Comparison of nucleocapsid antigen with strand-specific reverse-transcription PCR for monitoring SARS-CoV-2 infection

Alexandra L Chang-Graham; Malaya K Sahoo; ChunHong Huang; Daniel Solis; Mamdouh Sibai; Gianna August; Lira Calayag; Obadia M Kenji; Run-Zhang Shi; Heba H Mostafa; Guang-Sheng Lei; Ryan F Relich; Benjamin A Pinsky

doi:10.1016/j.jcv.2023.105468

Comparison of nucleocapsid antigen with strand-specific reverse-transcription PCR for monitoring SARS-CoV-2 infection

J Clin Virol. 2023 Jul:164:105468. doi: 10.1016/j.jcv.2023.105468. Epub 2023 Apr 24.

Authors

Affiliations

¹ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
² Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Clinical Virology Laboratory, Stanford Health Care, Stanford, CA, USA.
³ Clinical Virology Laboratory, Stanford Health Care, Stanford, CA, USA.
⁴ Special Chemistry and Immunology Laboratory, Stanford Health Care, CA, USA.
⁵ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Special Chemistry and Immunology Laboratory, Stanford Health Care, CA, USA.
⁶ Department of Pathology, Division of Medical Microbiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
⁷ Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA.
⁸ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; Clinical Virology Laboratory, Stanford Health Care, Stanford, CA, USA; Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA, USA. Electronic address: bpinsky@stanford.edu.

Abstract

Background: Tests that sensitively detect the presence of actively replicating SARS-CoV-2 may improve patient care by allowing the safe and timely discontinuation of isolation. Correlates of active replication include nucleocapsid antigen and virus minus-strand RNA.

Methods: Qualitative agreement of the DiaSorin LIAISON SARS-CoV-2 nucleocapsid antigen chemiluminescent immunoassay (CLIA) with minus-strand RNA was determined using 402 upper respiratory specimens from 323 patients previously tested using a laboratory-developed SARS-CoV-2 strand-specific RT-qPCR. Nucleocapsid antigen levels, minus-strand and plus-strand cycle threshold values, as well as virus culture, were used to evaluate discordant specimens. Receiver operating characteristic curves were also used to identify virus RNA thresholds for active replication, including values harmonized to the World Health Organization International Standard.

Results: Overall agreement was 92.0% [95% confidence interval (CI): 89.0 - 94.5], positive percent agreement was 90.6% (95% CI: 84.4 - 95.0), and negative percent agreement was 92.8% (95% CI: 89.0 - 95.6). The kappa coefficient was 0.83 (95% CI: 0.77 - 0.88). Discordant specimens contained low levels of nucleocapsid antigen and minus-strand RNA. 84.8% (28/33) were negative by culture. Sensitivity-optimized plus-strand RNA thresholds for active replication were 31.6 cycles or 3.64 log₁₀ IU/mL; resulting in 100.0% sensitivity (95% CI: 97.6 to 100.0) and 55.9 specificity (95% CI: 49.7 to 62.0).

Conclusions: Detection of nucleocapsid antigen by CLIA performs equivalently to minus-strand detection via strand-specific RT-qPCR, though these methods may overestimate replication-competent virus compared to culture. Careful implementation of biomarkers for actively replicating SARS-CoV-2 has the potential to inform infection control decision-making and patient management.

MeSH terms

COVID-19 Testing
COVID-19* / diagnosis
Humans
Nucleocapsid
Polymerase Chain Reaction
RNA, Viral / genetics
SARS-CoV-2 / genetics
Sensitivity and Specificity

Substances

RNA, Viral