COVID-19

In the last twenty years, several viral epidemics such as the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 to 2003, and H1N1 influenza in 2009, have been recorded. Most recently, the Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia in 2012.  An epidemic of cases with unexplained low respiratory infections detected in Wuhan, was first reported to the WHO Country Office in China, The etiology of this illness is attributed to a novel virus belonging to the coronavirus (CoV) family. On February 11, 2020, the WHO announced that the disease caused by this new CoV was a “COVID-19,” which is the acronym of “coronavirus disease. 2019 The CoVs have become the major pathogens of emerging respiratory disease outbreaks.

Coronaviruses appear crown-like structures under electron microscope hence named as coronavirus. They have positive-stranded RNA as their genomic material and have an outer envelope. Coronaviruses have the largest RNA genomes (27 to 32 kb) among the RNA viruses. The viral envelope is derived from the host cell and has glycoprotein spikes. The viral genome is protected within the nucleocapsid. The nucleocapsid is helical in shape when relaxed and spherical when inside the virus. The viral RNA replicates uniquely. It  replicates in the cytoplasm of the host cell. The RNA polymerase attached itself to the leader sequence of the viral genomic RNA, and in the event of repeated attachment and detachment, a nested set of mRNAs are generated with common 3’ ends. The coronavirus genome encodes for four to five structural proteins: spike (S), membrane (M), envelope (E), nucleocapsid (N), and hemagglutinin-esterase (HE) proteins.

In coronaviruses, the S gene codes for the receptor-binding spike protein that enables the virus to infect cells. This spike protein mediates receptor binding and membrane fusion, which determines host tropism and transmission capabilities. In SARS-CoV-2, the S gene is divergent with <75% nucleotide sequence similarity when compared to all previously described SARS-related coronaviruses. The other three structural proteins are more conserved than the spike protein and are necessary for general coronavirus function. These proteins are involved in encasing the RNA and/or in protein assembly, budding, envelope formation, and pathogenesis

Transmission

Because the first cases of the CoVID-19 disease were linked to direct exposure to the Huanan Seafood Wholesale Market of Wuhan, the animal-to-human transmission was presumed as the main mechanism. Nevertheless, subsequent cases were not associated with this exposure mechanism. it was concluded that the virus could also be transmitted from human-to-human, and symptomatic people are the most frequent source of COVID-19 spread. The possibility of transmission before symptoms develop seems to be infrequent, although it cannot be excluded. Moreover, there are suggestions that individuals who remain asymptomatic could transmit the virus.

Pathogenicity

For addressing pathogenetic mechanisms of SARS-CoV-2, its viral structure, and genome must be considerations. In CoVs, the genomic structure is organized in a +ssRNA of approximately 30 kb in length — the largest known RNA viruses — and with a 5′-cap structure and 3′-poly-A tail. The transcription works through the replication-transcription complex (RCT) organized in double-membrane vesicles and via the synthesis of subgenomic RNAs (sgRNAs) sequences. Of note, transcription termination occurs at transcription regulatory sequences, located between the so-called open reading frames (ORFs) that work as templates for the production of subgenomic mRNAs. In the atypical CoV genome, at least six ORFs can be present. Among these, a frameshift between ORF1a and ORF1b guides the production of both pp1a and pp1ab polypeptides that are processed by virally encoded chymotrypsin-like protease (3CLpro) or main protease (Mpro), as well as one or two papain-like proteases for producing 16 non-structural proteins (nsps). Apart from ORF1a and ORF1b, other ORFs encode for structural proteins, including spike, membrane, envelope, and nucleocapsid proteins. and accessory proteic chains. Different CoVs present special structural and accessory proteins translated by dedicated sgRNAs.

The pathogenic mechanism that produces pneumonia seems to be particularly complex. Clinical and preclinical research will have to explain many aspects that underlie the particular clinical presentations of the disease. The data so far available seem to indicate that the viral infection is capable of producing an excessive immune reaction in the host. In some cases, a reaction takes place which as a whole is labeled a ‘cytokine storm’. The effect is extensive tissue damage. The protagonist of this storm is interleukin 6 (IL-6). IL-6 is produced by activated leukocytes and acts on a large number of cells and tissues. It is able to promote the differentiation of B lymphocytes, promotes the growth of some categories of cells, and inhibits the growth of others. It also stimulates the production of acute phase proteins and plays an important role in thermoregulation, in bone maintenance and in the functionality of the central nervous system. Although the main role played by IL-6 is pro-inflammatory, it can also have anti-inflammatory effects. In turn, IL-6 increases during inflammatory diseases, infections, autoimmune disorders, cardiovascular diseases and some types of cancer. It is also implicated into the pathogenesis of the cytokine release syndrome (CRS) that is an acute systemic inflammatory syndrome characterized by fever and multiple organ dysfunction.

[ Cascella M, Rajnik M, Cuomo A, et al. Features, Evaluation and Treatment Coronavirus (COVID-19) [Updated 2020 Apr 6]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK554776/]

Current Diagnostic Tests for COVID-19

The symptoms expressed by COVID-19 patients are nonspecific and cannot be used for an accurate diagnosis. 44% of 1099 COVID-19 patients from China had a fever when they entered the hospital and that 89% developed a fever while in hospital.  Further it was found that patients had a cough (68%), fatigue (38%), sputum production (34%), and shortness of breath (19%). Many of these symptoms could be associated with other respiratory infections.

The development of molecular techniques is dependent upon understanding

(1) the proteomic and genomic composition of the pathogen or

(2) the induction of changes in the expression of proteins/genes in the host during and after infection.

Nucleic Acid Testing

Nucleic acid testing is the primary method of diagnosing COVID-19. Reverse transcription polymerase chain reaction (RT-PCR) kits have been designed to detect SARS-CoV-2 genetically. RT-PCR involves the reverse transcription of SARS-CoV-2 RNA into complementary DNA (cDNA) strands, followed by amplification of specific regions of the cDNA. The design process generally involves two main steps:

(1) sequence alignment and primer design, and

(2) assay optimization and testing.

Among the SARS-related viral genomes, they discovered three regions that had conserved sequences:

(1) RdRP gene (RNA-dependent RNA polymerase gene) in the open reading frame ORF1ab region,

(2) E gene (envelope protein gene), and

(3)  N gene (nucleocapsid protein gene).

Both the RdRP and E genes had high analytical sensitivity for detection (technical limit of detection of 3.6 and 3.9 copies per reaction), whereas the N gene provided poorer analytical sensitivity (8.3 copies per reaction). The assay can be designed as a two-target system, where one primer universally detects numerous coronaviruses including SARS-CoV-2 and a second primer set only detects SARS-CoV-2.

After designing the primers and probes, the next step involves optimizing assay conditions (e.g., reagent conditions, incubation times, and temperatures), followed by PCR testing. RT-PCR can be performed in either a one-step or a two-step assay. In a one-step assay, reverse transcription and PCR amplification are consolidated into one reaction. This assay format can provide rapid and reproducible results for high-throughput analysis. The challenge is the difficulty in optimizing the reverse transcription and amplification steps as they occur simultaneously, which leads to lower target amplicon generation. In the two-step assay, the reaction is done sequentially in separate tubes. This assay format is more sensitive than the one-step assay, but it is more time-consuming and requires optimizing additional parameters. Lastly, controls need to be carefully selected to ensure the reliability of the assay and to identify experimental errors.

There are three issues that have arisen with RT-PCR. First, the availability of PCR reagent kits has not kept up with demand. Second, community hospitals outside of urban cities lack the PCR infrastructure to accommodate high sample throughput. Lastly, RT-PCR relies on the presence of detectable SARS-CoV-2 in the sample collected. If an asymptomatic patient was infected with SARS-CoV-2 but has since recovered, PCR would not identify this prior infection, and control measures would not be enforced.

Computed Tomography

Due to the shortage of kits and false negative rate of RT-PCR. China temporarily used CT scans as a clinical diagnosis for COVID-19 Chest CT scans are non-invasive and involve taking many X-ray measurements at different angles across a patient’s chest to produce cross-sectional images The images are analyzed by radiologists to look for abnormal features that can lead to a diagnosis. The imaging features of COVID-19 are diverse and depend on the stage of infection after the onset of symptoms. The most common hallmark features of COVID-19 include bilateral and peripheral ground-glass opacities (areas of hazy opacity) and consolidations of the lungs (fluid or solid material in compressible lung tissue. The main caveat of using CT for COVID-19 is that the specificity is low (25%) because the imaging features overlap with other viral pneumonia. CT systems are expensive, require technical expertise, and cannot specifically diagnose COVID-19. Other technologies need to be adapted to SARS-CoV-2 to address these deficiencies.

Protein Testing

Viral protein antigens and antibodies that are created in response to a SARS-CoV-2 infection can be used for diagnosing COVID-19 immunoglobulin G and M (IgG and IgM) from human serum of COVID-19 patients using an enzyme-linked immunosorbent assay (ELISA). The SARS-CoV-2 Rp3 nucleocapsid protein, which has 90% amino acid sequence homology to other SARS-related viruses are used. The recombinant proteins adsorb onto the surface of 96-well plates, and the excess protein is washed away. Diluted human serum is added for 1 h, after which the plate is washed again. Antihuman IgG functionalized with horseradish peroxidase is added and allowed to bind to the target. The plate is washed, followed by the addition of the substrate 3,3′,5,5′-tetramethylbenzidine. The peroxidase reacts with the substrate to cause a color change that can be detected by a plate reader. If anti-SARS-CoV-2 IgG is present, it will be sandwiched between the adsorbed nucleoprotein and the antihuman IgG probe, resulting in a positive signal. The IgM test has a similar structure but uses antihuman IgM adsorbed to the plate and an anti-Rp3 nucleocapsid probe. Antibodies were detected in respiratory blood.

The challenge of using these biomarkers is that they are also abnormal in other illnesses. A multiplex test with both antibody and small molecule markers could improve specificity.

Point-of-Care Testing

Point-of-care tests are used to diagnose patients without sending samples to centralized facilities, thereby enabling communities without laboratory infrastructure to detect infected patients. In commercial lateral flow assays, a paper-like membrane strip is coated with two lines: gold nanoparticle-antibody conjugates are present in one line and capture antibodies in the other. The patient’s sample (e.g., blood and urine) is deposited on the membrane, and the proteins are drawn across the strip by capillary action. As it passes the first line, the antigens bind to the gold nanoparticle-antibody conjugates, and the complex flows together through the membrane. As they reach the second line, the complex is immobilized by the capture antibodies, and a red or blue line becomes visible. Individual gold nanoparticles are red in color, but a solution containing clustered gold nanoparticles is blue due to the coupling of the plasmon band. The lateral flow assay has demonstrated a clinical sensitivity, specificity, and accuracy of 57%, 100%, and 69% for IgM and 81%, 100%, and 86% for IgG, respectively. A test that detects both IgM and IgG yields a clinical sensitivity of 82%.

 Source: Udugama, B., Kadhiresan, P., Kozlowski, H. N., Malekjahani, A., Osborne, M., Li, V., Chen, H., Mubareka, S., Gubbay, J. B., & Chan, W. (2020). Diagnosing COVID-19: The Disease and Tools for Detection. ACS nano, 14(4), 3822–3835. https://doi.org/10.1021/acsnano.0c02624]

Treatment / Management

There is no specific antiviral treatment recommended for COVID-19, and no vaccine is currently available. The treatment is symptomatic, and oxygen therapy represents the major treatment intervention for patients with severe infection. Mechanical ventilation may be necessary in cases of respiratory failure refractory to oxygen therapy, whereas hemodynamic support is essential for managing septic shock.

On January 28, 2020, the WHO released a document summarizing WHO guidelines and scientific evidence derived from the treatment of previous epidemics from HCoVs. This document addresses measures for recognizing and sorting patients with severe acute respiratory disease; strategies for infection prevention and control; early supportive therapy and monitoring; a guideline for laboratory diagnosis; management of respiratory failure and ARDS; management of septic shock; prevention of complications; treatments; and considerations for pregnant patients

Intubation and protective mechanical ventilation

Special precautions are necessary during intubation. The procedure should be executed by an expert operator who uses personal protective equipment (PPE) such as FFP3 or N95 mask, protective goggles, disposable gown long sleeve raincoat, disposable double socks, and gloves. If possible, rapid sequence intubation (RSI) should be performed. Preoxygenation (100% O2 for 5 minutes) should be performed via the continuous positive airway pressure (CPAP) method. Heat and moisture exchanger (HME) must be positioned between the mask and the circuit of the fan or between the mask and the ventilation balloon.

Non-invasive ventilation 

Concerning HFNO or non-invasive ventilation (NIV), the experts’ panel, points out that these approaches performed by systems with good interface fitting do not create widespread dispersion of exhaled air, and their use can be considered at low risk of airborne transmission. Practically, non-invasive techniques can be used in non-severe forms of respiratory failure. However, if the scenario does not improve or even worsen within a short period of time (1–2 hours) the mechanical ventilation must be preferred.

Other therapies

Among other therapeutic strategies, systemic corticosteroids for the treatment of viral pneumonia or acute respiratory distress syndrome (ARDS) are not recommended. Moreover, unselective or inappropriate administration of antibiotics should be avoided, although some centers recommend it. Although no antiviral treatments have been approved, several approaches have been proposed such as lopinavir/ritonavir (400/100 mg every 12 hours), chloroquine (500 mg every 12 hours), and hydroxychloroquine (200 mg every 12 hours). Alpha-interferon (e.g., 5 million units by aerosol inhalation twice per day) is also used.

The WHO and other organizations have issued the following general recommendations:

  • Avoid close contact with subjects suffering from acute respiratory infections.
  • Wash your hands frequently, especially after contact with infected people or their environment.
  • Avoid unprotected contact with farm or wild animals.
  • People with symptoms of acute airway infection should keep their distance, cover coughs or sneezes with disposable tissues or clothes and wash their hands.
  • Strengthen, in particular, in emergency medicine departments, the application of strict hygiene measures for the prevention and control of infections.
  • Individuals that are immunocompromised should avoid public gatherings.

The most important strategy for the populous to undertake is to frequently wash their hands and use portable hand sanitizer and avoid contact with their face and mouth after interacting with a possibly contaminated environment.

Healthcare workers caring for infected individuals should utilize contact and airborne precautions to include PPE such as N95 or FFP3 masks, eye protection, gowns, and gloves to prevent transmission of the pathogen. {source who guidelines for covid-19} and [Cascella M, Rajnik M, Cuomo A, et al. Features, Evaluation and Treatment Coronavirus (COVID-19) [Updated 2020 Apr 6]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan-. Available from: ttps://www.ncbi.nlm.nih.gov/books/NBK554776/]

Updates to the Guidelines

Potential Antiviral Drugs Under Evaluation for the Treatment of COVID-19

Throughout the section, study descriptions were updated to clearly indicate a study’s publication status and to provide an assessment of a study’s limitations and results. Data were also updated as needed based on changes to preprints or post-publication changes.

The following recommendations were added or revised in this section:

Remdesivir:

On the basis of preliminary clinical trial data, the Panel recommends the investigational antiviral agent remdesivir for the treatment of COVID-19 in hospitalized patients with severe disease, defined as SpO2 ≤94% on ambient air (at sea level), requiring supplemental oxygen, mechanical ventilation, or extracorporeal membrane oxygenation (BI).

Remdesivir is not approved by the Food and Drug Administration (FDA); however, it is available through an FDA emergency use authorization for the treatment of hospitalized adults and children with COVID-19. Remdesivir is also being investigated in clinical trials, and it is available through an emergency access program for children and pregnant patients.

The Panel does not recommend remdesivir for the treatment of mild or moderate COVID-19 outside the setting of a clinical trial (AIII).

Chloroquine/Hydroxychloroquine:

The Panel recommends against using high-dose chloroquine (600 mg twice daily for 10 days) for the treatment of COVID-19 (AI), because the high dose carries a higher risk of toxicities than the lower dose.

The FDA warning that cautioned against the use of chloroquine or hydroxychloroquine for COVID-19 outside the setting of a hospital or clinical trial was added to this section.

Immune-Based Therapy Under Evaluation for Treatment of COVID-19

The following key changes were made to this section:

Convalescent Plasma and Immune Globulins:

New information has been added to the section on convalescent plasma and SARS-CoV-2-specific immune globulins.

A new section for non-SARS-CoV-2 intravenous immune globulin (IVIG) was created, in which the Panel recommends against the use of non-SARS-CoV-2-specific IVIG for the treatment of COVID-19, except in the context of a clinical trial (AIII). This should not preclude the use of IVIG when it is otherwise indicated for the treatment of complications that arise during the course of COVID-19.

Interleukin-6 Inhibitors:

New data from an interim review of a Phase 2/3 clinical trial for sarilumab have been included.

New preliminary results from a clinical trial for tocilizumab (CORIMUNO-TOCI) have been added.

There is no change to the Panel’s recommendation for IL-6 inhibitors. There are insufficient data to recommend either for or against the use of IL-6 inhibitors (e.g., sarilumab, siltuximab, tocilizumab) for the treatment of COVID-19 (AIII).

Content writer:  Anand