ABSTRACT
BackgroundTo limit viral transmission, COVID-19 testing strategies must evolve as new SARS-CoV-2 variants (and new respiratory viruses) emerge to ensure that the specimen types and test analytical sensitivities being used will reliably detect individuals during the pre-infectious and infectious periods. Our accompanying work demonstrated that there are extreme differences in viral loads among paired saliva (SA), anterior-nares swab (ANS) and oropharyngeal swab (OPS) specimens collected from the same person and timepoint. We hypothesized that these extreme differences may prevent low-analytical-sensitivity assays (such as antigen rapid diagnostic tests, Ag-RDTs) performed on a single specimen type from reliably detecting pre-infectious and infectious individuals. MethodsWe conducted a longitudinal COVID-19 household-transmission study in which 228 participants collected SA, ANS, and OPS specimens for viral-load quantification by RT-qPCR, and performed an ANS Ag-RDT (Quidel QuickVue At-Home OTC COVID-19 Test) daily. We evaluated the performance of the Ag-RDT (n=2215 tests) to detect infected individuals (positive results in any specimen type by RT-qPCR) and individuals with presumed infectious viral loads (at or above thresholds of 104, 105, 106, or 107 copies/mL). ResultsOverall, the daily Ag-RDT detected 44% (358/811) timepoints from infected individuals. From 17 participants who enrolled early in the course of infection, we found that daily Ag-RDT performance was higher at timepoints when symptoms were reported, but symptoms only weakly correlated with SARS-CoV-2 viral loads, so ANS Ag-RDT clinical sensitivity remained below 50%. The three specimen types exhibited asynchronous presumably-infectious periods (regardless of the infectious viral-load threshold chosen) and the rise in ANS viral loads was delayed relative to SA or OPS for nearly all individuals, which resulted in the daily ANS Ag-RDT detecting only 3% in the pre-infectious period and 63% in the infectious period. We evaluated a computationally-contrived combined AN-OP swab based on viral loads from ANS and OPS specimens collected at the same timepoint; when tested with similar analytical sensitivity as the Ag-RDT, this combined swab was predicted to have significantly better performance, detecting up to 82% of infectious individuals. ConclusionDaily ANS rapid antigen testing missed virtually all pre-infectious individuals, and more than one third of presumed infectious individuals due to low-analytical-sensitivity of the assay, a delayed rise in ANS viral loads, and asynchronous infectious viral loads in SA or OPS. When high-analytical-sensitivity assays are not available and low-analytical-sensitivity tests such as Ag-RDTs must be used for SARS-CoV-2 detection, an AN-OP combination swab is predicted to be most effective for detection of pre-infectious and infectious individuals. More generally, low-analytical-sensitivity tests are likely to perform more robustly using oral-nasal combination specimen types to detect new SASR-CoV-2 variants and emergent upper respiratory viruses.
ABSTRACT
BackgroundScreening 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 tests 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. MethodsWe 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. ResultsWe 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. ConclusionOur 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.
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 CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSReliable 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.
