Abstract
ABSTRACTEarly detection of SARS-CoV-2 infection is critical to reduce asymptomatic and presymptomatic transmission, curb the spread of variants, and maximize treatment efficacy. Low-analytical-sensitivity nasal-swab testing is commonly used for surveillance and symptomatic testing, but the ability of these tests to detect the earliest stages of infection has not been established. In this study, conducted between September 2020 and June 2021 in the greater Los Angeles County, California, area, initially SARS-CoV-2-negative household contacts of individuals diagnosed with COVID-19 prospectively self-collected paired anterior-nares nasal-swab and saliva samples twice daily for viral-load quantification by high-sensitivity reverse-transcription quantitative PCR (RT-qPCR) and digital-RT-PCR assays. We captured viral-load profiles from the incidence of infection for seven individuals and compared diagnostic sensitivities between respiratory sites. Among unvaccinated persons, testing saliva with a high-analytical-sensitivity assay detected infection up to 4.5 days before viral loads in nasal swabs reached concentrations detectable by low-analytical-sensitivity nasal-swab tests. For most participants, nasal swabs reached higher peak viral loads than saliva but were undetectable or at lower loads during the first few days of infection. High-analytical-sensitivity saliva testing was most reliable for earliest detection. Our study illustrates the value of acquiring early (within hours after a negative high-sensitivity test) viral-load profiles to guide the appropriate analytical sensitivity and respiratory site for detecting earliest infections. Such data are challenging to acquire but critical to designing optimal testing strategies with emerging variants in the current pandemic and to respond to future viral pandemics.
Highlights
ResultsWe first established and validated two independent quantitative assays to measure SARS-CoV-2 viral load: a RTqPCR based on the assay put forth by the U.S Centers for Disease Control and Prevention (CDC)[28] and a RTddPCR assay developed by Bio-Rad.[29] Both of these assays received an emergency use authorization (EUA) for qualitative, but not quantitative, detection of SARS-CoV-2
Detection of SARS-CoV-2 infection is critical to reduce asymptomatic and pre-symptomatic transmission, curb the spread of variants, and maximize treatment efficacy
We first established and validated two independent quantitative assays to measure SARS-CoV-2 viral load: a RTqPCR based on the assay put forth by the U.S Centers for Disease Control and Prevention (CDC)[28] and a RTddPCR assay developed by Bio-Rad.[29]
Summary
We first established and validated two independent quantitative assays to measure SARS-CoV-2 viral load: a RTqPCR based on the assay put forth by the U.S Centers for Disease Control and Prevention (CDC)[28] and a RTddPCR assay developed by Bio-Rad.[29] Both of these assays received an emergency use authorization (EUA) for qualitative, but not quantitative, detection of SARS-CoV-2. We optimized the extraction and each quantitative assay protocol (see Supplementary Methods) to obtain more reliable quantification of SARS-CoV-2 viral load. The LOD of the modified assay was determined to be 1,000 copies/mL or better by following FDA guidelines (see Methods, Fig. S1). Commercial, heat-inactivated SARS-CoV-2 virus were used to establish calibration curves to convert RT-qPCR quantification cycle values (Cq) to viral load
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