COVID-19 DIAGNOSIS

RT-PCR and rapid diagnostic kits or only RT-PCR?

by Mohammad Al Mamun | Published: 00:00, May 19,2020

 
 

ON JANUARY 30, the World Health Organisation designated an outbreak of a novel coronavirus not seen before in humans to be a ‘public health emergency of international concern’; this was followed by the declaration of a pandemic on March 11. Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for causing Coronavirus Disease 2019 (COVID-19). Infected hosts exhibit different clinical features, ranging from asymptomatic (producing or showing no symptoms) to severe symptoms in their respiratory organs. WHO refers to the virus as COVID-19 virus in its current documentation (WHO, 2020) to provide interim guidance to laboratories and stakeholders involved in COVID-19 virus laboratory testing of patients.

Bangladesh is now one of the many countries to have been hit by the COVID-19 wave that has swept across the world. At this point, it is extremely important to follow ‘stay at home’ orders without a second thought. From the epidemiological point of view, any deviation from this will likely to result in the formation of COVID-19 hotspots, which means clusters of infection in places around the country. Diagnosis of COVID-19 active cases (symptomatic) and their contacts, and isolation of those individuals to limit further spread, are key measures in this scenario. Over the course of the current COVID-19 crisis, the importance of reliable, rapid and accessible testing to screen for the disease has become increasingly important.

The SARS-CoV-2 virus is contagious and can spread from an infected person to a non-infected person during the incubation period.COVID-19 is a challenge to global public health. Therefore, rapid and an accurate identification of pathogenic viruses play a vital role in selecting appropriate treatments, saving people’s lives and preventing epidemics. It is important to establish a quick standard diagnostic test for the detection of the infection to prevent subsequent secondary spread. The two major methods for diagnosing the viral infection are the polymerase chain reaction, known as PCR, and immunoassays (antibody-antigen based assays). Until now, real-time reverse transcription polymerase chain reaction (RT-PCR) from a nasopharyngeal swab as specimen is the most popular testing method for the detection of SARS-CoV-2.Chest CT imaging may also be helpful for diagnosis in individuals where there is a high suspicion of infection based on symptoms and risk factors; however, experts do not recommend using it for routine screening.

The PCR is a routine laboratory technique used to amplify small samples of DNA into larger quantities that can be detected and analysed. For diagnosis, a viral RNA or DNA sample is taken by swab or blood draw, before being sent to a specialist laboratory for analysis. The RT-PCR technique, real-time reverse transcriptase-PCR, detection is currently favoured because of its advantages as a specific, sensitive and traditional quantitative assay, especially when we don’t have any alternative authentic means for a new case such as COVID 19. For RNA viruses, such as SARS-CoV-2, the polymerase chain reaction is preceded by an additional step to produce a complementary DNA template from RNA, by the addition of a reverse transcriptase enzyme, hence, reverse transcription-PCR.

However, inaccurate sample collection and preparation, laboratory conditions and technical errors can lead to false-negative results in RT-PCR. More importantly, detecting SARS-CoV-2 from pharyngeal swabs requires high-quality specimens that contain a sufficient amount of intact viral RNA. Yet, SARS-CoV-2 loads in the respiratory tract have shown to fluctuate considerably. This has not only led to high false-negative rates, with probable cases remaining negative after multiple swabs, but is further exposing healthcare workers to risk of infection. Moreover, testing for viral RNAs using this method, is time consuming, labour intensive and expensive, must be carried out only by experienced technicians in qualified laboratories. For resource-limited countries like ours, diagnostic infrastructure and training are even harder to come by, with many healthcare systems having only a handful of labs and technicians spread across vast geographical areas.

On the other hand, in this COVID-19 pandemic, beside RT-PCR, the most used diagnostic device for the detection of SARS-CoV 2 is antibody-antigen based colorimetric in-vitro diagnostic kit. Antibodies are Y-shaped proteins (also known as immunoglobulins of two types — IgM and IgG) produced by the immune system in response to an infection and are specific to that particular infection. Enzyme-linked immunoassay is able to detect viruses with high sensitivity and specificity through antigen and antibody. There are mainly two types of rapid colorimetric immunoassays associating enormous number of designs (eg direct, indirect, sandwich and competitive immunoassays) regarding the antigen-antibody reactions and enzyme linking strategies.

One type of this rapid diagnostic test detects the presence of viral proteins, antigens that is, expressed by the COVID-19 virus in a sample from the respiratory tract of a person. If the target antigen is present in sufficient concentrations in the sample, it will bind to specific antibodies fixed to a paper strip enclosed in a plastic casing and generate a visually detectable signal, typically within 30 minutes. The antigens detected are expressed only when the virus is actively replicating; therefore, such tests are best used to identify acute or early infection. There is another, more common type of rapid diagnostic test marketed for COVID-19; a test that detects the presence of antibodies in the blood of people believed to have been infected with COVID-19. This test detects either IgM antibodies, usually the first antibody produced by the immune system when the virus attacks or IgG antibodies that develop in most patients within 7–10 days after symptoms of COVID-19 begin. This means that a diagnosis of COVID-19 infection based on IgG antibody response will often only be possible in the recovery phase, when many of the opportunities for clinical intervention or interruption of disease transmission have already passed. In order to minimise this limitation, the colorimetric immunosensors based on an IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis has been introduced. However, antibody detection tests targeting COVID-19 may also cross-react with other pathogens; e.g. SARS and MERS, including other human coronaviruses and give false-positive results. ELISA based immunoassays can sometimes be quite expensive due to the cost of reagents (e.g. enzyme) being used.

WHO fixed a criteria for the scientists and researchers to set their research goal emphasising the benchmarks that is called ASSURED i.e., affordable, sensitive, specific, user-friendly, rapid, robust, equipment free, and deliverable systems during the development of any detection methods or any diagnostic devices. All those performance parameters possessing into the same device or method are very challenging. Scientists and researchers are continuously making effort to achieve those criteria in the same diagnostic device. Now, I would like to scientifically analyse the two main methods are currently draw attention in Bangladesh for the diagnosis of COVID-19 focusing on the WHO criteria (as demonstrated in the Table-1).

Why we need rapid diagnostic tools for the detection of COVID-19: According to WHO, as co-infections can occur, all patients that meet the suspect case definition should be tested for COVID-19 virus regardless of whether another respiratory pathogen (such as SARS-CoV or MERS-CoV) is found. On March 16, 2020 at a media briefing on COVID-19, WHO director-general also emphasised more number of tests to isolate the infected persons – which is the backbone of the response beside the social distancing measures. The most effective way to prevent infections and save lives is breaking the chains of transmission. And to do that, we must test and isolate. WHO also has provided a simple message for all countries: test, test and test. Test every suspected case. If they test positive, isolate them and find out who they have been in close contact with up to two days before they developed symptoms, and test those people too. In response to the growing COVID-19 infection and shortages of laboratory-based molecular testing like RT-PCR capacity and reagents, multiple diagnostic test manufacturers have developed and begun selling rapid and easy-to-use devices to facilitate testing outside of laboratory settings. These simple test kits are based either on detection of proteins from the COVID-19 virus in respiratory samples (eg sputum, throat swab) or detection, in blood or serum, of human antibodies generated in response to infection. However, before these tests can be recommended, they must be validated in the appropriate populations and settings. The Food and Drug Administration, USA has already authorised many companies to produce in-vitro diagnostic kits in this emergency public health crisis in order to conduct large scale diagnosis throughout the country.

In this circumstance, when the ongoingSARS-CoV-2 infection curve is climbing up continuously in Bangladesh, a rapid and affordable point-of-care diagnostic kit like antibody-antigen test kits after cross-validation with RT-PCR would be the best solution to face this extreme situation. However, the biggest advantage of antibody-assay is their ability to detect past infections. Once a patient has recovered from COVID-19 and virus is cleared from the body, viral RNA is no longer available for detection in the respiratory tract, leaving only a short window during the acute stage of infection in which SARS-CoV-2 can be detected.

By conducting random antibody sampling of the general public (known as a serosurvey), public health bodies could better estimate the true levels of exposure and resulting population immunity. Furthermore, by identifying potential geographical ‘hotspots’ of low population immunity, health systems could better allocate resources to prevent or manage transmission. Therefore, immunoassays incorporating specific antibody are a promising alternative for the detection of antigens. The advantages of this device are easy, inexpensive, equipment free, sensitive, rapid, and on-spot diagnosis without the need of highly skilled lab expert mandatory for RT-PCR. 

To conclude, we must need a comprehensive approach in this pandemic to survive. The antigen-antibody test kits after justification or cross-validation with RT-PCR might be the most suitable detection methods offering sufficient sensitivity, comprehensiveness, rapid detection and low cost for both the active and suspected SARS-CoV-2 viral infections in Bangladesh beside the RT-PCR based diagnosis.

 

Mohammad Al Mamun is a PhD candidate at the Biomedical Research Laboratory, Nanotechnology and Catalysis Research Centre, Institute of Advanced Studies, University of Malaya, Malaysia and an assistant professor, department of chemistry, Jagannath University.

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