COVID-19 Diagnostics Explained

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By : Rebecca Tan
Date : April 8, 2020

Access to testing is essential for containing COVID-19, but the accuracy of the tests used also matters a great deal.

Illustration: Oikeat Lam/Asian Scientist Magazine

AsianScientist (Apr. 8, 2020) – Say you have a runny nose, slight cough and maybe even a mild fever. With symptoms as generic as those, the only way to be sure that you have COVID-19 instead of a common cold is to get tested. On a national level, a country’s ability to test as many people as possible is key to containing the outbreak and finding out the true fatality rate of the disease.

Since the full genetic sequence of the SARS-CoV-2 virus was uploaded to the Global Initiative on Sharing All Influenza Data (GISAID) platform on January 10, companies and research groups across the globe have been working round the clock to develop diagnostic kits. While it took nearly six months to develop reliable diagnostics for severe acute respiratory syndrome (SARS) in 2003, COVID-19 tests kits were available in a matter of weeks—a testament to the technological advances over the last 17 years and the concerted efforts of the global scientific community.

But not all tests are alike, and each kind of diagnostic has its own strengths and weaknesses. Read on to find out about the different kinds of tests available and how each kind of diagnostic plays a role in the fight against COVID-19.

The gold standard: RT-PCR

Most of the tests being rolled out against COVID-19 are based on reverse transcriptase polymerase chain reaction (RT-PCR), and with good reason. An ideal diagnostic is both specific and sensitive, which means that people who test positive truly have the disease and none of the people carrying the virus slip through the test as a false negative. RT-PCR meets both criteria, with specificity and sensitivity rates of 90 percent and above.

The RT-PCR test starts with a throat or nasal swab, which is designed to capture virus genetic material. SARS-CoV-2 is an RNA virus, so its genetic material is more transient and fragile than DNA. Because of that, samples should ideally be transported to testing laboratories on ice or in special media to prevent them from degrading.

Once at the lab, the RNA must first be converted to DNA using an enzyme called reverse transcriptase. Then, specific sequences of DNA (primers) designed to recognize complementary virus sequences are added, so that another enzyme—usually a modified form of Taq polymerase—can make a copy of a short length of viral DNA. This process is repeated for 20-30 cycles, exponentially amplifying the amount of viral DNA so that it can be detected.

Having the entire virus genome was crucial for designing primers that would detect only SARS-CoV-2 and not SARS-CoV or any other closely related coronaviruses. Different tests target different parts of the SARS-CoV-2 genome, and the World Health Organization (WHO) has issued their own protocol specifying recommended primers and procedures.

While South Korea and Germany were able to develop and roll out their own COVID-19 RT-PCR tests based on the WHO protocol, the US Centers for Disease Control and Prevention (CDC) opted to issue their own version, which was later found to be problematic. Experts suggest that this initial lack of reliable diagnostics put the US’ COVID-19 response on the back foot.

Rapid tests kits: fast but not as accurate

The trade-off for RT-PCR’s accuracy is speed; even the fastest tests take at least a few hours, not counting the time taken for transporting samples back to the lab. For people anxiously waiting for their test results, the day or two it takes to turnaround an RT-PCR test can seem like eternity.

In contrast, rapid test kits can give results in under an hour, making point of care diagnostics possible. This means that people who suspect that they have been infected can get tested at their local general practitioner and receive their results at the same visit. Alternatively, rapid tests kits could even be used at home.

But before you rush out to get your hands on a rapid test kit, beware: rapid test kits are not as accurate at RT-PCR. In fact, although Shenzhen Bioeasy claimed that their rapid test kits (which received European Union CE certification in March 2020), had an accuracy rate of 80 percent, Spain ended up returning a shipment of the kits after finding that they had an accuracy of less than 30 percent.

Instead of detecting viral genetic material, rapid tests kits target the immune response of the person being infected, looking out specifically for antibodies against the virus or virus antigens. The trouble is that antibodies only develop several weeks after an infection, which means that antibody-based tests might miss asymptomatic cases or people in the earliest stage of the disease.

Although rapid tests like Shenzhen Bioeasy’s might report high sensitivity and specificity results, these tests are typically validated with samples from patients with severe disease; precisely the population that tends to develop a strong antibody response. People with mild symptoms tend to produce less virus-specific antibodies in their blood, so a test that is 80 percent accurate in severely ill patients might be less accurate at detecting patients with only mild symptoms.

That is not to say that antibody-based tests are not useful. On the contrary, antibody-based tests have proven to be crucial in linking clusters of infection, by detecting people who were infected but discovered too late to test positive via RT-PCR. As RT-PCR looks for virus RNA, it will only give a positive test result if there is an ongoing infection. On the other hand, antibodies can persist for months or years, allowing tests to identify anyone who has ever been infected.

Other approaches: CRISPR and beyond

In the race to develop much needed diagnostics, researchers are trying new approaches. Two companies—Mammoth Bioscience and Sherlock Biosciences—have started using the gene-editing tool CRISPR to detect SARS-CoV-2. Sherlock Biosciences, which was co-founded by CRISPR discoverer Zhang Feng, uses a variant of Cas9 called Cas13 that can cut reporter RNA sequences when activated by SARS-CoV-2-specific guide RNA. Similarly, Mammoth Biosciences also relies on the cleavage of reporter RNA sequence, but uses yet another Cas9 variant known as Cas12a.

Over in Singapore, researchers at the Agency for Science, Technology and Research (A*STAR) have developed a kit that combines the best of both worlds: the accuracy of RT-PCR and the speed of rapid diagnostics. Named Cepat, which means fast in Malay, the test uses an enzyme that can amplify viral genetic material at a single temperature rather than Taq polymerase which requires multiple rounds of heating and cooling. The test, which can be conducted on a portable device, can be completed in five to ten minutes, making it one of the fastest COVID-19 tests in the world if approved.

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