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 manufacturers 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.