Exploring beyond the limit: How comparative stochastic performance affects retesting outcomes in six commercial SARS CoV-2 nucleic acid amplification tests

Hiu Tat Chan; Marco H T Keung; Ivy Nguyen; Ellen L O Ip; Su M Chew; Danielle Siler; Marion Saville; David Hawkes

doi:10.1016/j.jcvp.2022.100079

Exploring beyond the limit: How comparative stochastic performance affects retesting outcomes in six commercial SARS CoV-2 nucleic acid amplification tests

J Clin Virol Plus. 2022 Aug;2(3):100079. doi: 10.1016/j.jcvp.2022.100079. Epub 2022 Apr 30.

Authors

Hiu Tat Chan^{1

2}, Marco H T Keung¹, Ivy Nguyen¹, Ellen L O Ip¹, Su M Chew¹, Danielle Siler³, Marion Saville^{1

4

5}, David Hawkes^{1

6

7}

Affiliations

¹ VCS Pathology, Australian Centre for the Prevention of Cervical Cancer, 265 Faraday Street, Carlton South, VIC 3053, Australia.
² Department of Physiology, Anatomy and Microbiology, La Trobe University, Bundoora, VIC 3086, Australia.
³ IPC Health, Deer Park, VIC 3023, Australia.
⁴ Department of Obstetrics and Gynaecology, University of Melbourne, Parkville, VIC 3010, Australia.
⁵ Department of Obstetrics and Gynaecology, University of Malaya, Kuala Lumpur 50603, Malaysia.
⁶ Department of Pharmacology and Biochemistry, University of Melbourne, Parkville, VIC 3010, Australia.
⁷ Department of Pathology, University of Malaya, Kuala Lumpur 50603, Malaysia.

Abstract

Objectives: To examine the comparative stochasticity profile of six commercial SARS-CoV-2 nucleic acid amplification tests (NAATs) and how this may affect retesting paradigms.

Methods: Commercial quality control (QC) material was serially diluted in viral transport media to create a panel covering 10-10,000 copies/ml. The panel was tested across six commercial NAATs. A subset of high cycle threshold results was retested on a rapid PCR assay to simulate retesting protocols commonly used to discriminate false positives.

Results: Performance beyond the LOD differed among assays, with three types of stochasticity profiles observed. The ability of the rapid PCR assay to reproduce a true weak positive specimen was restricted to its own stochastic performance at the corresponding viral concentration.

Conclusion: Stochastic performance of various NAATs overlap across low viral concentrations and affect retesting outcomes. Relying on retesting alone to discriminate false positives risk missing true positives even when a more sensitive assay is deployed for confirmatory testing.

Keywords: Comparative sensitivity; False negative; False positive; Limit of detection; Nucleic acid amplification tests; SARS-CoV-2; Stochastic performance.