Suzanne Leech, PhD
The emergence of a major global pandemic is far from unexpected. In fact, scientists have been warning of an event like the coronavirus outbreak for many years. In the book Disease Control Priorities: Improving Health and Reducing Poverty, published in 2007, the authors write, “the likelihood of pandemics has increased over the past century because of increased global travel and integration, urbanization, changes in land use, and greater exploitation of the natural environment.”
Unfortunately, our knowledge of the risks cannot help us to prepare a vaccine or treatment for an unknown pathogen. And until these measures can be taken, effective control strategies to prevent the spread of SARS-CoV-2 are essential to save lives. One of the most important strategies is the use of diagnostic tests. “We have a simple message for all countries: test, test, test,” said director-general Tedros Adhanom Ghebreyesus of the World Health Organization during a briefing on March 16, 2020. This article summarizes the available and emerging clinical coronavirus tests and their use in control strategies.
The RNA-based genome of SARS-CoV-2, of approximately 30,000 bases and 15 genes, was published by Chinese scientists in February 2020, allowing diagnostic companies worldwide to begin developing molecular-based tests. Those that have been or are being developed include reverse-transcriptase PCR (RT-PCR), quantitative or real-time RT-PCR (qRT-PCR), microfluidic panels, next generation sequencing (NGS), CRISPR-based diagnostics, and loop-mediated isothermal amplification (LAMP)-based testing.
One of the first widely used tests was the CDC’s 2019-nCoV Real-Time RT-PCR Diagnostic Panel for testing upper and lower respiratory tract specimens. Unfortunately, problems with some of the kit reagents meant the test had very high rates of false negatives and positives, and it is currently being redeveloped.
The FDA has since used emergency use authorization to fast-track the availability of PCR-based diagnostic kits from 18 companies as of March 27. The Roche cobas SARS-CoV-2 qRT-PCR test takes around 3.5 hours to run on the Cobas 6800/8800 system and targets both coronavirus and other sarbecovirus subgenus family members, while the ThermoFisher TaqPath COVID-19 Combo Kit, also based on qRT-PCR, can be run in four hours and targets only SARS-CoV-2.
When used correctly, PCR-based tests are incredibly sensitive and specific; however, a relatively high degree of technical knowledge is required to run them. Incorrect test operation can lead to false negatives, while cross-contamination of samples can lead to high rates of false positives. However, a new coronavirus diagnostic kit from BioFire, for use on the FilmArray PCR systems, promises a one-hour turnaround time and minimal specialist knowledge to operate.
Currently, RT-PCR-based tests are the only molecular coronavirus diagnostics commercially available in the US. However, Hong Kong University of Science and Technology created a microfluidic PCR platform that can detect coronavirus in 40 minutes and has achieved European Union CE approval. The technology facilitates much more rapid temperature changes during PCR than conventional thermocyclers. Tests based on CRISPR, such as those being developed by the University of Washington School of Medicine and Mammoth Biosciences Biotechnology from San Francisco, are still being evaluated. The technique has the advantage of very rapid turn-around (5-10 minutes) and high specificity.
Immunological tests that identify either antibodies to the coronavirus or viral antigens in serum can provide information not only on current but also historical infections. Based on the principles of the lateral flow immunoassay (LFIA), they are generally easier and faster to run than molecular-based tests (around 10-30 minutes); however, they are slower and more challenging to develop, which is why, in most countries, distribution of the first immunoassays has only recently begun.
SureScreen Diagnostic's lateral-flow COVID-19 IgG/IgM Rapid Test Cassette is CE marked and can test blood samples in 10 minutes for the presence of coronavirus antibodies. A similar test for anti-coronavirus IgM and IgG, produced and developed by Biomedomics Inc. and Jiangsu Medomics Medical Technology, is available in the US and has a reported 88.66 percent sensitivity and 90.63 percent specificity. Also FDA approved is the CoronaCheck ™ COVID-19 Rapid Antibody Test Kit from 20/20 BioResponse with a reported 97.2 percent sensitivity and 92 percent specificity. Tests with sensitivities and specificities around 90 percent are generally considered reliable. These point-of-care (POC) tests are simple to use and require minimal technical training.
Diagnostic tests and control of SARS-CoV-2
During pandemics, the rapid diagnosis of a virus can be hugely influential in controlling the speed and severity of an outbreak. In South Korea, the use of diagnostic tests to identify infected individuals among those who were either mildly ill or asymptomatic meant that strict and wide-spread social distancing measures were unnecessary. Tests are particularly significant for coronavirus control, as the infection has an incubation period of up to 14 days and around 30 percent of cases will remain asymptomatic.
Rapid diagnosis relieves pressure on health systems by allowing health workers with mild illnesses to return to work if uninfected, allowing quick decisions on patient isolation, and slowing the epidemic by identifying carriers. Correct diagnosis is also vital in providing essential epidemiological data, such as the speed of transmission and infection and fatality rates, which, along with statistical modeling, can be used to make predictions and design control measures. That said, the different types of test have various strengths and weaknesses in their utility during pandemic control. Therefore, choosing the right test for the right situation can lead to more effective control of the virus.
Generally, the great advantage of immunoassays is their suitability as POC tests. The tests are portable, rapid, and can be used with little or no training. As such, they are vital tools in hospitals, care homes, and in the field and are in high demand. Unfortunately, they are more time-consuming to develop, manufacture, and validate than PCR-based tests; therefore, many institutions have yet to receive these tests. Singapore has been using immunoassays since February, and more than 500,000 COVID-19 IgM/IgG Rapid tests have been sold in China. Immunoassays are also being distributed in Italy, Japan, and the Middle East. In the US, the FDA has permitted the use of antibody tests only in laboratories approved by the Centers for Medicaid and Medicare Services, but it warns that these tests “should not be used as the sole basis to diagnose or exclude SARS-CoV-2 infection or to inform infection status.” Typically, immunological tests are not as sensitive or specific as molecular techniques, which may lead to infected health workers being cleared to work with vulnerable patients. Furthermore, serum antibodies reach detectable levels around three to seven days after infection. Therefore, immunoassays should be followed up with more accurate tests for a definitive diagnosis.
To combat the disadvantages of immunoassays, there have been attempts to develop effective molecular-based POC tests, including the automated real-time PCR Xpert Xpress SARS-CoV-2 from Cepheid, which has received emergency use authorization by the FDA, and CRISPR-based tests. Still in development, the LAMP technique, which uses strand-displacement polymerase rather than denaturation during nucleic acid amplification, potentially has a high specificity (>95 percent) and is much faster to run than PCR. The developers of the technique claim it is much easier to operate than conventional molecular biology methods and could be applied in POC tests.
Along with better sensitivity and specificity, molecular methods are more readily adaptable to changes in pathogen characteristics. Some scientists believe there are already two strains of SARS-CoV-2, one more virulent than the other. Others believe that mutations are likely to create an entirely new strain in the future. As such, molecular methods are crucial in the very early stages to identify and control the emergence of new coronavirus strains.
Clearly, both molecular and antibody-based tests play important roles in pandemic control strategies. However, using the tests to their optimum effectiveness relies on as few limitations to their availability as possible. It is vital that governments, test developers, and health care bodies work together to ensure that test reagents and supplies are rapidly distributed to the sites where they are most needed.