key: cord-0925343-l4thjpb9
authors: Cohen, Yuval; Bamberger, Nadav; Mor, Orna; Walfisch, Ronen; Fleishon, Shay; Varkovitzky, Itay; Younger, Asaf; Levi, Danit Oz; Kohn, Yishai; Steinberg, David M.; Zeevi, Danny; Erster, Oran; Mendelson, Ella; Livneh, Zvi
title: Effective bubble-based testing for SARS-CoV-2 using swab-pooling
date: 2022-02-17
journal: Clin Microbiol Infect
DOI: 10.1016/j.cmi.2022.02.016
sha: 6a99c99d27702da83e2bbbe4850e86fd0e20a991
doc_id: 925343
cord_uid: l4thjpb9

OBJECTIVES: Despite the success in developing COVID-19 vaccines, containment of the disease is obstructed worldwide by vaccine production bottlenecks, logistics hurdles, refusal to be vaccinated, transmission through unvaccinated children, and the appearance of new viral variants. This underscores the need for effective strategies for identifying carriers/patients which was the main aim of this study. METHODS: We present a bubble-based PCR testing approach using swab-pooling into lysis buffer. A bubble is a cluster of people who can be periodically tested for SARS-CoV-2 by swab-pooling. A positive test of a pool mandates quarantining each of its members, who are then individually tested while in isolation, to identify the carrier(s) for further epidemiological contact tracing. RESULTS: We tested an overall sample of 25,831 individuals, divided into 1273 bubbles, with an average size of 20.3±7.7 swabs/test-tube, getting for all pools (≤37 swabs/pool) a specificity of 97.5% (lower bound 96.6%) and a sensitivity of 86.3% (lower bound 78.2%), and a post-hoc analyzed sensitivity of 94.6% (lower bound 86.7%) and a specificity of 97.2% (lower bound 96.2%) in pools with ≤25 swabs, relative to individual testing. CONCLUSIONS: This approach offers significant scale-up in sampling and testing throughput, savings in testing cost, while not reducing sensitivity and not affecting the standard PCR testing laboratory routine. It can be used in school classes, airplanes, hospitals, military units and workplaces, and may be applicable to future pandemics.

The success in developing COVID-19 vaccines raised the hope for containment of the disease. However the shortage in vaccine production, along with logistic issues and relaxation of preventive measures, led to a rise in the spread of the disease in many countries [1] . This underscores the notion that controlling the spread of the SARS-CoV-2 virus is a complex task involving a combination of preventive measures, diagnostic testing which involves complicated logistics, proper explanation to the public, and contact tracing. Moreover, it is confounded by issues of psychology of the masses, conspiracy theories, fake news and lack of public trust [2, 3] , and the significant number of asymptomatic carriers [4] [5] [6] .

Testing for the presence of the virus is a key to the termination of viral transmission. Simple and fast detection methods are available, but the gold standard is still qRT-PCR of nasal or nasopharyngeal swabs, which typically takes several hours and is carried out in an authorized laboratory [7, 8] . Several approaches were developed to increase the throughput of qRT-PCR testing, e.g., sample pooling, which enables a shorter turnaround time with a slight reduction in test sensitivity [9] [10] [11] [12] [13] [14] , as well as using lysis buffer for swab collection instead of viral preservation medium [15] . While numerous studies were published on sample pooling [e.g., [9] [10] [11] [12] [13] ], only a few studies were published on swab-pooling [16] [17] [18] [19] [20] , with only one large-scale study [21] .

Here we describe the combination of the bubble (termed 'capsule' in Israel) concept [22, 23] with a large-scale screening approach of swab-pooling into lysis buffer. Members of each bubble were assayed in parallel by swab-pooling and by individual testing, and the results of each swab-pool were compared to the individual testing results of its members. 

We defined a bubble as a group of people who spend time together, e.g., students in a class, workers in a specific factory space, etc., such that if one member of the bubble is identified as SARS-CoV-2 positive, all members of the bubble are sent into isolation. Here, the members of each bubble were assigned in advance, and tested by swab-pooling, namely a swab was taken from each person, and the swabs were all pooled into a single test-tube. To increase sensitivity and safety, we used lysis buffer for swab-pooling. Up to 37 swabs can be placed in a 50-ml test-tube containing 10 ml of lysis buffer, and up to 8 swabs can be placed in a standard 15-ml test-tube with 3 ml lysis buffer ( Supplementary Fig. S1 ). For comparison, all members of each bubble were also sampled into individual 3 ml viral transport medium (VTM) tubes. All the pools and individual samples were tested for SARS-CoV-2 using commercial qRT-PCR assays, as described under Methods. When a pool was declared PCR-positive, all bubble members were sent into isolation, and the carrier(s), identified by the individual test, underwent an epidemiology inquiry for contact tracing (Fig. 1 ).

The study was conducted with daycare nannies from the municipality of Bnei-Brak, students and personnel from yeshivas for orthodox Jewish boys/young men, and elementary and secondary schools for orthodox Jewish girls, as well as staff working in nursing homes for the elderly, geriatric hospitals, assisted living facilities, and welfare institutions for populations at risk. The study was coordinated with the relevant authorities, and all participants gave oral consent. The study enrolled a total of 27,348 subjects. Of these 1284 were excluded due to technical issues such as leakage from the test-tubes or inconsistency in the barcodes between a pool and its individuals' tests, and 233 of the samples were excluded because we did not have their test results in the computerized data system. Overall, 25,831 were included in the final analysis (94.5%),

including 1096 subjects (4.2%) for whom the age was not available. The gender and age distributions of the participants are presented in Table 1 .

The study included 654 daycare nannies, from different daycares. These were randomly 

To Clean copy preservative), as previous works showed an advantage in sensitivity to lysis buffer over VTM [15] .

This higher sensitivity of the pool may result from the accumulation effect of viral RNA from several positive samples in the pool, each having a viral load that was below detection level when tested individually, but rendering the pool positive.

The specificity of swab-pooling based on all pools was high (97.5%), and similar to the specificity calculated post-hoc for pools with ≤25 swabs (97.2%), with low false negative rates of 0.94% and 0.33%, respectively. In contrast, the sensitivity was significantly affected, with pools with ≤25 swabs exhibiting a post-hoc calculated high sensitivity of 94.6% (Supplementary Table   1 will stay unchanged, increase, or RNA will be undetected). Such an approach can help in the decision on whether to send an individual to isolation, and/or for how long.

Compared to sample pooling, our results demonstrate that swab-pooling does not result in loss of sensitivity, while pooling several liquid samples together typically results in a loss in sensitivity of 2-3 Ct compared to individual testing [25, 11, 12] . Importantly, the implementation of sample pooling in PCR testing laboratories requires significant modification in the standard operating procedures, notably the stage of preparing the pools, using a dedicated robotic platform, whereas swab-pooling is much simpler, and requires minimal adjustments in the lab procedures.

It does require adaptation during swab collection, appropriate barcoding for pool and individual samples, as well as corresponding data collection and transfer. This simplicity is a big advantage, which greatly facilitates implementation of this method.

The efficiency of swab-pooling, like any pooling methodology, depends on the prevalence of COVID-19 [26] [27] [28] . Supplementary Table 2 lists the recommended numbers of swabs/pool under various prevalence values. When prevalence is 0.02% or lower, pooling of up to 25 swabs/test-tube of 10 ml can be used, enabling up to over 20-fold increased sampling efficiency.

The efficiency strongly drops when prevalence is 2%, but it still provides a 3.65-fold better efficiency than individual testing. For a prevalence of 0.10-0.25%, 16-25 swabs can be pooled, and for 0.50-2.0% pools of 8-16 swabs are recommended (Supplementary Table 2 ).

Swab-pooling can be carried out in several configurations. In simple swab-pooling, a single swab is taken from each bubble member, and the swabs are pooled in a single test-tube which is delivered for testing. Individual testing of the members of a positive bubble is made while they are in isolation, and should preferably include all household members. A, individuals in a working place or a school are divided into bubbles, with each individual in a bubble (6 in this example) tested with two swabs: one is placed in the swab-pool test-tube, whereas the second in an individual tube. Black person image, healthy individual; Red image, SARS-CoV-2 infected individual, whose swab is colored red. B, Swab-pool A and swab-pool C are PCRnegative, and therefore all individuals belonging to bubbles A and C are regarded as negative.

Swab-pool B gave a positive PCR result, and therefore each individual belonging to swab-pool B is being isolated. C, the individual test-tubes for swab-pool B members are individually PCRtested to identify the SARS-CoV-2 positive individual. This is followed by contact tracing. 

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Lessons from applied largescale pooling of 133,816 SARS-CoV-2 RT-PCR tests

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Improved sensitivity, safety, and rapidity of COVID-19 tests by replacing viral storage solution with lysis buffer

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Ellison KA and Chinault AC Rapid identification of yeast artificial chromosome clones by matrix pooling and crude lysate PCR

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Dorfman R The detection of defective members of large populations

We thank the many devoted people who participated in sampling, analyzing and helped swab-pooling testing become a reality. 

(1) 1 Including geriatric hospitals, assisted living facilities, and welfare institutions for populations at risk. 2 Including students and personnel from regular yeshivas for orthodox boys and young man, and the Beis Yaakov elementary and secondary schools for orthodox Jewish girls 3 Daycare nannies from the city of Bnei Brak. 4 Mean age excluding a total of 1096 subjects whose age was unknown. These subjects were distributed as follows: Nursing homes for the elderly -246 females, 72 males, 8 of unknown; Yeshivas -27 females, 81 males, 8 unknowns; Nannies -654 females. Entire study -927 females, 153 males and 16 unknowns. 5 NA, not available. These data were not collected for the nannies. 6 Mean age of all subjects excluding the nannies for whom age was not available. 7 Mean numbers of swabs/pool for all pools, except those of the nannies group, which were not available. 8 Clean copy