RESUMO
Background: Little is known about the dynamics of SARS-CoV-2 antigen burden in respiratory samples in different patient populations at different stages of infection. Current rapid antigen tests cannot quantitate and track antigen dynamics with high sensitivity and specificity in respiratory samples. Methods: We developed and validated an ultra-sensitive SARS-CoV-2 antigen assay with smartphone readout using the Microbubbling Digital Assay previously developed by our group, which is a platform that enables highly sensitive detection and quantitation of protein biomarkers. A computer vision-based algorithm was developed for microbubble smartphone image recognition and quantitation. A machine learning-based classifier was developed to classify the smartphone images based on detected microbubbles. Using this assay, we tracked antigen dynamics in serial swab samples from COVID patients hospitalized in ICU and immunocompromised COVID patients. Results: The limit of detection (LOD) of the Microbubbling SARS-CoV-2 Antigen Assay was 0.5 pg/mL (10.6 fM) recombinant nucleocapsid (N) antigen or 4000 copies/mL inactivated SARS-CoV-2 virus in nasopharyngeal (NP) swabs, comparable to many rRT-PCR methods. The assay had high analytical specificity towards SARS-CoV-2. Compared to EUA-approved rRT-PCR methods, the Microbubbling Antigen Assay demonstrated a positive percent agreement (PPA) of 97% (95% confidence interval (CI), 92-99%) in symptomatic individuals within 7 days of symptom onset and positive SARS-CoV-2 nucleic acid results, and a negative percent agreement (NPA) of 97% (95% CI, 94-100%) in symptomatic and asymptomatic individuals with negative nucleic acid results. Antigen positivity rate in NP swabs gradually decreased as days-after-symptom-onset increased, despite persistent nucleic acid positivity of the same samples. The computer vision and machine learning-based automatic microbubble image classifier could accurately identify positives and negatives, based on microbubble counts and sizes. Total microbubble volume, a potential marker of antigen burden, correlated inversely with Ct values and days-after-symptom-onset. Antigen was detected for longer periods of time in immunocompromised patients with hematologic malignancies, compared to immunocompetent individuals. Simultaneous detectable antigens and nucleic acids may indicate the presence of replicating viruses in patients with persistent infections. Conclusions: The Microbubbling SARS-CoV-2 Antigen Assay enables sensitive and specific detection of acute infections, and quantitation and tracking of antigen dynamics in different patient populations at various stages of infection. With smartphone compatibility and automated image processing, the assay is well-positioned to be adapted for point-of-care diagnosis and to explore the clinical implications of antigen dynamics in future studies.
RESUMO
OBJECTIVE: The purpose of this study was two-fold: 1) to determine the effects of mechanical and chemical modalities treatments on periodontal clinical and microbiological parameters of mothers; and 2) to determine the subsequent colonization of periodontal pathogens in tongue samples from their infants. DESIGN: A total of 168 mothers met inclusion criteria to participate in a randomized double-masked placebo-controlled clinical trial. Of those, 121 mothers (and their infants) of 158 seen at baseline provided complete data during study protocols from when their infants were 3 months old until the infants were about 2 years old. Treatments consisted of a combination of xylitol chewing gum, fluoride and chlorhexidine varnishes. The control/placebo group received sorbitol chewing gum and placebo varnishes. The effect of these treatments on periodontal clinical and microbiological parameters of mothers and on microbiological parameters of their infants was assessed by generalized estimating equation models. RESULTS: Microbiological outcomes using the BANA Test were lower for the majority of the visits in the test group when compared to the control/placebo group. These differences, however, were not statistically significant. Similarly, mean PBS results were lower for all follow-up visits for test-group mothers when compared to the control/placebo group's mothers, but of no statistical significance. Colonization of the tongue in infants by periodontal pathogens as measured by the BANA Test showed no distinct patterns concerning the stability of colonization of periodontal pathogens throughout the study period, albeit slightly superior for the test group. CONCLUSIONS: We have demonstrated that a combined chemical modalities treatment consisting of xylitol chewing gum, fluoride varnish and chlorhexidine varnish was moderately superior to control/placebo treatments on periodontal clinical and microbiological parameters of mothers throughout the study period, but of no statistical significance. Similar results were found for the infants in the test group when compared to infants of the control/placebo groups.