COVID-19 Pool Testing: Is It Time to Jump In?

July 20, 2020

Pool testing, in which biological specimens (e.g., blood, urine, tissue swabs, etc.) from multiple subjects are combined and tested via a single test, can substantially improve the efficiency of public health screening and population-level surveillance of diseases. Introduced in the 1940s, pool testing has been used for applications like screening donated blood or regional human immunodeficiency virus (HIV) surveillance. 

I worked for a decade in the Texas Department of State Health Services Rabies and Arbovirus Unit within the Zoonosis Control Division, where I was introduced to the amazing world of zoonotic microbes. While much of my time was devoted to rabies diagnostics, those of us in the unit also tested for diverse zoonotic viruses, many of which are transmitted by mosquitoes. As one might guess, the logistics of looking for viral agents in the massive population of mosquitoes in Texas is literally like trying to find the proverbial needle in a haystack. Testing each mosquito separately for West Nile Virus, for example, would be a tremendous effort and consume a lot of testing resources.

Instead, we ground up batches of mosquitoes collected in a county or within a zip-code. Then, each pool was tested by a single immunoassay or molecular test to search for that needle in the haystack – West Nile Virus, St. Louis Encephalitis (SLE) virus, Western Equine Encephalitis virus (WEE), Eastern Equine Encephalitis virus (EEE) or other arbovirus of interest.

Likewise, pool testing COVID-19 specimens allows us to conserve both material and human resources (public health and medical laboratory personnel). Many countries, including the U.S., have ongoing shortages in testing kits and pre-analytical supplies, as well as laboratory personnel who are credentialed or licensed to perform testing. Conservation of resources is a definite strength of pool testing in light of the ever-expanding case count during this pandemic.

Pool testing for COVID-19 surveillance within a population.
Pool testing for COVID-19 surveillance within a population.
In a keynote address that kicked off ASM’s Microbe Online conference, Dr. Deborah Birx, the response coordinator for the White House coronavirus task force, urged labs to expand SARS-CoV-2 diagnostic testing in the U.S. through sample pooling and community engagement. Dr. Birx emphasized the ongoing testing capacity issues for conducting single tests on individuals in light of what many now believe to be one of the primary ways this virus and pandemic continues to move at warp speed – asymptomatic carriers and “lurking” transmission of the virus. Pool testing might prove to be a valuable way to increase our surveillance and ultimately help to improve our contact tracing.

Limitations of Pool Testing

Laboratories are faced with the importance and urgency of mass testing for COVID-19. However, pooled biomarker tests struggle with lower sensitivity (true positive probability) than single sample tests due to dilution effect. That is, the diagnostic target (in this case, SARS-CoV-2 viral RNA) from an infected specimen is diluted by target-free specimens in the pool and can approach the limit of detection for the test. For our example, try to visualize that 1 needle in a haystack when you add many, many more pieces of hay. It becomes very difficult, and sometimes impossible, for the particular target (needle) to be detected in such a massive volume of sample (hay). 

Pool testing for COVID-19 has already been used in Wuhan and Beijing, China, as well as the state of Nebraska. In a proof-of-principle paper from the Nebraska Public Health Laboratory, pools of 5 patient specimens (1 positive with 4 negative specimens) were shown to preserve the ability to detect SARS-CoV-2 RNA from the positive specimen. Previous studies in Israel, U.S. and Germany have shown that individual positive samples of SARS-CoV-2 can be detected in pools of up to 32 samples, and possibly even 64 samples with additional PCR amplification cycles. At a pool size of 16 samples, sensitivity was 96%, with a false negative rate of about 10%.

Another concern for pooling of samples is cross-contamination and the tracking of specimens. One of the primary reasons for medical (and laboratory) errors is the mishandling or labeling of specimens. With bench technologists handling more than 1 specimen at a time for pool testing, the chances of contaminating specimens increases. 

Lastly, there may be a need to seek Emergency Use Authorization (EUA) for pooled testing, which could take up to 3 weeks. The U.S. Food and Drug Administration (FDA) has created a template to make this process more accessible and smoother regarding the validation of molecular diagnostic tests for developers that intend their assay to be used for pooling patient samples or for screening asymptomatic individuals not suspected of having COVID-19. As of July 18, 2020, the FDA has issued an Emergency Use Authorization (EUA) for Quest Diagnostics to begin pooled testing, allowing up to 4 test samples to be tested at once.

Context is Important

Currently, laboratories return negative individual results for each patient represented in a negative pool test for SARS-CoV-2. If a pool test is positive, then individual tests are run to identify the (presumably) single positive specimen within that pool.  Again, negative results are returned for the negative specimens within the positive pool, and a positive result is returned for the single positive specimen.

Pool testing is most effective in increasing efficiency and output in situations where disease prevalence is low, since each negative pool test eliminates the need to individually test those specimens and maximizes the number of individuals who can be tested over a given amount of time. Nebraska's scheme to test pools of 5 specimens is estimated to increase their testing capacity by 69%, but only if prevalence of COVID-19 in the population remains less than 10%. That's because, as disease prevalence increases, more pools will test positive and require individual specimen tests. At some point, so many pools will test positive that in fact, labs will end up doing more tests using this strategy than an individual testing strategy.

Pooling Testing as Surveillance

At the University of Illinois at Urbana-Champaign and OSF HealthCare, Dr. Hadi Meidani and Dr. Daniel Lakeland are working together to develop pool testing techniques for surveillance of SARS-CoV-2. "There are various problems that can be addressed by testing, including individual diagnosis and medical care, of course. But perhaps a bigger issue is understanding the prevalence in a population and using it for prevention and quarantine. In this context, pooled test results can be obtained faster and more frequently, and therefore can be better than individual results," said Dr. Meidani. "We are trying to take a systems approach using information from pooled tests to inform management strategies without necessarily needing individual followup results. Right now it's like we're playing a chess game where we can't see the pieces, pooled testing can give us a view of the board and enable us to be more strategic," said Dr. Lakeland. They are currently using information theory and probabilistic modeling to determine the optimal design for pooled testing (the pooling ratio, frequency of testing, etc.), and how individual diagnosis can be facilitated with the fewest follow-up individual tests. 

While there are limitations to pool testing, there are some major advantages for utilizing this type of testing framework and strategy during shortages in the supply chain for SARS-CoV-2 testing. "[Pool testing] can be used in any of a number of circumstances, at a community level or even in schools if you wanted to do that," Dr. Anthony Fauci, head of the National Institute of Allergy and Infectious Disease,  said at a recent Senate hearing. While individual testing of each sample (subject) will always be more accurate, it may just be time for us to consider a trade-off — pooling lets you test a lot more people in a rapidly expanding pandemic.

Author: Rodney Rohde, Ph.D., SM(ASCP), SVCM, MBCM, FACSc

Rodney Rohde, Ph.D., SM(ASCP), SVCM, MBCM, FACSc
Rodney Rohde, Ph.D., is the Associate Director of the Translational Health Research Initiative at Texas State University.