{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,12]],"date-time":"2026-05-12T06:37:11Z","timestamp":1778567831674,"version":"3.51.4"},"reference-count":45,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,11,5]],"date-time":"2025-11-05T00:00:00Z","timestamp":1762300800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["CIMB"],"abstract":"<jats:p>Indeterminate serological results for SARS-CoV-2 antibodies create diagnostic uncertainty, requiring repeat testing after 14\u201321 days to establish seroconversion. This study evaluated whether direct viral detection methods could provide immediate diagnostic information in serum samples with indeterminate antibody results. We analyzed 163 serum samples from clinically healthy individuals collected during March\u2013December 2020 in Bulgaria. Samples were categorized by screening ELISA (IgA\/M\/G) as positive (n = 69), negative (n = 47), or indeterminate (n = 47). All samples underwent quantitative IgG ELISA, rapid antibody tests, rapid antigen detection (viral nucleoprotein), and RT-nested PCR. Among samples with indeterminate antibody results, 27.7% (13\/47; 95% CI: 15.6\u201342.6%) tested positive by rapid antigen detection and 12.8% (6\/47; 95% CI: 4.8\u201325.7%) by RT-PCR. All PCR-positive samples were also antigen-positive (Cohen\u2019s \u03ba = 0.69). Viral detection rates showed a gradient: antibody-positive samples 30.4% (antigen) and 16.4% (PCR), indeterminate samples 27.7% and 12.8%, antibody-negative samples 10.6% and 4.3%, respectively. The algorithm we proposed and the diagnostic methods used enable the application of certain approaches to differentiate infected from uninfected clinically healthy people, in case of intermediate antibody results. Direct viral detection identified evidence of potential SARS-CoV-2 infection in more than one-quarter of sera with indeterminate antibody results. These findings suggest immediate viral detection testing may complement standard serological approaches, though clinical validation through longitudinal studies is essential before routine implementation.<\/jats:p>","DOI":"10.3390\/cimb47110919","type":"journal-article","created":{"date-parts":[[2025,11,5]],"date-time":"2025-11-05T17:06:06Z","timestamp":1762362366000},"page":"919","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Molecular and Serological Tests for SARS-CoV-2 Detection in Indeterminate Serology: Can We Skip the Second Sample?"],"prefix":"10.3390","volume":"47","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4765-3231","authenticated-orcid":false,"given":"Ivo N.","family":"Sirakov","sequence":"first","affiliation":[{"name":"Department of Medical Microbiology \u201cCorr. Mem. Prof. Ivan Mitov, MD, DMSc\u201d, Faculty of Medicine, Medical University of Sofia, 2 \u201cZdrave\u201d Str., 1431 Sofia, Bulgaria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kalina","family":"Shishkova","sequence":"additional","affiliation":[{"name":"Laboratory of Virology, Faculty of Biology, University of Sofia \u201cSt. Kl. Ohridski\u201d, 1164 Sofia, Bulgaria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8203-3812","authenticated-orcid":false,"given":"Stoyan","family":"Shishkov","sequence":"additional","affiliation":[{"name":"Laboratory of Virology, Faculty of Biology, University of Sofia \u201cSt. Kl. Ohridski\u201d, 1164 Sofia, Bulgaria"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3186-1124","authenticated-orcid":false,"given":"Ivailo","family":"Alexiev","sequence":"additional","affiliation":[{"name":"National Reference Laboratory of HIV, National Center of Infectious and Parasitic Diseases, 1233 Sofia, Bulgaria"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2246","DOI":"10.1109\/JBHI.2024.3509630","article-title":"Early Time Series Classification Using Reinforcement Learning for Pre-Symptomatic COVID-19 Screening From Imbalanced Health Tracker Data","volume":"29","author":"Sarwar","year":"2024","journal-title":"IEEE J. Biomed. Health Inform."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"861","DOI":"10.3934\/mbe.2024036","article-title":"Quantifying the presymptomatic transmission of COVID-19 in the USA","volume":"21","author":"Zhang","year":"2024","journal-title":"Math. Biosci. Eng."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e220","DOI":"10.1136\/bmjspcare-2020-002813","article-title":"Asymptomatic COVID-19 infection: Diagnosis, transmission, population characteristics","volume":"14","author":"You","year":"2024","journal-title":"BMJ Support. Palliat. Care"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Marin, L., de Miranda, L.S., Carvalho, V.H.S., Voigt, M.E.F., Martire, J.P.L., Nunes, M.R.T., and Slhessarenko, R.D. (2025). Phylogeography of SARS-CoV-2 Omicron sublineages detected in asymptomatic blood donors during third epidemiological wave in Mato Grosso, Midwestern Brazil. Diagn. Microbiol. Infect. Dis., 111.","DOI":"10.1016\/j.diagmicrobio.2025.116693"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"377","DOI":"10.15585\/mmwr.mm6913e1","article-title":"James, A.; Taylor, J.; Spicer, K.; Bardossy, A.C.; Oakley, L.P.; Tanwar, S.; Chisty, Z.; et al. Asymptomatic and presymptomatic SARS-CoV-2 infections in residents of a long-term care skilled nursing facility\u2014King County, Washington, March 2020","volume":"69","author":"Kimball","year":"2020","journal-title":"MMWR Morb. Mortal. Wkly. Rep."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"845","DOI":"10.1038\/s41591-020-0897-1","article-title":"Antibody responses to SARS-CoV-2 in patients with COVID-19","volume":"26","author":"Long","year":"2020","journal-title":"Nat. Med."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Buitrago-Garcia, D., Egli-Gany, D., Counotte, M.J., Hossmann, S., Imeri, H., Ipekci, A.M., Salanti, G., and Low, N. (2020). Occurrence and transmission potential of asymptomatic and presymptomatic SARS-CoV-2 infections: A living systematic review and meta-analysis. PLoS Med., 17.","DOI":"10.1101\/2020.04.25.20079103"},{"key":"ref_8","unstructured":"World Health Organization Antigen-Detection in the Diagnosis of SARS-CoV-2 Infection Using Rapid Immunoassays: Interim Guidance. Available online: https:\/\/www.who.int\/publications\/i\/item\/antigen-detection-in-the-diagnosis-of-sars-cov-2infection-using-rapid-immunoassays."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1016\/S0140-6736(20)31483-5","article-title":"Prevalence of SARS-CoV-2 in Spain (ENE-COVID): A nationwide, population-based seroepidemiological study","volume":"396","author":"Oteo","year":"2020","journal-title":"Lancet"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"eabb6936","DOI":"10.1126\/science.abb6936","article-title":"Quantifying SARS-CoV-2 transmission suggests epidemic control with digital contact tracing","volume":"368","author":"Ferretti","year":"2020","journal-title":"Science"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2027","DOI":"10.1093\/cid\/ciaa344","article-title":"Antibody responses to SARS-CoV-2 in patients with novel coronavirus disease 2019","volume":"71","author":"Zhao","year":"2020","journal-title":"Clin. Infect. Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1016\/S0140-6736(20)30183-5","article-title":"Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China","volume":"395","author":"Huang","year":"2020","journal-title":"Lancet"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e57309","DOI":"10.7554\/eLife.57309","article-title":"SARS-CoV-2 (COVID-19) by the numbers","volume":"9","author":"Flamholz","year":"2020","journal-title":"Elife"},{"key":"ref_14","first-page":"1843","article-title":"Detection of SARS-CoV-2 in different types of clinical specimens","volume":"323","author":"Wang","year":"2020","journal-title":"JAMA"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1038\/s41586-020-2196-x","article-title":"Virological assessment of hospitalized patients with COVID-2019","volume":"581","author":"Corman","year":"2020","journal-title":"Nature"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1093\/ajcp\/aqaa112","article-title":"Review of current advances in serologic testing for COVID-19","volume":"154","author":"Espejo","year":"2020","journal-title":"Am. J. Clin. Pathol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"100040","DOI":"10.1016\/j.xcrm.2020.100040","article-title":"Rapid generation of neutralizing antibody responses in COVID-19 patients","volume":"1","author":"Suthar","year":"2020","journal-title":"Cell Rep. Med."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"533","DOI":"10.1056\/NEJMoa2034545","article-title":"Antibody status and incidence of SARS-CoV-2 infection in health care workers","volume":"384","author":"Lumley","year":"2021","journal-title":"N. Engl. J. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1339","DOI":"10.1126\/science.abe1107","article-title":"Preexisting and de novo humoral immunity to SARS-CoV-2 in humans","volume":"370","author":"Ng","year":"2020","journal-title":"Science"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"eabd4250","DOI":"10.1126\/science.abd4250","article-title":"Viral epitope profiling of COVID-19 patients reveals cross-reactivity and correlates of severity","volume":"370","author":"Shrock","year":"2020","journal-title":"Science"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2245","DOI":"10.1001\/jama.2020.7869","article-title":"COVID-19 and postinfection immunity: Limited evidence, many remaining questions","volume":"323","author":"Kirkcaldy","year":"2020","journal-title":"JAMA"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2372","DOI":"10.1016\/j.cell.2021.03.013","article-title":"Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity","volume":"184","author":"Lam","year":"2021","journal-title":"Cell"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e120","DOI":"10.1056\/NEJMp2025631","article-title":"Rethinking Covid-19 test sensitivity\u2014A strategy for containment","volume":"383","author":"Mina","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_24","first-page":"E31","article-title":"Health-related uncertainty among adults in the United States during the coronavirus disease 2019 pandemic","volume":"18","author":"Portnoy","year":"2021","journal-title":"Prev. Chronic Dis."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"438","DOI":"10.22207\/JPAM.18.1.27","article-title":"Retrospective Analysis of the Spread of SARS-CoV-2 in the Mediterranean Part of Bulgaria, During the First Wave of the Pandemic","volume":"18","author":"Sirakov","year":"2024","journal-title":"J. Pure Appl. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1080\/01621459.1927.10502953","article-title":"Probable inference, the law of succession, and statistical inference","volume":"22","author":"Wilson","year":"1927","journal-title":"J. Am. Stat. Assoc."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1177\/001316446002000104","article-title":"A coefficient of agreement for nominal scales","volume":"20","author":"Cohen","year":"1960","journal-title":"Educ. Psychol. Meas."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"World Medical Association (2013). World Medical Association Declaration of Helsinki: Ethical principles for medical research involving human subjects. JAMA, 310, 2191\u20132194.","DOI":"10.1001\/jama.2013.281053"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1781","DOI":"10.1001\/jama.2020.18796","article-title":"Change in antibodies to SARS-CoV-2 over 60 days among health care personnel in Nashville, Tennessee","volume":"324","author":"Patel","year":"2020","journal-title":"JAMA"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1085","DOI":"10.1056\/NEJMc2025179","article-title":"Rapid decay of anti-SARS-CoV-2 antibodies in persons with mild COVID-19","volume":"383","author":"Ibarrondo","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1093\/infdis\/jiaa659","article-title":"Longitudinal serological analysis and neutralizing antibody levels in coronavirus disease 2019 convalescent patients","volume":"223","author":"Muecksch","year":"2021","journal-title":"J. Infect. Dis."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"791","DOI":"10.1099\/jgv.0.001439","article-title":"The dynamics of humoral immune responses following SARS-CoV-2 infection and the potential for reinfection","volume":"101","author":"Kellam","year":"2020","journal-title":"J. Gen. Virol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"159","DOI":"10.2307\/2529310","article-title":"The measurement of observer agreement for categorical data","volume":"33","author":"Landis","year":"1977","journal-title":"Biometrics"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"e311","DOI":"10.1016\/S2666-5247(21)00056-2","article-title":"Comparison of seven commercial SARS-CoV-2 rapid point-of-care antigen tests: A single-centre laboratory evaluation study","volume":"2","author":"Corman","year":"2021","journal-title":"Lancet Microbe"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1931","DOI":"10.1038\/s41467-021-22072-9","article-title":"N-protein presents early in blood, dried blood and saliva during asymptomatic and symptomatic SARS-CoV-2 infection","volume":"12","author":"Shan","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1146\/annurev-med-042921-020956","article-title":"COVID-19: Challenges of viral variants","volume":"74","author":"Jacobs","year":"2023","journal-title":"Annu. Rev. Med."},{"key":"ref_37","first-page":"817","article-title":"Persistent virus infections and immune complex disease: Viral antigens, antibodies, and complement in the glomeruli of mice infected with lymphocytic choriomeningitis virus","volume":"118","author":"Perrin","year":"1977","journal-title":"J. Immunol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"316","DOI":"10.4049\/jimmunol.118.1.316","article-title":"The formation and fate of virus antigen-antibody complexes","volume":"118","author":"Perrin","year":"1977","journal-title":"J. Immunol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"116","DOI":"10.59393\/amb24400115","article-title":"SARS-CoV-2 Delta and Omicron Variants: Comparative Analysis of Confirmed Cases in Multi-Profile Hospital in Bulgaria","volume":"40","author":"Gotseva","year":"2024","journal-title":"Acta Microbiol. Bulg."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1126\/science.abb3221","article-title":"Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV-2)","volume":"368","author":"Li","year":"2020","journal-title":"Science"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"430","DOI":"10.1038\/s41586-020-2521-4","article-title":"Factors associated with COVID-19-related death using OpenSAFELY","volume":"584","author":"Williamson","year":"2020","journal-title":"Nature"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"e2016818","DOI":"10.1001\/jamanetworkopen.2020.16818","article-title":"Assessment of SARS-CoV-2 screening strategies to permit the safe reopening of college campuses in the United States","volume":"3","author":"Paltiel","year":"2020","journal-title":"JAMA Netw. Open"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1205","DOI":"10.1038\/s41591-021-01377-8","article-title":"Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection","volume":"27","author":"Khoury","year":"2021","journal-title":"Nat. Med."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"409","DOI":"10.1038\/s41579-021-00573-0","article-title":"SARS-CoV-2 variants, spike mutations and immune escape","volume":"19","author":"Harvey","year":"2021","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"eabd5393","DOI":"10.1126\/sciadv.abd5393","article-title":"Test sensitivity is secondary to frequency and turnaround time for COVID-19 screening","volume":"7","author":"Larremore","year":"2021","journal-title":"Sci. Adv."}],"container-title":["Current Issues in Molecular Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1467-3045\/47\/11\/919\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,7]],"date-time":"2025-11-07T05:19:26Z","timestamp":1762492766000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1467-3045\/47\/11\/919"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,5]]},"references-count":45,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["cimb47110919"],"URL":"https:\/\/doi.org\/10.3390\/cimb47110919","relation":{},"ISSN":["1467-3045"],"issn-type":[{"value":"1467-3045","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,5]]}}}