The novel coronavirus 2019 (COVID-19), also known as the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), is a single and positive-stranded RNA virus that belongs to the order Nidovirales and family Coronaviridae with its genome size varying from 29 to 32 kilobases [8, 9]. The SARS-CoV-2 shares 79.5% and 96% genome similarity with SARS-CoV and bat Coronavirus, respectively [10, 11]. The order of genes/coding regions of SARS-CoV-2 is as follows: replicase ORF1ab, spike (S), envelope (E), membrane (M), and the nucleocapsid (N). It has also been confirmed that SARS-CoV-2 uses the same cell entry receptor, angiotensin-converting enzyme II (ACE2), as SARS-CoV [10]. The earliest publications from Chinese laboratories which sequenced the viral RNA from the early clinical cases in Wuhan revealed that the 30,474 nucleotides long SARS-CoV-2 showed a nucleotide identity of 89.1% with a bat SARS-like coronavirus (CoV) isolate, bat SL-CoVZC45 (GenBank accession number MG772933), that had previously been sampled in China [11, 12].

Current RT-PCR (real-time reverse transcriptase-polymerase chain reaction) amplification of the viral DNA is considered the most reliable testing method. However, it is not always positive in the COVID-19 patients [13]. The computerized tomographic (CT) imaging of the chest could play an important role in detecting lesions in the parenchyma of lungs in the suspected patients. Hence, examination of a chest CT along with the RT-PCR results is recommended for precise diagnosis [14].

Based on the data gathered by Artis Ventures, as of 19^th August 2020, a total of 375 diagnostic tests have been authorized by regulatory bodies such as FDA (Food and Drug Administration) and EEA (Europe Economic Area). Out of the 375 tests, about 193 use PCR (Polymerase Chain Reaction), and around 138 are serological assays. Around 163 molecular assays have a downtime of 2 to 8 hours to show results, and 37 assays show results in 1 to less than 1 hour. Some of the assays include but are not limited to RealTime-SARS-CoV-2 assays from Abbot (giving results in 4-6 hours), Cobas-SARS-CoV-2 test from Roche (giving results in 3-8 hours), and Linea COVID-19 Assay kit from Applied DNA Sciences showing results in 1 hour [15]. The goal of all the diagnostic tests is to be accurate, quick, and cost-effective, which will help the patients get the care and the treatment they need in a timely manner and quickly rule out others who do not carry the disease [16]. In return, this could also help prevent the spread of disease by allowing the infected patients to be quarantined or in self-isolation.

So far, no drugs or biotherapeutics are approved by the FDA to prevent or treat COVID-19. Currently, Remdesivir [17], a broad-spectrum antiviral drug first used to treat the Ebola virus outbreak, has obtained emergency use authorization (EUA) from the FDA in May 2020, based on preliminary data showing a faster recovery time of patients with severe conditions [18, 19]. In June 2020, Russia approved the generic drug Favipiraviras the most promising anti-COVID-19 drug for its anti-viral properties [20]. Other drugs for treating symptoms, anti-inflammatory agents, and those for prevention are under various stages of clinical trials [15]. Prominent drugs and their mode of action are summarized in Table 1.

With the rapid spread of SARS-CoV-2, the search for an effective and permanent cure is still going on, with vaccines being developed by almost every country. Since SARS-CoV-2 shows similar pathways with SARS and MERS viruses, researchers use the knowledge gained from these two pathways as a basis to develop a vaccine against SARS-CoV-2. Researchers worldwide are working towards the development of vaccines. At least 20 teams aim to develop nucleic acid (DNA or RNA) vaccine coding for a coronavirus protein (mostly virus’s spike protein) to prompt an immune response. At the same time, many other researchers are engaged in making a sub-unit vaccine (a coronavirus protein directly injected into the body) [21]. Artis ventures in its website www.av.co/covid mentioned that 12 DNA, 10 inactivated, 3 live attenuated, 19 non-replicating viral vectors, 18 replicating viral vectors, 54 protein subunits, 17 RNAs, and 14 virus-like particle vaccines are under various stages of development. According to WHO, several vaccine candidates are in various stages of clinical trials, while 19 are approved by at least one country for emergency use purposes (covid19.trackvaccines.org/vaccines/). A version of the live and attenuated BCG (Bacillus Calmette – Guerin) vaccine is also considered the potential therapeutic approach to protect healthcare workers and vulnerable individuals by reducing the impact of the SARS-CoV-2 [22].

There are many research teams along with companies and universities worldwide in the race for an effective vaccine. Several groups have already begun phase III clinical trials; others have started testing in animals. At least seven teams have developed vaccines using the virus itself in a weakened or inactivated form. Around 25 groups claimed that they are working on viral-vector vaccines (a weakened virus is genetically engineered so that it can produce coronavirus proteins in the body). At least 20 teams aimed to develop nucleic acid (DNA or RNA) vaccine coding for a coronavirus protein (mostly virus’s spike protein) to prompt an immune response. At the same time, many other researchers are engaged in the process of making a sub-unit vaccine (a coronavirus protein directly injected into the body) [21].

According to WHO, at least 7 vaccine candidates have completed the phase III stage of the development (Table 2). A vaccine called ChAdOx1 nCoV-19, developed by Oxford university-UK and AstraZeneca, is reported to be highly promising by generating antibodies and T-cells against the SARS-CoV-2 virus successfully in 1077 volunteers [23]. It is made from a genetically engineered virus that causes the common cold symptoms in chimpanzees. Furthermore, Chinese firm Sinopharm in collaboration with United Arab Emirate’s artificial intelligence and cloud computing company called Group 42 and Abu Dhabi Department of Health, have announced phase IV trials for an inactivated vaccine (covid19.trackvaccines.org/vaccines/). The third one, CoronaVac (PiCoVacc), is developed by Sinovac, which has also begun phase IV trials for the inactivated form of the virus. This vaccine is inactivated by formalin and combined with an alum adjuvant. It is similar to the vaccine Sinovac invented against the SARS virus in 2002 [24]. Since India will be one of the biggest consumers of the COVID-19 vaccine, India’s Bharat Biotech Company, in alliance with the Indian Council of Medical Research (ICMR) – National Institute of Virology (NIV), is developing a COVAXIN vaccine candidate. Covaxin has demonstrated an encouraging positive safety profile in the phase-I trials (clinicaltrialsarena.com/news/bharat-biotech-covaxin-data/). Phase-II clinical trials of Covaxin were conducted at Gauhati Medical College & Hospital (GMCH), Assam. Recently, Russia has approved the world’s first COVID-19 vaccine, dubbed Sputnik V, tested in 76 people. The vaccine was developed by the Gamaleya Research Institute of Epidemiology and Microbiology in Moscow and is now in the phase III trials.mRNA-1273 is another one that has begun phase III trials, which is developed by Cambridge, Massachusetts-based company called Moderna, Inc., and the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH). The vaccine is a synthetic mRNA and is designed to produce antibodies against SARS-CoV-2 [25]. Finally, in recent days, a vaccine, BNT162b2, which is developed by Pfizer (United States of America) and BioNTech (Germany) in collaboration with Forsun Pharma (China), has begun phase III trials in Germany. Out of the 4 mRNA vaccines used for phase I and II trials, Pfizer-BioNTech-Forsun selected BNT162b2 because of its tolerability profile andT-cell responses against the receptor-binding domain (RBD) and the spike glycoprotein of the SARS-CoV-2 virus (SE, 2020). In March 2021, the FDA issued an emergency use authorization to Johnson and Johnson, USA, of a one-shot vaccine called Janssen Covid-19 vaccine, which is also an adenovirus-based vaccine.

Artis ventures in its website www.av.co/covid mentioned 12 DNA, 9 inactivated, 3 live attenuated, 19 non-replicating viral vectors, 17 replicating viral vectors, 50 protein subunits, 16 RNA, and 12 virus-like particle vaccines that are under various stages of development.

Convalescent Plasma therapy (CP) is a type of immunotherapy that has been used to effectively treat infectious diseases for over a century. Patients were treated with satisfactory efficacy and safety for diseases, like SARS, MERS, and H1N1 pandemic [26-29]. With the non-availability of an approved drug or vaccine to treat the COVID-19 disease, convalescent plasma therapy is being considered along with the standard treatment for the severe cases of COVID-19. Patients with a high neutralizing antibody titer against the SARS-CoV-2 virus are a valuable source of CP [30]. However, based on the published literature, high-titer convalescent plasma therapy has no significant impact on 28-day mortality or clinical recovery [31].

Convalescent plasma has the potential to show some benefit for patients with impaired humoral immunity. In a recent case series of 23 patients with prolonged COVID-19 on anti-CD20 agents who received a transfusion of a high-titer unit, 87% became PCR-negative and experienced full clinical recovery [32, 33]. In August 2020, the Food and Drug Administration (FDA) announced an Emergency Use Authorization for convalescent plasma in the COVID-19 patients. Later in February 2021, FDA limited its use only to hospitalized COVID-19 patients early in the course of disease and patients with impaired humoral immunity.

When more people are exposed to the virus, some manifest it clinically, whereas others may be sub-clinically, and thus a community starts building immunity at large; in either case, it is called herd immunity. Herd immunity, if developed, can have a multiplier effect. It can protect many people. However, herd immunity can work only when there is some vaccination also. However, recently, there is a report of pre-existing immune reactivity in the general population [34], which could be due to exposure to common cold coronaviruses. This finding may have implications for herd immunity and vaccine development. Most estimates had placed the herd-immunity threshold at 60–70% of the population, either through vaccinations or past exposure to the virus [35].

Detection for specific antibodies in blood samples is referred to as serological testing. Antibodies, also called immunoglobulins, are proteins produced in our body in response to an antigenic substance particularly from pathogenic microorganisms such as bacteria and viruses. They circulate in our system as a defense towards subsequent infection with the same pathogen. They are rapidly produced by the B cells of the immune system and can be detected in our blood approximately in a week’s time after symptoms begin to occur. There is an increased demand for serological testing for SARS-CoV-2 since it provides better ways to quantify the number of cases and has emerged as a promising solution to identify individuals exposed to the novel coronavirus and suffered from novel coronavirus disease. The presence of anti-COVID-19 antibodies can help medical practitioners make a conclusive diagnosis of the COVID-19, especially, when the nasopharyngeal swab tests are negative. Moreover, antibody testing can be useful in understanding those with mild symptoms of the flu and the asymptomatic carriers who may not show any sort of flu symptoms. Testing for the COVID-19 is typically done using polymerase chain reaction (PCR) with a nose or throat swab. However, the amount of detectable virus decreases after about five days and could be more difficult to test through this technique. Furthermore, the RT-PCR tests currently being used globally to diagnose the COVID-19 can only indicate the presence of viral material during infection and will not indicate if a person was infected and subsequently recovered. Also, this test is often positive in individuals without any symptoms.

Serological assays are easy to perform, less expensive, and have a short waiting period as compared to RT-PCR, nucleic acid assays. These assays, like enzyme-linked immunoassay (ELISA) for specific IgM and IgG antibodies, have a greater advantage such that they avoid false-negative results unlike RT-PCR [36]. In a recent report, patients who tested COVID-19 positive via the RT-PCR method were divided into two groups based on the time between the onset of disease and testing. It was found that seven out of ten patients in the first group and 13 out of 19 patients in the second group tested IgM positive within 9-17 days and 18-29 days, respectively. Nine out of ten patients in the first group and 18 out of 19 patients in the second group were tested IgG positive [37]. This indicates that the major limitation of serological assays is that the antibodies appear later during the disease. According to Zhong et al. [38], ELISA and chemiluminescence methods to detect IgG and IgM antibodies by the recombinant N and S proteins of SARS-CoV-2 were more consistent with the nucleic acid detection assay. In addition, a survey conducted by Long et al. [39] found that patients who tested PCR negative and had no COVID-19 symptoms showed a positive in IgG/IgM assay, indicating how important serological assays are to determine the extent of the COVID-19 pandemic, especially in asymptomatic cases.

Serology-based tests can give greater detail into the prevalence of a disease in a population by identifying individuals who have developed antibodies to the virus. Serological testing is comparatively rapid and is critical in understanding the spread of the virus via asymptomatic individuals and provides an understanding of the expected time of protection. It has the potential to contribute to the efforts in the current pandemic response [40]. However, the understanding of long-term protection via immune response to the COVID-19 is still in the early stages since not all antibodies produced are the same [41]. Also, reports suggest that the required performance characteristics of antibody tests will critically depend on individual-level vs. population-level studies [40]. Understanding the epidemiology of the disease, degree of prevalence, and the development of immunity is possible only from seroprevalence studies including testing for IgM and IgG against the virus due to long-lasting antibodies [42].

Furthermore, a minimum load of antibodies in our body is required to determine the level of protection. The level of protection directly correlates with the number of antibodies and this would necessitate procedures on how to integrate those who have recovered from the virus back into society. Also, long-term surveillance is still a problem since it is not yet established whether patients with antibodies can contract a second SARS-CoV-2 infection. In their review article, Zainol Rashid et al. [43], based on the performances of nine rapid detection tests (RDTs) including the evaluation reports concluded that antibody-based serological assays improve diagnosis of positive cases. In Italy, the patient was declared clear of the virus when the RT-PCR accompanied by the specific IgG antibody test showed negative results.

In one review from Stanford University, the author examined six systematic evaluations of seroprevalence data and concluded that the SARS-CoV-2 infection is widely spread and suggested only a 0.15% average global infection fatality rate (IFR) with substantial heterogeneity.

The in-vitro testing involves combining viral components such as viral spike proteins and others with the patient’s blood sample. If the blood contains antibodies to the virus, they will recognize and bind specifically to the viral component. Then the plate is washed, and the bound antibodies are determined. Most of the antibody tests detect IgG antibodies to the Spike (S) protein of the virus, although other isotypes and antigens are being investigated. The testing methods are based on ELISA, flow cytometry and chemiluminescence, and immuno-chromatography principles of which immunochromatographic assay is very well recognized for self-testing. The mere presence of antibodies may not signify immunity and does not necessarily mean that they are neutralizing the virus.

Several questions remain unclear at this stage. The reports may tend to be negative when there could be a deeper lower respiratory infection. Also, it is not yet clear a) whether people can get re-infected, or will the immune response give them long-term protection, and b) whether people infected will have resistance to other strains of the virus. Hence, though there could be short-term protection, there is no guaranteed long-term immunity at this point in time. With respect to a safe return to work, the protocol is never going to be 100% perfect. Thus, it depends on the answers that emerge in due course of time for the above queries. Antibody testing may help reduce false-negative results and provide reliable data for correlation purposes. According to a report, a foolproof return-to-work protocol is dependent on determining a minimal level of antibody protection [41]. With emerging COVID-19 data, we probably can determine the total risk and the amount of risk society is willing to accept. Multiple waves of the COVID-19 are anticipated [44]. In the meantime, health care workers are gathering as much data as possible to better inform standard operating procedures of sick patients, determine therapeutic interventions, and develop a vaccine.

Data from China and elsewhere suggested the fact that most people have shown mild to moderate symptoms when they contract COVID-19. Serum-based antibody studies are very important to quantify infection in the community and understand the number of mild and asymptomatic cases. This knowledge helps authorities plan for future interventions against the COVID-19. A response to a later reappearance could be tailored to protect only high-risk people if we know that a high percentage of people in a community were infected in its first wave of infection [45]. Reliable serologic evidence of immunity at the individual level will have important implications for the conditions under which individuals return to work, and their ability to work in settings at high risk of transmission, particularly certain health care settings.

However, there are still uncertainties about the characteristics of immunity, including durability and completeness of protection. These questions will need to be settled before serostatus could be considered for individual decision-making. Evidence on the sensitivity and reliability of new serologic tests must be evaluated before they are integrated into health care policies.

Decisions on how to contain the COVID-19 pandemic without completely shutting down business operations require reliable data and associated tools to understand the extent of transmission. Health care systems are heading to develop and implement serological testing for clinical use, evaluate social policy, and quantify risk at the level of the population. This has highlighted the challenges of surveillance programs globally. Hence, it is necessary to revisit and reevaluate surveillance data generated by our health systems and the way in which we can conveniently apply serological testing.

Rapid testing for active cases and tracing of contacts by directly testing for the presence of the virus is a key for containment strategies. There is a pressing need for real-time monitoring of changes in disease transmission across populations and geographies. For later phases of pandemic control, serological testing is important in understanding transmission intensity and population susceptibility which guide decisions on when, where, and how to lift lockdowns and relax social distancing constraints. As highlighted by Bryant et al., challenges to the assessment of performance characteristics of these serological rapid tests are threshold concerns on the sensitivity and specificity, the potential cross-reactivity with other coronaviruses, the use of neutralization assays as a gold standard, difficulties in harmonizing results reporting across different platforms, concerns for quality control in manufacturing, and lack of baseline data required for test interpretation. Moreover, the performance of different test formats may considerably vary, for instance, point of care lateral flow assay is likely to be marked with sensitivity and specificity concerns than ELISA format. However, the low cost and ease of use will facilitate ELISA format for rapid scale-up and widespread adoption. Despite efforts to study immune responses to the COVID-19, to date, there is a lack of sufficient and reliable data on the magnitude and duration of IgM, IgG, and IgA responses in severe, mild, and asymptomatic cases. Furthermore, it is not yet clear how antibody responses vary among diverse populations with different genetic backgrounds and comorbidities.

Use for individual versus population-level IgG testing applications emphasized the dangers of serologic testing for individual-level risk assessments but have highlighted the potential of population-level serological testing, referred to as seroepidemiology, even with assays of moderate sensitivity and specificity. For use at the individual level, many of the serological tests have concerns over the sensitivity and specificity characteristics. Hence, WHO has currently restricted antibody testing to research purposes only. Nevertheless, these tests provide valuable inputs on public health concerns such as the safety of relaxing lockdowns or evaluations of alternative intervention measures. Many fundamental questions relating to testing performance, the dynamics of antibody responses in relation to infection, and the link between antibody responses and immunity need to be addressed to realize the benefits of population-level seroepidemiological studies. Answers to these questions across different populations and epidemiologic contexts call for study designs that are key to the optimal interpretation of the growing number of population-based, cross-sectional serological surveys [40].

While many countries are gradually gearing up to learn to live with the virus, several tests, strategies, and research are being conducted to ensure a cure for the COVID-19. Serological tests can also serve to decode the immunity status in regional populations and answer questions on whether a country or region has attained the COVID-19 peak and whether a strategy, as a lockdown, is effective. For example, as indicated in Table 3, one such serological survey undertaken in the city of Mumbai, India, on the population of 6936 individuals revealed that 40.5% of the subjects have developed antibodies against SARS-CoV-2 [46, 47]. A similar survey conducted by the Delhi government again in India on random samples from 22823 individuals revealed that at least 23.48% of them were already infected. A high seroprevalence means that a larger population may be immune to SARS-CoV-2 and may break the chain of infection.

These surveys may inform us of the transmission pattern from region to region, especially from slums to plush localities and from country to country [52-54]. And if new vaccines practically prevent deaths, then the global IFR may decrease even below 0.1% [55-56].