Quantitative SARS-CoV-2 viral-load curves in paired saliva and nasal swabs inform appropriate respiratory sampling site and analytical test sensitivity required for earliest viral detection

Abstract

Early detection of SARS-CoV-2 infection is critical to reduce asymptomatic and pre-symptomatic transmission, curb the spread of variants by travelers, and maximize treatment efficacy. Low-sensitivity nasal-swab testing (antigen and some nucleic-acid-amplification tests) is commonly used for surveillance and symptomatic testing, but the ability of low-sensitivity nasal-swab tests to detect the earliest stages of infection has not been established. In this case-ascertained study, 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, high-sensitivity saliva testing detected infection up to 4.5 days before viral loads in nasal swabs reached the limit of detection of low-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-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 in the current pandemic and will be required for responding to future viral pandemics. As new variants and viruses emerge, up-to-date data on viral kinetics are necessary to adjust testing strategies for reliable early detection of infections.

Keywords: COVID-19; Case-ascertained; Diagnostics; Household study; Longitudinal sampling; Nasal swab; Pre-Symptomatic; RT-qPCR; Saliva; Surveillance; Transmission.

Figure 1.. SARS-CoV-2 viral load quantification for nasal-swab (A) and saliva (B) specimens from positive participants measured with RT-ddPCR and RT-qPCR.

Participant nasal swab (A) or saliva (B) SARS-CoV-2 N1 concentration (copies/mL) per detection method, RT-ddPCR (Blue circles) and RT-qPCR (orange circles) plotted against geometric mean of RT-qPCR and RT-ddPCR viral load concentrations. A total of 42 nasal swab and 63 saliva samples from study participants were quantified with both methods. Theoretical SARS-CoV-2 concentration input represents data from calibration curves created with a dilution series of contrived samples prepared using commercial, inactivated SARS-CoV-2 particles spiked into commercially available SARS-CoV-2 negative saliva or nasal fluid pooled from human donors (green circles), extracted and detected with RT-qPCR. Grey line represents x=y.

Figure 2.. Symptoms and SARS-CoV-2 viral loads in paired saliva and nasal-swab samples of seven participants who became SARS-CoV-2 positive during study participation.

(A-G) Self-reported twice-daily symptom data over the course of enrollment are shown as a top panel for each of the participants (see color-coded legend for symptom categories). Demographic data including any reported medical conditions are included in Table S1. Viral loads are reported for the N1 and N2 genes of SARS-CoV-2 for both saliva (black and grey circles) and nasal-swab samples (dark-green and light-green triangles); ND = not detected for Cqs ≥40; Samples with an indeterminate result by the CDC RT-qPCR assay are shown along the horizontal black dashed line. (see Methods for details). The limit of detection (LOD) of the Abbott ID NOW (300,000 NDU/mL) is indicated by the horizontal green dashed line; the range of LODs of antigen tests (horizontal green bar) are shown for reference (data are from Table S2 in ref.). A diagnostic test does not provide reliable detection for samples with viral loads below its LOD. For each participant, the first detected saliva point is emphasized with a pink circle and their first nasal-swab point above the LOD of the ID NOW is emphasized with a pink triangle. Vertical shading in grey indicates nighttime (8pm – 8am). Internal control of RNase P gene Cqs from the CDC primer set are provided for each sample to compare self-sampling consistency and sample integrity (failed samples, where RNase P Cq ≥40, are not plotted). Participant gender, age range, and SARS-CoV-2 variant are given in each panel’s title. Two regions of interest (ROI) are indicated by purple-shaded rectangles and discussed in the main text.

Figure 3.. Summary of Diagnostic Insights from Study Participants who became infected with SARS-CoV-2 while enrolled in the study

(A) Participant infection time courses were aligned to the first high-sensitivity (LOD of ≤1 × 10³ copies/mL) positive result from either saliva or nasal swab sample type (day 0) and the percentage of positive tests was calculated for each time point (0.5-day intervals) from the first positive sample. The predicted performance of low-sensitivity nasal swabs was determined using the individual SARS-CoV-2 N1 viral load values for each participant individually shown in Fig. 2, or above a viral load threshold of 1.9×10⁵ copies/mL, which is the most-sensitive of the low-sensitivity tests we included in shaded region of Fig. 2. We show the percentage of participants who were detected by our high sensitivity saliva test (black circle) or could be inferred to be detectable by a low-sensitivity nasal swab test (green triangle) at a given timepoint. (B) Quantitative SARS-CoV-2 N1 viral loads of paired samples collected during time windows of the infection (aligned to first positive result by high sensitivity testing of either sample type) are shown for saliva (grey circles) and nasal swabs (green triangles). Paired samples for a given time point are connected with grey lines, with emphasis on paired samples where only saliva (black connecting line) or nasal swab (green connecting line) were positive. ND = Not Detected, Ind = Indeterminate result. (C) Peak SARS-CoV-2 N1 viral loads measured in saliva (grey circles) and nasal swab (green triangles) for each of the seven participants are shown. Horizontal black line indicates the median. (D) Percentage of positive test results (out of total number of tests) are shown for the first 10 days for each participant (day 0 corresponds to the first positive test result in either sample type). Saliva (grey bars) and nasal swab (green bars) are shown. Positivity was either observed (by our high sensitivity test) or inferred to be positive by viral loads above a LOD threshold of 1.9e5 copies/mL for low-sensitivity tests. The symptomatic category includes any sample where one or more symptom was reported at the time of sample collection.