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.

Transmission of SARS-CoV-2 in community settings often occurs before symptom onset, therefore testing strategies that can reliably detect people in the early phase of infection are urgently needed. Early detection of SARS-CoV-2 infection is especially critical to protect vulnerable populations who require frequent interactions with caretakers. Rapid COVID-19 tests have been proposed as an attractive strategy for surveillance, however a limitation of most rapid tests is their low sensitivity. Low-sensitivity tests are comparable to high sensitivity tests in detecting early infections when two assumptions are met: (1) viral load rises quickly (within hours) after infection and (2) viral load reaches and sustains high levels (>105– 106 RNA copies/mL). However, there are no human data testing these assumptions. In this study, we document a case of presymptomatic household transmission from a healthy young adult to a sibling and a parent. Participants prospectively provided twice-daily saliva samples. Samples were analyzed by RT-qPCR and RT-ddPCR and we measured the complete viral load profiles throughout the course of infection of the sibling and parent. This study provides evidence that in at least some human cases of SARS-CoV-2, viral load rises slowly (over days, not hours) and not to such high levels to be detectable reliably by any low-sensitivity test. Additional viral load profiles from different samples types across a broad demographic must be obtained to describe the early phase of infection and determine which testing strategies will be most effective for identifying SARS-CoV-2 infection before transmission can occur.One sentence summaryIn some human infections, SARS-CoV-2 viral load rises slowly (over days) and remains near the limit of detection of rapid, low-sensitivity tests.

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.

The emergence of the novel human coronavirus, SARS-CoV-2, causes a global COVID-19 (coronavirus disease 2019) pandemic. Here, we have characterized and compared viral populations of SARS-CoV-2 among COVID-19 patients within and across households. Our work showed an active viral replication activity in the human respiratory tract and the co-existence of genetically distinct viruses within the same host. The inter-host comparison among viral populations further revealed a narrow transmission bottleneck between patients from the same households, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions.Author summaryIn this study, we compared SARS-CoV-2 populations of 13 Chinese COVID-19 patients. Those viral populations contained a considerable proportion of viral sub-genomic messenger RNAs (sgmRNA), reflecting an active viral replication activity in the respiratory tract tissues. The comparison of 66 identified intra-host variants further showed a low viral genetic distance between intra-household patients and a narrow transmission bottleneck size. Despite the co-existence of genetically distinct viruses within the same host, most intra-host minor variants were not shared between transmission pairs, suggesting a dominated role of stochastic dynamics in both inter-host and intra-host evolutions. Furthermore, the narrow bottleneck and active viral activity in the respiratory tract show that the passage of a small number of virions can cause infection. Our data have therefore delivered a key genomic resource for the SARS-CoV-2 transmission research and enhanced our understanding of the evolutionary dynamics of SARS-CoV-2.

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.

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.

An efficient laboratory-scale synthesis has been developed for the selective CCR5 antagonist 1. The convergent route has a longest linear sequence of nine steps (15 steps overall), and has overall yields of 18–25%. The route has enabled the preparation of 550 g of 1.

Importance: At-home rapid COVID-19 tests utilize nasal-swab specimens and require high viral loads to reliably give positive results. Longitudinal studies from the onset of infection have found infectious virus can present in oral specimens days before nasal. Detection and initiation of infection-control practices may therefore be delayed when nasal-swab rapid tests are used, resulting in greater exposure and transmission to contacts. Objective: We assessed whether index cases first identified by rapid nasal-swab COVID-19 tests had more transmission to household contacts than index cases who used other test types (tests with higher analytical sensitivity but longer turnaround times, and/or that utilize non-nasal specimen types). Design: In this observational cohort study, members of households with a recent COVID-19 case were screened for infection at least daily by RT-qPCR on one or more self-collected upper-respiratory specimen types. Participants reported demographic/medical information (including COVID-19 testing), symptom and exposure information, and household infection-control practices. A two-level random intercept model was used to assess the association between the infection outcome of household contacts and each covariable (household size, race/ethnicity, age, vaccination status, viral variant, infection-control practices, and whether a rapid nasal-swab test was used to initially identify the household index case). Setting: Southern California, September 2020-June 2021 and November 2021-March 2022. Participants: Cohort of 370 individuals from 85 households. Main Outcome(s) and Measure(s): Transmission was quantified by adjusted secondary attack rates (aSAR) and adjusted odds ratios (aOR). Results: An aSAR of 53.6% (95%CI 38.8-68.3%) was observed among households where the index case first tested positive by a rapid nasal-swab COVID-19 test, which was significantly higher than the aSAR for households where the index case utilized another test type (27.2% [19.5-35.0%], P=0.003 pairwise comparisons of predictive margins). We observed an aOR of 4.90 (95%CI 1.65-14.56) for transmission to household contacts when a nasal-swab rapid test was used to identify the index case, compared to other test types. Conclusions and Relevance: Use of nasal-swab rapid COVID-19 tests for initial detection of infection and initiation of infection control may not limit transmission as well as other test types.