Extreme differences in SARS-CoV-2 Omicron viral loads among specimen types drives poor performance of nasal rapid antigen tests for detecting presumably pre-infectious and infectious individuals, predicting improved performance of combination specimen antigen tests (preprint)

Background. To 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. Methods. We 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 10^4, 10^5, 10^6, or 10^7 copies/mL). Results. Overall, 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. Conclusion. Daily 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.

SARS-CoV-2 exhibits extreme differences in early viral loads among specimen types suggesting improved detection of pre-infectious and infectious individuals using combination specimen types (preprint)

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.

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

Background. The 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. Methods. From Sept. 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). Findings. Viral 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. Interpretation. Collecting 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.

SARS-CoV-2 is detectable using sensitive RNA saliva testing days before viral load reaches detection range of low-sensitivity nasal swab tests (preprint)

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.