It’s been almost 20 months since COVID-19 first surfaced in China in November 2019. From then until now, our arsenal to bring this pandemic under control comprises mainly of safe distancing, testing (for infectious cases), contact tracing, treatment (mostly symptomatic treatment) and now vaccination. Testing is key as a false negative means that infectious people are going around spreading the disease unknowingly.
I became curious when I’ve read reports of people being tested positive months after they were initially tested negative and the reports suggested that their infection may have already occurred during the initial tests! Effectively, these infectious cases had been active in the community thinking that they were well but passing the disease to others. If the tests are not 100% accurate, is there a point in testing? After all, it takes only one person to set off a super-spreader event, whether or not that person shows any symptoms. With all the new variants circulating today, should we be more concerned about testing?
The below is a summary of my research into the COVID-19 tests. While the sensitivity and specificity of the tests are not low, in absolute numbers, a 1% drop in accuracy may easily translate to 1.56 million undetected cases (based on the total number of cases in the world as at May 7, 2021: 156,707,444 (https://www.worldometers.info/coronavirus/)). The key takeaway is still to practice the safe distancing measures diligently as there is no conclusive way to isolate all infectious cases and as the number of variants grow (particularly virulent variants), it may be wiser to maintain a physical distance for now.
There are two main category of tests: diagnostic tests and antibody test.
Diagnostic tests check for active COVID-19 infection. Molecular and antigen tests are the main types of diagnostic tests. Molecular tests (also called PCR tests, viral RNA tests, nucleic acid tests) look for genetic material that could come only from the virus while antigen tests identify protein fragments (antigens) from the virus.
Antibody tests look for antibodies produced by the immune system in response to SARS-CoV-2, and as antibodies take several days or weeks to develop after an infection and may stay in the blood even after recovery, such tests is often used to test for past infection.
Definitions
To understand the differences between the tests, there are some key terms to take note:
Polymerase chain reaction: molecular biology technique to amplify and detect DNA and RNA sequences.
Amplification: A process where a particular RNA/DNA sequence is repeatedly amplified until there is sufficient amount for detection.
Cycle threshold (Ct): The number of amplification cycles necessary to spot the virus. Beyond certain Ct values (37 to 40 cycles), the test result is deemed negative.
False positive: Positive COVID-19 detection even when the test subject does not have COVID-19.
False negative: Negative COVID-19 detection even when the test subject does have COVID-19.
True positive: Positive COVID-19 detection and test subject does have COVID-19.
True negative: Negative COVID-19 detection and test subject does not have COVID-19.
Specificity: measures how well a test can identify true negatives.
Sensitivity: measures how well a test can identify true positives.
Diagnostic: Molecular Tests
Molecular tests look for genetic material (such as RNA) that comes ONLY from the virus. Samples are collected from the subject using nasal swabs, throat swabs, but saliva or other bodily fluids can also be tested.
How it works?
Molecular tests amplify bits of the viral RNA so that the viral infection can then be detected with a specialized test, hence these tests are also known as as nucleic acid amplification tests (NAAT).
1. As the virus mainly infected the respiratory system, a sample from a potentially infected person’s nose, throat or mouth (saliva) is taken.
2. In the case of SAR-COV-2, its viral RNA is fragile and its extraction and usage must be done carefully to preserve the genetic material and prevent it from contamination and destruction. So a reverse transcription to convert the RNA to DNA using proteins known as reverse transcriptase enzymes is developed as the DNA is a more stable molecule.
A small piece of single-stranded DNA called “primer” that precisely matches a specific section of the viral genome is then designed. These primers then attach to the specific areas of the viral genome and provide the basis for amplification of that region. As the viral genome is too small to visualize and detect in such small quantities, signal amplification is needed.
3. The region in which the primers are attached is then amplified in repeated cycles. These cycles are designed to closely mimic the natural DNA replication processes. This process repeats until enough genetic material have been synthesized to be read using molecular diagnostic assays.
4. A positive test result is when the viral RNA is detected.
5. A negative test result is when no viral RNA has been copied or detected after many rounds of amplification (cycle threshold).
Most molecular tests for SARS-CoV-2 use the process of real-time reverse transcriptase quantitative polymerase chain reaction (rRT-PCR), and there are rapid molecular tests today that are highly sensitive and can provide results in minutes. Molecular tests are thought to have high sensitivity and specificity.
Diagnostic: Antigen
Antigen tests look for proteins (antigens) on the surface of the virus to ascertain the presence of the pathogen. Like molecular tests, the sample is usually taken from a nasal or throat swab. The sample is mixed with a solution that unleashes specific viral proteins and the combination is then applied to a paper strip that contains a bespoke antibody optimized to bind the specific viral proteins if they are present and the result is reflected as a band on the paper strip.
Unlike most molecular tests, antigen tests can be done quickly and hence, they are also known as “rapid tests” until the emergence of rapid molecular tests. Another advantage of the antigen test is the cost and ease of deployment, and is particularly useful when mass testing is required.
Antibodies Test
The test for antibodies is also known as a serological test. A sample of blood is taken and a blood test is carried out to identify antibodies that the body’s immune system has produced in response to the infection. The test cannot determine if you have an infection now but it can accurately identify past infection as it takes a week or two after infection for the immune system to produce antibodies.
Studies suggested antibody levels may wane over just a few months. While a positive antibody test proves exposure to the virus, such results are not a definite indication that an infectious person is no longer contagious or the presence of long-lasting, protective immunity. There have been reports of re-infection of COVID-19 and with more variants sprouting up these days, recovery from past infections may not offer any protection at all.
Sensitivity and Specificity
An ideal COVID-19 test should have high sensitivity and specificity with quick turnaround for containment to be carried out effectively. The true accuracy of tests for COVID-19 is however uncertain.
False negatives means that a infectious person is not treated or quarantined and may lead to more infections. False positives may lead to unnecessary treatment and given that, many who tested positive for COVID-19 are clustered in the same facility, the false positive may eventually test really positive because of prolonged exposure in the facility.
A Cochrane study on antigen tests showed that it correctly identified COVID-19 infection in an average of 72% of people with symptoms, compared to 58% of people without symptoms. Tests were most accurate when used in the first week after symptoms first developed and this is likely due to a higher viral load in their system. In people who did not have COVID-19, antigen tests correctly ruled out infection in 99.5% of people with symptoms and 98.9% of people without symptoms.
Molecular tests are generally reported to have higher specificity and sensitivity compared to antigen tests.
What affects the accuracy?
– How carefully a specimen is collected and stored.
– Viral load in the body. The rate of false negatives was found to vary with how long the infection has been present. In one study, the false-negative rate was 20% when testing was performed five days after symptoms began, but much higher (up to 100%) earlier in infection. However, if the test was done too early, there may not be sufficient viral load for a positive test result. There have been many cases of people who initially tested negative who tested positive when the test was repeated several days later.
– Test parameters such as the primer used and the definition of the cycle threshold.
– Human error in computation.
– Lab contamination or other problems with the lab procedures.
References
[1] How accurate are rapid tests for diagnosing COVID-19?
https://www.cochrane.org/CD013705/INFECTN_how-accurate-are-rapid-tests-diagnosing-covid-19
Retrieved May 7, 2021.
[2] Antigen and Molecular Tests
https://www.centerforhealthsecurity.org/covid-19TestingToolkit/testing-basics/types-of-COVID-19-tests/antigen-and-molecular-tests.html
Retrieved May 7, 2021.
[3] Explained: Why are several people with Covid-19 symptoms testing negative?
https://indianexpress.com/article/explained/covid-tests-rt-pcr-coronavirus-negative-result-7297329/
Retrieved May 7, 2021.
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