However, some SARS-CoV-2 rapid Ag tests, such as that from Sensing Self, Singapore, have also shown good analytical performance for the qualitative detection of SP antigens. But as most of these tests targeting the SP were developed last year, there is a need to demonstrate that they can work in subjects affected by various spike mutations. The preference for clinical decision-making is certainly the NP detection-based rapid Ag tests. Apart from nasal swabs, several companies have demonstrated the use of saliva, sputum, and stool samples to detect SARS-CoV-2 viral Ag.

#### **3.3 Antibodies detection**

Various IVD companies have developed serological IAs to detect anti-SARS-CoV-2 Ab in the serum, plasma, or whole blood samples. They enable identifying individuals with an adaptive immune response to SARS-CoV-2 either due to prior or recent infection [31]. Despite several reports stating the persistence of immunity after SARS-CoV-2 infection for several months [32, 33], it is still unclear how long the anti-SARS-CoV-2 Ab persists and whether they confer protective immunity. Therefore, serology tests are not used to diagnose acute SARS-CoV-2 infection. In the case of SARS-CoV-2, IgM, IgG, and IgA antibodies appear in the subjects at nearly the same time and have the seroconversion between 14 and 23 days post onset of symptoms. SARS-CoV-2 IgG and IgM antibodies may be below the detectable levels in COVID-19 patients who are within 14 days after the onset of symptoms. However, the COVID-19 samples should be handled with care as there is a possibility of detectable SARS-CoV-2 in samples even after seroconversion. Almost all assays have a poor PPA with RT-PCR in patient samples taken from subjects within 14 days from the onset of symptoms. But they have good PPA for samples taken from subjects more than 14 days post onset of symptoms. However, there is always a risk of false-positive results due to the presence of pre-existing antibodies or other possible causes. The most widely used IAs to detect anti-SARS-CoV-2 Ab are automated CLIA, manual ELISA, and rapid LFIA tests that are specified in more detail below.
