key: cord-277841-7sp8ftbc
authors: Kumari, Pratibha; Singh, Archana; Rinchui Ngasainao, Moses; Shakeel, Ilma; Kumar, Sanjay; Lal, Seema; Singhal, Anchal; Singh Sohal, Sukhwinder; Kumar Singh, Indrakant; Imtaiyaz Hassan, Md.
title: Potential diagnostics and therapeutic approaches in COVID-19
date: 2020-08-12
journal: Clin Chim Acta
DOI: 10.1016/j.cca.2020.08.013
sha: 
doc_id: 277841
cord_uid: 7sp8ftbc

Abstract The most important aspect of controlling COVID-19 is its timely diagnosis. Molecular diagnostic tests target the detection of any of the following markers such as the specific region of the viral genome, certain enzyme, RNA-dependent RNA polymerase, the structural proteins such as surface spike glycoprotein, nucleocapsid protein, envelope protein, or membrane protein of SARS-CoV-2. This review highlights the underlying mechanisms, advancements, and clinical limitations for each of the diagnostic techniques authorized by the Food and Drug Administration (USA). Significance of diagnosis triaging, information on specimen collection, safety considerations while handling, transport, and storage of samples have been highlighted to make medical and research community more informed so that better clinical strategies are developed. We have discussed here the clinical manifestations and hospital outcomes along with the underlying mechanisms for several drugs administered to COVID-19 prophylaxis. In addition to favourable clinical outcomes, the challenges, and the future directions of management of COVOD-19 are highlighted. Having a comprehensive knowledge of the diagnostic approaches of SARS-CoV-2, and its pathogenesis will be of great value in designing a long-term strategy to tackle COVID-19.

The ongoing coronavirus pandemic is the most catastrophic global crisis after the Second World War [1] . COVID-19 is a contagious disease, caused by a novel severe acute respiratory syndrome Coronavirus (SARS-CoV-2). The genome analysis of this new virus showed ~79.5% similarity with SARS-CoV and middle east respiratory syndrome Coronavirus (MERS-CoV) [2] . However, the highest sequence similarity (~96%) was observed for the bat Coronavirus. Therefore, it has been speculated that COVID-19 was transmitted from bats to humans. Recent studies suggest an intermediary animal host, which potentially could be pangolin [3] or dog [4] .

Earlier studies on COVID-19 suggest that this disease spreads through close contact through the infected person and with small respiratory droplets released while coughing, sneezing, or talking [5] . Tiny droplets of saliva or sputum released from the mouth may carry heavy loads of viruses that are known to stay in the air for a long time and hence potentially can act as carriers of infection [6] . The inhalation of these minute droplets leads to the spreading of viral infection from the sick to a healthy person even when a person is not in direct physical contact with the infected person. Even though COVID-19 is not an airborne disease, some medical practices such as cardiopulmonary resuscitation and intubation may trigger respiratory secretions that can get aerosolized and hence resulted in its spread [7] . The virus gets its way inside the human body through eyes, nose, and mouth and thus spread through contacting the virus from contaminated surfaces and then touching these body parts [8] . However, the extent of viral transmission through contaminated surfaces depends upon environmental conditions such as temperature and humidity [9] . The entry of SARS-CoV-2 into host cells is facilitated by the binding of homo-trimer spike protein (S) that is on the surface of the virus to ACE2 on the host's cell membrane [10] . The recognition of the host cell receptor is a key determinant of the tissue tropism and pathogenicity of the virus [11] . A schematic representation of the life cycle of SARS-CoV-2 is illustrated in Figure 1 . The life cycle of SARS-CoV-2 is similar to the SARS-CoV, MERS-CoV [12] .

Sometimes, animal viruses get accidentally transferred to humans and can evolve itself causing sickness. Thus, it becomes a human host-virus, such as SARS-CoV, MERS-CoV, and SARS-CoV-2. The current COVID-19 pandemic has intensified the interest of scientists all over the world for developing treatment options to mitigate the impact of the disease on human life [12, 13] . More than 300 clinical trials are currently in progress for the treatment of COVID-19.

Different strategies have been adopted to combat COVID-19. However, there is no approved treatment for the same till date. In this article, we evaluated literature for reports informing various diagnostic methods, potential antiviral chemical therapeutics, and effective treatment strategies towards clinical management of COVID-19 patients.

Diagnosis of COVID-19 is a critical step in tracing the virus and understanding its epidemiology. This helps to block the transmission and ensure proper patient care. The preliminary step of COVID-19 diagnosis is through observation of signs and symptoms such as initial loss of smell or taste or both [14] , cough, mild to high fever, myalgia, or fatigue [15] , etc. In addition, several gastrointestinal symptoms of vomiting, diarrhoea, and nausea in some individuals are observed [16] . However, inconsistencies in the manifestation of symptoms from asymptomatic to severe cases like, septic shock, imbalanced metabolic acidosis, coagulation dysfunction, and acute respiratory pneumonia-like syndrome are mostly reported [17] . The presentation of signs and symptoms should be considered as the basis for further tests and not for diagnostic purposes.

The prime consideration in the diagnosis of COVID-19 is the detection of symptoms in clinical situations. Conventionally, samples for pathology are collected from upper and lower respiratory regions (throat, oropharyngeal, nasopharyngeal, bronchoalveolar fluid, and sputum) through swabs for RT-PCR test. In the blood and urine of the infected, there is still an absence of the virus; hence, these are not considered a useful clinical specimen. Reports of inconsistency in RT-PCR test results for CoV-SARS-2 in various tissues [18] and temporal variation of test results from the same tissues [19] suggests that the inter-link between the temporal surge of viral load and its bio-distribution in different tissues of the body would have a critical implication on the accuracy of various tests for diagnosis.

Many assays or tools have been commercialized for the diagnosis of COVID-19 and many more are currently in development. Various molecular assays and immunological assays are currently validated in different laboratories. We discuss here some techniques for the diagnosis of SARS-CoV-2 pathogenesis:

The knowledge of the viral genome sequence has made it possible for us to use molecular techniques in the detection of SARS-CoV-2. Molecular diagnostic methods target to detect either specific regions of the viral genome or RNA-dependent RNA polymerase (RdRP) and/or structural proteins of SARS-CoV-2 (Table 1) .

Currently, the RT-PCR test is a single discrete standard method for the diagnosis of SARS-CoV-2. The RT-PCR protocol involves the extraction of RNA from the specimen and reverses the transcription to cDNA followed by PCR amplification of a specific region with SARS-CoV-2 specific primers and detection through specific probes. Evaluation of RdRp and Envelope (E) genes was originally devised [20] which was adopted by Charité Germany for the diagnosis of COVID-19. The assay screens the E gene which is a pan-SARS-betacoronavirus gene. The confirmatory test is done by targeting the RdRp gene using specific primers and probes listed in Table 2 . The limit to detection is 3.6 copies (RdRp gene) and 3.9

copies (E gene) per reaction and cycle threshold value of less than 37.0 is treated as a positive test. Specific probes and primers target the 1ab gene (ORF1 ab gene or Transcriptase/Replicase gene) as a confirmatory assay. While the level of E gene confirms the presence of SARS related virus. The minimum limit of detection is taken as 1000 copies/ml [18] . The cycle threshold value of less than 40 is set as positive confirmation test criteria.

Three nucleocapsid protein gene (N gene) regions are targeted using primer and probe ( Table   2 ). The assay uses individual probe and primers for three N genes (N1, N2, N3 gene) with an additional primer and probe for detecting human RNase P. N3 gene primer and probe sets are for detection of all SARS-like Coronaviruses. The sensitivity of this assay is lower than other assays as it has a limit of detection of 8.3 copies per reaction. [21] .

For the qualitative detection of the specific gene sequence of SARS-CoV-2, the sample is generally collected as nasopharyngeal or oropharyngeal swabs, sputum, lower respiratory tract aspirates, bronchoalveolar lavage, or nasopharyngeal wash/aspirate as recommended by the FDA. In addition, swabs of upper respiratory specimens including nasopharyngeal, nasal swabs, or mid-turbinate are collected from an individual, with or even without symptoms of COVID-19. Despite the great advantages of these methods, a well-trained technical person is required to carry out such diagnostic procedures. Potential of these molecular tools are restricted to the samples obtained from the respiratory tracts of the suspected individuals.

Sputum, nasopharyngeal aspirates, BAL fluid, nasal aspirates, nasopharyngeal or oropharyngeal swabs can only be tested through this approach. Also, the chances of falsenegative results become high when the lab reagents are contaminated, used past their expiry date, or samples are not timely collected from the right region. False-negative results are also obtained with improper storage and transport of specimen, the presence of amplification inhibitors in samples, and if the mutation rate of the virus is high during the PCR cycle [63].

DETECTOR assay is an RNA-sensing assay that uses synthetic SARS-CoV-2 RNA fragments to recognize the signature of E and N gene sequences of SARS-CoV-2. Viral RNA targets are reversed transcribed to cDNA and amplified which subsequently transcribed back to RNA isothermally. The RNA fragments in the reaction combine with Cas13a protein (ribonuclease) that binds with the amplified RNA product forming a SHERLOCK. Ribonuclease (Cas13a) then cleaves the surrounding fluorophore-quencher probes emitting signals. This CRISPR-Cas13 is a working protocol for SARS-CoV-2 detection that has been rolled out for clinical trials [22] .

For the diagnosis of COVID-19 by immunological technique, viral antigen (antigen test) present in the specimen or antibodies or immunoglobulins produced (antibody test) against specific components of SARS-CoV-2 are detected. These are blood-based tests commonly used to detect antibodies generated against SARS-CoV-2 after the infection. Antibody response to SARS-CoV-2 infection is similar to any typical viral infection [23] . Therefore, the chance of antibody cross-reactivity from infections of other pathogens can exist and sometimes be misinterpreted. Like most immunological diagnostic protocols, Enzyme-Linked Immunosorbent Assay (ELISA) for COVID-19 detection uses IgM and IgG antibody against nucleocapsid (N) and receptor binding domain spike proteins (S) of SARS-CoV-2.

The immunochromatographic test is a rapid chromogenic double antigen/double antibody sandwich ELISA developed in the wake of the SARS-CoV outbreak during 2004 [24] .

Recombinant N protein of SARS-CoV-2 was taken as an antigen which is immobilized on a nitrocellulose strip with IgG coupled to colloidal gold particles. Anti-SARS-CoV-2 N protein antibody IgG binds to the SARS-CoV-2 antigen to form red-coloured antibody-antigen-gold complex ( Figure 2 ). The specificity of the immunochromatographic test is around 98.2% in various kits.

Most tests for rN are done through an indirect ELISA method using IgG where N protein is bounded with IgG/IgM antibody to a microplate or nitrocellulose membrane. A secondary antibody conjugated with horseshoe reddish peroxidase (HRP) binding to the complex is detected by the generation of colour on the addition of substrate TMB (3,3'5, 5' tetramethylbenzidine). The sensitivity for the IgG tests is reported to be 99.0% [23] .

Ab-ELISA is a double-antigen sandwich immunoassay for the detection of total antibodies that binds to the SARS-CoV-2 spike protein receptor domain (RBD) in plasma or serum ( Figure   3 ). In this method, immobilized mammalian cell-expressed recombinant antigen containing the receptor-binding domain of the virus spike proteins and HRP-conjugate antigen were used. The specificity of the Ab-ELISA test kit is 99.1% [23] . IgM specific for recombinant spike protein RBD detection is done using HRP-conjugate and TMB as a substrate for the colorimetric test following IgM µ-chain capture method. The specificity of IgM ELISA is 98.2% [23] . A sensitivity value of above 68% is taken as SARS-CoV-2 nucleocapsid protein test positive [25] . In principle, the detection of viral antigen would be the virus-specific marker for the diagnosis of COVID-19. These diagnostic methods are having a great advantage of convenience and rapidness but limited by inadequate sensitivity and specificity compared to standard RT-PCR. Due to these reasons, results from these methods need to be interpreted with caution.

Change in physio-pathological parameters such as hematological and histopathological can act as a measure to detect SARS-CoV-2. A blood sample of suspected patient is taken for hematological diagnosis. Many studies have shown that individuals with SARS-CoV-2 exhibit clinical characteristics of systemic inflammation such as lymphopenia, neutrophilia, thrombocytopenia, lowered serum albumin and increased C-reactive proteins (CRPs), lactate dehydrogenase, aspartate aminotransferase, alanine transferase, cardiac troponin, erythrocyte sedimentation rate, D dimer, ferritin, creatine kinase, etc [26] . Until now, a well-established hematological parameter/ pathophysiological novel marker for COVID-19 is still lacking.

However, monitoring of hematological parameters could serve as a clinical indicator of severity and prognostication of the disease for early intervention of treatment, but not as a diagnostic tool.

Computed Tomography (CT) is serving as a valuable technique for the diagnosis and progression of the disease. CT scan results of COVID-19 infected individual shows a variety of abnormalities that are unique from other viral pneumonia infections of SARS, MERS, and Adenoviruses [27] . CT scan was found to be more appropriate in providing insights about pneumonia but the results were variable in different patients highlighting the different stages the virus might have been at that time [28] . The scan manifestation unique to COVID-19 patients includes patchy lesion-that are nodular to irregularly shaped have ground-glass opacity (GGO) and are consolidated in the subpleural region of the central lung lobes [29] .

They have distinct reversed halo sign and pulmonary nodules with halo sign [27] . A Followup CT scan revealed that the lesions were migratory with the phenomenon of absorption of the primary lesion to the emergence of new lesions in the pleural and sub-pleural regions [30] .

Since the varied representation of pneumonia is obtained by CXR and CT scan in different patients, therefore it was difficult to conclude the actual pathogenic mechanism [31] . Also, certain asymptomatic patients showed early changes under CT scan which is an excellent approach to identify the patients without symptoms however; this was not always the case [32] .

The crucial factors of diagnosis and treatment of virus-related diseases can be effectively addressed through nanotechnological platforms. The nanomaterials and microfabrication technologies offer valuable diagnostic tools for viral detection in ultralow concentration in blood, serum, and plasma ( Figure S1) . Nanomaterials respond to a minor stimulus and provide a signal even to ultra-low concentrations. Plasmonic metal nanoparticles, such as gold nanoparticles (AuNPs), offer high surface area, which immobilizes various analytes and induces a significant change in plasmonic signal. A colorimetric detection methodology has been reported for the detection of influenza virus A (IVA) using gold nanoparticles (NPs) modified with monoclonal anti-hemagglutinin antibodies (mAb). The AuNPs-mAb probes are sensitive for surface recognition sites of the virus and induce the plasmon effect in the attached AuNPs, leading to visual detection of viral strains [33] . The enveloped virus proteins like hemagglutinin primarily may be utilized for detection parameters for the rapid and easy detection of viral infections.

Zhao et al. [34] reported the fluorescent quantum dots (QDs) and superparamagnetic nanoparticle beads functionalized with mAb for diagnosis of avian influenza virus (AIV). The detection of infectious viruses using various other nanomaterials such as SiNPs [35] , AgNPs [36] , CuNPs [37] , and CNTs [38] 

Despite the challenges associated with COVID-19 therapy, there are still several approaches currently being undertaken which show significant outcomes [40] . In this section, we shall discuss the positive impacts of some of the clinically used medications for the treatment of severe COVID-19 patients ( Figure S2, Table S1 ). Several drugs are in clinical trials and some of them have shown significant promise in addressing COVID-19 patients [13, 41, 42] . The site of action of these drugs is illustrated in Figure 4 . In addition, to drugs under clinical trials, several vaccines are in the pipeline which is expected to play a significant role in controlling the COVID-19 pandemic (Table S2) . This section provides a brief discussion of some of the promising drugs which are used to fight SARS-CoV-2 infection.

Remdesivir (GS-5734™) is a novel small molecule, a broad-spectrum antiviral nucleoside analog that is now being investigated as a potential treatment option for COVID-19 [43] . It belongs to the class of polymerase inhibitors [44] . It is reported to show activity against different RNA viruses, including SARS-CoV, MERS-CoV, Nipah virus, Hendra viruses, and Ebola viruses. Remdesivir is a prodrug of its parent adenosine analog, GS-441524. Both of these medicines metabolized into the active component as nucleoside triphosphate (GS-443902) after ingestion and they show antiviral activity against SARS-CoV [45] . Being a nucleoside analog, Remdesivir targets the viral genome replication process by acting as an RdRp inhibitor. Yin et al. [46] established the structural basis leading to inhibition of the RNAdependent RNA polymerase of SARS-CoV-2 using Remdesivir. On metabolization of remdesivir into active nucleoside triphosphate (NTP) which competes with ATP for incorporation into nascent RNA strand resulting in premature RNA synthesis leading to termination and ceasing of growth of RNA strands [45] . It has been reported that Remdesivir and chloroquine are highly effective in the control of SARS-CoV-2 infection in-vitro. Recently, WHO has claimed that antiviral drug Remdesivir failed in the first trials for the treatment of COVID-19 disease [47] . However, the drug now has become a matter of debate between the clinical experts, scientists, and US-based pharmaceutical companies. There are still controversies regarding the results obtained from this trial, as clinicians suggest no benefit in COVID-19 treatment using Remdesivir; whereas, the company claims it as a promising drug for the same [48]. 

Lopinavir/ritonavir has been used in combination with other anti-retroviral medicinal products for the treatment of HIV-1 infected people. During the present pandemic of COVID-19, this combination of drugs has shown potential for the treatment, and many trials are under progress.

Lopinavir/ritonavir act as a protease inhibitor drug and inhibit the action of 3CLpro, chymotrypsin-like protease enzyme, that plays a vital role in the processing of virus and thus interfere with the process of viral replication and its release from host cells. In an adaptive, randomized controlled clinical trial, Lopinavir/Ritonavir has been administered twice orally as tablets (200 mg and 50 mg) for 14 days to patients. While the others who cannot swallow, 5 mL oral suspension is provided twice daily, followed by a daily assessment. The study is in phase 2 clinical trial [50] . In another phase-2/phase-3 clinical trial, a random, double-blind, multi-centric investigation is being carried out on COVID-19 patients. Four different groups of patients are receiving lopinavir/ritonavir and hydroxychloroquine. The study started on the 6 th of April 2020 and due completion in April 2021 [51].

Novaferon has shown great potential as an antiviral drug against COVID-19 [52] . It is a 

Chloroquine is a widely used antimalarial drug that was observed to have a broad-spectrum antiviral activity [53] . It has been reported to block SARS-CoV-2 infection at low micromolar concentrations [54] . Chloroquine has the inhibition capability of replication of several intracellular micro-organisms, including SARS-CoV-2. The use of chloroquine increases endosomal pH and thereby interferes with the glycosylation of the cellular receptor of SARS-CoV. Therefore, it has the potential to block viral infection. Hydroxychloroquine is a 4aminoquinoline with immunosuppressive, anti-autophagy with antimalarial effects [55] . It has been purposed that HCQ interferes with the processing and presentation of antigens and production of cytokines and hence suppresses immune functions. During the pandemic of COVID-19, HCQ has been studied and under numerous trials for the treatment of coronavirus disease [56] . In two small, uncontrolled studies, HCQ and its congener, chloroquine, were reported to be effective against COVID-19, however, the publishing journal later declared that the trial did not meet the Society's expected standard [57] . Due to severe complications observed in the COVID-19 patients, WHO recently discouraged the use of both CQ and HCQ [58] .

Auranofin is an FDA approved lipophilic organogold compound which has been used to treat rheumatoid arthritis. It exhibits anti-inflammatory and potential antineoplastic activities. These 

Famotidine is a competitive histamine H2-receptor antagonist that inhibits gastric secretion.

Famotidine is the active compound in the heartburn drug, Pepcid. According to a report, on the 27 th of April 2020, Northwell Health in the New York City area began a clinical trial to test famotidine against SARS-CoV-2. The trials have been underway since 7 th of April with 1,174 patients, including 187 who were critically ill. Famotidine is structured in such a way that it could prevent the replication of SARS-CoV-2 protease [61] . Huaier Granule is under phase 2 and phase 3 clinical trial for the treatment of COVID-19, in a study started by Chen Xiaoping at Tongji Hospital on 1 st of April 2020, Over 550 patients are under investigation primarily for their mortality rate, and ICU stays [62] . This study is likely to be completed by September 2020. 

Another example of nucleoside analog used to stop viral RNA synthesis and viral mRNA capping with broad-spectrum antiviral activity is ribavirin which has been analysed for its action against SARS-CoV-2 [65] . It is a prodrug, which when metabolized resembles purine RNA nucleotides that interferes with RNA metabolism required for viral replication. It has been observed during a comparative study on SARS-CoV-2 patients treated with combined therapy of lopinavir/ritonavir and ribavirin; that they are at lower risk of death than when receiving ribavirin monotherapy [66] .

Ivermectin is an FDA-approved anti-parasitic drug having broad-spectrum antiviral activity in vitro. A laboratory level study demonstrated that ivermectin has antiviral action against the SARS-CoV-2 clinical isolate in vitro in the Vero-hSLAM cells. Researchers have claimed that a single dose of ivermectin was able to cause ~5000-fold reduction in viral RNA at 48 h. It has led to the hypothesis that this is likely through inhibition of IMPα/β1-mediated nuclear import of viral proteins [67] . However, on the 10 th of April 2020, the FDA issued guidance not to use ivermectin intended for animals as a treatment for COVID-19 in humans [68].

It is an antiviral widely used to treat the influenza virus. Arbidol can effectually prevent SARS-CoV-2 infection in vitro [41] . Lopinavir/ritonavir and Arbidol have been recently recommended by the National Health Commission and National Administration of Traditional Chinese Medicine for dealing with COVID-19 [69] . A recent study, data suggest that Arbidol monotherapy is more effective than lopinavir/ritonavir in treating COVID-19. No viral load was observed in the Arbidol group than the 44.1% viral load in patients having lopinavir/ritonavir on the 14 th day of treatment. Additionally, no apparent side effects were found in both groups [70] .

Darunavir is a second generation of HIV-1 protease inhibitors used to prevent SARS-CoV-2 infection in-vitro [71] by inhibiting viral replication at 300 μM and this inhibition efficiency was 280-fold compared to the untreated group.

Carmofur is a pyrimidine analog used as an antineoplastic agent. It is an orally delivered 

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SPICA = Solid Phase Immunochromatographic Assay, NA = Neutralization Assay)

Table 2: Primers and probes for targeting SARS-Cov-2 genes in an RT-PCR test for COVID-19 diagnosis. The sequence of the primers and probes are written in 5՜ to 3՜-end pattern (Left to Right). Probes labelled at the 5'-end = Reporter (FAM = 6-carboxyfluorescein, HEX = Hexachloro-fluorescein) and at 3'-end = Quencher (BHQ1 = Black Hole Quencher1

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper