Relevance of rapid, reliable and low-cost diagnostics in the current COVID-19 pandemic

Coronaviruses are a family of RNA viruses originally identifi ed in 1960s. The -coronaviruses are known to cause respiratory diseases that can range from common cold to severe disease conditions like Severe Acute Respiratory Syndrome (SARS-CoV) and Middle East Respiratory Syndrome (MERSCoV) and are likely to be of zoonotic origin. The lethal virus was named “SARS-CoV-2” as it was found to be related to Severe Acute Respiratory Syndrome (SARS-CoV) [1].


Introduction
Coronaviruses are a family of RNA viruses originally identifi ed in 1960s. The -coronaviruses are known to cause respiratory diseases that can range from common cold to severe disease conditions like Severe Acute Respiratory Syndrome (SARS-CoV) and Middle East Respiratory Syndrome (MERS-CoV) and are likely to be of zoonotic origin. The lethal virus was named "SARS-CoV-2" as it was found to be related to Severe Acute Respiratory Syndrome (SARS-CoV) [1].
The disease induced by the virus is known as 'COVID-19'.
The early cases of COVID-19 were identifi ed in Wuhan City of the Hubei Province, China [2]. On March 11 th, the Director of WHO declared COVID-19 as a "global pandemic" [3]. The virus has high transmissibility with an estimated reproductive number of 2.2, which connotes that an infected person can spread the virus to another 2.2 individuals within an incubation period of 5.8 days [4].
The disease has spread globally and has infected over 6.0 million people with around 3.63 lakh deaths till the end of May 2020. Many asymptomatic cases are reported which challenges the control of infection. This virus outbreak has challenged the economic, medical and public health infrastructure all over the world [5]. It has pushed the global economy into severe recession and several countries have resorted to lockdowns to fl atten the curve of infection causing shutting of many businesses and disruption in public services [6]. The economic losses in the Asia ranges from $1.7 trillion to $2.5 trillion during containment scenario, accounting to 30 percent of overall decline in the global output [7]. In India, the steep drop-in service activity has accounted to 52 percent of its GDP [8]. The initial 21 days of lockdown costed nearly $4.5 billion every single day [9]. The outbreak has led to an increase in the demand for hospital beds and medical equipments, while the medical and other supporting staffs are themselves getting infected [10]. Vaccine developments are underway at an accelerated speed, yet it might take a year to come out with a good vaccination programme which makes diagnostics and therapeutics are the only option available for containment of the disease and bringing the economy back in track.
SARS-CoV-2 is large enveloped, positive-sense singlestranded virus ((+) ssRNA) that encode four essential proteins like Spike (S) glycoprotein, small Envelope (E) protein, Membrane (M) protein, Nucleocapsid (N) protein and several accessory proteins that interfere with the host innate immune response [11]. Based on these structural information, researchers across the globe are working round the clock to come up with diagnostic kits for the early detection of the infection and development of appropriate novel vaccine and therapeutics. The review discusses about the probable vaccine and therapeutic approaches in current situation and various diagnostic assays and kits based on PCR, Isothermal assays and various types of immunoassays that are commercially available worldwide.

Probable vaccine and therapeutic approaches for virus containment
World-wide efforts for developing novel therapeutics and vaccines is at war front. Vaccine development is a lengthy, complex and expensive process [12]. It involves enormous research, followed by trials, regulatory clearances and huge co-operation from both public and private sectors. The study of the virus is a very important component as the virus is prone to frequent mutations [13].
Numerous strategies are implemented for the development of coronavirus vaccines. Spike glycoprotein (S protein) is targeted as it plays a major role in inducing protective immunity during infection by eliciting neutralizing antibodies and T-cell response [14]. Recombinant protein containing receptor binding domain (RBD) and recombinant vectors encoding RBD can be used for the development of effective SARS-CoV-2 vaccines.
Immunoinformatics methods are utilized for the identifi cation of epitopes for the inclusion in SARS-CoV-2 vaccine [15].  protease inhibitor) are under phase III trial for COVID-19 disease [18]. Repurposed drugs are the potential therapeutic option for COVID-19 disease management. Lopinavir/ritonavir and interferon-1 possess in vitro anti-MERS-CoV activity. The in vivo study conducted in non-human primate model showed better outcomes [19].
Recently, the anti-viral effi ciency of remdesivir and chloroquine were found to be highly effective in controlling COVID-19 in vitro [20]. Quantitative PCR (Q-PCR) is now considered the gold standard for the diagnosis of viral respiratory disease [21]. Globally this technique is currently in use for the detection of SARS-CoV-2.
It is a simple and specifi c quantitative assay. Fluorescent dye or fl uorescently labelled DNA probe or a quencher molecule is used to monitor the amplifi cation of the DNA [22]. One-step procedure is the chosen approach for the detection of SARS-CoV-2. It is rapid, requires less bench time and reduces the chances of pipetting errors and cross-contamination. The only constrain of this technique is the risk of many false-positive and false-negative results [23]. However, the sensitivity and specifi city of this technique is not hundred percent reliable.
The decrease in assay performance can be attributed towards the generation of mutated strains due to rapid evolution and genetic diversity, causing mismatches with the probe [24,25. The overall procedure of RT-PCR requires highly skilled personnel, high installation cost and a long time to deliver the test results.
Several types of RT-PCR kits are being developed that promises to overcome the inherent limitations. Globally diversifi ed healthcare company, Abbott has developed a fully automated amplifi cation and detection system for nucleic acids using 5´-nuclease technology [26]. Abbott's Real-Time SARS-CoV-2 assay is engineered for dual targeting of RdRp and N-genes [27]. CE certifi ed "ViroQ SRS-CoV-2" kit developed by BAG diagnostics from Germany, is designed for the detection of E gene and RdRp in the suspected samples. The RNA is converted to cDNA in one-step PCR procedure and then amplifi ed according to the Real-Time PCR protocol. The test results are delivered in 90 minutes [28]. DiaSorin Molecular from Cypress, South California has developed "SimplexaTM COVID-19 Direct assay system" which is a Real-Time PCR system that enables the direct amplifi cation of the virus from nasal or nasopharyngeal swabs. The assay targets the ORF1ab Citation: Gayathri   CoV-2 pathogens [32]. The Pixel by LabCrop COVID-19 utilizes multiplex PCR using two primers and probe sets to detect two regions of N gene and one primer and probe set for detection of human RNase P (RP) [33].
DNAzymes is yet another low-cost, reliable and rapid diagnostic assay. DNAzyme is a nucleic acid G-quadruplex structure that binds hemin to yield a complex that mimics  The test has received Emergency Use Authorisation (EUA) in the US [37].

Isothermal assays
LAMP assay is a widely attempted isothermal assay that has been standardized for the detection of large number of microorganisms specifi cally for disease diagnostics. The assays conventionally employ four set of primers to recognise eight distinct regions of target. Strand displacement is carried out by using Bst polymerase enzyme at standardized temperature that ranges between 60-65°C for an amplifi cation time of close to 1 hour. In order to detect RNA viruses, LAMP assay can be modifi ed by incorporation of reverse transcription step. This technique has generated a great interest among investigators and is believed to have the potential to be used as point-ofcare diagnostics with higher sensitivity and can be performed in a decentralized test facility [38]. It has been used for the  diagnosis. The technique was named Mango after the aptamer, because it is specifi c for the RNA sequence bind and the brightly coloured fl uorescent dye become excited and glow. It allows the detection of RNA and can be studied under a microscope [41].
RCA is an isothermal enzymatic amplifi cation assay, developed for sensitive diagnosis for a variety of targets including nucleic acids, DNA or RNA. A short RNA primer and RNA polymerase are employed to amplify circular RNA or DNA template to give rise to concatemer product containing tens to hundreds of tandem repeats that are complementary to the circular template. RCA is a unique procedure among isothermal reactions, requires a very fi ne approach for standardisation that needs to be further explored for large number of viral diseases. However, it has not been deployed for the detection of SARS-CoV-2 at this point [42].
RPA is relatively a new technique for developing isothermal assays and holds a better promise compared to other isothermal based assays due to its rapidity and specifi city. It is a fi eld deployable assay with minimal sample requirement, low operation temperature ranging from 25-42°C and is user friendly [43]. The assay is carried out by employing recombinase Biosciences company has developed SHERLOCK method that uses Cas13 that cleaves the fl uorescent RNA reporter when activated by target sequence [44]. Another assay developed by Mammoth Biosciences "DETECTR" uses Cas12 to specifi cally detect E and N genes of the RNA sequence. The target then undergoes isothermal amplifi cation, resulting in visual readout with fl uorophore. This technique does not require complex instrumentation and can be read using paper strip to detect SARS-CoV-2 without the loss of specifi city or sensitivity. These tests have a great potential for point-of-care diagnosis and have the advantage of being low cost and less detection time of one hour [45].

Viral RNA extraction kit using magnetic beads
The fi rst step of any detection test involves isolation of high quality and good concentration of RNA without degradation.
This step is crucial as it is critical for the proper outcome of PCR or any another assays. Isolating viral genome using magnetic beads has risen a lot of interest among many researchers. The basic principle in viral isolation by magnetic beads involves addition of magnetic beads conjugated with specifi c binding agent, followed by magnetic separation of the bound particles from the mixture. This is a popular approach for extraction of nucleic acids due to high potential for automation. The extraction effi ciency is much higher, and the time taken for the isolation is less. Simple procedure and avoidance of organic solvents makes it an attractive method for routine diagnostic purposes [46].

Serological and immunological assays
Immunoassays for the recognition of antigens of microorganisms has emerged as an important tool for diagnosing and managing infectious diseases. Antigen detection is a useful diagnostic tool as it is rapid, simple one-step assay  can then be detected using additional tracer antibody to produce a colorimetric or fl uorescent-based readout [24].
"Platelia SARS-CoV-2 Total Ab" is a one-step antigen capture ELISA (Enzyme-linked Immunosorbent Assay) for qualitative detection of IgM/ IgA/ IgG in human serum or plasma [56].

CoV-2. The test is used on Abbott's i1000SR and i20000SR
laboratory instruments, which can run up to 100-200 tests per hour [57]. for detection of antibodies, serum proteins, antigens from human samples. This assay was fi rst utilized during Ebola Haemorrhagic fever outbreak in Central Africa targeting monoclonal antibodies to the EBOV matrix protein (VP40), which previously had been found to work in a conventional enzyme-linked immunosorbent assay [65]. Immunofi ltration combined with magnetic detection has various advantages such as quantifi cation and sensitive detection. This assay is found to have a sensitivity similar to that of the widely used antigen-detection ELISA and relatively less sensitive than RT-PCR assay. Turklab, a Turkish fi rm based in Aegean province of Izmir has developed a test kit relying on immunofi ltration assay which will yield results within fi ve minutes [66]. The main disadvantage of fl ow virometry is the inability to distinguish between infectious and non-infectious particles [68].
The antibody tests do not have much value for disease diagnostics but do have merits in retrospectively confi rming infection, for epidemiological studies to asses community spread and to evaluate herd immunity.

Indian efforts for development of diagnostics
India has been greatly dependent on other countries for diagnostics kits. Many imported kits are faulty and are sent back as they reported inaccurate results in many places [69].