Dive into the deep end of pooled testing for COVID-19

Reviewed by: Rebekah Moehring, MD, MPH, FSHEA; Duke University Hospital and Apara Dave, MD; Exeter Hospital

Two recent reports address the technique of pooling specimens from different individuals into a single PCR run.  This technique is one potential solution to widespread limitations of laboratory resources during high demand for COVID-19 testing, as well as an option for more efficient population level screening.

A recent Emerging Infectious Diseases article from Stanford evaluated the dilutional effect of pooled testing using different testing methods.  Investigators tested 1,648 prospectively pooled samples using three different nucleic acid application tests, with pool sizes of 8 and 4. Samples were OP/NP, from inpatients and outpatients, both symptomatic and asymptomatic, from tertiary academic hospitals and affiliated outpatient facilities in the San Francisco area. Sample from each pool, and each individual, was run on the laboratory developed test QIAGEN QIA symphony. On the same day, sample from each pool was run on Hologic Panther Fusion and Hologic Panther Aptima. PPA and NPA were calculated using individual testing as reference.  The PPA ranged from 71-82% for pools of 8, and 82-100% for pools of 4. NPA was 98-100%.  For pools with false negative results, the false negative samples were obtained from patients previously known to be positive; none were initial diagnostic specimens.  Investigators also developed a stochastic simulation model to estimate dilutional effect on PPA. PPA varied with proportion of positive individual tests in the pool, cycle threshold, and assay limit of detection. The dilutional effect varied with infection prevalence, assay characteristics, and pooling size. Overall, pooled testing decreased PPA relative to individual samples.  PPA also varied with testing method used for pooled samples; something not typically accounted for in pooled testing studies. 

The November MMWR describes the use of pooled testing to screen students at Duke University returning to campus for the fall 2020 semester. Investigators describe a multifaceted plan to limit the spread of COVID-19 in a university congregate setting.  Students signed the “Duke Compact” indicating their agreement to entry testing and quarantine, routine testing for asymptomatic students, universal masking and social distancing, daily symptom screening and reporting via a mobile app, and following all instructions for quarantining and contact tracing. Most notable was the strategy for large-scale routine asymptomatic testing, which was accomplished using 5:1 pooled testing with a quantitative, in-house, laboratory-developed RT-PCR. Routine tests were 2 times per week for on-campus undergraduates, 1-2 times per week for off-campus undergraduates, and once weekly for graduate students. Adherence to testing was 95% and enforced through restricting access to campus facilities. From August through October 2020, >68,000 specimens from >10,000 students were tested: 84 specimens were positive, around half of which were from asymptomatic students. Several asymptomatic students had high viral loads, suggesting that identifying these students likely prevented further infections. In October, average weekly per-capita incidence among students was 0.08%, compared with the surrounding county incidence of 1%. The pooled testing strategy reduced the need for laboratory resources, but still allowed high volume throughput with high sensitivity and rapid result turnaround — fast enough to identify positives, initiate contact tracing, and reduce transmission events. Duke undergrads are a committed group in a well-resourced, private university, thus generalization of this experience to other settings may be limited. 

A pooled test strategy doesn’t solve every resource issue – for example, swabs have also become a scarce commodity during the pandemic as much as reagents. Despite this, pooled testing can be an effective strategy to save resources, especially when used for large scale testing of asymptomatic individuals.


Denny TN, Andrews L, Bonsignori M, et al. Implementation of a Pooled Surveillance Testing Program for Asymptomatic SARS-CoV-2 Infections on a College Campus — Duke University, Durham, North Carolina, August 2–October 11, 2020. MMWR Morb Mortal Wkly Rep 2020;69:1743–1747. DOI: http://dx.doi.org/10.15585/mmwr.mm6946e1  

Wang H, Hogan CA, Miller JA, Sahoo MK, Huang C, Mfuh KO, et al. Performance of nucleic acid amplification tests for detection of severe acute respiratory syndrome coronavirus 2 in prospectively pooled specimens. Emerg Infect Dis. 2021 Jan. https://doi.org/10.3201/eid2701.203379 

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