key: cord-1008491-4otj7d1s
authors: Salimnia, Hossein; Mitchell, Robert; Gundel, Angela; Cambell, Alicia; Gammou, Fadi; Chopra, Teena; Fairfax, Marilynn
title: Pooling samples: A testing option for SARS-CoV-2 during a supply shortage
date: 2020-09-11
journal: Diagn Microbiol Infect Dis
DOI: 10.1016/j.diagmicrobio.2020.115205
sha: 69e90e7d45e3b46e1f7556fdab4db7db5fd9c654
doc_id: 1008491
cord_uid: 4otj7d1s

Pooling of one positive sample with up to 5 negative samples prior to testing with the Cepheid GenXpert SARS-CoV-2 assay did not adversely impact detection of positive samples. At our current prevalence of 2%, it could save up to 70% of the test kits.

Shortages of reagents and kits for SARS-CoV-2 (CoV-2) tests may limit testing as hospitals reopen to patients at low risk of COVID-19. We wished to validate pooling nasopharyngeal samples collected in viral transport medium from such patients to allow large-scale CoV-2 testing while conserving reagents (Abdulhamad, et al, 2020; Hogan, et al., 2020) . U.S. blood collection agencies routinely pool 16 donor samples to perform molecular-based screening for Hepatitis B and C and human immunodeficiency viruses (Dwyre, et al., 2011) . The U.S. Food and Drug Administration has not issued emergency use authorization for pooling samples for CoV-2, although they say, "we realize that pooling and asymptomatic testing are critical to ending this pandemic and we want to ensure that recommended validation approaches are appropriately designed to provide sufficient data in a least burdensome manner" (email from Yvonne Shea, on 06/12/2020 (yvonne.shea@fda.hhs.gov)). In addition, they have recommended a comment to be added if sample pooling is utilized (https://www.fda.gov/emergency-preparedness-andresponse/coronavirus-disease-2019-covid-19/covid-19-frequently-asked-questions 6/16/2020).

We wanted to validate the use of pooling using the Cepheid GenXpert SARS-CoV-2 assay (CoVassay), a reverse transcriptase PCR assay, in an atmosphere of low positivity rates. This assay detects two targets genes, E (envelope) and N2 (nucleocapsid). A result is interpreted as positive if N2 is positive, regardless of whether E is detected (Cepheid, 2020 ).

Our medical system (7 hospitals; one each specializing in pediatrics and cancer; 2000 beds) experienced a CoV-2 positivity rate >50% at the peak of the Detroit epidemic, when we were testing up to 150 samples/day from symptomatic patients. This rate has fallen to around 2 % as our hospitals reopen to patients at lower risk of COVID-19, but we are testing approximately 400 samples/day. Testing 100 samples, in pools of five, as opposed to individually, would require 20 test kits, followed by 10 more to test individual samples in the two anticipated positive pools, J o u r n a l P r e -p r o o f Journal Pre-proof conserving 70 kits. This may prove essential as Cepheid is limiting the amount of testing supplies they will send us.

We determined the impact of sample pooling on detection by retesting 15 previously frozen CoV-2-positive samples initially submitted for patient testing. The samples were chosen based on the presumed concentrations of virus, reflected by their initial cycle or crossing threshold (Ct) value with the E target. Ct values decrease as the viral load decreases. Five had Ct <25 (high virus concentration), five had Ct 25-33 (intermediate), and five had Ct >33 (low). The manufacturer provides a Ct value for positive but not negative samples, and the cut-off value is proprietary (Cepheid, 2020) .

Each sample was first retested to ensure that storage and one freeze/thaw cycle had not altered the positivity. The apparent viral load occasionally increased (the Ct decreased), possibly due to random fluctuations or to disaggregation of sample and tissue clumps in the freeze thaw process.

Samples were de-identified and given a testing number. Each sample was then retested repeatedly after pooling with 2-5 negative samples (Table) . To prepare a pool of three samples, 0.5 ml from a positive sample was pooled with an equal volume taken from each of two negative samples.

Pools containing one positive and up to five added negative samples were prepared similarly.

Negative samples were used only once. Each pool was vortexed for 5 seconds, and 300 µL was used for CoV-2 testing according to the manufacturer's instructions (Cepheid, 2020) Each pool remained positive, regardless of initial Ct or of whether the pool contained two, four, or five negative samples. However, the E target became undetectable in the three samples with the highest initial Ct values (lowest virus titers) when they were pooled with five negative samples (pool size 6 samples; Table) . This suggested that further dilution could cause samples with low titers of CoV-2 to become undetectable.

There are two general groups of CoV-2 molecular diagnostic assays from multiple manufacturers.

The first requires extraction and purification of the nucleic acid prior to the assay, which could theoretically facilitate concentration and pooling of larger numbers of specimens, but may exhibit prolonged turn-around time (TAT). The second group, which includes our assay, is designed for direct from the sample testing and should have a shorter TAT. Sample concentration is not an option, so pooling requires sample dilution and may be more consequential. Before pooling is utilized, initial studies should be performed with each assay to determine how many samples can be pooled without impacting the detection of positive samples. A theoretical calculation by Abdalhamad, et al. (2020) concluded that with a sensitivity of 95% or 100%, a specificity of 100%, a lower limit of detection of 1-3 copies/µL and a prevalence of 5%, the optimal pool size was 5 samples, and their experimental validation supported this. Hogan, et al., pooled 10 samples, but our data suggests that such a pool size may be too great for samples with low viral loads tested in our assay. A further consideration is that approximately half of our current testing is ordered STAT, as the person being tested is going for a procedure or to the operating room or is in the process of delivering a baby. If such a sample is in a pool that exhibits a positive result, the required retesting would double the in-lab testing time. Thus pooling of samples may not be appropriate for STAT tests. 

Assessment of Specimen Pooling to Conserve SARS CoV-2 Testing Resources

Hepatitis B, hepatitis C and HIV transfusion-transmitted infections in the 21st century

Sample pooling as a strategy to detect community transmission of SARS-CoV-2

Acknowledgements: