Detection of SARS-CoV-2 RNA in Upper Respiratory Swap Samples by Pooling Method

Background: Widespread and effective use of molecular diagnostic tests is indispensable for protecting public health and containing the severe respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. More than 1 year into the pandemic, as resources have reached a point of depletion, grouping samples in pools of certain sizes appears to be a reasonable method to reduce both the costs and the processing time without necessitating additional training, equipment, or materials. Aims: To assess whether the pooling strategy that was used in past outbreaks and is used in blood tests prior to transfusion for screening large populations can also be used in SARS CoV-2 tests. Study Design: Diagnostic accuracy study. Methods: This prospective study was conducted with 2815 samples, sent to the coronavirus disease 2019 (COVID-19) Laboratory of our hospital between February 12 and 21, 2021, to be tested for the presence of SARS-CoV-2. The samples were examined individually and in pools of five 100 µl taken from each sequential sample, using 3 different SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) kits, the Allplex™ 2019-nCoV Assay kit (Seegene, Republic of Korea), the GeneMAP™ 2019-nCoV detection V.3 kit (GenMark, Türkiye), and the Bio-Speedy™ SARS-CoV-2 Double Gene™ RT-qPCR kit (Bioeksen, Türkiye) on the BioRAD CFX96™ Touch (Bio-Rad Laboratories Inc., Hercules, CA, USA) platform available in our laboratory. Results: Following the extraction of serial dilutions prepared from the SARS-CoV-2 RNA positive (cycle of threshold: 20) sample, the standard curves of RT-PCR were analyzed. By evaluating the efficiency (E) values, all 3 kits showed high sensitivity and similar results; while the highest level was detected with the Allplex™ 2019- nCoV Assay kit in the nucleocapsid (N) gene (E: 124%), the lowest was detected with the Double Gene™ RT-qPCR kit in the N and ORF 1ab genes (E: 90%). Of the samples included in the study, only 1 positive sample with low viral load was found to be negative when studied by pooling. The total number of kits to be used in pooled tests and then to individually retest the 5 samples in positive pools was calculated as 827 and the savings rate as 69.91% (1968/2815). Conclusion: The pooling strategy is an effective approach to extend the impact of limited testing resources and reagents available in certain periods of the COVID-19 pandemic. Testing by pooling samples requires improvement of RNA extraction methods and careful monitoring of RT-PCR test sensitivity to avoid missing low-positive entities. Therefore, based on the prevalence of COVID-19 in their regions, laboratories should conduct their own validation of pooling studies for RNA extraction and amplification methods they use.

PDF

___

1. Del Rio C, Malani PN. COVID-19: new insights on a rapidly changing epidemic. JAMA. 2020;323:1339-1340. [CrossRef]
2. World Health Organization (WHO). Country & Technical Guidance - Coronavirus Disease (COVID-19). 2020. Available at: https://www.who.int/emergencies/diseases/ novel-coronavirus-2019/technical-guidance.
3. Fomsgaard AS, Rosenstierne MW. An alternative workflow for molecular detection of SARS-CoV-2 - escape from the NA extraction kit-shortage, Copenhagen, Denmark, March 2020. Euro Surveill. 2020;25(14). [CrossRef]
4. KalikiriMKR, Hasan MR, Mirza F, Xaba T, Tang P, Lorenz S. High-throughput extraction of SARS-CoV-2 RNA from nasopharyngeal swabs using solid-phase reverse immobilization beads. medRxiv. 2020. [CrossRef]
5. Aldridge M, Johnson O, Scarlett J. Group testing: an information theory perspective. Found Trends Commun Inf Theory. 2019;15:196-392. [CrossRef]
6. Shani-Narkiss H, Gilday OD, Yayon N, Landau ID. Efficient and practical sample pooling high-throughput PCR diagnosis of COVID-19. medRxiv. 2020. [CrossRef]
7. 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;50:891-896. [CrossRef]
8. Dwyre DM, Fernando LP, Holland PV. Hepatitis B, hepatitis C and HIV transfusion transmitted infections in the 21st century. Vox Sang. 2011;100:92-98. [CrossRef]
9. Dorfman R. The detection of defective members of large populations. Ann Math Statist. 1943;14:436-440. [CrossRef]
10. Gupta E, Padhi A, Khodare A, et al. Pooled RNA sample reverse transcriptase real time PCR assay for SARS CoV-2 infection: a reliable, faster and economical method. PLoS One. 2020;15:e0236859. [CrossRef]
11. Ministry of Health and Family Welfare, Government of India. Advisory on Feasibility of Using Pooled Samples for Molecular Testing of COVID-19. Available at: https:// www.mohfw.gov.in/.
12. Abdalhamid B, Bilder CR, McCutchen EL, Hinrichs SH, Koepsell SA, Iwen PC. Assessment of specimen pooling to conserve SARS CoV-2 testing resources. Am J Clin Pathol. 2020;153:715-718. [CrossRef]
13. Centers for Disease Control and Prevention (U.S.). Research Use Only Real-Time RT-PCR Protocol for Identification of 2019-nCoV Available at: https://www.cdc.gov/ coronavirus/2019-ncov/about/testing.html.
14. Li C, Debruyne D, Spencer J, et al. High sensitivity detection of SARS-CoV-2 using multiplex PCR and a multiplex-PCR-based metagenomic method. bioRxiv. 2020. [CrossRef]
15. Guha P, Guha A, Bandyopadhyay T. Application of pooled testing in screening and estimating the prevalence of Covid-19. Health Serv Outcomes Res Method. 2021. [CrossRef]
16. Goyal S, Bist P, Sharma R. Optimal sample pooling: an efficient tool against SARS CoV-2. medRxiv. 2020. [CrossRef]
17. Ben-Ami R, Klochendler A, Seidel M, et al. Large-scale implementation of pooled RNA extraction and RT-PCR for SARS-CoV-2 detection. Clin Microbiol Infect. 2020;26:1248-1253. [CrossRef]
18. Randazzo W, Truchado P, Cuevas-Ferrando E, Simón P, AllendeA, Sánchez G. SARS CoV-2 RNA in wastewater anticipated COVID-19 occurrence in a low prevalence area. Water Res. 2020;181:115942. [CrossRef]
19. Orive G, Lertxundi U, Barcelo D. Early SARS-CoV-2 outbreak detection by sewage based epidemiology. Sci Total Environ. 2020;732:139298. [CrossRef]