Morning SARS-CoV-2 testing yields better detection of infection due to higher viral loads in saliva and nasal swabs upon waking

Abstract

BackgroundThe analytical sensitivities of SARS-CoV-2 diagnostic tests span 6 orders of magnitude. Optimizing sample-collection methods to achieve the most reliable detection for a given sensitivity would increase the effectiveness of testing and minimize COVID-19 outbreaks.MethodsFrom September 2020 to April 2021 we performed a household-transmission study in which participants self-collected samples every morning and evening throughout acute SARS-CoV-2 infection. Seventy mildly symptomatic participants collected saliva and, of those, 29 also collected nasal-swab samples. Viral load was quantified in 1194 saliva and 661 nasal-swab samples using a high-analytical-sensitivity RT-qPCR assay (LOD, 1,000 SARS-CoV-2 RNA copies/mL).FindingsViral loads in both saliva and nasal-swab samples were significantly higher in morning-collected samples than evening-collected samples after symptom onset. We used these quantitative measurements to infer which diagnostic tests would have detected infection (based on sample type and test analytical sensitivity). We find that morning collection would have resulted in significantly improved detection and that this advantage would be most pronounced for tests with low to moderate analytical sensitivity, which would likely have missed infections if sampling in the evening.InterpretationCollecting samples for COVID-19 testing in the morning offers a simple and low-cost improvement to clinical diagnostic sensitivity of low- to moderate-analytical-sensitivity tests. The phenomenon of higher viral loads in the morning may also have implications related to when transmission is more likely to occur.FundingBill & Melinda Gates Foundation, Ronald and Maxine Linde Center for New Initiatives (Caltech), Jacobs Institute for Molecular Engineering for Medicine (Caltech)RESEARCH IN CONTEXTEvidence before this studyReliable COVID-19 diagnostic testing is critical to reducing transmission of SARS-CoV-2 and reducing cases of severe or fatal disease, particularly in areas with limited vaccine access or uptake. Saliva and anterior-nares nasal swabs are common sample types; however, different diagnostic tests using these sample types have a range of analytical sensitivities spanning 6 orders of magnitude, with limits of detection (LODs) between 102 and 108 genomic copy equivalents of SARS-CoV-2 RNA (copies) per mL of sample. Due to limitations in clinical laboratory capacity, many low-resource settings rely on COVID-19 tests that fall on the moderate (LODs of 104 to 105 copies/mL) to lower (LODs of 105 to 108 copies/mL) end of this spectrum of analytical sensitivity. Alterations in sample collection methods, including time of sample collection, may improve the performance of these diagnostics.Added value of this studyThis study quantifies viral loads from saliva and nasal-swab samples that were longitudinally self-collected by symptomatic patients in the morning immediately after waking and in the evening just prior to sleeping throughout the course of acute SARS-CoV-2 infection. The study cohort was composed of mildly or moderately symptomatic individuals (outpatients). This analysis demonstrates significantly higher viral loads in samples collected in the morning, relative to those collected in the evening. When using moderate to lower analytical sensitivity test methods, these loads are inferred to result in significantly better detection of infected individuals in the morning.Implications of available evidenceThese findings suggest that samples collected in the morning immediately after waking will better detect SARS-CoV-2 infection in symptomatic individuals tested by moderate to lower analytical sensitivity COVID-19 diagnostic tests (LODs at or above 104 viral copies per mL of sample), such as many rapid antigen tests currently available.

Extreme differences in SARS-CoV-2 viral loads among respiratory specimen types during presumed pre-infectious and infectious periods

“…N4-52286 Lot 70034991). To achieve higher concentrations and greater dynamic range in the standard curve, volume from a participant saliva specimen previously quantified to have a viral load of 6.44x10 9 copies/mL 53 was used to generate 4 additional points. Diluted particles or volume from the participant specimen was spiked into pooled matrix from freshly collected SA, ANS, or OPS specimens from SARS-CoV-2 negative donors, collected as described above.…”

“…Each day, participants reported symptoms, then self-collected saliva, anterior-nares (hereafter nasal) swab, and posterior oropharyngeal (hereafter throat) swab specimens for RT-qPCR testing in Zymo Research's SafeCollect devices (CEmarked for EU use), following manufacturer's instructions (53,54). Participants collected specimens immediately upon enrollment, then daily every morning upon waking, as morning sample collection has been shown to yield higher viral loads than evening collection (51).…”

“…56 57 Participants collected specimens immediately upon enrollment, then daily every morning upon waking, as morning sample collection has been shown to yield higher viral loads than evening collection. 53…”

“…A university study 26 captured daily saliva and nasal-swab samples for 2 weeks from 60 individuals, only 3 of whom were negative for SARS-CoV-2 upon enrollment, allowing only limited inferences about earliest detection. In our prior study, we captured twice-daily viral-load timecourses from 72 individuals for 2 weeks, 53 seven of whom were negative upon enrollment. 16 In 6 of 7 individuals, we inferred from viral-load quantifications that a high-analytical-sensitivity saliva assay would detect infection earlier than a low-analytical-sensitivity ANS test.…”

Index Cases First Identified by Nasal-Swab Rapid COVID-19 Tests Had More Transmission to Household Contacts Than Cases Identified by Other Test Types

“…We conducted a case-ascertained COVID-19 household transmission observational cohort study in Southern California in two phases: between September 2020 and June 2021, 44,54 prior to the predominance of the Delta variant 55 , and between November 2021 and March 2022, 43 during the emergence and subsequent predominance of the Omicron variant 55 (Table S1A). The study was approved by the California Institute of Technology IRB (protocol #20-1026).…”