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Swab pooling for large-scale RT-qPCR screening of SARS-CoV-2

Ana Paula Christoff PhD
1, Giuliano Netto Flores Cruz BPharm
1, Aline Fernanda Rodrigues Sereia

PhD1, Dellyana Rodrigues Boberg PhD1, Daniela Carolina de Bastiani BSc1, Laís Eiko Yamanaka MSc1,

Gislaine Fongaro PhD2, Patrícia Hermes Stoco PhD3, Maria Luiza Bazzo PhD4, Edmundo Carlos Grisard

PhD3, Camila Hernandes PhD5, Luiz Felipe Valter de Oliveira PhD1*

1
BiomeHub Biotechnologies , Florianópolis-SC, Brasil
2
Laboratório de Virologia Aplicada, Departamento de Microbiologia, Imunologia e Parasitologia,

Universidade Federal de Santa Catarina (UFSC), Florianópolis-SC, Brazil.

3
Laboratório de Protozoologia, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade

Federal de Santa Catarina (UFSC), Florianópolis-SC, Brazil.

4
Laboratório de Biologia Molecular, Microbiologia e Sorologia – LBMMS, Universidade Federal de

Santa Catarina (UFSC), Florianópolis-SC, Brazil.

5
Hospital Israelita Albert Einstein (HIAE), São Paulo, SP, Brasil

These authors contributed equally.

*Correspondence author: Luiz Felipe Valter de Oliveira, BiomeHub Biotechnologies. Av. Luiz Boiteux

Piazza, 1302, Sapiens Parque, Canasvieiras, Florianópolis, SC, Brazil

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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-ND 4.0 International license .

ABSTRACT

Pool testing has been proposed as an alternative for large-scale SARS-CoV-2 screening. However,

dilution factors proportional to the number of pooled samples have been a source of major concern

regarding its diagnostic performance. Further, sample pooling can lead to increased laboratory workload

and operational complexity. Therefore, pooling strategies that minimize sample dilution, loss of

sensitivity, and laboratory overload are needed to allow reliable and large-scale screenings of SARS-

CoV-2. Here, we describe a pooling procedure in which nasopharyngeal swabs are pooled together at the

time of sample collection (swab pooling), decreasing laboratory manipulation and minimizing dilution of

the viral RNA present in the samples. Paired analysis of pooled and individual samples from 613 patients

revealed 94 positive individual tests. Having individual testing as a reference, no false-positives or false-

negatives were observed for swab pooling. A Bayesian model estimated a sensitivity of 99% (Cr.I. 96.9%

to 100%) and a specificity of 99.8% (Cr.I. 99.4% to 100%) for the swab pooling procedure. Data from

additional 18,922 patients screened with swab pooling were included for further quantitative analysis.

Mean Cq differences between individual and corresponding pool samples ranged from 0.1 Cq (Cr.I. -0.98

to 1.17) to 2.09 Cq (Cr.I. 1.24 to 2.94). Overall, 19,535 asymptomatic and presymptomatic patients were

screened using 4,400 RT-qPCR assays, resulting in 246 positive patients (positivity rate 1.26%). This

corresponds to an increase of 4.4 times in laboratory capacity and a reduction of 77% in required tests.

Finally, these data demonstrate that swab pooling can significantly minimize sample dilution and

sensitivity issues commonly seen in its traditional counterpart. Therefore, swab pooling represents a

major alternative for reliable and large-scale screening of SARS-CoV-2 in low prevalence populations.

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medRxiv preprint doi: https://doi.org/10.1101/2020.09.03.20187732.this version posted September 5, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-ND 4.0 International license .

INTRODUCTION

The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2

(SARS-CoV-2), has dramatically impacted public health worldwide in this year of 20201,2. Rapid

identification and isolation of infected individuals is essential, but it can be particularly challenging given

the infectious potential of both asymptomatic and presymptomatic cases3,4. In this scenario, massive

population SARS-CoV-2 testing is an urgent need to allow the isolation of infected individuals and,

ultimately, the pandemic control.

The most sensitive and recommended test is based on the RT-qPCR method, which detects an

active infection through the identification of the viral RNA in nasopharyngeal samples5–8. However,

several limitations have hampered large-scale population screenings using RT-qPCR, mainly related to

the worldwide shortage of supplies and their relatively high cost. To overcome these limitations and

scale-up testing capability, some research groups have proposed pooling samples for testing, in which

several individuals are simultaneously analyzed using a single test9–15.

In the many ways that pool testing was proposed so far, all of them are based on individual

sample mixing by the laboratory (sample pooling). This procedure involves substantial sample

manipulation, leading to operational challenges and, more importantly, to substantial dilution of viral

RNA present in any of the pool samples. Such a dilution effect directly impacts the analytical sensitivity

of the RT-qPCR assay, potentially leading to reduced diagnostic sensitivity9. Here, we describe a pooling

procedure in which nasopharyngeal swabs are pooled together at the time of sample collection (swab

pooling), decreasing laboratory manipulation and minimizing dilution of the viral RNA present in the

sample.

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medRxiv preprint doi: https://doi.org/10.1101/2020.09.03.20187732.this version posted September 5, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-ND 4.0 International license .

METHODS

Study design

A retrospective study was performed using de-identified results from nasopharyngeal samples

subjected to RT-qPCR-based SARS-CoV-2 testing from May 5th to July 31st, 2020. Sample collection was

performed focusing on low-prevalence COVID-19 populations (e.g. asymptomatic or presymptomatic

individuals). A total of 45 pool samples and their 613 corresponding individual samples were analyzed in

parallel to assess correspondence between individual and pool qualitative results, i.e., individual samples

were tested regardless of the results from their corresponding pools. Further, 18,922 additional patients

were tested using swab pooling, corresponding to 1,344 pools. Among these, only positive pools had their

respective individual samples tested. Individuals from negative pools were considered negative for

SARS-CoV-2 RNA detection. Comparison of paired cycle quantification (Cq) values from all positive

samples obtained was carried out to assess potential quantitative biases due to swab pooling. This study

was approved by the Hospital Israelita Albert Einstein Ethics Committee (number

36371220.6.0000.0071).The patient informed consent was waived off by the ethics committee as the

research was performed on de-identified, anonymised samples.

Specimen collection and swab pooling for SARS-CoV-2 screening

Nasopharyngeal samples were collected by trained healthcare professionals using nylon flocked

swab, stored in tubes containing sterile saline solution and submitted to laboratory processing within a

maximum of 48h after sample collection.

For the swab pooling method, two swabs were collected from the same individual (Figure 1). The

first swab, collected through one nostril, was stored in an individual tube containing 3 mL of saline

solution. A second swab, collected from the second nostril, was stored in a pool tube containing 5 mL of

saline solution. As a general rule, each pool tube was allowed to contain up to 16 swabs, from 16 different

patients, collected apart within a maximum of 1h. In this way, the pooling of swabs is performed at the

time of sample collection, dismissing further manipulation, mixing and dilution of the samples by the

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medRxiv preprint doi: https://doi.org/10.1101/2020.09.03.20187732.this version posted September 5, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-ND 4.0 International license .

laboratory. When a given pool tested positive, all the corresponding individual samples were also tested

to identify the infected patients. If a pool yielded a negative result, all individuals within that pool were

considered negative for SARS-CoV-2 RNA detection.

SARS-CoV-2 RT-qPCR detection

RNA isolation was performed from nasopharyngeal samples using guanidine thiocyanate lysis

solution followed by magnetic beads capture and purification (BiomeHub, Brazil). Samples were eluted

in 40 µL of RNAse-free water. RNA reverse transcription (RT) was performed using SupesScriptTM IV

(Invitrogen, USA) and random hexamers, according to the manufacturer instructions.

Real-Time PCR detection of SARS-CoV-2 was performed using the following genetic markers: a

region of the gene encoding the viral envelope protein (E) with P1 probe and the RNA-dependent RNA

polymerase gene (RdRp) with P2 probe for discriminatory assay, as described in the Charité-Berlin

protocol8,16. Also, data from the detection of the surface glycoprotein gene (S) using SYBR Green

intercalating fluorophore as previously described17 were included. Amplifications below cycle

quantification (Cq) 40 were considered positive. Amplifications were performed in 7500 Fast,

QuantStudio 6 Pro Real Time PCR (Applied Biosystems, USA), or in a CFX 384 (BioRad, USA). Cycle

quantification values from the RT-qPCR amplifications were used for data analysis.

Statistical analysis

All statistical analyses were performed using R statistical software (v. 3.6.3)18. Data wrangling

and visualization were performed using the tidyverse package suite (v. 1.3.0)19. Modeling was performed

using the brms R package (v. 2.12.0) and the Stan probabilistic programming language (v. 2.19.1)20,21.

Additional R packages included ggpubr (v. 0.2.5), RColorBrewer (v. 1.1.2), binom (v. 1.1.1), and

patchwork (v. 0.0.1)22–25.

Concordance between pool and individual tests was determined by considering their

corresponding qualitative results, i.e., a test was considered concordant if the individual result matched

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medRxiv preprint doi: https://doi.org/10.1101/2020.09.03.20187732.this version posted September 5, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-ND 4.0 International license .

the result from its corresponding pool. Among positive tests, we quantified the mean Cq difference

between individual samples and their corresponding pools. We employed a Bayesian hierarchical model

with patient-specific intercepts as follows:

Cqi ~ N (μi, σ2)

μi = α + α patient[i] + βpool *Pooli + βE *Ei + βRdRp *RdRpi

+ βPool:E *Pooli *Ei + βPool:RdRp *Pooli *RdRpi

α patient ~ N (0, σα)

(1)
α ~ N (25, 3)

β. ~N (0, 5)

σα ~Exponential (1)

σ ~Exponential (1)

where Pooli is an indicator variable which equals 1 when the ith observation is from a pool sample and 0

otherwise. The Ei and RdRpi variables adjust for variation in the genetic marker used for the RT-qPCR

assay. In these settings, the population-level intercept α represents the average Cq value for an individual

test using the S gene, whereas the patient-specific intercept α patient[i] accounts for patient-to-patient

variability. The β. coefficients allow quantification of mean Cq differences between individual and pool

tests across different genetic markers. We set weakly informative priors for all parameters. Results were

reported as posterior means and 95% credible intervals.

For the sample dilution in swab pooling experiment, the same model as in (1) was employed,

except that varying intercepts varied with inoculating samples instead of with patients; also, only E and

RdRp genes were used. Credible intervals for proportions were obtained using a Beta(1, 1) prior for the

binomial likelihood. Observed correlations were reported as Spearman’s rank correlation as well as

Pearson’s correlation coefficient.

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medRxiv preprint doi: https://doi.org/10.1101/2020.09.03.20187732.this version posted September 5, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-ND 4.0 International license .

RESULTS

Sample dilution in swab pooling - Proof of concept

In a laboratory experiment, 16 positive nasopharyngeal samples were selected as inoculating

samples to be mixed in equal volumes into 16 negative pool samples as well as into 16 negative

individual samples, according to a dilution factor of 1.67. This dilution factor corresponds to volumes

between samples collected in swab pooling tubes (with 5 mL saline solution) and samples collected in

individual tubes (with 3 mL saline solution). For this paired experiment, we observed a mean Cq

difference between pool and individual samples of 0.42 Cq (95% Cr.I. -0.22 to 1.09) for the E gene and

0.6 Cq (95% Cr.I. -0.05 to 1.24) for the RdRP gene (Figure 2A).

In Figure 2B we show the expected slopes (ΔCq) in RT-qPCR amplifications with variable

amplification efficiencies and considering different dilution scenarios. In sample pooling, the dilution

factor is equal to the number of individual samples within a pool, yielding expected mean Cq differences

of at least 3.32 Cq and as high as 7.37 Cq depending on the number of pooled samples and the

amplification efficiency. For swab pooling, on the other hand, the dilution factor is kept fixed at 1.67 so

that the expected variation due to dilution alone is constrained between 0.73 and 1.08 Cq.

Paired analysis of pool and individual tests

To investigate any loss of diagnostic sensitivity due to swab pooling, we analyzed individual and

pool samples from 613 patients regardless of their pool results (i.e., positive and negative pools). All the

individual and pool samples were analyzed in parallel resulting in 94 positive individual tests and 20

positive pools (Figure 3A). Among the 20 positive pools, at least one individual sample in each pool

tested positive for SARS-CoV-2. Positive patients per pool varied from 1 to 11 (Figure 3B). We observed

no clear evidence of correlation between the pool Cq values and the number of positive samples within

each pool (Figure 3C). Paired comparisons of the pool and their respective individual Cq values can be

visualized in Figure 3D. Further analysis of Cq variation is performed in the next section.

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medRxiv preprint doi: https://doi.org/10.1101/2020.09.03.20187732.this version posted September 5, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-ND 4.0 International license .

Qualitatively, we did not observe any positive individual test paired with a negative pool, i.e., no

false-negatives due to swab pooling. In fact, we observed complete agreement (100%) between

qualitative results from the pool and individual paired samples. Hence, we employed a simple beta-

binomial model with flat priors on performance estimates that would otherwise reach 100%. Having

individual testing as a reference, the current data supports a sensitivity of 99% (95% Cr.I. 96.9% to

100%) and a specificity of 99.8% (95% Cr.I. 99.4% to 100%) for the swab pooling procedure, indicating

evidence of strong similarity in diagnostic performance.

Large-scale screening for SARS-CoV-2 using swab pooling

To investigate any biases in quantitative results, we included data from additional 1,344 pools and

their respective individual tests. In total, 19,535 patients (1,389 pools) were screened using the swab

pooling method herein described. Considering all combined results, we observed 246 positive patients for

SARS-CoV-2 distributed in 163 pools, resulting in a positivity rate of 1.26%. For 12 pools (0.86%),

amplification of both E and RdRp genes was detected but no associated positive individual sample was

identified. In such cases, a new sample collection was requested by the laboratory.

Among the 163 positive pools, 100 (61.3%) contained exactly 16 pooled swabs (Figure 4A).

Also, 104 pools (63.8%) corresponded to exactly one positive individual test each. Over 81% of positive

pools presented at most 3 correspondent positive individual tests. Some Cq values above 40 were

individually inspected and considered positive for 3 pools and 3 individual samples in the E gene (41.06,

40.58, 40.58 for pools and 41.93, 41.61, 40.99 for individuals) given that their respective RdRp gene

amplification was also positive but with lower Cqs (34.58 to 37.52). Additionally, one pool sample with a

Cq value of 44.79 for the RdRp gene was considered positive as its E gene counterpart showed a Cq value

of 34.83.

Correlation between Cq values from individual tests and their corresponding pools was strongest

for pools associated with one or two positive samples, seemingly diminishing with the increase in the

number of positive samples within the pools (Figure 4B). To estimate the Cq variation due to swab

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medRxiv preprint doi: https://doi.org/10.1101/2020.09.03.20187732.this version posted September 5, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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pooling, we assigned to each patient the Cq value from their individual test and the Cq value from their

respective pool. Using a hierarchical model with patient-specific intercepts, we estimated the mean Cq

difference between individual tests and their corresponding pools for each genetic marker (Figure 5). For

the S gene (94 patients), the mean Cq difference was estimated to be 0.1 Cq (95% Cr.I. -0.98 to 1.17).

Differences for the E and RdRp genes (152 patients) were estimated to be 1.8 Cq (95% Cr.I. 0.93 to 2.66)

and 2.09 Cq (95% Cr.I. 1.24 to 2.94), respectively.

DISCUSSION

Pool testing has gained importance to fight the COVID-19 pandemic, as challenges involving cost

and logistics are at the core of shared struggles to promote large-scale screenings worldwide10. Traditional

pooling methods proposed9–15 rely on the combination of multiple individual samples prior to RNA

extraction or RT-qPCR, leading to a dilution factor directly related to the number of samples in the

pool9,11–15. This dilution effect has been of major concern over the diagnostic performance of pool testing

procedures26. Here, we report a pooling strategy that readily minimizes such an effect and enables large-

scale screening for SARS-CoV-2.

Assessing data from 19,535 screened patients, swab pooling and individual testing showed hardly

distinguishable performances both qualitatively and quantitatively. With complete agreement between

paired qualitative results from 613 patients, the presented data indicates evidence of strong similarity in

diagnostic sensitivity and specificity. We did not observe a clear correlation between pool Cq values and

number of positive individuals within the pools, as previously suggested considering other pooling

methods14. Also, the correlation between Cq values from individual tests and their corresponding pools

seems to be stronger for pools with no more than two positive individuals. This is mainly reflecting the

potentially wide range of individual Cq values for samples composing a single pool.

Although we do use a larger volume for swab pooling (5 mL of saline solution versus 3 mL in

individual tubes), the corresponding dilution factor of 1.67 will lead to an expected mean increase of 1.08

Cq even under sub-optimal amplification efficiencies. In a laboratory-controlled experiment, we did not

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medRxiv preprint doi: https://doi.org/10.1101/2020.09.03.20187732.this version posted September 5, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-ND 4.0 International license .

detect clear differences due to dilution alone, with point estimates from 0.43 to 0.61 Cq. In practice,

observational data from 246 positive patients generated point estimates of mean Cq differences between

individual tests and their corresponding pools ranging from 0.1 to 2.09 Cq. While such values are hardly

significant in terms of analytical sensitivity, the expected counterparts for traditional pooling would range

from 3.3 to 5 Cq under optimal amplification conditions. This range corresponds to dilution factors from

10 to 32, when equivolumetric pools from 10 to 32 samples, respectively, are formed post-collection by

the laboratory as traditionally proposed9,11,13,15. In a worst-case scenario for swab pooling, a mean Cq

difference of 2.94 Cq (RdRp gene, upper limit of 95% credible interval) would still be considerably lower

than the expected differences for sample pooling with 10 samples and perfect amplification efficiency.

Nonetheless, there is always a limitation towards samples with Cq’s higher than 35, in which case mean

differences as small as 1 Cq could still result in false-negative tests regardless of the pooling strategy.

Operationally, the major difference between swab pooling and traditional methods regards sample

collection: while in swab pooling we combine multiple swabs in the same tube at the time of sample

collection, traditional strategies pool equal volumes from individually collected samples. Beyond dilution,

the latter methodology adds complexity to laboratory operations and may lead to increased workload to

already saturated laboratory facilities. Traditional pooling requires significant sample manipulation with a

risk of contamination and even sample exchange during the laborious pooling process.

On the other hand, collecting two swabs from the same patient can be operationally trivial. While

one swab goes into the pooling tube, the other one will only be processed by the laboratory if the pool

tests positive. A critical step, this sample collection process can still represent an important limitation of

swab pooling as it can cause variation between pooled and individual swabs. In this study, we detected 12

pools with positive results but no positive associated individual test. Of these, 8 pools were associated

with two specific collection events (4 pools collected each day). Thus, it is likely that such inconsistencies

are attributable to the sample collection process. Still, these cases represented 0.86% of all 1,389 tested

pools. Notably, the proper training of sample collection staff represents a cheaper and easier-to-

implement alternative to increased laboratory complexity. Any laboratory capable of routine processing

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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-ND 4.0 International license .

of diagnostic samples for SARS-CoV-2 can also perform swab pool analysis using the same detection

methods and infrastructure already in use.

Using swab pooling during sample collection, laboratories in which traditional pooling is

currently unfeasible become readily able to contribute to large-scale screenings. Swab pooling, therefore,

represents a gain in operational performance for reliable testing of SARS-CoV-2 at scale. As it is well-

known, however, any pooling strategy only boosts testing capability for low positivity rates27. Swab

pooling does not address this matter and is, therefore, suitable for screening populations a with low

expected prevalence of COVID-19.

The data in the present study comes from the application of swab pooling in asymptomatic or

presymptomatic populations, yielding a 1.26% positivity rate. The proposed method was used with pools

containing a majority of 16 individuals, but the optimum pool size can be determined by each laboratory

during internal validation. Pools with 8, 10, 16, or even 32 swabs may be desirable depending on local

epidemiological status and target populations. Upon validation, swab pooling may be applied to any

reasonable pool size traditionally proposed to optimize testing scale. Here, over 19,500 patients were

screened using approximately 4,400 RT-qPCR assays, corresponding to an increase of 4.4 times in

laboratory capacity and a reduction of 77% in the total of required tests.

Finally, identification of infected patients is essential to contain the spread of SARS-CoV-2. This

has been hampered by the fact that several people carrying the virus remain asymptomatic or

presymptomatic4,28. Thus, massive and sensitive testing of asymptomatic and presymptomatic individuals

is of utmost importance to fight the COVID-19 pandemic, especially at the moment in which the world

attempts to resume economic and social activities.

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medRxiv preprint doi: https://doi.org/10.1101/2020.09.03.20187732.this version posted September 5, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-ND 4.0 International license .

CONCLUSION

Pool testing is a major alternative for large-scale screening of SARS-CoV-2 in low prevalence

populations. Here, we demonstrate that the swab pooling minimizes sample dilution, can be as sensitive

as individual testing and reduces laboratory workload. A total of 77% of tests were saved in the screening

of 19,535 asymptomatic or presymptomatic patients.

ACKNOWLEDGEMENTS

We would like to thank all BiomeHub, HIAE, and UFSC staff who were involved in all stages of sample

collection, laboratory processing, and results discussion. We are grateful for their countless efforts to

persist in high-quality research during such difficult times for science in our country, especially during the

COVID-19 pandemic. Conflict of interest disclosures: all authors from BiomeHub are currently full-time

employees of this research and consulting company specialized in microbiome biotechnologies.

BiomeHub funded the study design, analysis and data submission for publication.

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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-ND 4.0 International license .

REFERENCES

1. Yang P, Wang X. COVID-19: a new challenge for human beings. Cell Mol Immunol. 2020;17(5):555-
557. doi:10.1038/s41423-020-0407-x

2. Lai C-C, Shih T-P, Ko W-C, Tang H-J, Hsueh P-R. Severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) and corona virus disease-2019 (COVID-19): the epidemic and the challenges. Int J
Antimicrob Ag. 2020;55(3):105924. doi:10.1016/j.ijantimicag.2020.105924

3. He X, Lau EHY, Wu P, et al. Temporal dynamics in viral shedding and transmissibility of COVID-19.
Nat Med. 2020;26(5):672-675. doi:10.1038/s41591-020-0869-5

4. Lee S, Kim T, Lee E, et al. Clinical Course and Molecular Viral Shedding Among Asymptomatic and
Symptomatic Patients With SARS-CoV-2 Infection in a Community Treatment Center in the Republic of
Korea. Jama Intern Med. 2020;180(11). doi:10.1001/jamainternmed.2020.3862

5. Vogels CBF, Brito AF, Wyllie AL, et al. Analytical sensitivity and efficiency comparisons of SARS-
CoV-2 RT–qPCR primer–probe sets. Nat Microbiol. Published online 2020:1-7. doi:10.1038/s41564-020-
0761-6

6. Sethuraman N, Jeremiah SS, Ryo A. Interpreting Diagnostic Tests for SARS-CoV-2. Jama.
2020;323(22):2249-2251. doi:10.1001/jama.2020.8259

7. Caruana G, Croxatto A, Coste AT, et al. Diagnostic strategies for SARS-CoV-2 infection and
interpretation of microbiological results. Clin Microbiol Infec. 2020;26(9):1178-1182.
doi:10.1016/j.cmi.2020.06.019

8. World Health Organization. Laboratory testing for coronavirus disease 2019 (COVID -19) in suspected

human cases: interim guidance, 2 March 2020. World Health Organization. Published online March 2,
2020. https://apps.who.int/iris/handle/10665/331329

9. Yelin I, Aharony N, Tamar ES, et al. Evaluation of COVID-19 RT-qPCR test in multi-sample pools.
Clin Infect Dis. Published online 2020. doi:10.1093/cid/ciaa531

10. Lohse S, Pfuhl T, Berkó-Göttel B, et al. Pooling of samples for testing for SARS-CoV-2 in
asymptomatic people. Lancet Infect Dis. Published online 2020. doi:10.1016/s1473-3099(20)30362-5

11. Ben-Ami R, Klochendler A, Seidel M, et al. Large-scale implementation of pooled RNA extraction
and RT-PCR for SARS-CoV-2 detection. Clin Microbiol Infec. 2020;26(9):1248-1253.
doi:10.1016/j.cmi.2020.06.009

12. Hogan CA, Sahoo MK, Pinsky BA. Sample Pooling as a Strategy to Detect Community Transmission
of SARS-CoV-2. Jama. 2020;323(19):1967-1969. doi:10.1001/jama.2020.5445

13. Torres I, Albert E, Navarro D. Pooling of nasopharyngeal swab specimens for SARSCoV2
detection by RTPCR. J Med Virol. Published online 2020. doi:10.1002/jmv.25971

13
medRxiv preprint doi: https://doi.org/10.1101/2020.09.03.20187732.this version posted September 5, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-ND 4.0 International license .

14. Gupta E, Padhi A, Khodare A, et al. Pooled RNA sample reverse transcriptase real time PCR assay
for SARS CoV-2 infection: A reliable, faster and economical method. Plos One. 2020;15(7):e0236859.
doi:10.1371/journal.pone.0236859

15. Perchetti GA, Sullivan K-W, Pepper G, et al. Pooling of SARS-CoV-2 samples to increase molecular
testing throughput. J Clin Virol. 2020;131:104570. doi:10.1016/j.jcv.2020.104570

16. Corman VM, Landt O, Kaiser M, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-
time RT-PCR. Eurosurveillance. 2020;25(3):2000045. doi:10.2807/1560-7917.es.2020.25.3.2000045

17. Chan JF-W, Yuan S, Kok K-H, et al. A familial cluster of pneumonia associated with the 2019 novel
coronavirus indicating person-to-person transmission: a study of a family cluster. Lancet.
2020;395(10223):514-523. doi:10.1016/s0140-6736(20)30154-9

18. Team RC. R: A Language and Environment for Statistical Computing. R Foundation for Statistical
Computing; 2019. http://www.r-project.org

19. Wickham H, Averick M, Bryan J, et al. Welcome to the Tidyverse. J Open Source Softw.
2019;4(43):1686. doi:10.21105/joss.01686

20. Bürkner P. brms: An R Package for Bayesian Multilevel Models Using Stan. J Stat Softw.
2017;80(1). doi:10.18637/jss.v080.i01

21. Carpenter B, Gelman A, Hoffman M, et al. Stan: A Probabilistic Programming Language. J Stat
Softw. 2017;76(1). doi:10.18637/jss.v076.i01

22. Kassambara A. Ggpubr: “ggplot2” Based Publication Ready Plots. R Package Version 0.4.0.; 2020.
https://CRAN.R-project.org/package=ggpubr

23. Neuwirth E. RColorBrewer: ColorBrewer Palettes. R Package Version 1.1-2.; 2014. https://CRAN.R-
project.org/package=RColorBrewer

24. Dorai-Raj S. Binom: Binomial Confidence Intervals For Several Parameterizations. R Package
Version 1.1-1.; 2014. https://CRAN.R-project.org/package=binom

25. Pedersen TL. Patchwork: The Composer of Plots. R Package Version 1.0.1.; 2020. https://CRAN.R-
project.org/package=patchwork

26. Cherif A, Grobe N, Wang X, Kotanko P. Simulation of Pool Testing to Identify Patients With
Coronavirus Disease 2019 Under Conditions of Limited Test Availability. Jama Netw Open.
2020;3(6):e2013075. doi:10.1001/jamanetworkopen.2020.13075

27. Eberhardt JN, Breuckmann NP, Eberhardt CS. Multi-Stage Group Testing Improves Efficiency of
Large-Scale COVID-19 Screening. J Clin Virol. 2020;128:104382. doi:10.1016/j.jcv.2020.104382

28. Moghadas SM, Fitzpatrick MC, Sah P, et al. The implications of silent transmission for the control of
COVID-19 outbreaks. Proc National Acad Sci. 2020;117(30):17513-17515.
doi:10.1073/pnas.2008373117

14
medRxiv preprint doi: https://doi.org/10.1101/2020.09.03.20187732.this version posted September 5, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-ND 4.0 International license .

29. Ramakers C, Ruijter JM, Deprez RHL, Moorman AFM. Assumption-free analysis of quantitative
real-time polymerase chain reaction (PCR) data. Neurosci Lett. 2003;339(1):62-66. doi:10.1016/s0304-
3940(02)01423-4

30. Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative
PCR and the 2−ΔΔCT Method. Methods. 2001;25(4):402-408. doi:10.1006/meth.2001.1262

31. Kralik P, Ricchi M. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions,
Parameters, and Everything. Front Microbiol. 2017;8:108. doi:10.3389/fmicb.2017.00108

15
medRxiv preprint doi: https://doi.org/10.1101/2020.09.03.20187732.this version posted September 5, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-ND 4.0 International license .

FIGURE LEGENDS

Figure 1. Molecular screening for SARS-CoV-2 through swab pooling method. 1) From each

individual, two swabs are collected. One nasopharynx swab is collected from one nostril and stored in an

individual 3 mL tube. Then, another swab is collected through the other nostril and stored in a 5 mL tube

containing up to 15 other individuals (pool tube). 2) In the laboratory, the RNA from pooled swabs is

extracted, and SARS-CoV-2 detection is performed using RT-qPCR. If a given tested pool presents a

positive result, all corresponding individual samples are then processed to identify infected patients.

Figure 2. Swab pooling and dilution effects. (A) In a laboratory controlled experiment, 16 positive

samples were inoculated in negative individual and pool samples maintaining the dilution factor of 1.67

between individual (3 mL) and swab pooling (5 mL) samples. Mean Cq differences between individual

and pool samples estimated for E and RdRp genes along with 95% credibility intervals are shown in the

top-left corner of the graphs. (B) Expected Cq variations (ΔCq) for sample pooling methods with 10, 16

or 32 samples compared to swab pooling in different amplification efficiencies. Expected ΔCq was

calculated using Efficiencyslope = dilution factor29–31. In swab pooling, the number of samples are not

related to the dilution factor.

Figure 3. Paired analysis of pool and individual tests. (A) Results for samples analyzed in parallel as

pools and as individual tests. (B) Total number of positive samples for each positive pool. (C) Correlation

between the number of positive samples within a given pool and the corresponding pool Cq (R: Pearson’s

corr. coefficient;
: Spearman’s rank corr. coefficient). Points were colored by the total number of

individuals within the pool (pool size). (D) Cycle quantification values for positive pools and

corresponding positive individual samples.

Figure 4. Large-scale molecular screening of SARS-CoV-2 with swab pooling. (A) Number of

positive pools related to the original pool size and the number of positive samples within the pool. (B)

16
medRxiv preprint doi: https://doi.org/10.1101/2020.09.03.20187732.this version posted September 5, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-ND 4.0 International license .

Correlation between Cq values from pools and their respective individual samples stratified by the

number of positive samples within the pool (1, 2, and 3 or more positive individual samples). Point color

represents the pool size (from less than 10 to 16 individuals). Cq values for the three marker genes tested

were included. Correlation coefficients are presented in the figure (R: Pearson’s corr. coefficient;
:

Spearman’s rank corr. coefficient).

Figure 5. Paired cycle quantification comparison and estimated mean differences for pool or

individual samples. Cq values from individual tests and corresponding pools were assigned to each

patient. A hierarchical model with patient-specific intercepts was used to estimate mean variations

between individual and pool samples across varying genetic markers. Estimates for mean Cq differences

along with 95% credible intervals are shown in the top-left corner of each graph.

17
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