There is a considerable health threat to humans by the emergence and spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the world (Chawla et al., 2020).
To help advance the diagnosis of COVID-19, we describe here different testing methods with greater emphasis on next-generation sequencing (NGS) technology, which caused to sequence discovery of a novel human coronavirus. In the following, we will explain the standard methods of treating patients and give a brief reference to the use of new treatment technologies that might be used to cure this disease.
2. The Overview of SARS-CoV-2 Virus
The experts at the People’s Republic of China (PRC) Centers for Disease Control recognized a virulent coronavirus as novel coronavirus pneumonia (NCP) for the first time (Wang, Wang, Ye, & Liu, 2020). Afterward, the name of the disease was introduced by World Health Organization (WHO) as coronavirus disease-2019 (COVID-19) (Wan et al., 2020). On 7 January 2019, scientists called it a 2019 novel coronavirus (2019-nCoV). Meanwhile, it was renamed SARS-CoV-2 by the International Committee on Taxonomy of Viruses (Yuen, Ye, Fung, Chan, & Jin, 2020). This virus belongs to the β-coronavirus family, a significant class of viruses that are widespread (Li, Liu, Yu, Tang, & Tang, 2020). COVID-19 is a biological hazard and becomes a political and economic threat to healthcare worldwide (e Silva & de Carvalho, 2020). SARS-CoV-2 has potential definitive and intermediate hosts like several viruses, but its transmissibility is higher than the Middle East respiratory syndrome coronaviruses (MERS-CoV). Thus, there are significant challenges for the elimination and treatment of COVID-19 (Liu, Gayle, Wilder-Smith, & Rocklöv, 2020).
The main entry point of the virus to the cell is Angiotensin Convertase Enzyme 2 (ACE2) receptor. It is located on the surface of the nasal mucosa, bronchus, lung, heart, esophagus, kidney, stomach, bladder, and ileum cells, so all of these organs are susceptible to SARS-CoV-2. ACE2 is the primary receiver of the spike protein of the SARS-CoV-2 (Fekrazad, 2020). The Spike protein of the COVID-19 consists of two parts: S1 and S2. The first is for primary attachment, while the second is for viral infusion (Belouzard, Millet, Licitra, & Whittaker, 2012).
3. Definitive Diagnosis of COVID-19
The growing concerns regarding the COVID-19 pandemic cause pathogen discovery of novel CoVs now consider even more important than in the past (Xiu et al., 2020). As 2019-nCoV is a newly discovered virus, there is limited availability of laboratory-based diagnostic tests for it. Among all of the laboratory tests with their pros and cons (Table 1), viral nucleic acid testing can prevent the more spread of the COVID-19. It is commercially available for the detection of pathogenic organisms (Phan, 2020).
Metagenomics analysis using next-generation sequencing technology (mNGS) leads to sequence discovery of several CoV strains of four genera. These strains are genetically similar to human viruses within the beta coronaviruses (β-CoV), such as the bat SARS-related Coronaviruses; the new sequence also is added to the GenBank (Gorbalenya et al., 2020). Briefly, a sample of RNA based-mNGS approach may be collected from bronchoalveolar lavage fluids (BALF) in which nucleic acids are extracted, and synthesized cDNA is prepared for the sequencing library. Then, high-throughput sequencing is performed on each library.
Table 1. Current and upcoming SARS-CoV-2 diagnostic tests.
LAMP: Loop-mediated isothermal amplification; RT-LAMP: Reverse transcription LAMP; ELISA: Enzyme-linked immunosorbent assay; GICA: Colloidal gold-immunochromatographic assay; NLR: Neutrophil-to-lymphocyte; qRT-PCR: Quantitative reverse transcription polymerase chain reaction; CRISPR/Cas: Clustered Regularly Interspaced Short Palindromic Repeats; POCTs: Point-of-care tests; ICG: immunochromatographic; CT: Computed tomography.
Sequenced reads are mapped to the reference 2019-nCoV genomic sequences. These reads are translated into the single intact open reading frame gene. Afterward, the aligned amino acid sequences are categorized based on the same mutations/deletions in the S1/S2 region and ranked by frequency of occurrence. In this method, complete and partial 2019-nCoV genome sequences can be obtained (Zhou et al., 2020).
NGS can result in increased surveillance for infection prevention and patient management due to the investigation of infectious agents from original clinical samples directly. Primarily, RNA based-mNGS approach could determine concomitant the whole “infectome” (i.e., RNA/DNA viruses, Prokaryotes, and Eukaryotes) present within an organism (Chen et al., 2020). This technology shows higher sensitivity than conventional RT-PCR. It can be used in various aspects of virus detection, including double-check detection, secondary diagnosis, and suspected sample detection to be RT-PCR false-negative (Xiao et al., 2020).
NGS may be used unbiased evolutionary analysis of bat CoVs that considers their high genetic diversity because of its high-throughput sample processing and need the compilation of whole-genome sequences (WGS) (Li et al., 2020). It can also be a laboratory-confirmatory 2019-nCoV infection test for patients or pregnant women with suspected cases of COVID-19 pneumonia (Deng et al., 2020). As defective viral genomes (DVGs) containing genomic deletions are generated during CoV replication, direct analysis of these recombined CoV RNAs is feasible with the advent of NGS technology. This technology minimizes the risk of handling infectious viral cultures and enables new scientific inquiry (Gribble et al., 2021). Genome Detective Coronavirus Typing Tool is a software application of NGS to assemble all known virus genomes that can monitor novel viral mutations as the outbreak expands globally. It may improve new diagnostics, medicines, and vaccines to prevent the COVID-19 infection (Cleemput et al., 2020).
Activities have been carried out to decrease labor and overall turn-around time and increase the cost-effectiveness of this technique. One of them is the usage of the bioinformatics process for physically separating reads derived from host DNA and RNA. The other activity is related to Virome Capture Sequencing to off-target enrichment of viral nucleic acid relies on predesigned viral probes considering balancing depth and length of coverage that share more than 60% homology with the target virus sequence resulting in decreasing essential metagenomics sequencing effort. Amplicon- or PCR-based and hybridization capture-based enrichment (hybrid capture) are two major enrichment strategies for targeted sequencing (Nasir et al., 2020).
The other strategy is based on testing only the minimal region of the virus essential for interacting with the host receptor, where dramatically reduces cost and synthesis production time for testing several spikes for entry in the system (Letko & Munster, 2020).
On the other hand, despite all of the efforts to apply high-throughput sequencing technology in clinical diagnosis and become more inexpensive each consecutive year, the high expense is still the most indispensable barrier to generalize virus sequencing these days (Xiao et al., 2020).
Equipment dependency, time-consuming, sophisticated library preparations, and data processing and missing low-abundance CoV sequences due to the high background level of nonviral sequences present in surveillance field samples are the other its limitation. Also, an inherent deficiency of sensitivity with an unbiased approach increases data analysis burden and decreases the chance of detection in field samples with low viral loads.
It is highly recommended that employing novel coronavirus surveillance, including pan-CoV assay and other pan-viral assays, among people who exposure to animals for a long time because there are some common viruses between humans and animals. This surveillance can be through cell culture, full genome sequencing, and seroepidemiology of both the worker and the animals if indicated (Xiao et al., 2020).
Based on the items stated above, it can be concluded that combined screening strategies may be the more efficient way to identify new CoVs.
4. Novel Therapies
Since the molecular mechanism of pathogenicity of this virus remains to be fully determined, there is still presently no vaccine or specific antiviral drug regime used to treat critically ill patients. Currently, treatment provided to the affected individuals is the main symptom-based (Vellingiri et al., 2020). Oxygenation, ventilation, and fluid management are used as the provision of supportive care. The recent catastrophic prevalence of COVID-19 highlights the immediate introduction of potential therapeutics targeting SARS-CoV-2. Forasmuch as it seems unlikely that the development of drugs specific for COVID-19 will take at least in the coming months, medications that have been proven to be safe for humans can be repurposed to treat this disease. Drugs used to treat SARS-CoV-2 are mostly based on their effectiveness on primary strains of coronavirus, SARS-CoV, and MERS-CoV (Rismanbaf, 2020). The vast majority of the medicines utilized for treatment around the world are discussed below.
Drugs under anti-viral category generally follow the inhibition of one of the following three mechanisms: viral replication, ion channel, and serine proteases (Razonable, 2011).
Remdesivir is an adenosine triphosphate analog first described in 2016 as a potential effector on the treatment Ebola. This drug is a nucleotide analog, which was used to treat against Ebola virus, Marburg virus, SARS-CoV and MERS-CoV (Elfiky, 2020; Warren et al., 2016). In order to treatment COVID-19, Remdesivir was granted an FDA Emergency Use Authorization on 1 May 2020 (Ison, Wolfe, & Boucher, 2020). However, this is not the same as an FDA approval (Sung et al., 2020).
Protease inhibitors, are an integral component of highly active antiretroviral therapy (HAART) (Kohlrausch, de Cássia Estrela, Barroso, & Suarez-Kurtz, 2010). Lopinavir/ritonavir is a protease inhibitor combination used for the treatment of HIV infection (Gupta et al., 2008).
Lopinavir generates little systemic concentrations when used alone, it is extensively metabolized by CYP3A4. Ritonavir can increase Lopinavir concentration by potently inhibiting the CYP3A4 (Corbett, Lim, & Kashuba, 2002). It may be applied as a component of initial or salvage therapy.
Literature has demonstrated, after prescribing this composition, β-coronavirus viral loads notably decreased and no or little coronavirus titers were observed (Shen et al., 2020). In contrast, other studies have shown that the use of lopinavir-ritonavir to treat hospitalized adult patients with severe Covid-19, was not associated with clinical improvement or mortality in seriously ill patients (Cao et al., 2020).
Ribavirin is a broad-acting antiviral drug whose therapeutic potential was uncovered during 1972. It is a guanosine analog that interferes with the replication of RNA and DNA viruses and is used to treat different viruses in combination with immunomodulators (interferon α) or direct-acting antivirals (Gane et al., 2013; Manns et al., 2001). It inhibits natural guanosine generation by directly inhibiting inosine monophosphate dehydrogenase (Khalili, Zhu, Mak, Yan, & Zhu, 2020). Ribavirin was the standard of care therapy in hepatitis C virus (HCV) infection (Ampuero & Romero-Gómez, 2016; Cusato et al., 2018). This drug is phosphorylated by adenosine kinase and cytosolic 5'-nucleotidase and it is transported into cells by concentrative nucleoside transporters (Allegra et al., 2015). The wide availability and low cost of ribavirin support its potential to significantly impact the treatment of COVID-19 infections.
Favipiravir is a pyrazine carboxamide derivative (6-fluoro-3-hydroxy-2-pyrazine-carboxamide) that is considered a purine nucleic acid analog and a potent RNA polymerase inhibitor. It is a broad-spectrum antiviral drug approved for the treatment of influenza (Du & Chen, 2020). This drug has obtained approval from Shenzan Health Commission for treating COVID-19 patients (Wu et al., 2020). According to a study by Cai et al., patients with COVID-19 who were treated with Favipiravir have a significantly higher improvement rate in chest imaging and furthermore, they showed significantly better treatment effects on COVID-19 in terms of disease progression and viral clearance (Cai et al., 2020).
Sofosbuvir is a potent nucleotide HCV Non-structural protein 5 (NS5B) polymerase inhibitor (Cusato et al., 2018). Sofosbuvir is an approved drug by the FDA against HCV in the year 2013 (Elfiky, Gawad, & Elshemey, 2016; Lam et al., 2012). It gives excellent results against other viruses including the Zika virus (Bullard-Feibelman et al., 2017). It was used in combination with interferon or Ribavirin. Additionally, studies have shown sofosbuvir as a potent inhibitor against the newly emerged COVID-19 strain (Elfiky, 2020).
4.7. Anti-Malarial Agent
Chloroquine is an amine acidotropic form of quinine (Winzeler, 2008) and hydroxychloroquine by the presence of a hydroxyl group at the end of the side chain, differs from chloroquine. For decades, chloroquine/hydroxychloroquine was widely-used as an anti-malarial and autoimmune disease drug (Devaux, Rolain, Colson, & Raoult, 2020). Previous studies reported that chloroquine/ hydroxychloroquine possesses the potential of inhibiting the exacerbation of pneumonia due to its anti-viral and anti-inflammatory activities (Cunningham, Goh, & Koh, 2020; Zhang et al., 2020). Chloroquine/hydroxychloroquine is known to block virus infection by increasing endosomal pH, in addition, can prevent SARS-CoV from effect? The glycosylation of a virus cell surface receptor, ACE2 (Lajoie, Mwangi, & Fowke, 2017; Wang et al., 2020). A very recent publication of results showed that chloroquine can reduce the SARS-CoV-2 viral load and shorten the duration of viremia (Vellingiri et al., 2020). There are side effects to these medications, serious retinopathies and cardiopathies associated with bioaccumulation of the drugs are described in the literature (Palmeira, Costa, Perez, Ribeiro, & Lanza, 2020).
4.8. Immunologically Based Strategies
Uncontrolled and excessive cytokine release, also known as cytokine storm (Zhu et al., 2020). Cytokine storm syndrome can be caused by a variety of diseases, including infectious and non-infectious diseases. Accumulating evidence revealed that patients hospitalized with severe COVID-19 have an elevated cytokine profile, similar to cytokine storm (Ye, Wang, & Mao, 2020). The immune system attacks the body by the cytokine storm, which in turn will lead to Acute Respiratory Distress Syndrome (ARDS) and multiple organ failure, at least in the most severe cases of SARS-CoV-2 infection, the final result being death. Interferons, interleukins, chemokines and TNF-alpha represent the main ingredients involved in the expansion of the Cytokine storm (Coperchini, Chiovato, Croce, Magri, & Rotondi, 2020; Xu et al., 2020). As far as we know, there are a diversity of anti-inflammatory medicines, including non-steroidal anti-inflammatory drugs, glucocorticoids, inflammatory cytokines antagonists (such as IL-6R monoclonal antibodies, TNF inhibitors, IL-1 antagonists, Literature suggested that tailored anti-inflammatory therapy can reduce systemic inflammation before it overwhelmingly results in multi-organ dysfunction (Vickers, 2017; Zhang et al., 2020).
However, there is a dilemma of anti-inflammatory therapy and creating a balance between balancing the risk and benefit ratio is a critical issue. It is important that we know whether anti-inflammation therapy is helpful in treating COVID-19 patients, at what stage and for how long should we use anti-inflammation therapy? Also, which anti-inflammatory medications are helpful? (Zhang et al., 2020) Our major concern is that anti-inflammatory therapy, such as corticosteroid, may delay the elimination of virus and increase the risk of secondary infection.
4.9. Convalescent Plasma
Passive immunization has been successfully used to treat infectious diseases. As we know, the SARS-CoV-2 entry into the target host cells by binding the S protein to ACE2 receptors. By employing neutralizing antibodies against the ACE2 receptors, there is a high possibility for reducing the severity of the disease (Zheng & Song, 2020). Neutralizing antibody titers increase in the plasma of patients who have recently completely recovered from the COVID-19.
A systematic review describes a considerable decrease in mortality and viral load in studies using convalescent plasma for the treatment of severe acute viral respiratory infections, including those caused by related coronaviruses (SARS-CoV and MERS-CoV) (Cunningham et al., 2020).
But on the contrary, it is not yet clear whether convalescent plasma can cure critically ill patients with COVID-19 and ARDS. An important point to consider when using this technique, the need for adequate selection of donors with high neutralizing antibody titers (Lim et al., 2020).
4.10. Novel Treatment Strategies
Our modern world inaccessibility to a method that can destroy the virus or stop its speed brings us to the fact that today’s world needs new techniques to deal with emerging diseases (Boluki et al., 2017; Pourhajibagher et al., 2016). COVID-19 pandemic proves the ineffectiveness of current methods to deal with coronavirus. Here, we will discuss some of the newer ways to deal with microorganisms.
Light-based antimicrobial therapy strategy relies on the ability to eradicate microbes regardless of antibiotic resistance (Pourhajibagher et al., 2016). Briefly, the basis of the photodynamic therapy (PDT) mechanism is a combination of non-toxic photosensitizers (PSs) and harmless visible light that produce reactive oxygen species (ROS). The ROS’s can oxidize biomolecules and thereby kill cells. Nucleic acids (DNA or RNA), virus proteins and if present, viral lipids are three main molecular targets for PDT and reaction with the generated ROS (Wiehe, O’Brien, & Senge, 2019). SARS-CoV-2 with lipids and a protein envelope, in general, seems to be more vulnerable to PDT than those without (Baptista et al., 2017; Girotti, 2001). PDT is a clinically approved, minimally invasive therapeutic procedure that is used for the treatment of infections and can exert a selective cytotoxic activity toward malignant cells (Agostinis et al., 2011). It is an exceptional treatment strategy, So that in addition to its direct effect on biomolecules, the PS may activate the immune system to attack target cells (Taylor, 2007).
One of the major challenges with COVID-19 pandemics is that the virus SARS-CoV-2 is transmitted between people through direct contact and bio-aerosols. Discovery of antiviral procedure for indoor air purification can be beneficial to solve this problem. One of the new technologies that have recently been used to kill microorganisms in the air is the photocatalyst technique (Moshfegh, Khosraviani, Moghaddasi, Limoodi, & Boluki, 2021). Photocatalysis has recently emerged as an effective green solution for antimicrobial disinfection applications (Boluki, Pourhajibagher, & Bahador, 2020; Ganguly, Byrne, Breen, & Pillai, 2018). Utilizing air purification systems based on photocatalyst method can easily disrupt the transmission chain of this virus through the air.
The interaction of nanomaterials with Infectious agents is fast-revolutionizing the biomedical field by offering advantages in both diagnostic and therapeutic applications. Nanoparticle offers unrivaled Physico-chemical properties that have linked benefits for drug delivery as ideal tools for viral treatment (Cojocaru et al., 2020). The notable features of these compounds include, the particle size (which affects bioavailability and circulation time), the large surface area to volume ratio (raise solubility), contains a hollow volume that can accommodate a drug payload, surface charge tunable, and the possibility of encapsulation (de Vries et al., 2020; Kerry et al., 2019; Tebaldi, Belardi, & Montoro, 2016).
These properties generate great expectations for the nanoparticulate drug. Progresses of nanotechnology in antiviral therapy can deliver new therapeutic strategies to explore to achieve and improve therapeutic effects.
SARS-CoV-2 is spreading rapidly and has a high rate of mutagenesis and changes in structure, which scientists are endeavoring to discover antivirals specific for its efficacious treatment (Vellingiri et al., 2020). Confronting the challenge of the outbreak of COVID-19 should sharpen the focus on global drug access as a key issue in antiviral therapy. The testing and adoption of effective therapies for novel coronaviruses are hampered by the challenge of conducting controlled studies during a state of emergency (Khalili et al., 2020). The pathology of COVID-19 resembles that of the 2013 MERS-CoV and 2003 SARS-CoV infections such that the extrapolation of treatment guidance from those prior clinical experiences can provide guidance for the current outbreak of 2019-nCoV (Liu et al., 2020).
SARS-CoV-2 diagnosis can be based on detection of the virus presence by nucleic acid test and antibodies (serological tests) produced in response to infection as well as medical imaging.
Obviously, combining assessment of imaging features with clinical and laboratory findings could facilitate early diagnosis of COVID-19. The critical need for treatment and patient care in outbreak settings, on the frontlines of nCoV outbreaks, will place stress on any medical system and clinical research mechanism. Accordingly, in this review, we have summarized recent progress in treatment strategies of COVID-19 and discussed various diagnostic methods for SARS-CoV-2 which may lead to help clinicians make more efficient, data-informed decisions and battle against SARS-CoV-2.
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