An Evaluation of Pooling Strategies for RT-qPCR testing for SARS-CoV-2 Infection A Pragmatic Multi-site Parallel Operational Study

Main Article Content

Raymundo Lo
Agnes Barrientos
Bernadette Espiritu
Farrah Kristine Santiago
Amado Tandoc III
http://orcid.org/0000-0002-2186-4989
Januario Veloso
Socorro Yanez

Abstract

Background and Objectives. Sample pooling of COViD-19 PCR tests has been recently proposed as a low-cost alternative to individual tests.  This multi-site, laboratory-based, proof-of-concept study explores the feasibility of pooled SARS-CoV-2 RT-qPCR testing, by demonstrating the effect of pooling on sensitivity, specificity, accuracy, number of tests saved, and turnaround time. 


Methodology.  The research was conducted in two experiments.  In Experiment 1, archival nasopharyngeal (NPS) and oropharyngeal (OPS) swab samples were diluted to simulate 5, 10, and 20 sized pools, and tested for SARS-CoV-2 RNA using RT-qPCR.   In Experiment 2, actual nasopharyngeal and oropharyngeal swab samples were collected from asymptomatic low-risk volunteers.  Aliquots of the samples were pooled following the 5, 10-5, and 20-10-5 multi-staged Dorfman pooling methods and tested.  The sensitivity, specificity, accuracy, test savings, and turnaround time for each pooling method were documented. 


Results and Conclusions.  The study provided evidence that pooling of NP and OP samples for SARS-CoV-2 RNA detection using RT-qPCR is feasible and can be implemented in the Philippines. A 2-stage Dorfman 5 pooling strategy appears to be the best method, because it has the highest over-all accuracy, while still achieving acceptable test savings, and turnaround time. Pooling of nasopharyngeal and oropharyngeal swab samples prior to RT-qPCR testing may be considered by select molecular diagnostic laboratories to further increase testing capacity and at the same time reduce the cost of testing.

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How to Cite
Lo, R., Barrientos, A., Espiritu, B., Santiago, F. K., Tandoc III, A., Veloso, J., & Yanez, S. (2021). An Evaluation of Pooling Strategies for RT-qPCR testing for SARS-CoV-2 Infection: A Pragmatic Multi-site Parallel Operational Study . Philippine Journal of Pathology, 5(2), 12-33. https://doi.org/10.21141/PJP.2020.12
Section
Original Articles
Author Biographies

Raymundo Lo, Philippine Children's Medical Center

Head, COVID-19 Testing Laboratory

Agnes Barrientos, University of Perpetual Help System DALTA Medical Center, Las Pinas

Department of Pathology and Laboratory Services

Farrah Kristine Santiago, Philippine Children's Medical Center

Pathology Division

Amado Tandoc III, Research Institute for Tropical Medicine

Chief, Laboratory Research Division

Januario Veloso, University of the Philippines-Philippine General Hospital

Division Chief, Medical Research Laboratory

Socorro Yanez, University of the East Ramon Magsaysay Memorial Medical Center

Head, Section of Anatomic Pathology

References

1. Fetalco M. DOH confirms 32,000 estimated testing capacity. PTV News; 2020. https://ptvnews.ph/doh-confirms-32000-estimated-testing-capacity/.

2. Department of Health. Philippines now has 100 licensed testing labs. https://www.doh.gov.ph/doh-press%20release/PHILIPPINES-NOW-HAS-100-LICENSED-TESTING-LABS-%E2%80%93-DOH.

3. Ritchie H, Ortiz-Ospina E, Beltekian D, et al. Coronavirus pandemic (COVID-19). Our World in Data; 2020. https://ourworldindata.org/coronavirus.

4. Lancet COVID-19 Commissioners, task Force Chairs and Commission Secretariat. Lancet COVID-19 Commission Statement on the occasion of the 75th session of the UN General Assembly. Lancet. 2020;396(10257):1102-24. PMID: 32941825. PMCID: PMC7489891. https://doi.org/10.1016/S0140-6736(20)31927-9.

5. Lesmoras M. Pres. Duterte places NCR, Region 2, 3, 4A, Davao City, Pangasinan, Albay under GCQ. PTV News; 2020. https://www.ptvnews.ph/pres-duterte-places-ncr-region-2-3-4a-davao-city-pangasinan-albay-under-gcq/.

6. Padua Jr. R. Diagnostic testing strategies to manage COVID-19 pandemic: proposed by the Philippine Society of Pathologists, Inc. Philipp J Pathol. 2020; 5(1), 5-8. https://doi.org/10.21141/.PJP.2020.08.

7. Caoili SEC, King RAN, Dungog CC, Reyes JCB, Sarol Jr. JN, de Castro RJ. Sample pooling in a resource-limited situation. PAASE Bulletin 34. On PAASE strategic action group 3: mass testing & fast-tracking. 2020.

8. Shani-Narkiss H, Gilday OD, Yayon N, Landau ID. Efficient and practical sample pooling for high-throughput PCR diagnosis of COVID-19. medRxiv. 2020. https://doi.org/10.1101/2020.04.06.20052159.

9. Abdalhamid B, Bilder CR, McCutchen EL, et al. Assessment of specimen pooling to conserve SARS CoV-2 testing resources. Am J Clin Pathol. 2020;153(6):715-8. PMID: 32304208. PMCID: PMC7188150. https://doi.org/10.1093/ajcp/aqaa064.

10. Bilder CR, Tebbs JM. Pooled-testing procedures for screening high volume clinical specimens in heterogeneous populations. Stat Med. 2012;31(27):3261-8. PMID: 22415972. PMCID: PMC3500568. https://doi.org/10.1002/sim.5334.

11. Deckert A, Bärnighausen T, Kyei NN. Stimulation of pooled-sample analysis strategies for COVID-19 mass testing. Bull World Health Organ. 2020;98(9):590-8. PMID: 33012859. PMCID: PMC7463190. https://doi.org/10.2471/BLT.20.257188.

12. Hirotsu Y, Maejima M, Shibusawa M, et al. Pooling RT-PCR test of SARS-CoV-2 for large cohort of ‘healthy’ and infection-suspected patients: a prospective and consecutive study on 1,000 individuals. medRxiv. 2020 https://doi.org/10.1101/2020.05.04.20088146.

13. Hogan CA, Sahoo MK, Pinsky BA. Sample pooling as a strategy to detect community transmission of SARS-CoV-2. JAMA. 2020;323(19):1967-9. PMID: 32250394. PMCID: PMC7136853. https://doi.org/10.1001/jama.2020.5445.

14. Perchetti GA, Sullivan KW, Pepper G, et al. Pooling of SARS-CoV-2 samples to increase molecular testing throughput. J Clin Virol. 2020;131:104570. PMID: 32805524. PMCID: PMC7396208. https://doi.org/10.1016/j.jcv.2020.104570.

15. Mulu A, Dawit HA, Fekadu A, et al. Evaluation of Sample Pooling for Screening of SARS-CoV-2. medRxiv. 2020. https://doi.org/10.1101/2020.06.10.20123398.

16. Litvak E, Tu XM, Pagano M. Screening for the presence of a disease by pooling sera samples. J Am Stat Assoc. 1994;89(426):424-34. https://doi.org/10.1080/01621459.1994.10476764.

17. Noriega R, Samore MH. Increasing testing throughput and case detection with a pooled-sample Bayesian approach in the context of COVID-19. bioRxiv. 2020. https://doi.org/10.1101/2020.04.03.024216.

18. Phatarfod RM, Sudbury A. The use of a square array scheme in blood testing. Stat Med. 1994;13(22):2337–43. PMID: 7855467. https://doi.org/10.1002/sim.4780132205.

19. Shental N, Levy S, Wuvshet V, et al. Efficient high throughput SARS-CoV-2 testing to detect asymptomatic carriers. medRxiv. 2020. https://doi.org/10.1101/2020.04.14.20064618.

20. Sterrett A. On the detection of defective members of large populations. Ann Math Stat. 1957;28(4):1033-6. https://www.jstor.org/stable/2237067.

21. Torres I, Albert E, Navarro D. Pooling of nasopharyngeal swab specimens for SARS-CoV-2 detection by RT-PCR. J Med Virol. 2020;92(11):2306-7. PMID: 32369202. PMCID: PMC7267454. https://doi.org/10.1002/jmv.25971.

22. Van TT, Miller J, Warshauer DM, et al. Pooling nasopharyngeal/throat swab specimens to increase testing capacity for influenza viruses by PCR. J Clin Microbiol. 2012;50(3):891-6. PMID: 22205820. PMCID: PMC3295167. https://doi.org/10.1128/JCM.05631-11.

23. Yelin I, Aharony N, Tamar ES, et al. Evaluation of COVID-19 RT-qPCR test in multi-sample pools. Clin Infect Dis. 2020;71(16):2073-8. PMID: 32358960. PMCID: PMC7197588. https://doi.org/10.1093/cid/ciaa531.

24. Lohse S, Pfuhl T, Berkó-Göttel, et al. Pooling of samples for testing for SARS-CoV-2 in asymptomatic people. Lancet Infect Dis. 2020;20(11):1231-2. PMID: 32530425. PMCID: PMC7194818. https://doi.org/10.1016/S1473-3099(20)30362-5.

25. Dorfman R. The detection of defective members of large populations. Ann Math Stat. 1943;14(4):436-40. https://webpages.tuni.fi/uta_statistics/tilasto/liski-arkisto/mtt-perusteet10/mttp-kurssi10/Materiaalia/Dorfman-Ann1943.pdf.

26. Roth WK. History and future of nucleic acid amplification technology blood donor testing. Transfus Med Hemother. 2019;46:67-75. https://doi.org/10.1159/000496749.

27. US Department of Health and Human Services Food and Drug Administration Center for Biologics Evaluation and Research. Guidance for industry: use of nucleic acid tests on pooled and individual samples from donors of whole blood and blood components (including source plasma and source leukocytes) to adequately and appropriately reduce the risk of transmission of HIV-1 and HCV;2004. https://www.fda.gov/media/124349/download.

28. Seli HM, ElBashaar MA,. ElWakil SG. Donor minipool NAT screening for HBV, HCV and HIV: a 2-year experience in a private hospital in Saudi Arabia. Comp Clin Pathol. 2014;23:1125-32. https://doi.org/10.1007/s00580-013-1751-3.

29. Department of Health. Updated interim guidelines on expanded testing for COVID-19. 2020. https://www.doh.gov.ph/node/22681.

30. Kim SY, Lee J, Sung H, et al. Pooling upper respiratory specimens for rapid mass screening of COVID-19 by real-time RT-PCR. Emerg Infect Dis. 2020;26(10):2469-72. PMID: 32844739. PMCID: PMC7510748. https://doi.org/10.3201/eid2610.201955.

31. Mina MJ, Parker R, Larremore DB. Rethinking Covid-19 Test sensitivity — a strategy for containment. New Engl J Med. 2020;383(22):e120. PMID: 32997903. https://doi.org/0.1056/NEJMp2025631.

32. Philippine Society of Pathologists Inc. "Recommendations in the DOH Interim Guidelines for COVID-19 Pooled Testing". Aug 15 2020.

33. Standard Method for Verification of Pooled Testing: Interim Guidance for Laboratories in the COVID-19 Laboratory Network. Aug 17, 2020.

32. Ceron JJ, Lamy E, Martinez-Subiela S, et al. Use of saliva for diagnosis and monitoring the SARS-CoV-2: a general perspective. J Clin Med. 2020;9(5):1491. PMID: 32429101. PMCID: PMC7290439. https://doi.org/10.3390/jcm9051491.

33. Czumbel LM, Kiss S, Farkas N, et al. Saliva as a candidate for COVID-19 diagnostic testing: a meta-analysis. Front Med (Lausanne). 2020;7:465. PMID: 32903849. PMCID: PMC7438940. https://doi.org/10.3389/fmed.2020.00465.

34. Fakheran, O, Dehghannejad M, Khademi A. Saliva as a diagnostic specimen for detection of SARS-CoV-2 in suspected patients: a scoping review. Infect Dis Poverty 2020;9(1):100. PMID: 32698862. PMCID: PMC7374661. https://doi.org/10.1186/s40249-020-00728-w.

35. Iwasaki S, Fujisawa S, Nakakubu S, et al. Comparison of SARS-CoV-2 detection in nasopharyngeal swab and saliva. J Infect. 2020;81(2):e145-7. PMID: 32504740. PMCID: PMC7270800. https://doi.org/10.1016/j.jinf.2020.05.071.

36. Ott IM, Strine MS, Watkins AE, et al. Simply saliva: stability of SARS-CoV-2 detection negates the need for expensive collection devices. medRxiv 2020.08.03.20165233. PMID: 32793924. PMCID: PMC7418742. https://doi.org/10.1101/2020.08.03.20165233.

37. Pasomsub E, Watcharananan SP, Boonyawat K, et al. Saliva sample as a non-invasive specimen for the diagnosis of coronavirus disease-2019 (COVID-19): a cross-sectional study. Clin Microbiol Infect 2020. S1198-743X(20)30278-0. PMID: 32422408. PMCID: PMC7227531. https://doi.org/10.1016/j.cmi.2020.05.001.

38. Ranoa DRE, Holland RL, Alnaji FG, et al. Saliva-based molecular testing for SARS-CoV-2 that bypasses RNA extraction. bioRxiv. 2020. https://doi.org/10.1101/2020.06.18.159434.

39. Vogels CBF, Brackney D, Wang J, et al. SalivaDirect: simple and sensitive molecular diagnostic test for SARS-CoV2 surveillance. medRxiv. 2020. https://doi.org/10.1101/2020.08.03.20167791.

40. UI receives FDA approval for saliva-based COVID-19 test first offered on campus. 2020. https://www.wcia.com/news/ui-receives-fda-approval-for-saliva-based-test-covid-19-test-first-offered-on-campus/.

41. Wyllie AL, Fournier J, Casanovas-Massana A, et al. Saliva or nasopharyngeal swab specimens for detection of SARS-CoV-2. N Engl J Med. 2020;383(13):1283-6. PMID: 32857487. PMCID: PMC7484747. https://doi.org/10.1056/NEJMc2016359.

42. Xu J, Li Y, Gan F, Du Y, Yao Y. Salivary glands: potential reservoirs for COVID-19 asymptomatic Infection. J Dent Res. 2020; 99(8):989. PMID: 32271653. https://doi.org/10.1177/0022034520918518.