ORIGINAL RESEARCH article
Front. Microbiol.
Sec. Virology
Volume 16 - 2025 | doi: 10.3389/fmicb.2025.1675605
Improving the Diagnostic Performance for Prior COVID-19 with T-SPOT, an Interferon-Gamma Release Assay
Provisionally accepted
Masaki Yamamoto1*
Michael White2
Takahisa Kawaguchi1Kazuya Setoh3Yusuke Tsuda1
Koh Shinohara1
Yasuhiro Tsuchido1
Yasufumi Matsumura1Fumihiko Matsuda1
Miki Nagao1- 1Kyoto University Graduate School of Medicine, Kyoto, Japan
- 2Institut Pasteur, Paris, France
- 3Kyoto Prefectural University of Medicine, Kyoto, Japan
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Abstract Introduction: The precise diagnosis of a prior COVID-19 infection remains challenging. This study aimed to evaluate the efficacy of T-SPOT assays for diagnosing prior SARS-CoV-2 infections by using frozen peripheral blood mononuclear cells (PBMCs) combined with antibody tests. Methods: The study included 122 participants with PCR-confirmed COVID-19 (the positive control cohort) and 67 participants with no evidence of prior infection (the negative control cohort). Antibody testing was conducted using iFlash-SARS-CoV-2 IgG (YHLO, iF_N) and MAGPIX® assays (Luminex, Lumi_N), which target the nucleocapsid protein. T-SPOT® Discovery SARS-CoV-2 assays (Oxford Immunotec) were used to detect cell-mediated immune responses against nucleocapsid (Tspot_N) and membrane (Tspot_M) proteins. Results: Antibody tests had similar sensitivities (if_N: 67.2% and Lumi_N: 64.8%) and specificities (> 98.4%). The Tspot_N assay demonstrated comparable performance to the antibody tests, with a sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) of 62.5% (95% confidence interval: 52.0%-72.2%), 98.4% (95% CI: 91.2%-100.0%), and 0.923, respectively. The Tspot_M assay had lower sensitivity (15.3%). The combination of the Tspot_N test and the Lumi_N antibody test significantly improved the sensitivity and AUC to 88.0% and 0.979, respectively (p = 0.012). Net reclassification improvement and integrated discrimination improvement analyses further supported the improved diagnostic performance of the combination assay. Conclusion: Frozen PBMCs were useful for performing T-SPOT assays. The combination of T-SPOT assays targeting nucleocapsid protein and antibody tests improved the diagnosis of past SARS-CoV-2 infections in vaccinated participants. These findings suggest that integrating cellular and humoral immunity assays can facilitate COVID-19 prevalence studies.
Keywords: COVID-19, diagnosis, antibody test, IGRA, T-SPOT
Received: 29 Jul 2025; Accepted: 09 Sep 2025.
Copyright: © 2025 Yamamoto, White, Kawaguchi, Setoh, Tsuda, Shinohara, Tsuchido, Matsumura, Matsuda and Nagao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Masaki Yamamoto, Kyoto University Graduate School of Medicine, Kyoto, Japan
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