The Food and Drug Administration (FDA) Veterinary Laboratory Investigation and Response Network (Vet-LIRN) conducted and funded two inter-laboratory comparison exercises to assess the reliability of methods designed to test animals for SARS-CoV-2, the virus identified as the cause of COVID-19. Detailed information on the first of these two exercises was recently published in the Journal of Veterinary Diagnostic Investigation.
The inter-laboratory comparison exercise also helped laboratories have confidence in their method performance and laboratory competency. In addition to assuring that methods for testing animals for SARS-CoV-2 were reliable, Vet-LIRN supported 20 participating veterinary diagnostic laboratories in obtaining the certification needed to test human samples for SARS-CoV-2. Those laboratories have tested more than 5 million human samples. Veterinary diagnostic laboratories responded to the public health emergency by providing increased capacity to test human samples. This work aligns with the FDA Center for Veterinary Medicine's (CVM) mission to protect human and animal health.
Coronavirus 2019 (COVID-19) is a zoonotic disease, meaning that it is thought to have originated in an animal before infecting humans. To protect humans and animals from getting sick, it was important to find out what (if any) species of animals could be sickened, transmit, and/or serve as a reservoir for infection with the virus.
Early in 2020, veterinary diagnostic laboratories across the U.S. began to develop tests to detect SARS-CoV-2 virus in animals with suspected infections. They understood the importance of monitoring animals for SARS-CoV-2 and had experience with the unique aspects of developing diagnostic tests for animals. Their methods had to handle diverse materials – samples ranging from nasal swabs to feces, collected from cats, dogs, horses, lions, tigers, minks, and other animals. In addition, they needed to avoid kits or other supplies needed for human testing, so as not to create additional demand on those limited resources.
The laboratories quickly developed methods to detect SARS-CoV-2 by detecting the virus’s unique genetic fingerprint. RNA is the genetic material of SARS-CoV-2; the methods measured RNA sequences that are unique to the virus. Although they used a similar approach, the SARS-CoV-2 testing methods independently developed by different veterinary diagnostic laboratories used different equipment and materials, and each had only been evaluated using internal validation protocols. When new diagnostic tests are developed, they need to be evaluated to make sure they deliver reliable results. This is especially important during a pandemic when information is being rapidly shared among laboratories across the entire public health system.
How can you test the reliability of a method for detecting a virus? One way is to perform an inter-laboratory comparison exercise. During an inter-laboratory comparison exercise, blinded samples are sent to participating laboratories for testing. The word “blinded” means that the folks performing the testing don’t know the concentration of the material in the samples and they need to use their methods to figure it out. The organizer of an inter-laboratory comparison exercise prepares the samples that get tested, and they hold the answer key. In this case, the answer key listed the concentration of SARS-CoV-2 virus RNA in each sample tested.
After all participating labs reported their results, reported values were compared to the expected responses and labs were provided feedback on their performance to make improvements, if necessary.
Vet-LIRN, a CVM-funded partnership among 46 veterinary diagnostic laboratories in North America, was established in 2010 to ensure that participating laboratories are ready, willing, and able to investigate emerging problems with animal feed and animal drugs by providing a rapid response to reports of adverse events. Vet-LIRN also provides funding to help develop and improve laboratory testing capability.
To meet its mission, Vet-LIRN has extensive experience conducting inter-laboratory comparison exercises across many laboratories to test the reliability of methods for detecting molecules of interest. Three times each year, Vet-LIRN conducts inter-laboratory comparison exercises or proficiency tests where samples are prepared and sent to network laboratories for testing. Although Vet-LIRN typically focuses on methods to detect contaminants in food and drugs, the same skills, resources, and experience was needed to evaluate methods to detect SARS-CoV-2 virus in animals.
Vet-LIRN funded and organized two inter-laboratory comparison exercises for methods of detecting SARS-CoV-2 in animals. The first exercise was conducted in August 2020 in collaboration with six other teams:
(1) CFSAN’s Moffett Proficiency Testing Laboratory
(2) the Institute for Food Safety and Health at the Illinois Institute of Technology
(3) the US Department of Agriculture’s (USDA) National Animal Health Laboratory Network
(4) the United States Geological Survey (USGS)
(5) the statistical company QuoData, and
(6) Cornell University College of Veterinary Medicine.
During the planning and implementation of the 2020 inter-laboratory comparison exercise, Vet-LIRN shared information with the Centers for Disease Control and Prevention (CDC) and with the Integrated Consortium of Laboratory Networks (ICLN).
To create the samples tested, organizers extracted RNA from the SARS-CoV-2 virus sampled from the first U.S. patient with COVID-19 (confirming no viable virus was present), spiked that RNA into different solutions, and shipped those samples along with solutions containing no viral RNA (i.e. “blank” samples) to participants for a blinded analysis. Each of the 42 participating laboratories analyzed 19 samples. Results were received and analyzed according to the principles of International Organization for Standardization.
The analysis showed that 100% sensitivity was achieved for detecting the SARS-CoV-2 RNA, meaning that all samples with SARS-CoV-2 RNA were identified as containing SARS-CoV-2 RNA.
A second inter-laboratory comparison exercise was conducted in June 2021 to evaluate how well methods could distinguish between SARS-CoV-2 and other coronaviruses. It was also designed to find out how few copies of virus RNA need to be present for a method to detect it, which is called the limit of detection for a method. In addition, it tested whether laboratory methods could detect SARS-CoV-2 variants in animals. The results of that comparison exercise are currently being evaluated.
Accurate detection of SARS-CoV-2 in animals is vital to protecting both animals and humans from COVID-19 infection. There have been a relatively small number of reports of animals that have tested positive for the SARS-CoV-2 virus worldwide compared to human positives. The list of animals includes domestic cats, dogs, ferrets, big cats in zoos or sanctuaries (i.e., lions, tigers, pumas, cougars, and snow leopards), gorillas in zoos, white-tailed deer, and mink on farms. Animals continue to be monitored for COVID-19 infection and results are publicly reported and regularly updated.
The COVID-19 pandemic highlighted the importance of seeking optimal health outcomes for humans and animals in their shared environment, a public health strategy referred to as the One Health approach. The mission of the FDA’s Center for Veterinary Medicine is to protect human and animal health and we apply the One Health approach to everything we do.
Vet-LIRN worked to assure that the methods used to test animals for SARS-CoV-2 reliably detect the virus in animals. Vet-LIRN has had a more direct impact on human health, as well, by providing funding for veterinary diagnostic laboratories to help obtain Clinical Laboratory Improvement Amendments (CLIA) certification to be qualified to test human samples. During the COVID-19 pandemic, more than 20 participating veterinary diagnostic laboratories obtained provisional CLIA certification to support the public health efforts with respect to COVID-19 testing. Those laboratories have tested >5 million human samples.