Early detection of SARS-CoV-2 infection is critical to reduce asymptomatic and pre-symptomatic 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 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 design optimal testing strategies with emerging variants in the current pandemic and to respond to future viral pandemics.

Early detection of SARS-CoV-2 infection is critical to reduce asymptomatic and pre-symptomatic spread of COVID-19, curb the spread of viral variants by travelers, and maximize efficacy of therapeutic treatments. We designed a study to evaluate the preferred test sensitivity and sample type (saliva and nasal swab) for detecting early infections of COVID-19. We performed a case-ascertained study to monitor household contacts of individuals recently diagnosed with a SARS-CoV-2 infection. From those individuals, we obtained twice-daily self-collected anterior-nares nasal swabs and saliva samples and quantified SARS-CoV-2 RNA viral loads in those samples using high-sensitivity RT-qPCR and RT-ddPCR assays. We found that SARS-CoV-2 RNA first appears in saliva and then in nasal-swab samples. A high-sensitivity (limit of detection of ~103 copies/mL) RNA test detected SARS-CoV-2 virus in saliva 1.5 to 4.5 days before the viral load in the paired nasal-swab samples exceeded the limit of detection of low-sensitivity tests. It was possible to observe a high (>107-108 copies/mL) viral load in saliva samples while the paired nasal swab was either negative or had low (~103 copies/mL) viral load. Our results indicate that both sampling site and test sensitivity must be considered to ensure early detection of SARS-CoV-2 infection: high-sensitivity tests that use saliva can detect SARS-CoV-2 infection days earlier than low-sensitivity tests that use nasal swabs. Furthermore, early in the infection, low-sensitivity tests that use nasal swabs may miss SARS-CoV-2-positive individuals with very high and potentially infectious viral loads in saliva.

SARS-CoV-2 viral-load measurements from a single-specimen type are used to establish diagnostic strategies, interpret clinical-trial results for vaccines and therapeutics, model viral transmission, and understand virus–host interactions. However, measurements from a single-specimen type are implicitly assumed to be representative of other specimen types. We quantified viral-load timecourses from individuals who began daily self-sampling of saliva, anterior-nares (nasal), and oropharyngeal (throat) swabs before or at the incidence of infection with the Omicron variant. Viral loads in different specimen types from the same person at the same timepoint exhibited extreme differences, up to 109 copies/mL. These differences were not due to variation in sample self-collection, which was consistent. For most individuals, longitudinal viral-load timecourses in different specimen types did not correlate. Throat-swab and saliva viral loads began to rise as many as 7 days earlier than nasal-swab viral loads in most individuals, leading to very low clinical sensitivity of nasal swabs during the first days of infection. Individuals frequently exhibited presumably infectious viral loads in one specimen type while viral loads were low or undetectable in other specimen types. Therefore, defining an individual as infectious based on assessment of a single-specimen type underestimates the infectious period, and overestimates the ability of that specimen type to detect infectious individuals. For diagnostic COVID-19 testing, these three single-specimen types have low clinical sensitivity, whereas a combined throat–nasal swab, and assays with high analytical sensitivity, was inferred to have significantly better clinical sensitivity to detect presumed pre-infectious and infectious individuals.

Background. Screening testing, often via self-collected specimens, remains a key strategy to detect infections early and prevent SARS-CoV-2 transmission, and to enable earlier initiation of treatment. However, which specimen type best detects the earliest days of infection remains controversial. Further, the analytical sensitivity of diagnostic tests must also be considered, as viral loads below a test's limit of detection (LOD) are likely to yield false-negative results. Comparisons of quantitative, longitudinal SARS-CoV-2 viral-load timecourses in multiple specimen types can determine the best specimen type and test analytical sensitivity for earliest detection of infection. Methods. We conducted a COVID-19 household transmission study between November 2021 and February 2022 that enrolled 228 participants and analyzed 6,825 samples using RT-qPCR to quantify viral-load timecourses in three specimen types (saliva [SA], anterior-nares swab [ANS], and oropharyngeal swab [OPS]). From this study population, 14 participants enrolled before or at the incidence of infection with the Omicron variant. We compared the viral loads in specimens collected from each person at the same timepoint, and the longitudinal viral load timecourses from each participant. Using these viral loads, we inferred the clinical sensitivity of each specimen type to detect infected, pre-infectious and infectious individuals (based on presumably infectious viral load levels) using assays with a range of analytical sensitivities. We also inferred the clinical sensitivity of computationally contrived specimen types representing combinations of single specimen types. Results. We found extreme differences (up to 109 copies/mL) in viral loads between paired specimen types in the same person at the same timepoint, and that longitudinal viral load timecourses across specimen types did not correlate. Because of this lack of correlation, infectious viral loads were often observed in different specimen types asynchronously throughout the course of the infection. In the first 4 days of infection, no single specimen type was inferred to achieve >95% detection of infected or infectious individuals, even with the highest analytical sensitivity assays. In nearly all participants (11/14), a rise in ANS viral loads was delayed (as many as 7 days) relative to SA and OPS. We also observed that ANS and OPS had the most complementary viral load timecourses, resulting in optimal inferred performance with a computationally contrived combined anterior nares-oropharyngeal (AN-OP) swab specimen type. The combination AN-OP swab had superior inferred clinical sensitivity the first 8 days of infection with both high- and low-analytical-sensitivity assays. This AN-OP swab was also inferred to significantly improve detection of pre-infectious and infectious individuals over any single specimen type. Conclusion. Our work demonstrates that the viral load in one specimen type cannot reliably predict the viral load in another specimen type. Combination specimen types may offer a more robust approach for earliest detection of new variants and respiratory viruses when viral kinetics are still unknown.

Our findings suggest that collecting saliva and nasal swab specimens in the morning immediately after waking yields higher SARS-CoV-2 viral loads than collection later in the day. The higher viral loads from morning specimen collection are predicted to significantly improve detection of SARS-CoV-2 in symptomatic individuals, particularly when using moderate- to low-analytical-sensitivity COVID-19 diagnostic tests, such as rapid antigen tests.