# What is the principal goal in pooled COVID-19 testing?

Pooled testing for COVID-19 is being considered as a method to ramp up testing Stat News. Apparently this is being considered by the White House coronavirus team.

It's not clear to me what the goal of this approach is as it seems to be contradictory to the notion of rapidly detecting persons who may be contagious so as to place that person in immediate isolation.

Using the example from the Stat News article, say that a company has 100 employees that need to be "observed" on an on-going basis. Pooling, (as I think I understand it) might consist of taking 100 swabs and combining groups of ten swabs, then subsequently conducting just ten analyses. For the sake of example, let's agree that of the 100 employees only ten employees are actually contagious.

Under the best of circumstances, as luck would have it, all ten of those contagious employees might be in just one pool. In that case 9 of the tests would come back negative and 1 test would be positive. In the worst case, those ten contagious employees are uniformly distributed, resulting in 10 tests all coming back positive. More likely, however, those swabs of the ten infected employees will be randomly distributed among the ten pools.

So, for argument's sake, let's suggest that 5 of the pools are analyzed as positive and 5 pools are negative. Assuming that the analysis is perfect (that is no false positives or false negatives), it would seem that the 50 employees in the 5 negative pools could be deemed safe to return or continue to work. The other 50 employees would have to be retested, either by pools or individually. If they are tested by pools (and the pool size is maintained at 10 swabs per pool), the ten infected employees will still not have been positively identified, making a third round and fourth round of testing a necessity.

The obvious advantage to pooled testing is that a smaller number of analysis could be performed, however the number of swabs (supposedly in short supply). Some non-infected persons will be tested multiple times.

On the other hand, the length of time to detect only those who are infected, will double, triple and possibly quadruple.

So this seems to be a classic case of cost (of testing) versus urgency (to mitigate spread of the virus)

If a company (or county or country) feels an urgency to attempt to isolate infected persons, it would seem sensible to deploy individual (non-pooled) testing. On the other hand, if there is no sense of urgency, then a pooled approach (because it involves less analytical work) is appropriate.

If my perspective or understanding of how pooled testing would be deployed, I certainly would like someone to correct me, in the meanwhile my question is:

What is the principle goal of pooled COVID-19 testing for the United States?

• Jun 28, 2020 at 8:43
• The same swab can be used, according to colleagues at pcr-pooling Jun 30, 2020 at 22:03
• @chongman re: same swab. perhaps you could explain. What I envision is that (for example) ten people are swabbed with 10 sterile swabs and placed in 10 sterile carriers. To pool those swabs, the swabs are placed in an eleventh carrier, and biologic material washed out of the combined swabs to form a "pool" of biologic material that represents the 10 original specimens. That pool is then analyzed. The original swabs are now cross-contaminated, and discarded. The only way I could envision the original swabs to be re-usable (as a single original individual), is if the original swab...
– BobE
Jul 1, 2020 at 3:15
• @chongman (continued) were either dissected, or that an aliquot of the each of the original 10 swab washings were used to create the pooled specimen. Any insight???
– BobE
Jul 1, 2020 at 3:24
• @BobE Why "consume" 100% of the original swab when pooling? Jul 1, 2020 at 3:26

The principal goal of pooled testing is to rapidly clear many cases in low prevalence (or incidence) situations.

Long version:

Disclaimer: I'm analytical chemist, i.e. someone who could by profession be involved in developing such tests, but I'm not involved in SARS-CoV2 testing or SARS-CoV2 test development. What I write here is basically my general professional knowledge and what I read in newspapers.

As you notice, pooling works only if the fraction of positive cases is sufficiently low, otherwise all or nearly all pooled samples are positive and the procedure does not save tests*, time and money.

I use "tests*" to denote the RT-PCR testing procedure done in the lab. For this question, we need to distinguish this "lab test in the narrower sense" (which I mark with a *) from the overall testing procedure in the wider sense that starts with swabbing and ends with reporting the results.

If a pooled test* is positive, it is usually not necessary to redo the whole testing from sampling on: the lab sampling procedure has steps where diluted sample is prepared (or more precisely: aliquots of the sample are taken, i.e. a known volume that is smaller than the sample volume), and thus only a fraction of this solution actually goes into the PCR vial. So doing a second test* here means that another aliqot of the already prepared sample is taken, put into a 2nd PCR vial and goes into the next (or another) batch of PCR samples. This PCR procdure "test*" is in order of magnitude of an hour, whereas the overall testing procedure from swabbing the patient over shipping the samples, entering into the lab sample logistics, preparation, actual "test*", signing of the results, getting the results back to health authorities and call the patient can take a few days. Doing the lab PCR analysis a 2nd time if the pooled sample was positive is quick compared to the overall time from swabbing to results are known to the patient

The pooling protocol can be adapted to the expected fraction of positive cases: the lower this fraction is, the more samples can be pooled.
From the 2nd diagram in my answer to a similar question you can see that in your scenario of 10 % of the cases, pooling 4 cases would in the end save about 40 % of the tests*. Pooling 10 cases in that scenario would save about 25 % of the tests*

But: 10 % positive cases for a workplace screening would be really high prevalence, in the order of magnitude of the current outbreaks in German meat plants. To put it into some context: the Tönnies meat plant that recently made news as the worst hotspot in the whole of Europe had 1500+ of 6500 workers positive, so 20 - 25 % (in the actual slaughtering part even 65 % positive). In another meat plant, Westfleisch, they apparently caught the outbreak earlier with 33 positive out of 1250 workers (2.5 %).
In contrast, the general population in Germany now has about 3.5 positive tests per 100000 inhabitants and week, so <0.01 %. At such low fractions of positive cases, always pooling 10 samples saves more than 90 % of the tests. In other words, within the same time, you can tell 9x as many people that they are negative, and still have only a slightly slower results for the remaining 10 % of tested people.

Whether it makes sense to test at such low incidences if you do not have any indication of elevated risk, is a different question: a recent ring trial found roughly 1.5 - 2 % of false positive tests* or 98 - 98.5 % specificity. Testing a population with incidence << (1 - specificity) means that almost all positives are false positives.
(I did not include false positive rate in the calculations above)

• A 2nd requirement is that the test* is sufficiently sensitive to detect a single positive sample in the pool. As I understand, this is less of a concern with the discussed pooling protocols for SARS-CoV2 since only few cases (10) are pooled, and I suspect that for the pooled tests* the sample material is diluted less than for single sample tests*.

Update: Yelin et al.: Evaluation of COVID-19 RT-qPCR Test in Multi-Sample Pools, Clin Infect Dis, 2020, DOI: 10.1093/cid/ciaa531 discuss a pooling protocol that uses the standard kits.
They start by taking an aliquot of 130 μl of the swab transport buffer (typical volume: 1 ml) and add 270 μl of lysis buffer. As I understand, these 400 μl are a typical sample volume to go into the RT-qPCR machine.
Note that this already offers the possibility to take additional aliquots from the transport buffer, i.e. a solution optimized to keep the sample analyzable for much longer times and under less controlled conditions than sample storage in the lab to wait whether it needs to go into the next batch of tests*. They found that 1 positive in 32 samples could be identified 90 % of the time. Typical definition for lower limit of detection is getting a positive response 95 % of the time. Still, this looks as if pools of size 10 (the size I've seen discussed in the local media here) may work without or without too much adjustments to the standard test*ing procedure.

Without knowing details, I'd expect that the "analyze quickly after putting into test kit vial" refers to such a lysis buffer.

• Couple of questions to help me clearly understand: The "dilution" factor first. Is dilution really a washing of the swab, as opposed to "dilute by a factor of six, based on the weight or volume of the biologic to be tested". If it is actually a washing, the amount of virus (or RNA) in the specimen would be unknown at that time, so I'm not sure what the "dilution" criteria might be. (My uninformed suspicion is that the "dilution" is really getting the biologic material in a suspension - rather than a classic dilution).
– BobE
Jul 1, 2020 at 3:54
• second is more of a comment than a question. The Abbott NOW test platform (15 minute turn around time) was criticized as being subject to as much as a 30% failure rate (false negatives or positives). I recall Abbott defending their platform by saying that the specimens should be analyzed quickly (within 30 minutes) after being procured and placed in a test kit vial. If an aliquot approach is used with the original specimens (before pooling), such that a second round (smaller pool) to refine the first pool, it would not appear that the Abbott platform is amenable because of time elapse.
– BobE
Jul 1, 2020 at 4:07
• @BobE: dilution here is not washing a swab, it is dilute by factor of six. More precisely, we're talking of aliquoting steps: taking a known volume of the sample solution or suspension (after washing the swab), e.g. as part of what the analysis machine/robot is doing. As for Q2, the pooled test protocol needs to be established and valiated as well. Some analysis platforms or testing protocols may be fine with this, others not. In some circumstances, only the result of the pooled test is needed to guide the practical decision without going into who exactly is positive (e.g. one may decide to... Jul 6, 2020 at 15:24
• ... quarantine the whole kindergarden group with their families, regardless of who had negative swabs at the time of swabbing since further transmission may have occured.), in other situations, one needs to know whom exactly to quarantine and who could get a clean bill (e.g. if the pool is not coming from a physically closeby group of people but from a random collection of samples that are pooled to enhance testing capacity). The answers to these demands are diffent tests, there is not "the one SARS-CoV2 test" that meets all demands (there rarely is in medical diagnostics). Jul 6, 2020 at 15:27
• If I understand you correctly: swab with biologic specimen is washed with (say) 10 cc. An aliquot of that 10 cc is taken (say 2cc) to be analyzed. So far there has been no quantifiable dilution of the biologic because the original amount of biologic is still unknown. Is there a dilution step beyond this stage? Taking an aliquot is not the same as diluting - at least in the classic sense.
– BobE
Jul 6, 2020 at 17:58

There are several use cases.

But the main reason is always to operate in a situation where the number of tests is a bottleneck or constraint. In the USA, this is usually because of limited test reagents or limited laboratory techs and machines to run the PCR.

If you have unlimited testing, you would not use pooled testing.

One use case is surveillance. You pool test 100 people. The amount of RNA copies may tell you roughly if there are 0, 1, 2 or N people in that group that are positive.

Another is reopening. For schools, you could swab everyone on Friday, run the PCR, and say I'd you likely have 0 active carriers. If so, you open on Monday. If not, you close and do more testing and tracing.

Another is to save on testing. (With a speed tradeoff). If you have 16 people, and your goal is to get a YES or NO answer for each, you can pool test all 16. Then, if it's positive, then test in sets of 4,4,4,4. Then 1,1,1,1. In the worst case, yes, you use more tests. But if the positive rate is lower than about 30%, this will save tests overall. But this technique takes 3x the time vs test everyone.

SOURCES I've been working tangentially on this issue for 2 months. It's already in use in Germany and has been used at Stanford and in labs a few other places.

Or the subReddit for CoronaVirusTesting. I'm u/chongman99

A NYT article, a few research reports cited on there.

## UPDATE 08/01/2020

See https://arxiv.org/pdf/2004.14934.pdf for a detail of one procedure used in Uganda. Apparently, they devised a way to do the PCR's in parallel, which doesn't have the slowdown effect of sequential.

• I want to be sure that I understand what your use case (where you refer to 'pool test 100 people'). Are you starting with a total population of 100 people to be tested and that from that 100 there would be N numbers of pools (say N=20, therefore five specimens (swabs) per pool). Then, after each of the pooled specimens is analyzed, by examining the number of RNA copies in each pool one can deduce that there are 1 or 2 or 4 persons that are shedding virus at the test time. Does that assume that each contributor (person) is shedding equally? Can you expand on that
– BobE
Jul 1, 2020 at 3:40
• @BobE. Sorry for my imprecision. I meant total population of 100. And each pool size is 20. Though, the microbiologists say that a pool size of 100 is detectable. It just might be too large to be useful (will always come in positive). I'm not that clear on the bench procedures. See arxiv.org/pdf/2004.14934.pdf for a detail of one procedure used. Apparently, they devised a way to do the PCR's in parallel, which doesn't have the slowdown effect. Aug 1, 2020 at 20:49

Group testing was introduced by Robert Dorfman during WWII to "weed out all syphilitic men":

• So it would seem that your answer could be summarized by Dorfman's statement: "... a different statistical approach can... yield significant savings in effort and expense**...". That would lead me to conclude that your answer to the principle goal question is a 'reduction in cost' over other forms of testing. Is that your answer?
– BobE
Jun 28, 2020 at 16:55
• Jun 29, 2020 at 12:21