The Importance of Diagnosis in the COVID-19 Pandemic Initiative

Large-scale testing and contact tracing are central to efforts to control pandemics.It relies on diagnostic tests that can be detected quickly, accurately and reliably. SARS-CoV-2.

In December 2019, the new coronavirus SARS-CoV-2 first appeared in China and has since spread rapidly around the world. The disease it causes, COVID-19, has killed more than one million people and puts tremendous pressure on the healthcare system and economy.

“Until a vaccine is developed, our main approach is to test and prevent viral infections,” he says. Chaz Langerie, Associate Medical Director of UCSF.

Researchers can quickly and accurately identify people currently infected with SARS-CoV-2 through the detection of specific viral nucleic acids (molecular tests) or proteins (antigen tests), or by whether they have been infected. We have been working on the development of inspections. Previous exposure to the virus by detection of anti-SARS-CoV-2 antibody (serological examination).

“SARS-CoV-2 has an RNA genome surrounded by a lipid membrane containing several different proteins,” says Langelier. “Probably the best known is the” spike “protein, which is important for mediating the entry of the virus into the host cell. “

Many clinical diagnostic laboratories currently rely on polymerase chain reaction (PCR) -based molecular tests to detect SARS-CoV-2 infections in patient samples such as swabs in the throat and nose.

“PCR-based testing is probably the most widely used and is considered the’gold standard’in terms of sensitivity and specificity,” says Langelier.

However, PCR testing requires specialized equipment and trained personnel, so attention is shifting to the development of low-cost portable technologies that can provide faster results. Antigen testing is already beginning to be deployed as a low-cost screening tool, and alternative molecular testing offers great potential in the future.

PCR test is “gold standard”

By the end of January 2020 SARS-CoV-2 genome sequence is now available, Allows researchers to design PCR assays that are currently in daily use.

This technique extracts viral RNA from a patient’s sample. This is usually a cotton swab taken from the upper respiratory tract. After converting it to complementary DNA, a specific section of the viral genome is amplified with a thermal cycler to detect the product.

“The PCR test is designed to detect sections of the viral genome,” says Langelier. “But the exact target may vary from assay to assay.”

Researchers are paying attention to the evolution of the virus, as the emergence of new strains can potentially lead to false negatives.

“SARS-CoV-2 mutates about once every two weeks, which is relatively slow for the virus,” he said. Emily Crawford, Part-time assistant professor at UCSF, group leader at Chan Zuckerberg BioHub. “But a single point mutation in the primer or probe binding site can essentially kill the ability of the PCR assay to detect the virus.”

To counter this, most assays are based on at least two different targets, dramatically reducing the likelihood of spontaneous mutations occurring simultaneously at both locations.

“The tests we use in our lab target both the N and E genes of the virus,” explains Crawford. “Therefore, if you see a sample where one gene is detected very reliably and the other gene is detected at a low level or not at all, you will immediately be warned.” N gene Contains virus instructions Nucleocapsid protein And the E gene Envelope protein..

Ongoing issues

The· PCR assay sensitivity and specificity can be over 95%, When administered under ideal conditions. However, in the real world, other factors, such as when and how samples were collected, can reduce swab test performance.

“Viral load peaks about a day before a person develops symptoms and decreases when symptoms occur,” says Langelier. “Therefore, collecting a sample two weeks after the first symptom will result in a much lower amount of virus, which will affect the sensitivity of the test.”

The specificity of the PCR test can be a major problem as the sample cross-contaminates with other viral substances.For example, earlier this year Contamination of test kits assembled at the US Centers for Disease Control and Prevention laboratory I disabled them.

“Some patient samples have very high viral load and can contaminate other samples if not treated with great care,” explains Langelier. “It’s probably a more general issue than we understand.”

Another major challenge is that the viral load threshold required for a person to infect is currently unknown.

“We saw significant differences in viral load in different samples, from about 100 viral genome copies per milliliter to about 100 billion copies,” says Crawford. “This is a challenge because we need an assay that can accurately detect both very high and low titers of the virus, but whether a low titer can infect others. I don’t know. “

Rapid Antigen Test is a Potential Game Changer

To delay the spread of SARS-CoV-2, most experts agree that the focus should be on the identification and isolation of infectious people. Theoretically, this can be achieved by using a new rapid antigen test. The first of these recently received an emergency license from the US Food and Drug Administration (FDA).

Typical antigen tests usually start with a swab in the nose or throat, but saliva samples have also been developed. After adding the solution to break down the virus, the mixture is added to a laminar flow strip containing antibodies that bind to specific viral antigens.

“These assays can be placed in something like a small card that provides readings,” explains Langerie. “Therefore, as with pregnancy tests at home, you may see bands that appear only in the presence of the SARS-CoV-2 antigen.”

Antigen-based testing is much cheaper and faster than PCR-based assays. It also does not require specialized equipment and can be used in point-of-care settings. But the cost is reduced sensitivity.

“Current estimates suggest that the detection limit of antigen testing can be 10 to 100 times higher than PCR-based assays,” said Langelier. “Therefore, a small amount of SARS-CoV-2 in the body can result in false negatives.” Negative results obtained using an antigen test may need to be confirmed by a molecular test to signal a treatment decision.

However, this may not be so important if the purpose of the test is to identify a contagious person.

“There is increasing data suggesting that most people with mild or moderate illness are no longer infectious after 7 to 10 days,” explains Langelier. “Therefore, antigen-based testing can be very effective in identifying people when they are most likely to be able to pass it, as long as it is done frequently enough.”

Antibody-based testing offers another diagnostic strategy. These are blood-based immunoassays designed to detect specific antibodies produced by the immune system in response to SARS-CoV-2 infection.

“It doesn’t really play a role in diagnosing acute infections because it takes about 7-10 days for someone to produce enough antibodies to detect in the assay,” says Langelier. “However, COVID-19 is highly suspected, but may help identify cases where PCR tests remain negative.” Antibody tests show samples more than 8 days after the onset of human symptoms. It may be useful to confirm SARS-CoV-2 infection when PCR does not give accurate results, such as when collected.

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New molecular test in pipeline

Alternative technology known as Loop-mediated isothermal amplification (LAMP) It is gaining more and more attention because it may be cheaper, faster, and easier to perform than PCR. This technique amplifies the genetic material of the virus in a targeted manner similar to PCR, but does not require thermal cycling.

“Theoretically, that means you can use it in a Point of Care setting where you don’t have access to luxury machines,” Crawford explains. “But these techniques have drawbacks because they are generally not as sensitive or specific as PCR.”

CRISPR, the most well-known gene editing technology, has great potential as a diagnostic tool. This approach binds proteins to specific sequences in the viral genome and detects their interactions.

“It’s very specific, but not as sensitive as PCR,” says Crawford. “But what people are doing is combining isothermal amplification with CRISPR, which means they can get very good sensitivity and specificity.”

However, many researchers are cautious about preparing for CRISPR-based tests as a means of dealing with current pandemics.

“I think there is a temptation to find a cool new toy that solves all our problems,” says Crawford. “But in reality, there are still many challenges.”

Another potential approach involves detecting changes in the host’s immune response rather than the presence of the virus itself. For example, according to a study by the Langelier team. The pattern of genetic activity in the upper respiratory tract is unique to SARS-CoV-2 compared to other respiratory illnesses., He states, “it was available in the assay.”

Diagnosis is central

Large-scale rapid diagnostic testing of people with COVID-19 symptoms is essential to identify SARS-CoV-2 infection, contain its spread, and prevent death.

“Until vaccination, our main approach is to test and prevent infections,” says Langerie. “Easy and frequent access to diagnostic tests can help overcome many of today’s challenges in infection control and prevention.”

The long-term goal is to reach a point where extensive testing for SARS-CoV-2 infection is no longer needed. However, this is unlikely to happen soon.

“Even if vaccinated, I think diagnostic tests will continue to play an important role in identifying and preventing infections,” says Langerie.