A Case Report of a COVID-19 Infection with Positive Sputum and Negative Nasopharyngeal Rapid Antigen-Based Testing: A Practical Method to Explore Sputome

Current COVID-19 antigen testing is primarily carried out by obtaining a specimen via nasopharyngeal swab and performing a rapid lateral flow immunoassay (LFIA) or related immunoassays. On average, a nasopharyngeal antigen-based LFIA for COVID-19 remains positive for approximately one week from symptom onset, and levels of infectivity and duration of the symptoms may depend primarily on carrying a high viral load enough to infect others. It has been proposed that patients with long-COVID, a syndrome in which patients continue to have complications of COVID with ongoing symptoms, may have occurring viral replication, despite testing negative via rapid COVID antigen testing. We therefore propose a modified antigen-based method that exposes hidden or masked antigenic sites of viral specimens, or lingering fragments of viral proteins, present in sputum using a home-based rapid immunoassay for COVID-19. Almost all protocols for testing were performed according to LFIA kit manufacturer’s instructions for the detection of SARS-CoV-2 nucleocapsid protein antigen, one of the most predominant proteins encoded by the SARS-CoV-2 virus. However, in challenging the manufacturer instructions, one or more digestive enzymes and a detergent were added to the collected biosamples (nasopharyngeal, oropharyngeal, saliva, buccal, gargle, and sputum); this modified procedure expose hidden or masked antigenic sites of the coronavirus or cross-reactant antigenic sites of related or non-related viruses, or some of the plethora of epitopes generated by the sample corresponding microbiota to accomplish an optimal binding to the commercial antibody used in the diagnostic test. The modified protocol can enhance detection sensitivity by making the resultant test band in sputum samples visible, that would otherwise not be seen, and consequently may generate a false negative result, in a nasopharyngeal sample from a patient with mild symptoms of COVID-19 and/or low viral load. Therefore, a need exists for an improved sample pre-treatment extraction procedure that allows optimization of sample preparation to attain a more accurate test result. Although the experiments described here were performed using commercial platforms, with antibodies directed to SARS-CoV-2 nucleocapsid antigen, this method may also be viable for the detection of any other pathogen in sputum by using antibodies directed to the key antigens present in the pathogen of interest. Furthermore, this modified method to expose the content of sputum can be used as a simple protocol to study the sputome, the proteome of sputum, and other omics (sputomics). In summary, this simple method is non-invasive, rapid, inexpensive, accurate, and may provide increased sensitivity and specificity in the detection of COVID-19 antigens for several weeks or even months. ### Competing Interest Statement Competing interest N.A.G. is the inventor of patents pending on this subject ### Funding Statement Funding This work did not receive any specific grant from funding agencies in the public, commercial, or non-for-profit sectors ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Compliance with ethical standards Biological samples were obtained from one volunteer from the start of the symptoms for 15 weeks with written and informed consent. The dated and signed consent document has been archived. Ethics Committee of Princeton Biochemicals, Inc. approved this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Data Availability Statement All data generated or analyzed during the study are included in the article