Editorial Article: Limit of detection: The key to preventing false negative COVID-19 tests, regardless of patient viral load

Learn the importance of low LoD tests and discover the most sensitive kits available, as determined by the FDA’s SARS-CoV-2 Reference Panel

19 Oct 2020



Stephanie Dallaire, Principal Scientist for the Molecular Diagnostics group at PerkinElmer

In this exclusive SelectScience interview, we speak with Stephanie Dallaire, Principal Scientist for the Molecular Diagnostics group at PerkinElmer, about the current challenges of COVID-19 testing, the need for sensitive detection kits, and how limit of detection (LoD) affects COVID-19 testing and reporting. 

What are the current challenges for those testing for COVID-19?

SD: COVID-19 testing has many challenges for our society. Widespread testing needs to happen for everyone to go back to work, school, and other activities. Unfortunately, not everyone who wants to get tested appears to have access to accurate testing. Our objective at PerkinElmer is to resolve this challenge by equipping labs with the necessary instruments and reagents.    

SARS-CoV-2 tests are generally PCR-based assays for viral detection or serological assays for antibody detection. The PCR test is the most accurate and will determine if a patient is currently infected, whereas antibody testing is useful to see if someone has been infected in the past. Both types are useful in determining if an individual has been exposed to the virus. 

There is also the issue of false positive tests and false negative tests. A false-positive result mistakenly identifies a person as infected, which can result in unnecessary quarantine and contact tracing. False-negative results are far more dangerous because infected persons may not isolate themselves and can infect others.

What does the SARS-CoV-2 Reference Panel comparative data show?

SD: The FDA SARS-CoV-2 Reference Panel is comprised of common, well-characterized reference materials used to allow for a precise comparison of the many different molecular IVD assays that have been developed to detect SARS-CoV-2. Early in the pandemic, the FDA issued emergency use authorization (EUAs) to many molecular tests based on contrived positive samples for analytical and clinical performance evaluations. While these contrived samples illustrated confidence in the diagnostic tests, it is not feasible to compare the various tests this way.  Each molecular test granted an EUA to detect SARS-CoV-2 may focus on different genes, so establishing a panel from the live virus allows for direct comparison of all these tests in terms of analytical sensitivity and specificity. 

Each of the tests that received an EUA were asked to participate in this study. The FDA provided the reference samples and a protocol to follow and asked the kit developers to relay the results and confirmatory tests. The reference panel material contains one heat inactivated SARS-CoV-2 and one MERS-CoV strain in media. This viral material is in a known concentration of nucleic acid amplification testing (NAAT) detectable units/mL (NDU/ML) in one tube to determine LoD range in the kit. The rest of the panel concentrations are blinded to the kit developers and are used to confirm the LoD as well as evaluate the cross reactivity of the kit. In molecular diagnostic assays, the LoD is the lowest concentration of target that can be detected in 95% of measurements taken repeatably.  This means that 1 in 20 individuals who have a viral load at this level will not be detected.

Why is being able to compare LoD between kits important?

SD: During the current COVID-19 pandemic, we have had three priorities: persons infected, persons infectious, and protecting exposure of persons susceptible to infection. Experts estimate that as little as 1,000 copies of SARS-CoV-2 are needed to cause an infection. Viral loads can vary as much as a billion-fold between patients, with the highest viral load per patient occurring 5-6 days following infection. High-titer patients are easily detected by most commercially available kits due to the large amounts of virus present in the sample. However, there are also affected individuals who have much lower viral loads and need a more sensitive test to detect their infections. These patients are more of a concern because if they have a negative COVID-19 test, they may go about their everyday life with less caution, which undermines public health efforts.

Having a low LoD is beneficial in two other regards. The first being that the time to detection is sooner. The first few days after exposure to someone with COVID-19 are when the infection takes root. During this incubation period, before experiencing symptoms, the virus is multiplying exponentially. If you know that you have been exposed and can be tested reliably in this short window, the chances of you infecting another person lessens and, with a test that can detect low viral load, the early detection can save lives.

The other benefit of a test with low LoD is in pooling patients for screening. The amount of testing being done globally is far below the level necessary to open back up again. Pooling patient samples is a strategy being deployed to increase the number of patients tested with limited SARS-CoV-2 detection kits. A test with lower LoD is beneficial in this instance as being able to detect a lower viral titer means testing multiple individuals in the same reaction is feasible and accurate. Pooling samples by nature dilutes each individual sample input to the assay. Having a sensitive assay in this instance really makes a difference in making sure there are no false negative tests.

How does LoD affect COVID-19 testing and reporting?

SD: A recent study has shown that every 10-fold increase in LoD is expected to increase the false negative rate of a test by 13%. This translates into an additional 1 in 8 patients not being detected. Based on the FDA reference panel data, some of the tests on the market could potentially have a false negative rate of 70%. This could have disastrous effects of epidemiological and clinical consequence and impact identification, control and containment of the SARS-CoV-2 pandemic.

What does this data mean for PerkinElmer’s Coronavirus Nucleic Acid Detection Kit?

SD: With a 3-fold greater sensitivity than the nearest competitors, the PerkinElmer® New Coronavirus Nucleic Acid Detection kit was determined to be the most sensitive kit granted EUA by the FDA. We are proud and have worked hard to establish the best-in-class detection of COVID-19. Years of experience and development of RT-qPCR assays along with high quality nucleic acid extraction has proven to be the right combination to detect the SARS-CoV-2 virus.

PerkinElmer’s mission of innovating for a healthier world is at the heart of everything we do. We will continue to enable customers to make smarter decisions, diagnose patients earlier, and develop more effective treatments and scientific breakthroughs.  

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