Since the outbreak of COVID-19 in Wuhan, China last December, the disease has now spread to more than 70 countries worldwide. While much effort has been put into containing the disease by speeding up the testing for the severe acute respiratory syndrome-coronavirus-2 ( SARS-CoV-2), the news last week about the first test kits distributed by the United States CDC being found unable to produce consistent conclusive results from the negative control, is a bit bizarre .1
COVID-19 real time RT-PCR test kit by CDC of the United States.
This may arise your interest in knowing what test methods have been used by your own country for the newly emerged coronavirus. How do the tests work? How accurate are the test results from the test kits, and how the test results usually been validated and verified?
RT-PCR test for SARS-CoV-2
Until now, real time RT-PCR (reverse transcription polymerase chain reaction) technology has been the only method to detect the virus globally. PCR is an amplification method by which a targeted nucleotide sequence of an organism can be multiplied exponentially. By first converting the RNA sequence of the virus genome into complementary DNA and subsequently amplifying the target sequence using the complementary DNA as template, even a tiny amount of the virus genome in collection sample can be detected.
Overview of Reverse Transcription-Polymerase Chain Reaction
(From Wikimedia Commons photo: licensed under the Creative Commons Attribution-Share Alike 4.0 International license.)
In early January, a genetic sequence of the newly emerged coronavirus was first released by China.2 The analysis of the genome structure revealed that SARS-CoV-2 has 79% identity with the SARS-CoV.3 The whole viral genome contains genes encoding non-structural polyprotein, S (Spike) protein, E (Envelope) protein, M (Membrane) and N (Nucleocapsid) protein (S, E and M proteins together form the viral envelope). Based on the viral genome data, researchers from different countries developed test kits by designing primers, short stretches of DNA, to amplify mainly S, N, and E gene regions of new virus.4 The nucleotide regions on the virus which encode the 3 proteins are less prone to mutation and are therefore usually picked for virus detection in RT-PCR test.
3D medical animation still shot showing 2019 novel Coronavirus Structure. From Scientific Animation: www.scientificanimations.com. Click for full-size image.
How reliable are tests using RT-PCR?
RT-PCR tests have been widely used in diagnosis of other viruses such as mumps, HIV, and influenza, and are normally highly reliable. In order to validate the results of each PCR experiment, negative control and positive control are included in every experiment for sample testing. Simply speaking, the positive signal in positive template control is an indication to show that the experiment works. The clear negative signal in the negative control is an indication of no contamination for the experiment. This is used to validate the positive test result of a sample in the same experiment.
For the new coronavirus, every country uses different criteria to make final diagnosis. These include the clinical observations and epidemiological data. If the RT-PCR result did not match with the clinical observation, in most cases RT-PCR will be repeated. Therefore, the chance of misdiagnosis is lower even the RT-PCR test result is wrong.
What are the possible causes of false-negative from RT-PCR test?
Although controls are used in RT-PCR test experiment to validate the positive results and to prove the experiment is working, false-negative from RT-PCR test is unavoidable. According to experience from China, the false-negative accounts for 3% of RT-PCR tests for COVID-19 patients.5
The false-negative from RT-PCR test may come from technical handling errors such as inappropriate specimen collection, storage, and transport. Viral RNA is very much prone to degradation with higher temperature. Therefore, once the sample is collected, it should be placed in a designated collection tube and be kept and transported at 4°C to 8°C. If the test is not going to be done in 24 hours upon sample collection, it should be kept frozen. In addition, the way the tests are being conducted may also cause problems. A dangle or a good rub could mean a big difference in the amount of virus material being collected.
Moreover, insufficient viral material in the specimen collected can also lead to false-negative results. A patient in the early state of infection will shed much less virus; tests taken at this stage have a higher chance of showing negative results. In addition, if the virus is drawn toward the lower respiratory tract for example, then a test from throat or nose swab may miss the virus.6,7 Samples collected from tracheal aspirate or sputum are alternatives for a highly suspicious patient with negative test result.
However, there is the possibility that the tests are accurate and the patients do not have coronavirus at the time of testing, according to Dr MacDermott of King’s College London. The early signs of coronavirus are very similar to other respiratory viruses. The patients may not be actually infected with the new coronavirus, therefore the test result is negative. But they can became infected and later test positive for the coronavirus.7
What we can learn so far from the countries worldwide in their handling of the outbreak of COVID-19?
Since the outbreak in South Korea in late February, the country has now managed to test more than 10,000 people a day for the newly emerged coronavirus, using kits provided by 4 local biotech companies, with sensitivity rates of over 95%. This powerful, fast testing ability is mostly attributed to its painful experience in handling the outbreak of Middle East Respiratory Syndrome (MERS) in 2015. Since then, the country has set up a system to allow rapid approval of testing kits for viruses that may cause pandemics.8 While the shortage of test kits in Japan and the US has jeopardized the containing of the virus in those countries, the effective collaboration system between the regulator and the local biotech companies in South Korea has provide a good example for countries worldwide in handling the outbreak of new disease.
The number of patients with COVID-19 is now surging in European countries over the last two weeks. Let us hope that these countries do not have bureaucratic processes that prevent them from providing a high capacity of tests that can quickly identify and treat COVID-19 patients.
References
1. https://web.archive.org/web/20200306034835/https://www.sciencemag.org/news/2020/02/united-states-badly-bungled-coronavirus-testing-things-may-soon-improve
2. https://web.archive.org/web/20200307043124/https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/
3. Roujian Lu, Xiang Zhao, Juan Li et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 2020; 395: 565–74.
4. https://web.archive.org/web/20200303000654/https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/laboratory-guidance
5. XingzhiXie, Zheng Zhong, and Wei Zhao et al. Chest CT for Typical 2019-nCoV Pneumonia: Relationship to Negative RT-PCR Testing. Radiology, Published Online:Feb 12 2020. https://doi.org/10.1148/radiol.2020200343
6. “What actually happens during a coronavirus test?” Arman Azad, CNN, 5 March 2020. https://web.archive.org/web/20200306034011/https://edition.cnn.com/2020/03/04/health/coronavirus-test-what-happens-explainer/index.html
7. “Are Coronavirus tests flawed?” James Gallagher. BBC news, 13 February 2020.
8. “Virus Testing Blitz Appears to Keep Korea Death Rate Low”. Heejin Kim, Sohee Kim, and Claire Che. Bloomberg, 4 March 2020.
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