key: cord-0705206-pbvya19c
authors: Perchetti, Garrett A.; Sullivan, Ka-Wing; Pepper, Greg; Huang, Meei-Li; Breit, Nathan; Matthias, Patrick; Jerome, Keith R.; Greninger, Alexander L.
title: Pooling of SARS-CoV-2 samples to increase molecular testing throughput
date: 2020-08-02
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104570
sha: 588940c0493e7d6c76d49524f757e52d4089828b
doc_id: 705206
cord_uid: pbvya19c

BACKGROUND: SARS-CoV-2 testing demand has outpaced its supply. Pooling samples for lower risk populations has the potential to accommodate increased demand for SARS-CoV-2 molecular testing. OBJECTIVE: To evaluate the sensitivity, specificity, and reproducibility of 4-way pooling of SARS-CoV-2 specimens for high-throughput RT-PCR. STUDY DESIGN: Individual samples were pooled 1:4 through automated liquid handling, extracted, and assayed by our emergency use authorized CDC-based RT-PCR laboratory developed test. Positive samples were serially diluted and theoretical and empirical PCR cycle thresholds were evaluated. Thirty-two distinct positive samples were pooled into negative specimens and individual CTs were compared to pooled CTs. Low positive samples were repeated for reproducibility and 32 four-way pools of negative specimens were assayed to determine specificity. RESULTS: Four-way pooling was associated with a loss of sensitivity of 1.7 and 2.0 CTs for our N1 and N2 targets, respectively. Pooling correctly identified SARS-CoV-2 in 94 % (n = 30/32) of samples tested. The two low positive specimens (neat CT > 35) not detected by pooling were individually repeated and detected 75 % (n=6/8) and 37.5 % (n = 3/8) of the time, respectively. All specimens individually determined negative were also negative by pooling. CONCLUSION: We report that 1:4 pooling of samples is specific and associated with an expected 2 CT loss in analytical sensitivity. Instead of running each sample individually, pooling of four samples will allow for a greater throughput and conserve scarce reagents.

1

Widespread COVID-19 infections have placed extraordinary demand on molecular 30 diagnostics. COVID-19 cases continue to rise and laboratory capacities to detect SARS-CoV-2 31 RNA are becoming increasingly strained, causing delays in testing turnaround times [1] [2] [3] . To 32 accommodate demand for increased testing volumes, on July 18 th , 2020, the FDA issued its first 33

Emergency Use Authorization (EUA) for sample pooling in diagnostic testing [4] . 34

Early testing during the COVID-19 pandemic focused primarily on symptomatic 35

individuals, but as we expand the populations tested to asymptomatic patients, overall positivity 36 rate declines and pooling methods become increasingly favorable [5] . Sample pooling provides 37 improved benefits as SARS-CoV-2 incidence rates decline; higher incidence rates (i.e. >10%) 38 provide little advantage, but pooling with incidence rates <5% can substantially increase testing 39 capacity [6, 7] . 40

Modeling suggests that for asymptomatic or mild cases based on overall lower SARS-41 Initial water and VTM templates were used to confirm pipetting accuracy with 384 54 samples into a 96-well deep well plate. Artificial sample IDs were attributed to each respective 55 pool and the eluted volume was manually confirmed for accuracy by pipette. Prior to pooling, 56 neat samples were assayed by LDT and stored at 4°C for <24hrs. HeLa cells were included as 57 a negative extraction control and water as a negative PCR template on every run. 58

We created a web application for converting Hamilton output files into plate maps that 59 can be imported into the Applied Biosystems 7500 software. It concatenates the container IDs 60 that were mixed in each well into sample names for the 7500 application so that they can be 61 tracked. The web application also provides a form where the experiment output from the Applied 62

Biosystems 7500 software is uploaded and edited to produce extracts for other downstream 63 systems and processes. 64

To evaluate lower positivity levels of pooled samples, we first ran an initial 10-fold 71 dilution series on a positive nasopharyngeal swab with an initial mean cycle threshold CT of 31.4 72 corresponding to 2,500-5,000 copies/mL by our LDT. The sample was serially diluted with 73 phosphate-buffered saline 1:10, then 1:100, and then 1:1,000, corresponding to 1:40, 1:400, 74 and 1:4,000 respective dilutions for pooling. 75

Next, we verified that low positive samples with CTs >33indicative of a low viral load -76

were not missed by pooling of samples (Table 1 ). Our data indicate that even though individual 77 samples are diluted by the process of pooling four separate specimens together, the effect is 78 not substantial enough to push borderline positives beyond the limit of detection. 79

We expanded this experiment to include 32 additional SARS-CoV-2 positive specimens 80 by the CDC-based Washington state EUA assay pooled into negative samples. Our results 81 confirm the empirical CTs of pooled samples are not dissimilar from the theoretically calculated 82 values ( Table 2) . As expected, diluted samples with theoretical CTs beyond our limit of detection Here, four-way pooling correctly identified SARS-CoV-2 in 94% of samples, only missing 120 low viral load specimens with CT >35. We report that 1:4 pooling of samples is associated with 121 an acceptable 2 CT loss in analytical sensitivity. Pooling samples for SARS-CoV-2 molecular 122 detection can be performed efficiently without sacrificing substantial accuracy or specificity. . https://www.ajc.com/news/state--regional/georgia-sees-testing-system-138

strained-with-rise-covid-cases/pyb5miiLT74o8FUP9BsCAK/. 139 

A Dire Warning From COVID-19 Test Providers

Optimal sample pooling: an efficient tool against SARS-CoV-186 2, Health Policy

Evaluation of Sample Pooling for Screening 189 of SARS-CoV-2, Genetic and Genomic Medicine

Large-scale implementation of pooled RNA 204 extraction and RT-PCR for SARS-CoV-2 detection

Interpreting Diagnostic Tests for SARS-CoV-2

Viral RNA load as determined by cell culture as a management tool for discharge of

SARS-CoV-2 patients from infectious disease wards

A strategy for finding people infected with SARS-CoV-2: optimizing 221 pooled testing at low prevalence

Poolkeh Finds the Optimal Pooling Strategy for a 224

Population-wide COVID-19 Testing (Israel, UK, and US as Test Cases), Public and Global 225