ABSTRACT
ObjectivesRapid diagnostics is pivotal to curb SARS-CoV-2 transmission, and saliva has emerged as a practical alternative to naso/oropharyngeal (NOP) specimens. We aimed to develop a direct RT-LAMP workflow for viral detection in saliva, and to provide more information regarding its potential in COVID-19 diagnostics. MethodsClinical and contrived specimens were used to screen/optimize formulations and sample processing protocols. Salivary viral load was determined in symptomatic patients to evaluate clinical performance (n = 90) and to characterize saliva based on age, gender and time from onset of symptoms (n = 49). ResultsThe devised workflow achieved 93.2% sensitivity, 97% specificity, and 0.895 Kappa for salivas containing >102 copies/L. Further analyses in saliva showed peak viral load in the first days of symptoms and lower viral loads in females, particularly among young individuals (<38 years). NOP RT-PCR data did not yield relevant associations. ConclusionsThis novel saliva RT-LAMP workflow can be applied to point-of-care testing. This work reinforces that saliva better correlates with transmission dynamics than NOP specimens, and reveals gender differences that may reflect higher transmission by males. To maximize detection, testing should be done immediately after symptom onset, especially in females. HIGHLIGHTS- Development of DGS, a dithiothreitol/guanidine-based solution for stabilization of the viral genome that increases sensitivity for SARS-CoV-2 detection in saliva; - Rapid, cost-effective RT-LAMP assay workflow for viral detection in saliva without need of RNA extraction; - Insights into the differences in viral load between saliva and naso-oropharyngeal specimens, and correlation with age, gender and time from symptom onset;
ABSTRACT
The SARS-CoV-2 pandemic has already killed more than 800,000 people worldwide. To gain entry, the virus uses its spike protein to bind to host hACE-2 receptors on the host cell surface and mediate fusion between viral and cell membranes. As initial steps leading to virus entry involves significant changes in protein conformation as well as in the electrostatic environment in the vicinity of the spike-hACE-2 complex, we explored the sensitivity of the interaction to changes in ionic strength through computational simulations and surface plasmon resonance. We identified two regions in the receptor-binding domain (RBD), E1 and E2, which interact differently with hACE-2. At high salt concentration, E2-mediated interactions are weakened but are compensated by strengthening E1-mediated hydrophobic interactions. These results provide a detailed molecular understanding of spike RBD/hACE-2 complex formation and stability under a wide range of ionic strengths. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=192 SRC="FIGDIR/small/267351v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@1a7dc02org.highwire.dtl.DTLVardef@15d3c78org.highwire.dtl.DTLVardef@2d09c1org.highwire.dtl.DTLVardef@db78a9_HPS_FORMAT_FIGEXP M_FIG C_FIG
