A COVID-19 Infection with Positive Sputum and Negative Nasopharyngeal/Oropharyngeal Rapid Antigen-Based Testing: A Case Report and Pilot Study

Abstract

Current COVID-19 antigen testing is primarily carried out by obtaining a specimen via nasopharyngeal/oropharyngeal swab and performing a rapid lateral flow immunoassay (LFIA) disposable micro-device or related immunoassays. In this case report and pilot study, we propose the application of a modified pretreatment antigen-based method for sputum, using at least one detergent and one digestive enzyme. This method exposes hidden or masked antigenic sites of viral specimens or lingering fragments of viral proteins present in a complex biological matrix using a home-based rapid immunoassay for COVID-19. The modified technique can enhance detection sensitivity of LFIA by making visible the resultant test band, from viralcontaining sputum samples, that would otherwise not be seen.

Human Sputum Proteomics: Advancing Non-Invasive Diagnosis of Respiratory Diseases with Enhanced Biomarker Analysis Methods

“…Today, sputum specimens are collected by one of the two non-invasive methods-either the expectorated or the induced procedure. The expectorated or spontaneous mucous secretion is taken by coughing, following standardized protocols to collect the best sample and avoid contaminating the specimen [40,41]. The feasibility for patients with severe airflow obstruction to expel phlegm is low; consequently, an induced sputum sample is required with the aid of an ultrasonic wave nebulizer [42].…”

“…Acetone-precipitated samples are then dissolved in 8M urea, reduced with Tris-(2-carboxyethyl)phosphine, and alkylated with iodoacetamide [17]. A summary of protocols for extracting proteins from sputum is found in Table 1 [17,34,41,[51][52][53][54][55][56]. Regardless of the procedure used, fluctuations may occur in all methods due to the non-standard selective approach to sample preparation.…”

“…These techniques use a sampling protocol that can be stressful, especially in children, particularly when repeated testing is required. Unfortunately, these procedures have a rate of false-negative results that might enable convalescent COVID-19 patients to meet the criteria for discharge from the hospital and/or release from quarantine, resulting in the spread of the disease [41].…”

“…Recently, a rapid and affordable pretreatment protocol for releasing constituents of sputum was published. As depicted in Figure 1, this process consists of adding Triton X-100 and/or another detergent, Alcalase (subtilisin), and/or another proteolytic or digestion enzyme to a sputum sample, thereby exposing hidden or masked antigenic sites of viral specimens or lingering fragments of viral proteins present in the sputum meshwork [41].…”

“…The antigen tests do not require trained healthcare professionals and provide results in 15 to 20 min. Unfortunately, many LFIA tests that use nasopharyngeal and oropharyngeal samples have poor analytical sensitivity unless there is a high viral load, but a sputum specimen can provide a positive COVID-19 diagnostic result with a rapid antigen test when nasopharyngeal and oropharyngeal samples yield a negative finding [41].…”