key: cord-0928436-h3vc5x09
authors: Borillo, Gwynngelle A; Kagan, Ron M; Baumann, Russell E; Fainstein, Boris M; Umaru, Lamela; Li, Hai-Rong; Kaufman, Harvey W; Clarke, Nigel J; Marlowe, Elizabeth M
title: Pooling of Upper Respiratory Specimens Using a SARS-CoV-2 Real Time RT-PCR Assay Authorized for Emergency Use in Low Prevalence Populations for High-Throughput Testing
date: 2020-09-29
journal: Open Forum Infect Dis
DOI: 10.1093/ofid/ofaa466
sha: 9677bfbd5c33c9a0cea51aaad0bd68313f1c257f
doc_id: 928436
cord_uid: h3vc5x09

BACKGROUND: Nucleic acid amplification testing is a critical tool for addressing the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. Specimen pooling can increase throughput and conserve testing resources but requires validation to ensure that reduced sensitivity does not increase the false-negative rate. We evaluated the performance of a real time reverse transcription PCR (RT-PCR) test authorized by the U.S. Food and Drug Administration (FDA) for emergency use for pooled testing of upper respiratory specimens. METHODS: Positive specimens were selected from three prevalence groups, 1-3%, >3-6%, and >6-10%. Positive percent agreement (PPA) was assessed by pooling single-positive specimens with three negative specimens; performance was assessed using Passing-Bablok regression. Additionally, we assessed the distributions of RT-PCR cycle threshold (Ct) values for 3,091 positive specimens. RESULTS: PPA was 100% for the 101 pooled specimens. There was a linear relationship between Ct values for pooled and single-tested specimens (r: 0.96-0.99, slope ≈ 1). The mean pooled Ct shifts at 40 cycles were 2.38 and 1.90 respectively for the N1 and N3 targets. The median Cts for 3,091 positive specimens were 25.9 (N1) and 24.7 (N3). The percent of positive specimens with Cts between 40 and the shifted Ct was 1.42% (N1) and 0.0% (N3). CONCLUSIONS: Pooled and individual testing of specimens positive for SARS-CoV-2 demonstrated 100% agreement and demonstrate the viability of pooled specimens for SARS-COV-2 testing using a dual-target RT-PCR system. Pooled specimen testing can help increase testing capacity for SARS-CoV-2, with a low risk of false-negative results.

M a n u s c r i p t 3

The ongoing SARS-CoV-2 pandemic has resulted in an unprecedented worldwide demand for laboratory testing. As of 19 July 2020, more than 3.7 million people in the U.S. and over 14 million worldwide have been diagnosed with COVID-19 (https://coronavirus.jhu.edu/map.html). Timely access to SARS-CoV-2 nucleic acid testing is a critical tool for patient management, controlling the spread of the epidemic and informing public health efforts, highlighting the key role of the clinical laboratory in the healthcare system. The average number of daily tests for SARS-CoV-2 performed in the U.S. has reached more than 700,000 (https://covidtracking.com/data/us-daily), however this increased demand for testing has put pressure on the laboratory supply chain, resulting in shortages of testing materials and instrumentation, leading to delays in obtaining test results.

Pooled diagnostic testing offers a means to reduce utilization of testing supplies and reagents while increasing laboratory testing throughput. In a simple Dorfman (1, 2) pooled testing scheme, a number of individually collected specimens are combined in a single well or tube and tested together. If the pooled test result is negative, results for all individual specimens may be immediately reported as negative. If a pool is positive, then each specimen in the pool must be tested individually before the patient results can be reported. The optimal number of specimens that can be included in a pool to maximize efficiency is determined by the prevalence of positive specimens (1, 3, 4) in the population being tested and is further constrained by the sensitivity of the test to reliably detect a positive signal in a diluted negative specimen pool. It is critical therefore, to validate pooling strategies for diagnostics tests to ensure that the false negative rate remains below an acceptable threshold.

Pooling specimens to increase testing efficiency and conserve testing resources has been used for human immunodeficiency virus (HIV) and hepatitis B and C viruses screening (5, 6) . Pooling has also been evaluated for the detection of influenza virus and bacterial pathogens from nasopharyngeal or throat swab specimens (7, 8) and for SARS-CoV-2 testing using pool sizes of between 4 and 64 specimens (3, 4, (9) (10) (11) . Here we describe the performance of pooled testing of A c c e p t e d M a n u s c r i p t 4 upper respiratory specimens for a U.S. FDA emergency use authorized (EUA) real-time reverse transcription PCR (RT-PCR) test performed in low prevalence populations for high throughput testing. This study was performed as part of an FDA EUA application to authorize the use of pooled testing for detecting SARS-CoV-2 RNA.

Clinical specimens. Deidentified specimens collected between May 2020 and early July 2020 that had been previously tested using the Quest Diagnostics SARS-CoV-2 RNA, Qualitative Real-Time RT-PCR EUA were recovered from frozen (-10°C to -30°C) storage. Specimen types included upper respiratory specimens (nasopharyngeal, mid-turbinate, nasal swabs) collected in viral transport media (UTM, UTM-RT), PBS, or normal saline. Swabs were limited to those with a synthetic tip such as Dacron®, Flocked, or Nylon and an aluminum or plastic shaft as specified in the FDA EUA instructions for use (https://www.fda.gov/media/136231/download). Sequentially tested singlepositive and single-negative specimens were selected.

Pooled testing. A pool size of four specimens was selected based on the estimated prevalence of SARS-CoV-2 positive specimens submitted for testing (7-11%) Study design. The Quest Diagnostics Informatics database was utilized to geographically stratify specimens according to SARS-CoV-2 testing positivity rate into three groups: group 1: positivity rate of 1-3%; group 2: positivity rate of 3-6% and group 3: positivity rate of 6-10%. Each prevalence group included specimen remnants selected from at least two separate geographic locations.

Sensitivity studies were performed using single-positive pools by combining one positive sample with three negative samples and evaluated in all three prevalence groups. All single-positive specimens were repeated at the same time as the single-positive pools to ensure that there was no degradation of the archived specimens. The linearity and the shift in the Ct values between pooled and singlicate results was assessed using Passing-Bablok regression (12, 13) . The shifted upper range for the Ct values for the N1 and N3 targets was defined as (40 -y intercept)/slope. Specificity studies were performed in all three prevalence groups by combining four negative samples per pool.

The informatics database was further used to select 3,091 deidentified positive test results from the three defined prevalence groups selected from U.S. counties for in silico analyses. The percent of positive test results in the range between the upper limit of a positive result for the singlicate assay (40 cycles) and the upper limit minus the Ct shift seen in the pools (for example, 37.0 for the N1 detector in prevalence group 1) was then calculated to predict the potential number of high Ct value false negatives in a pooled testing design. Human Subjects. This study utilized deindentified specimen remnants and retrospectively collected deindentified data from previously tested specimens. No human subjects were utilized in this study and thus patient consent was not applicable.

Sensitivity of pooled testing. Three groups of single-positive pooled specimens were prepared using sequentially tested single-positive and single-negative specimen remnants from three prevalence populations as described in Materials and Methods. Of the single-positive samples, 30 were included from group 1, 36 were included from group 2, and 35 were included from group 3 (Supplementary Materials, Table S1 ). Overall, 44.6% of the positive specimens were from women and 52.5% were from men, and the median patient age was 38 years (Supplementary Materials, Table S1 ). We obtained 100% percent positive agreement (PPA) between the pooled and the singlicate tests (Table 1 ). There were two inconclusive pools (Ct values of only one of the two detectors <40) in prevalence group 3, which were positive for SARS-CoV-2 based on singlicate retesting.

We performed Passing-Bablok regression for the N1 and N3 detector Ct values in each of the three groups to assess the linearity between the pooled and singlicate Ct values and to determine the shift in Ct as a measure of the reduction in sensitivity resulting from the dilution of positive samples in a negative pool (Figure 1a-f ). The average regression correlation coefficients for the N1 and N3 targets in the three groups were 0.97 ± 0.007 (SD) and 0.97 ± 0.016 (SD), respectively, indicating good linearity. The average slopes were 1.02 ± 0.033 (SD) for the N1 target and 0.993 ± 0.030 (SD) for the N3 target indicative of minimal proportional bias ( Table 2 ). The average reduction in sensitivity for the pooled tests as measured by the shift in Ct values was 1.71 ± 0.64 (SD) for N1 and 2.17 ± 0.52 (SD) for N3 (Table 2 ). These shifts are close to the shift of 2 Cts which would be the expected estimate for a 1:4 dilution of a positive specimen.

A c c e p t e d M a n u s c r i p t 7 Specificity of pooled testing. Pools were prepared from negative specimens using sequentially tested single-negative specimen remnants from three prevalence populations as described in Table S3 ). The negative percent agreement (NPA) for pooled sample testing was 99% for group 1 (102/103) with one inconclusive pool and 100% for distributions. While less than 1.5% of the specimens had an N1 Ct value in the shifted Ct range, none of the Ct values for the N3 target were in the shifted Ct range. Interestingly, specimens from the highest (6-10%) prevalence group in this population had a slightly lower median Ct for both the N1 and N3 targets than for the two lower prevalence groups. Although this difference was statistically significant, the difference was small and the Ct distributions largely overlapped between the three groups. It is therefore difficult to ascribe any biological or clinical significance to these differences which may be related to geography or other factors, rather than to prevalence. Abdalhamid et al (4) utilized a pool size of five to assess 60 randomly selected specimens drawn from a population with an estimated prevalence of 5%. They identified two positive pools that were subsequently tested individually, resulting in a savings of 38 extractions and RT PCR reactions compared to single sample testing.

However, as the prevalence of SARS-CoV-2 increases in the tested population, the efficiency of pooled testing will decrease. The current positivity rate for SARS-CoV-2 specimens submitted for laboratory testing as of July 2020 is much higher than that for low prevalence screening or asymptomatic populations and the national testing positivity rate is estimated at 7-8% as of early July 2020 (https://covidtracking.com/). At prevalences between 1% and 3%, an eight-specimen pool will yield greater testing efficiencies than a four-specimen pool: for example, at a 3% prevalence a A c c e p t e d M a n u s c r i p t 10 four-specimen pool would require 36 tests per 100 specimens whereas an eight-specimen pool would require only 34 tests (https://bilder.shinyapps.io/PooledTesting/) (4). In contrast, at 8% prevalence, the eight-specimen pool would require 61 tests per 100 specimens compared to only 53 tests per 100 specimens for the four-specimen pool. At a 10% prevalence at least 40% fewer tests would be run with a four-specimen pool strategy compared to single specimen testing (https://bilder.shinyapps.io/PooledTesting/) (4) . The balance between pool size and sensitivity is also a key consideration for pooling strategies. For a pool size of four the dilution factor would be 1:4, Our study had several limitations. First, this study utilized previously tested samples that had been stored frozen and then thawed for retesting in the study. In a previous study in our laboratory (10) , we demonstrated that SARS-CoV-2 specimens in a variety of transport media remain stable while stored under refrigerated or frozen conditions. However, in the current study, specimens were stored frozen for longer periods than previously assessed. The greatest impact would have been to increase Ct values for high-Ct positive specimens. Nevertheless, we had no false-negative results in this study. As pooled testing is intended for use on freshly obtained specimens under the same conditions used for single-specimen testing, retesting of frozen specimens is unlikely to affect pooled testing.

Second, not all transport media or specimen types were evaluated in this pooling study. For example, specimens collected in COPAN Eswab™ were excluded from the pooling studies. Internal A c c e p t e d M a n u s c r i p t 11 stability studies with specimens spiked in COPAN Eswab™ have demonstrated reduced stability (loss of > 3 Cts) following increased freeze-thaw cycles (≥ 2 times; data not shown). Given that this study utilized archived specimens, the optimal conditions for such media may not have been utilized. A variety of collection media, including ESwab™, are approved for CoV testing using commercially available assays. Pooling of specimens in the study was not limited to the use of like media for a pool of four specimens, however the media pooled was limited to UTM, PBS and saline. Since PBS and saline are commonly used as a diluent the impact of the pooling of the different transport media was likely minimum. Further work is needed with prospective specimens before additional media types can be included in a pooling protocol.

Third, no clinical information was available for the patient specimens that were utilized in this study. Thus we were not able to correlate the SARS-CoV-2 test result with clinical diagnoses of COVID 19, stage of disease or presence of symptoms. However given the results we have presented here for pooled testing as well as our retrospective analysis of patient Ct values, pooling of four samples is unlikely to have a significant impact on clinical management. Patients undergoing testing soon after exposure, asymptomatic patients being screened for COVID 19 and positive patients who never go on to develop symptoms may have lower SARS-CoV-2 viral loads that could theoretically impact the clinical accuracy of pooling. In a follow up retrospective analysis (manuscript in preparation) we found that although SARS-CoV-2 positive patients in some of these categories do have higher Ct values, only a very small fraction (<0.15%) of these Cts would fall above the shifted Ct value for the more sensitive N3 RT-PCR target.

Finally, it is important to stress that the implementation of pooled testing in a high-throughput laboratory adds additional operational challenges and complexity to the testing process. It requires the validation of automated liquid handling processes to pool the specimens for testing as well as robust support from one's information technology department to enable laboratory information system (LIS) reporting of pooled results to the LIS. These considerations need to be addressed prior to implementing a pooled testing approach in the laboratory.

A c c e p t e d M a n u s c r i p t 12 In summary, we have demonstrated that pooled testing of four upper respiratory specimens from populations with a prevalence ≤ 10% with a sensitive dual-target RT-PCR test for SARS-CoV-2 is highly correlated to single specimen testing and does not generate false negative test results. This pooling strategy can improve testing capacity while reducing reagent and supply utilization and therefore afford better access and a more rapid turnaround time for patients in need of testing and help to combat the SAR-CoV-2 pandemic. M a n u s c r i p t 16 1 Ct values from positive specimens tested in pools and in singlicate were analyzed by Passing-Bablok regression analysis as shown in Figure 1a -f. 2 The Ct shift at 40 cycles was defined as 40 -(40 -intercept)/slope A c c e p t e d M a n u s c r i p t 18 

The Detection of Defective Members of Large Populations

Pooled-testing procedures for screening high volume clinical specimens in heterogeneous populations

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

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

Pooling of sera for human immunodeficiency virus (HIV) testing: an economical method for use in developing countries

High throughput screening of 16 million serologically negative blood donors for hepatitis B virus, hepatitis C virus and human immunodeficiency virus type-1 by nucleic acid amplification testing with specific and sensitive multiplex reagent in Japan

Cost-effective pooling of DNA from nasopharyngeal swab samples for large-scale detection of bacteria by real-time PCR

Pooling nasopharyngeal/throat swab specimens to increase testing capacity for influenza viruses by PCR

Pooling of samples for testing for SARS-CoV-2 in asymptomatic people

Evaluation of COVID-19 RT-qPCR test in multi-sample pools

Pooled RNA sample reverse transcriptase real time PCR assay for SARS CoV-2 infection: A reliable, faster and economical method

A new biometrical procedure for testing the equality of measurements from two different analytical methods. Application of linear regression procedures for method comparison studies in clinical chemistry, Part I

Comparison of several regression procedures for method comparison studies and determination of sample sizes. Application of linear regression procedures for method comparison studies in Clinical Chemistry, Part II

M a n u s c r i p t 13 ACKNOWLEDGMENTS Financial support. This work was supported by Quest Diagnostics Incorporated (Secaucus, NJ USA).

Incorporated (Secaucus, NJ USA), a diagnostics laboratory that provides SARS-CoV-2 testing.A c c e p t e d M a n u s c r i p t 14 A c c e p t e d M a n u s c r i p t