Since the detection of the first patient infected by the novel coronavirus on November 17, 2019 (Covid-19), it has been causing an unprecedented havoc in the entire world. As of April 26, 2020, the Covid-19 pandemic has affected almost every corner of the world. The total number of affected and deaths are 2,940,993 and 203,822 respectively and they are rapidly rising every day.
There is no vaccine or specific antiviral treatment options available for Covid-19 so far. Therefore, social distancing and contact minimizing of the infected patients are the only viable tools available to curve the spread of this ominous virus.
In order to screen people for Covid-19 infection, we must rely on the most reliable detection method. It must be deployed promptly and made readily available for mass screening. Without this simple task, there is no chance that the spread of this virus could be stopped. The success story of South Korea in curving the spread of this virus very early and minimizing the death toll points to this simple fact.
Precision Diagnostics of Covid-19
Currently two methods play central role in detecting the infection of this virus and understanding the spread among population in community.
One is Reverse Transcription - Polymerase Chain Reaction (RT-PCR), and the other is Antibody or Serological Test. These two methods are fundamentally different - in the ways test samples are collected and used (nasal swab vs blood drop) and the target of analysis (genetic material - RNA vs free floating antibodies in blood).
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Real time reverse transcription–polymerase chain reaction (real time RT-PCR) is one of the most accurate laboratory methods for detecting, tracking, and studying the coronavirus. It detects the presence of the virus by identifying its RNA, which is the genetic material of this virus.
This virus can be considered as nano-meter size particle with genetic material - RNA packed inside the protein envelope. It can’t replicate without using the replication machineries of other living cells. Therefore, in order to survive and sustain, it must constantly infect others. It has evolved a very sneaky way of entering other cells. Once inside the cell, the virus uses its own genetic code (RNA) to take control of and ‘re-programme’ the cell so that it becomes a virus-making factory and very quickly it can produce millions of its own copies.
In RT-PCR method, the RNA is first converted to DNA. This is a process called ‘reverse transcription’. The resultant DNA is copied/replicated millions of times and measured in real-time which is the key part of the real time RT-PCR process for detecting the virus.
A sample is collected from parts of the body where the coronavirus gathers, such as a person’s nose. The sample is then treated with chemical solution to be able to extract the RNA molecules present in the sample. The RNA is selectively reverse transcribed to its compliment DNA using a specific enzyme, and then it is added with short fragments of DNA designed specific for the virus RNA sequence.
The latter allow amplification of the DNA segments specific to the virus by a process called Polymerase Chain Reaction (PCR). The process of DNA replication can be monitored in real-time, that’s why this method is called real-time RT-PCR.
The method is highly sensitive and specific and can deliver a reliable diagnosis. Although traditional RT-PCR can take 2-3 hours, a number of new technologies have recently emerged which can do much faster, as fast as 15 minutes and at high through puts (single machine can do thousands of samples in a day).
There are also advanced PCR based machines available that can do most of the work without manual intervention eliminating human errors and health risk of the technicians. This method is very sensitive because the virus RNA molecule is amplified by millions of times. It continues to be the most accurate method available for the detection of the Covid-19. This is the reason why most of the coronavirus detection reports are exclusively based on this method.
To detect past infections and determine immunity, which are important for understanding the development and spread of the virus, real time RT-PCR cannot be used as viruses are only present in the body for a specific window of time. Other methods, such as antibody or antigen tests are necessary to detect, track and study past infections, particularly those that may have developed and spread without symptoms.
Antibody or Serological Test
An antibody test relies on detecting antibodies produced in body in response to infection. It can detect if a person has had coronavirus infection before.
The basis of commercially available test kits – rapid diagnostic test (RDT), and enzyme-linked immunosorbent assay (ELISA), etc are based on antibody test.
The antibody test is also known as a "serological test". The test is carried out by taking a drop of blood and analysing the presence of antibodies produced specific to the virus infection as shown in Figure 1.
It can be done with a modest lab setting and results are usually obtained in about 15 minutes. This method does not check for virus itself, rather it checks body’s response to the virus. Therefore, in order to be effective, the patient must have gained some immunity, which can take 7-15 days depending on body’s immune response. Sometimes, it may not detect at all when infection was not so severe. This test can also check if a person has had past infection but recovered.
Figure 1. Function of antibody test kit: (a) collection of blood sample, (b) addition of blood sample to sample well, (c) Placement of 2-3 drops of buffer in sample well and (d) results reading after 15-30 minutes. (Source: https://www.telegraph.co.uk/news/2020/04/25/)
This kind of test usually looks for two types of antibodies -IgM antibodies, which develop early in an infection and IgG antibodies, which are more likely to show up later, usually after a patient recovered.
In order for full use of this detection method, the scientists must accurately know when each of these classes of antibodies are sufficiently generated and how long they live in body. If the test is carried out before sufficient quantity of antibodies are produced in body, this method can give false negative. And past infection of the same virus and related ones can produce false positive. Because of these, this method is used only for qualitative surveillance measures.
A major use of this method is to find out roughly what fraction of the population has been already infected. This method is not a reliable one to find out who is infected, because the antibodies are produced in body after a week or two, when the virus should be mostly cleared, if not all, from the system. Therefore, this method is not the primary method of choice for detecting virus among Covid-19 suspected patients.
Other disadvantage of this method is that it can’t be quickly established, compared with PCR based method. That’s why for rapid response to any emerging diseases, the PCR method is usually the first one deployed on the frontline.
Ultimately, it boils down to these simple facts - the PCR based tests are highly accurate and reliable and much more so than antibody based tests. It can determine active infection of virus much early on. On the other hand, the antibody based test are error prone and can determine only past infection and body’s response to it. This is true for all antibody based tests, including the method proposed by Gonoshasthya Kendro. These two methods cannot be replaced by one another, rather they have different areas of applications at different situations.
Lessons for Bangladesh
The world is facing an unprecedent crisis because of this invisible enemy – coronavirus. The situation in Bangladesh is grave. With a huge population and large population density (over 160 million people and 1,265.19 people per km2 population density), the country is at a great risk of massive community infection.
As of April 26, 2020, over 5,500 people have been infected with total death of 145. The number of tests capability is only 283 per 1 million population which is one of the lowest in the world. Therefore, the gof Bangladesh is desperate to increase the capability of testing. In this time of crisis, decision making based on valid, proven science is of paramount importance.
Recently, Gonoshasthaya Kendra has taken initiative for producing testing kits locally, called Rapid Dot Blot. According to its claim, Covid-19 can be diagnosed within five minutes by using this kit which is based on antibody test. Its work has not been published in peer review journal or validated by third parties and the government regulatory bodies. Like any other antibody based tests, this method is expected to experience common drawbacks – low sensitivity and selectivity, and greater rates of false negative and false positive.
In this regard two aspects are important
One, selecting the method of testing is the most important one. Millions of human lives could be at risk if we decide to use pre-mature and unvalidated technologies. It is very important that we stick to internationally recognized method, in this case real-time RT-PCR method as a primary method of choice for diagnosis of Covid-19.
At the same time, we must also recognize the fact that antibody based tests broadly, may play an important role in the near future such as in checking percentage of infection or immunity among the large population, as well as selecting blood donor for treating using convalescent plasma from already recovered patients. However, any new technology including the one that Gonoshasthaya Kendra wants to deploy, must undergo rigorous validation and quality control process as per international standard, checked by independent third parties and the government regulatory bodies. Without this, it will be very risky to jump to a new technology – be it a foreign or domestic one.
And secondly, the commercial kits used in RT-PCR can be relatively expensive. But such kits can be manufactured at the cost fraction using commercial reagents already validated by regulatory bodies, such as CDC of USA and ECDC of EU, etc. Asian countries such as South Korea, China and Japan were able to deploy such technologies rapidly at the crisis period due to their preparedness and ability to manufacture such kits on their own. We should follow their paths.
This is an important lesson for the country, one way or another. Medical sccience research has not been a top national priority for our country, which is why we have found ourselves completely unprepared. We must wake up now, because due to climate change and other factors, we will, unfortunately but almost certainly, face crisis like this more often in the future.
Dr Mazharul Islam Rana is working at the Department of Chemistry of Bath University, UK. He is one of the coordinators of Forum Vision 2041, a forum of Bangladesh-born academics/scientists working at home and abroad. This forum is publishing three books on three main pillars of sustainable development of Bangladesh, on the occasion of the ‘Mujib-Borsho’. He is also the Founder member of the newly established University of Skill Enrichment and Technology (USET), Narayangonj, Dhaka.
Dr Mong Sano Marma is a Principal Scientist at Miltenyi Biotec, Boston, USA. He was one of the lead scientists of the team which successfully sequenced the genome sequence of Hilsha fish. He is an experienced scientist in the molecular diagnostic products research and development, and inventor of a number of US and International patents relevant to new generation DNA sequencing technologies.