An Informative Review on screening of COVID-19 (SARS-COVID-II)
Nensi Raytthatha, Isha Shah, Dr. Jigar Vyas, Dr Umesh Upadhyay
Sigma Institute of Pharmacy, Ajwa-Nimeta Road, Waghodia, Baroda, Gujarat, 390019 India.
*Corresponding Author E-mail: drjigarvyas@gmail.com, ishashah3498@gmail.com
ABSTRACT:
Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) is a highly transmissible and pathogenic coronavirus that emerged in late 2019 and has caused a pandemic of acute respiratory disease, named ‘coronavirus disease 2019’ (COVID-19), which threatens human health and public safety. The disease caused by SARS‐CoV‐2, coronavirus disease 2019 (COVID‐19), presents flu‐like symptoms which can become serious in high‐risk individuals. During the early phase of the coronavirus (COVID-19) pandemic, design, development, validation, verification and implementation of diagnostic tests were actively conveyed by a large number of diagnostic test manufacturers. In this Review, we give an outline of the crucial role of diagnostic tests during the first world-wide indication of COVID-19. The severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) and its associated coronavirus (COVID-19) pandemic has demanded rapid up scaling of in-vitro diagnostic assays to enable mass screening and testing of high-risk groups. To encounter the exponential demand in testing, there has been an expedite development of both molecular and serological assays across a superfluity of manifestos. The adjacent review discusses the current information on these modalities, including nucleic acid amplification tests, direct viral antigen tests. In the analytic stage, real-time reverse transcription-PCR (RT-PCR) assays remain the molecular test of choice for the etiologic diagnosis of SARS-CoV-2.
KEYWORDS: Rt-pcr testing, SARS-CoV-2, Antibody testing, Molecular testing, COVID‐19 diagnosis.
1. INTRODUCTION:
The name "coronavirus" is derived from Latin corona, meaning "crown" or "wreath", itself a borrowing from Greek "garland, wreath"1. They are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry.2 The genome size of coronaviruses ranges from approximately 26 to 32 kilobases, one of the largest among RNA viruses.3
1.1 Origin and transmission of covid-19:
The first cases were seen in Wuhan City of Hubei Province China in December 2019, and have been linked to the Huanan Seafood Market (South China) and the infection has spread to several countries around the world.4 The recombination of DNA was found to be involved at spike glycoprotein which assorted SARS-CoV (CoVZXC21 or CoVZC45) with the RBD of another Beta CoV, thus could be the reason for cross-species transmission and rapid infection.5 The virus that causes coronavirus disease 19 (COVID-19) is a highly transmittable and pathogenic viral infection and mainly transmitted through contact with respiratory droplets rather than through the air. A single cough can circulate up to 3.000 droplets. These droplets can land on other people, and covering surfaces around them, however, several smaller particles will stay within the air the virus is also shed for extended in faecal matter, thus anyone who not washing their hands thoroughly after visiting the toilet, bathroom could contaminate anything they touch like many respiratory viruses, including flu, Covid-19 can be spread by close contact with small droplets released from infected individuals upper respiratory tract secretions6, e.g. sneezing, common cold or coughing from the nose and mouth.4
1.2 Causes of covid-19 virus:
A day-by-day breakdown of coronavirus symptoms shows how symptoms progress among typical patients, how the disease, COVID-19, goes from bad to worse.
Table 1: Days with onset of symptoms
|
Day |
Symptoms |
|
Day 1 |
In the starting day of the symptom, the patient suffers from fever along with fatigue, muscle pain, and a dry cough. Few of them may experience nausea and diarrhoea a few days before the arousal of symptoms. |
|
Day 5 |
Patients may suffer from breathing problem especially if they are elderly or have some pre-existing health condition. |
|
Day 7 |
According to the Wuhan University study, these are the symptoms of the patient that lead the patient to be admitted in the hospital. |
|
Day 8 |
On the 8th day, patients (15%, according to the Chinese CDC) develop acute respiratory distress syndrome (ARDS), a condition where the fluid fills up in the lungs and this is mostly fatal. This usually happens in severe cases. |
|
Day 10 |
The progression of the disease leads to worsening of the symptom and at this point the patient is shifted to ICU. Patients with milder symptoms probably have more abdominal pain and loss of appetite. |
|
Day 17 |
On average, after two-and-a-half weeks patients who recover are discharged from the hospital. However, it's difficult to find out the symptoms in the earlier days of the infection. This is usually seen after 5-6 days. 7 |
2. Types of treatment:
2.1 Allopathy Treatment:
· Chloroquine and hydroxychloroquine
· Lopinavir
· Remdesivir
· Favipiravir (Avigan)8
2.2 Homeopathic Treatment:
2.3 Naturopathy:
In this review paper, testing of COVID-19 is enlightened:
3. Diagnosis:
3.1 Testing kits:
This COVID-19 Rapid Test Kit is suitable for the qualitative detection of SARS-CoV-2 IgM/IgG antibodies in human serum, plasma, or whole blood. 9
3.1.1 Accuracy and specificity:
IgG sensitivity: 90.0%, specificity: 100.0%., IgM sensitivity: 100.0%, specificity: 98.8%.
Overall positive agreement rate of 80.8%, negative agreement rate of 100.0%.
Sample Type:
Fingertip blood, whole blood sample, serum and plasma.
Detection Method: Colloidal Gold, Detection Time: 10 – 15 minutes.
FDA EUA approved; CE Certified 9
3.1.2 Mechanism of Testing:
IgM provides the first line of defence during viral infections, followed by the generation of adaptive, high affinity Immunoglobulin G. (IgG) responses for long-term immunity and immunological memory. IgG is usually detectable about 7 days after the IgM appears.
Both Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies are produced during the primary immune response. As the body’s largest antibody, IgM is the first antibody to appear in response to an initial exposure to antigens.
Fig.1 Instructions of use interpretation of test results
3.1.3 Limitations:
1. The product is designed only for use with human serum, plasma, or whole blood samples for the qualitative detection of SARS-CoV-2 IgM and IgG antibody.
2. A false negative may occur if the amount of SARS-CoV-2 IgM or IgG antibody is below the detection level of the kit.
3. If the product gets wet prior to use, or is stored improperly, it may cause incorrect results.
4. The test is for qualitative detection of SARS-CoV-2 IgM or IgG antibody in human serum, plasma, or blood samples, it does not indicate the quantity of the antibodies present.9
3.2 Testing methods:
3.2.1. Molecular testing:
3.2.1.1. Rt-pcr testing:
Polymerase chain reaction (PCR) is a process that amplifies (replicates) a small, well-defined segment of DNA many hundreds of thousands of times, creating enough of it for analysis. Test samples are treated with certain chemicals10,11 that allow DNA to be extracted. Reverse transcription converts RNA into DNA. Reverse transcription polymerase chain reaction (RT-PCR) first uses reverse transcription to obtain DNA, followed by PCR to amplify that DNA, creating enough to be analysed.10 RT-PCR can thereby detect SARS-CoV-2, which contains only RNA. The RT-PCR process generally requires a few hours.13 Real-time PCR (qPCR) 14 provides advantages including:
Higher-throughput
It has become the preferred method.12
Average sensitivity for rapid molecular tests were 95.2% (ranging from 68% to 100%) and average specificity was 98.9% (ranging from 92% to 100%) between test results of different company brands and sampling methods15 Samples can be obtained by various methods, including a nasopharyngeal swab, sputum (coughed up material) throat swabs, deep airway material collected via suction catheter16 or saliva. Many companies and labs have developed tests to diagnose COVID-19 based on detection of the virus’s genetic material in a sample from the patient’s nose or throat. These steps may change as new technology becomes available, but currently the typical steps in molecular testing are:
1. A doctor, pharmacist, or other health professional orders a COVID-19 test. All COVID-19 tests, including those used with a home collection kit, require a prescription or order from a health professional.
2. You or a health care professional use a specialized swab to collect mucus from your nose or throat.
3. You or a health care professional put the swab in a sterile container and seal it for transport to a lab.
4. During the shipping process, most molecular test swabs must be kept within a certain temperature range so that the test will be accurate. The sample must arrive at the lab within 72 hours.
Fig.2 RT-PCR Testing
5. A lab technician mixes liquids with the swab to extract the genetic material of any virus that may be on the swab.
The lab technician uses special reagents, called primers and probes, and a high-tech machine to conduct several controlled heating and cooling cycles to convert the virus's RNA into DNA, and then make millions of copies of the DNA. Some tests use only one warming cycle to make copies of the DNA.
When specific probes bind to DNA, a special type of light is produced that can be seen by the machine and the test shows a "positive" result for infection with SARS-CoV-2, the virus that causes COVID-19.
Mechanism of RT-PCR testing:
Fig.3 Mechanism of RT-PCR Testing
RT-PCR starts with laboratory conversion of viral genomic RNA into DNA by RNA-dependent DNA polymerase. In real-time RT-PCR, the amplification of DNA is monitored in real time as the PCR reaction progresses. This is done using a fluorescent dye or a sequence-specific DNA probe labelled with a fluorescent molecule and a quencher molecule, as in the case of TaqMan assays. An automated system then repeats the amplification process for about 40 cycles until the viral cDNA can be detected, usually by a fluorescent or electrical signal.17
3.2.1.2. Isothermal nucleic acid amplification:
RT-PCR requires multiple temperature changes for each cycle, involving sophisticated thermal cycling equipment.18 Isothermal nucleic acid amplification is an alternative strategy that allows amplification at a constant temperature and eliminates the need for a thermal cycler.
(i) Reverse transcription loop-medicated isothermal lamp (RT-LAMP)
RT-LAMP has been developed as a rapid and cost-effective testing alternative for SARS-CoV-2. RT-LAMP requires a set of four primers specific for the target gene/region to enhance the sensitivity and combines LAMP with a reverse transcription step to allow for the detection of RNA. The reaction can be followed in real time either by measuring the turbidity or by fluorescence using intercalating dyes. Since real-time RT-LAMP diagnostic testing requires only heating and visual inspection, its simplicity and sensitivity make it a promising candidate for virus detection.
3.2.2 Antibody Testing:
Fig.4 Overview of Antibody Testing
The body responds to a viral infection by producing antibodies that help neutralize the virus. Blood tests (serology tests) can detect the presence of such antibodies. Antibody tests can be used to assess what fraction of a population has once been infected, which can then be used to calculate the disease's mortality rate. 19 the most notable antibodies are IgM and IgG. IgM antibodies are generally detectable several days after initial infection, although levels over the course of infection and beyond are not well characterized. IgG antibodies generally become detectable 10 –14 days after infection and normally peak around 28 days after infection. Average specificity of antigen tests is 99.5% and average sensitivity is 56.8%.20
3.2.3 Serological testing:
Serological testing is defined as an analysis of blood serum or plasma and has been operation-ally expanded to include testing of saliva, sputum, and other biological fluids for the presence of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies.21
Fig.5 ELISA Technique
A. ELISA:
ELISA is a microwell, plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies, and hormones. The test can be qualitative or quantitative, and the time to results is typically 1–5 h, In the case of SARS-CoV-2 the plate wells are typically coated with a viral protein. If present, antiviral antibodies in the patient samples will bind specifically, and the bound antibody–protein complex can be detected with an additional tracer antibody to produce a colorimetric or fluorescent-based readout.
B. Lateral flow immunoassay:
In practice, fluid samples are applied to a substrate material that allows the sample to flow past a band of immobilized viral antigen. If present, anti-CoV antibodies are collected at the band, where, along with co-collected tracer antibodies, a colour develops to indicate the results. When used in conjunction with symptomology, a diagnosis of infection may be feasible. Samples move via capillary flow on the nitrocellulose membrane. When anti-SARS-CoV-2 antibodies are present, they bind to the labelled antigen and continue to move until they are captured by the immobilized antihuman antibodies. The presence of the captured antibody–antigen complex is visualized as a colored test band.
3.2.4. Rapid antigen testing:
An antigen is the part of a pathogen that elicits an immune response. Antigen tests look for antigen proteins from the viral surface. In the case of a coronavirus, these are usually proteins from the surface spikes.22 SARS-CoV-2 antigens can be detected before onset of COVID-19 symptoms (as soon as SARS-CoV-2 virus particles) with more rapid test results, but with less sensitivity than PCR tests for the virus.23 Samples may be collected via nasopharyngeal swab, a swab of the anterior nares, or from saliva. The sample is then exposed to paper strips containing artificial antibodies designed to bind to coronavirus antigens. Antigens bind to the strips and give a visual readout. The process takes less than 30 minutes, can deliver results at point of care, and does not require expensive equipment or extensive training. [22] According to the WHO the sensitivity of similar antigen tests for respiratory diseases like the flu ranges between 34% and 80%.
Fig.6 Molecular testing vs Paper based testing
3.2.5. Paper based testing:
An accurate and low-cost paper-based coronavirus test will soon be available in India. Developed by a research team of the Council of Scientific and Industrial Research (CSIR) - Institute of Genomics and Integrative Biology (IGIB) and Tata Group, the test has been named ‘Feluda’- a fictional private detective in a series of popular Bengali novels, short stories Written by renowned filmmaker Satyajit Ray. The paper-strip uses cutting-edge CRISPR gene-editing technology to detect SARS-CoV-2 virus in less than an hour.
4. Sample collection method:
4.1 Respiratory Sample Collection Method:
4.1.1 Upper Respiratory Tract Samples:
Patients with viral pneumonia do not typically produce purulent sputum; therefore, the most common collection method used to obtain a specimen for respiratory pathogen testing is the use of NP swabs (NPS). NPS and oropharyngeal swabs (OPS) (also called throat swabs) were among the first specimens suggested for COVD-19 testing. Mid-turbinate swabs (MTS) and anterior-nares (i.e., nasal) swabs (ANS) are now also accepted for testing. Despite its popularity, an NPS sample is challenging to collect from some patients, and suboptimal sampling may result in false-negative results. 24 For this reason, MTS may be considered.
4.1.2 Saliva: Saliva is an offshoot of OPS; however, it can be collected in larger volumes than that collected for an OPS. Saliva was evaluated as another option for non-invasive sample collection. Because saliva collection is non-invasive, it has been adopted as a specimen in some settings; however, caution in result interpretation is warranted, as sensitivity using saliva samples is lower than for NPS and OPS.25
4.2. Lower Respiratory Tract Samples:
4.2.1 Sputum:
Because of the false-negative OPS and in some cases NPS documented early in the pandemic, the diagnostic value of analysing sputum samples to improve accuracy was assessed. Using an RT-PCR assay, the positive rates from sputum specimens and OPS were 76.9% and 44.2%, respectively, with sputum having a significantly higher positive rate than OPS.26
4.2.2 Bronchoalveolar lavage (BAL):
One study assessed the detection of SARS-CoV-2 in a variety of sample types, including BAL, sputum, and bronchoscope brush biopsy samples; blood; faeces; urine; and nasal and pharyngeal swabs. BAL specimens showed the highest positive rates, followed by sputum and nasal swabs.27
4.3. Gastrointestinal and Anal Samples:
4.3.1. Stool:
Detection of viral shedding in the stool was found to be equally accurate as OPS testing in a small case series in which a positive stool test was not correlated with gastrointestinal symptoms.28 Active virus replication was observed for sputum and OPS, but not stool, despite high viral loads.29
4.4 Blood Samples:
In some reports, viral RNA was not detected in blood samples; however, SARS-CoV-2 nucleic acid was detected in sera collected from critically ill COVID-19 patients.30 These patients also exhibited extremely high interleukin 6 levels, which suggested a close correlation with the detection of viral RNA in the serum samples.
5. Type of swabs during collection of samples:
5.1 Oropharyngeal swab (e.g. throat swab):
Tilt patient’s head back 70 degrees. Rub swab over both tonsillar pillars and posterior oropharynx and avoid touching the tongue, teeth, and gums. Use only synthetic fiber swabs with plastic shafts. Do not use calcium alginate swabs or swabs with wooden shafts. Place swabs immediately into sterile tubes containing 2-3 ml of viral transport media.31
5.2 Combined nasal & throat swab:
Tilt patients head back 70 degrees. While gently rotating the swab, insert swab less than one inch into nostril (until resistance is met at turbinates). Rotate the swab several times against nasal wall and repeat in other nostril using the same swab. Place tip of the swab into sterile viral transport media tube and cut off the applicator stick. For throat swab, take a second dry polyester swab, insert into mouth, and swab the posterior pharynx and tonsillar areas (avoid the tongue). Place tip of swab into the same tube and cut off the applicator tip.31
5.3 Nasopharyngeal swab:
Tilt patient’s head back 70 degrees. Insert flexible swab through the nares parallel to the palate (not upwards) until resistance is encountered or the distance is equivalent to that from the ear to the nostril of the patient. Gently, rub and roll the swab. Leave the swab in place for several seconds to absorb secretions before removing.31
6. CONCLUSIONS:
At present, NAAT based methodologies remain the cornerstone of in-vitro diagnostic assays for SARS-CoV-2. Appropriate measures are required to keep laboratory staff safe while producing reliable test results. In the analytic stage, real-time reverse transcription-PCR (RT-PCR) assays remain the molecular test of choice for the etiologic diagnosis of SARS-CoV-2 infection while antibody-based techniques are being introduced as supplemental tools.
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Received on 03.03.2021 Modified on 16.03.2021
Accepted on 23.03.2021 ©A&V Publications All right reserved
Res. J. Pharma. Dosage Forms and Tech.2021; 13(3):259-265.
DOI: 10.52711/0975-4377.2021.00043