Challenges and issues of SARS-CoV-2 pool testing – Authors' reply
Authors: Stefan Lohse, Thorsten Pfuhl, Barbara Berkó-Göttel, Barbara Gärtner, Sören L Becker, Sophie Schneitler, Sigrun Smola
Journal: The Lancet Infectious Diseases (2020)

We appreciate the comments on our letter,1Lohse S Pfuhl T Berkó-Göttel B et al.Pooling of samples for testing for SARS-CoV-2 in asymptomatic people.Lancet Infect Dis. 2020; (published online April 28.)https://doi.org/10.1016/S1473-3099(20)30362-5Summary Full Text Full Text PDF PubMed Scopus (231) Google Scholar in which we described a strategy to identify asymptomatic people infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in large populations of uninfected people when testing capacity is low and laboratory infrastructure is overwhelmed. We proposed pool testing to screen for individuals who might spread SARS-CoV-2 without showing any symptoms. He and colleagues2He X Lau EHY Wu P et al.Temporal dynamics in viral shedding and transmissibility of COVID-19.Nat Med. 2020; 26: 672-675Crossref PubMed Scopus (2883) Google Scholar reported temporal patterns of viral shedding and inferred from their data that viral load peaks 0·7 days before symptom onset and estimated that 44% of SARS-CoV-2 infections occur during the pre-symptomatic stage of the index case. Studies indicate a steady decline of viral loads and infectiousness after symptom onset.2He X Lau EHY Wu P et al.Temporal dynamics in viral shedding and transmissibility of COVID-19.Nat Med. 2020; 26: 672-675Crossref PubMed Scopus (2883) Google Scholar, 3To KK Tsang OT Leung WS et al.Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study.Lancet Infect Dis. 2020; 20: 565-574Summary Full Text Full Text PDF PubMed Scopus (2309) Google Scholar We recommended restricting pool testing to asymptomatic people, excluding patients with severe acute respiratory illness and high-risk contacts, and for when testing capacity is limited.1Lohse S Pfuhl T Berkó-Göttel B et al.Pooling of samples for testing for SARS-CoV-2 in asymptomatic people.Lancet Infect Dis. 2020; (published online April 28.)https://doi.org/10.1016/S1473-3099(20)30362-5Summary Full Text Full Text PDF PubMed Scopus (231) Google Scholar For symptomatic individuals we use individual testing so not to miss patients with low or borderline SARS-CoV-2 viral loads and to obtain diagnostic results as quickly as possible. We proposed that pool sizes should "accommodate different infection scenarios", which should "be optimised according to infrastructure constraints", and suggested that "sub-pools can further optimise resource use when infection prevalence is low."1Lohse S Pfuhl T Berkó-Göttel B et al.Pooling of samples for testing for SARS-CoV-2 in asymptomatic people.Lancet Infect Dis. 2020; (published online April 28.)https://doi.org/10.1016/S1473-3099(20)30362-5Summary Full Text Full Text PDF PubMed Scopus (231) Google Scholar We investigated 164 pools in total and mainly used pool sizes of five (81 pools) or ten (45 pools), as further illustrated in the appendix. Beyond that, we explored the pooling of 30 samples with sub-pools of ten samples (five pools; appendix). During the period of our study (March 13–21, 2020), the positivity rate was below 2% in samples examined by pool testing. We agree with Jens Eberhardt and colleagues that pool sizes should be smaller than 30 when prevalence is higher than 2%. We agree with Baijayantimala Mishra and colleagues that the swab sampling procedure is of paramount importance. The testing for host nucleic acid might help to confirm accurate sampling and improve the accuracy of a negative test result. Yet, as SARS-CoV-2 is released in mucous or saliva,3To KK Tsang OT Leung WS et al.Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study.Lancet Infect Dis. 2020; 20: 565-574Summary Full Text Full Text PDF PubMed Scopus (2309) Google Scholar the absence of cellular control signals does not necessarily prove that mucous or saliva is missing and that swab taking needs to be redone. Jaehyeon Lee and colleagues highlighted another point in our letter. In our study, we used the Copan Liquid Amies Elution Swab Collection and Preservation System for sampling and pooled media before RNA extraction. We performed nucleic acid extraction from 400 μL of our single or pooled samples. From theoretical considerations one would expect dilution effects and a resulting increase of Ct values in pools. We observed a puzzling increase of detection sensitivity in pools containing single samples with high Ct values (appendix). This increase did not occur randomly but was reproducible, systematic, and significantly associated with higher Ct value samples for both E-gene and S-gene RT-PCRs (appendix). To explain our observation, we hypothesised that samples with higher Ct values might have gained detection efficiency through an RNA carrier effect in pools from the other negative samples with potentially higher cellular RNA content. We are currently addressing this interesting question in a further study, as well as whether different swab collection systems affect this phenomenon. The concern raised by Lee and colleagues that this phenomenon might cause false-positive results is not supported by our data obtained with now more than 3900 pools assessed in our institution since March, 2020. In a broader context, several distinct steps contribute to accurate test results. Major contributing factors are adequate sample collection, quality of swabs, transport media, efficient nucleic acid extraction from a sufficient amount of material, and a highly sensitive detection method. All these steps need to be optimised and validated within the laboratory to obtain optimal pool testing efficiency and accuracy. Owing to highly diverse laboratory settings, it might be difficult to harmonise worldwide pool testing protocols for SARS-CoV-2. However, we would be grateful if national authorities could guide SARS-CoV-2 pool testing procedures as has been done for blood donor pool testing in Germany4Paul-Ehrlich-InstitutDonor screening.https://www.pei.de/EN/regulation/marketing-authorisation-human/blood-components/bc-node.html?cms_tabcounter=3Date accessed: May 22, 2020Google Scholar and recently been announced by the US Food and Drug Administration for SARS-CoV-2.5US FDACoronavirus (COVID-19) update: facilitating diagnostic test availability for asymptomatic testing and sample pooling.https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-facilitating-diagnostic-test-availability-asymptomatic-testing-andDate accessed: June 23, 2020Google Scholar We declare no competing interests. Intramural funding was obtained from Saarland University Medical Center. Download .pdf (.79 MB) Help with pdf files Supplementary appendix Challenges and issues of SARS-CoV-2 pool testingWe read with interest the Correspondence by Stefan Lohse and colleagues,1 who evaluated the practicability of pool testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Pooling of samples yields considerable savings of test kits when the prevalence of infection is low because pools with all-negative samples can be discarded with a single test. Lohse and colleagues' findings suggest that pooling up to 30 samples is technically feasible with currently used and commercially available SARS-CoV-2 RT-PCR kits. Full-Text PDF Pooling of samples for testing for SARS-CoV-2 in asymptomatic peopleThe ongoing coronavirus disease 2019 (COVID-19) pandemic is a substantial challenge for health-care systems and their infrastructure. RT-PCR-based diagnostic confirmation of infected individuals is crucial to contain viral spread because infection can be asymptomatic despite high viral loads. Sufficient molecular diagnostic capacity is important for public health interventions such as case detection and isolation, including for health-care professionals.1 Full-Text PDF Challenges and issues of SARS-CoV-2 pool testingWe read with interest Stefan Lohse and colleagues' Correspondence about sample pooling for testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in asymptomatic people.1 Some of the findings Lohse and colleagues report do not seem to be consistent with other research results2,3 nor our experiences. Full-Text PDF Challenges and issues of SARS-CoV-2 pool testingStefan Lohse and colleagues1 described a sample pooling strategy for testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via RT-PCR to meet the unprecedented demand for laboratory testing. Lohse and colleagues evaluated a range of pool sizes (four to 30 samples per pool) in asymptomatic people. The additional time to deconvolute the larger pools yielding a positive result into sub-pools precludes the use of this strategy in patients with severe acute respiratory illness and high-risk contacts. Full-Text PDF