The New Normal: Delayed Peak SARS-CoV-2 Viral Loads Relative to Symptom Onset and Implications for COVID-19 Testing Programs (preprint)

Background: Early in the COVID-19 pandemic, peak viral loads coincided with symptom onset. We hypothesized that in a highly immune population, symptom onset might occur earlier in infection, coinciding with lower viral loads. Methods: We assessed SARS-CoV-2 and influenza A viral loads relative to symptom duration in recently-tested adults. Symptomatic participants [≥]16y presenting to testing sites in Georgia (4/2022- 4/2023; Omicron variant predominant) provided symptom duration. Nasal swab samples were tested by the Xpert Xpress SARS-CoV-2/Flu/RSV assay and Ct values recorded. Nucleoprotein concentrations in SARS-CoV-2 PCR-positive samples were measured by Single Molecule Array. To estimate hypothetical antigen rapid diagnostic test (Ag RDT) sensitivity on each day after symptom onset, percentages of individuals with Ct value [≤]30 or [≤]25 were calculated. Results: Of 621 SARS-CoV-2 PCR-positive individuals (64.1% women, median 40.9y), 556/621 (89.5%) had a history of vaccination, natural infection, or both. By both Ct value and antigen concentration measurements, median viral loads rose from the day of symptom onset and peaked on the fourth day. Ag RDT sensitivity estimates were 35.7-71.4% on the first day, 63.9-78.7% on the third day, and 78.6-90.6% on the fourth day of symptoms. In 74 influenza A PCR-positive individuals (55.4% women; median 35.0y), median influenza viral loads peaked on the second day of symptoms. Conclusions: In a highly immune adult population, median SARS-CoV-2 viral loads peaked on the fourth day of symptoms. Influenza A viral loads peaked soon after symptom onset. These findings have implications for ongoing use of Ag RDTs for COVID-19 and influenza.

Sensitivity of Rapid Antigen Tests Against SARS-CoV-2 Omicron and Delta Variants (preprint)

Rapid Antigen Tests (RAT) have become an invaluable tool for combating the COVID-19 pandemic. However, concerns have been raised regarding the ability of existing RATs to effectively detect emerging SARS-CoV-2 variants. We compared the performance of eight commercially available, emergency use authorized RATs against the Delta and Omicron SARS-CoV-2 variants using individual patient and serially diluted pooled clinical samples. The RATs exhibited lower sensitivity for Omicron samples when using PCR Cycle threshold (CT) value (a proxy for RNA concentration) as the comparator. Interestingly, however, they exhibited similar sensitivity for Omicron and Delta samples when using quantitative antigen concentration as the comparator. We further found that the Omicron samples had lower ratios of antigen to RNA, which offers a potential explanation for the apparent lower sensitivity of RATs for that variant when using CT value as a reference. Our findings underscore the complexity in assessing RAT performance against emerging variants and highlight the need for ongoing evaluation in the face of changing population immunity and virus evolution.

The clinical utility and epidemiological impact of self-testing for SARS-CoV-2 using an-tigen detecting diagnostics: a systematic review and meta-analysis (preprint)

Introduction Self-testing for COVID-19 (C19ST) based on antigen detecting diagnostics could signifi-cantly support controlling the SARS-CoV-2 pandemic. To inform the World Health Organiza-tion in developing a C19ST guideline, we performed a systematic review and meta-analysis of the available literature. Methods We electronically searched Medline and the Web of Science core collection, performed secondary reference screening, and contacted experts for further relevant publications. Any study published between December 1, 2020 and November 30, 2021 assessing the epidemio-logical impact and clinical utility of C19ST was included. Study quality was evaluated using the Newcastle Ottawa Scale (NOS). The review was registered on PROSPERO (CRD42022299977). Results 11 studies only from high-income countries with an overall low quality (median of 3/9 stars on the NOS) were found. Pooled C19ST positivity was 0.2% (95% CI 0.1% to 0.4%; eight data sets) in populations where otherwise no dedicated testing would have occurred. The impact of self-testing on virus transmission was uncertain. Positive test results mainly resulted in people having to isolate without further confirmation of results (eight data sets). When testing was voluntary by study design, pooled testing uptake was 53.2% (95% CI 36.7% to 68.9%; five data sets. Outside direct health impacts, C19ST reduced quarantine duration and absenteeism from work, and made study participants feel safer. Study participants favored self-testing and were confident that they performed testing and sampling correctly. Conclusions The present data suggests that C19ST could be a valuable tool in reducing the spread of COVID-19, as it can achieve good uptake, may identify additional cases, and was generally perceived as positive by study participants. However, data was very limited and heterogenous, and further research especially in low- and middle-income countries is needed to assess the clinical utility and epidemiological impact of C19ST in more detail.

Accuracy of rapid point-of-care antigen-based diagnostics for SARS-CoV-2: an updated systematic review and meta-analysis with meta regression analyzing influencing factors (preprint)

BackgroundComprehensive information about the accuracy of antigen rapid diagnostic tests (Ag-RDTs) for SARS-CoV-2 is essential to guide public health decision makers in choosing the best tests and testing policies. In August 2021, we published a systematic review and meta-analysis about the accuracy of Ag-RDTs. We now update this work and analyze the factors influencing test sensitivity in further detail. Methods and findingsWe registered the review on PROSPERO (registration number: CRD42020225140). We systematically searched multiple databases (PubMed, Web of Science Core Collection, medRvix, bioRvix, and FIND) for publications evaluating the accuracy of Ag-RDTs for SARS-CoV-2 until August 31, 2021. Descriptive analyses of all studies were performed, and when more than 4 studies were available, a random-effects meta-analysis was used to estimate pooled sensitivity and specificity with reverse transcription polymerase chain reaction (RT-PCR) testing as a reference. To evaluate factors influencing test sensitivity, we performed 3 different analyses using multivariate mixed-effects meta-regression models. We included 194 studies with 221,878 Ag-RDTs performed. Overall, the pooled estimates of Ag-RDT sensitivity and specificity were 72.0% (95% confidence interval [CI] 69.8 to 74.2) and 98.9% (95% CI 98.6 to 99.1), respectively. When manufacturer instructions were followed, sensitivity increased to 76.4% (95%CI 73.8 to 78.8). Sensitivity was markedly better on samples with lower RT-PCR cycle threshold (Ct) values (sensitivity of 97.9% [95% CI 96.9 to 98.9] and 90.6% [95% CI 88.3 to 93.0] for Ct-values <20 and <25, compared to 54.4% [95% CI 47.3 to 61.5] and 18.7% [95% CI 13.9 to 23.4] for Ct-values [≥]25 and [≥]30) and was estimated to increase by 2.9 percentage points (95% CI 1.7 to 4.0) for every unit decrease in mean Ct-value when adjusting for testing procedure and patients symptom status. Concordantly, we found the mean Ct-value to be lower for true positive (22.2 [95% CI 21.5 to 22.8]) compared to false negative (30.4 [95% CI 29.7 to 31.1]) results. Testing in the first week from symptom onset resulted in substantially higher sensitivity (81.9% [95% CI 77.7 to 85.5]) compared to testing after 1 week (51.8%, 95% CI 41.5 to 61.9). Similarly, sensitivity was higher in symptomatic (76.2% [95% CI 73.3 to 78.9]) compared to asymptomatic (56.8% [95% CI 50.9 to 62.4]) persons. However, both effects were mainly driven by the Ct-value of the sample. With regards to sample type, highest sensitivity was found for nasopharyngeal (NP) and combined NP/oropharyngeal samples (70.8% [95% CI 68.3 to 73.2]), as well as in anterior nasal/mid-turbinate samples (77.3% [95% CI 73.0 to 81.0]). ConclusionAg-RDTs detect most of the individuals infected with SARS-CoV-2, and almost all when high viral loads are present (>90%). With viral load, as estimated by Ct-value, being the most influential factor on their sensitivity, they are especially useful to detect persons with high viral load who are most likely to transmit the virus. To further quantify the effects of other factors influencing test sensitivity, standardization of clinical accuracy studies and access to patient level Ct-values and duration of symptoms are needed.

Where is Omicron? Comparison of SARS-CoV-2 RT-PCR and Antigen Test Sensitivity at Commonly Sampled Anatomic Sites Over the Course of Disease (preprint)

Background: Upper respiratory samples for SARS-CoV-2 detection include the gold standard nasopharyngeal (NP) swab, and mid-turbinate (MT) nasal swabs, oropharyngeal (OP) swabs, and saliva. Following the emergence of the omicron (B.1.1.529) variant, limited preliminary data suggest that saliva may be more sensitive than nasal swab, highlighting the need to understand differences in viral load across different sites. Methods: MT, OP, and saliva samples were collected from symptomatic individuals presenting for evaluation in Atlanta, GA, in January 2022. Longitudinal samples were collected from a family cohort following COVID-19 exposure to describe detection of viral targets over the course of infection. Results: SARS-CoV-2 RNA and nucleocapsid antigen measurements demonstrated a nares-predominant phenotype in a familial cohort. A dominant location for SARS-CoV-2 was not found among cohort of 54 individuals. Positive percent agreement for virus detection in MT, OP and saliva specimens were 66.7 [54.1-79.2], 82.2 [71.1-93.4], and 72.5 [60.3-84.8] by RT-PCR, respectively, and 46.2 [32.6-59.7], 51.2 [36.2-66.1], and 72.0 [59.6-84.4] by ultrasensitive antigen assay. The composite of positive MT or OP assay was not significantly different than either alone for both RT-PCR and antigen assay (PPA 86.7 [76.7-96.6] and 59.5 [44.7-74.4], respectively). Conclusions: Our data suggest that SARS-CoV-2 nucleocapsid and RNA exhibited similar kinetics and diagnostic yield in three upper respiratory sample types across the duration of symptomatic disease. Collection of OP or combined nasal and OP samples does not appear to increase sensitivity versus validated nasal sampling for rapid detection of viral antigen

Predictive value of isolated symptoms for diagnosis of SARS-CoV-2 infection in children tested during peak circulation of the delta variant (preprint)

BackgroundCOVID-19 testing policies for symptomatic children attending U.S. schools or daycare vary, and whether isolated symptoms should prompt testing is unclear. We evaluated children presenting for SARS-CoV-2 testing to determine if the likelihood of having a positive SARS-CoV-2 test differed between participants with one versus [≥]2 symptoms, and to examine the predictive capability of isolated symptoms. MethodsParticipants [≤] 18 years presenting for clinical SARS-CoV-2 molecular testing in six sites in urban/suburban/rural Georgia (July-October, 2021; delta variant predominant) were queried about individual symptoms. Participants were classified into three groups: asymptomatic, one symptom only, or [≥]2 symptoms. SARS-CoV-2 test results and clinical characteristics of the three groups were compared. Sensitivity, specificity, and positive/negative predictive values (PPV/NPV) for isolated symptoms were calculated by fitting a saturated Poisson model. ResultsOf 602 participants, 21.8% tested positive and 48.7% had a known or suspected close contact. Children reporting one symptom (n=82; OR=6.00, 95% CI: 2.70-13.33) and children reporting [≥]2 symptoms (n=365; OR=5.25: 2.66-10.38) were significantly more likely to have a positive COVID-19 test than asymptomatic children (n=155), but they were not significantly different from each other (OR=0.88: 0.52-1.49). Sensitivity/PPV were highest for isolated fever (33%/57%), cough (25%/32%), and sore throat (21%/45%); headache had low sensitivity (8%) but higher PPV (33%). Sensitivity/PPV of isolated congestion/rhinorrhea were 8%/9%. ConclusionsWith high delta variant prevalence, children with isolated symptoms were as likely as those with multiple symptoms to test positive for COVID-19. Isolated fever, cough, sore throat, or headache, but not congestion/rhinorrhea, offered highest predictive value. Key pointsIn an area with high community prevalence of the delta variant, children presenting with one symptom were as likely as those with two or more symptoms to test positive for SARS-CoV-2 infection. Isolated symptoms should be considered in testing decisions.

Measurement of SARS-CoV-2 antigens in plasma of pediatric patients with acute COVID-19 or MIS-C using an ultrasensitive and quantitative immunoassay (preprint)

BackgroundDetection of SARS-CoV-2 antigens in blood has high sensitivity in adults with acute COVID-19, but sensitivity in pediatric patients is unclear. Recent data suggest that persistent SARS-CoV-2 spike antigenemia may contribute to multisystem inflammatory syndrome in children (MIS-C). We quantified SARS-CoV-2 nucleocapsid (N) and spike (S) antigens in blood of pediatric patients with either acute COVID-19 or MIS-C using ultrasensitive immunoassays (Meso Scale Discovery). MethodsPlasma was collected from inpatients (<21 years) enrolled across 15 hospitals in 15 US states. Acute COVID-19 patients (n=36) had a range of disease severity and positive nasopharyngeal SARS-CoV-2 RT-PCR within 24 hours of blood collection. Patients with MIS-C (n=53) met CDC criteria and tested positive for SARS-CoV-2 (RT-PCR or serology). Controls were patients pre-COVID-19 (n=67) or within 24h of negative RT-PCR (n=43). ResultsSpecificities of N and S assays were 95-97% and 100%, respectively. In acute COVID-19 patients, N/S plasma assays had 89%/64% sensitivity, respectively; sensitivity in patients with concurrent nasopharyngeal swab cycle threshold (Ct) [≤] 35 were 93%/63%. Antigen concentrations ranged from 1.28-3,844 pg/mL (N) and 1.65-1,071 pg/mL (S) and correlated with disease severity. In MIS-C, antigens were detected in 3/53 (5.7%) samples (3 N-positive: 1.7, 1.9, 121.1 pg/mL; 1 S-positive: 2.3 pg/mL); the patient with highest N had positive nasopharyngeal RT-PCR (Ct 22.3) concurrent with blood draw. ConclusionsUltrasensitive blood SARS-CoV-2 antigen measurement has high diagnostic yield in children with acute COVID-19. Antigens were undetectable in most MIS-C patients, suggesting that persistent antigenemia is not a common contributor to MIS-C pathogenesis. Key pointsIn a U.S. pediatric cohort tested with ultrasensitive immunoassays, SARS-CoV-2 nucleocapsid antigens were detectable in most patients with acute COVID-19, and spike antigens were commonly detectable. Both antigens were undetectable in almost all MIS-C patients.

Implementation of SARS-CoV2 Screening in K-12 Schools using In-School Pooled Molecular Testing and Deconvolution by Rapid Antigen Test (preprint)

What is already known about this topic?SARS-CoV2 testing is a key component of a multi-layered mitigation strategy to enable safe return to in-person school for the K-12 population. However, costs, logistics, and uncertainty about effectiveness are potential barriers to implementation. What is added by this report?Over three months, 259,726 individual swabs were tested across 50,636 pools from 582 schools. Pool positivity rate was 0.8%; 98.1% of pools tested negative and 0.3% inconclusive, and 0.8% of pools submitted could not be tested. In reflex testing, 92.5% of fully deconvoluted pools with N1 or N2 target Ct [≤]30 yielded a positive individual using the BinaxNOW antigen rapid diagnostic test (Ag RDT) performed 1-3 days later. With sufficient staffing support and low pool positivity rates, pooled sample collection and reflex testing were feasible for schools. What are the implications for public health practice?Screening testing for K-12 students and staff is achievable at scale and at low cost with a scheme that incorporates in-school pooling, RT-PCR primary testing, and Ag RDT reflex/deconvolution testing. Staffing support is a key factor for program success.

Performance and Operational Evaluation of the Access Bio CareStart Rapid Antigen Test in a High-throughput Drive-through Community Testing Site in Massachusetts (preprint)

BackgroundTo facilitate deployment of point-of-care testing for SARS-CoV-2, we evaluated the Access Bio CareStart COVID-19 Antigen test in a high-throughput, drive-through, free community testing site using anterior nasal (AN) swab RT-PCR for clinical testing. MethodsConsenting symptomatic and asymptomatic children ([≤]18 years) and adults received dual AN swabs. CareStart testing was performed with temperature/humidity monitoring. All tests had two independent reads to assess inter-operator agreement. Patients with positive CareStart results were called and instructed to isolate pending RT-PCR results. The paired RT-PCR result was the reference for sensitivity and specificity calculations. ResultsOf 1603 participants, 1245 adults and 253 children had paired RT-PCR/CareStart results and complete symptom data. 83% of adults and 87% of children were asymptomatic. CareStart sensitivity/specificity were 84.8% (95% confidence interval [CI] 71.1-93.7)/97.2% (92.0-99.4) and 85.7% (42.1-99.6)/89.5% (66.9-98.7) in adults and children, respectively, within 5 days of symptoms. Sensitivity/specificity were 50.0% (41.0-59.0)/99.1% (98.3-99.6) in asymptomatic adults and 51.4% (34.4-68.1)/97.8% (94.5-99.4) in asymptomatic children. Sensitivity in all 234 RT-PCR-positive people was 96.3% with cycle threshold (Ct) [≤]25, 79.6% with Ct [≤]30, and 61.4% with Ct [≤]35. All 21 false positive CareStart tests had faint but normal bands. Inter-operator agreement was 99.5%. Operational challenges included identification of faint test bands and inconsistent swab elution volumes. ConclusionsCareStart had high sensitivity in people with Ct [≤]25 and moderate sensitivity in symptomatic people overall. Specificity was unexpectedly lower in symptomatic versus asymptomatic people. Excellent inter-operator agreement was observed, but operational challenges indicate that operator training is warranted.

Performance and Implementation Evaluation of the Abbott BinaxNOW Rapid Antigen Test in a High-throughput Drive-through Community Testing Site in Massachusetts (preprint)

BackgroundRapid diagnostic tests (RDTs) for SARS-CoV-2 antigens (Ag) that can be performed at point-of-care (POC) can supplement molecular testing and help mitigate the COVID-19 pandemic. Deployment of an Ag RDT requires an understanding of its operational and performance characteristics under real-world conditions and in relevant subpopulations. We evaluated the Abbott BinaxNOW COVID-19 Ag Card in a high-throughput, drive-through, free community testing site in Massachusetts (MA) using anterior nasal (AN) swab RT-PCR for clinical testing. MethodsIndividuals presenting for molecular testing in two of seven lanes were offered the opportunity to also receive BinaxNOW testing. Dual AN swabs were collected from symptomatic and asymptomatic children ([≤] 18 years) and adults. BinaxNOW testing was performed in a testing pod with temperature/humidity monitoring. One individual performed testing and official result reporting for each test, but most tests had a second independent reading to assess inter-operator agreement. Positive BinaxNOW results were scored as faint, medium, or strong. Positive BinaxNOW results were reported to patients by phone and they were instructed to isolate pending RT-PCR results. The paired RT-PCR result was the reference for sensitivity and specificity calculations. ResultsOf 2482 participants, 1380 adults and 928 children had paired RT-PCR/BinaxNOW results and complete symptom data. 974/1380 (71%) adults and 829/928 (89%) children were asymptomatic. BinaxNOW had 96.5% (95% confidence interval [CI] 90.0-99.3) sensitivity and 100% (98.6-100.0) specificity in adults within 7 days of symptoms, and 84.6% (65.1-95.6) sensitivity and 100% (94.5-100.0) specificity in children within 7 days of symptoms. Sensitivity and specificity in asymptomatic adults were 70.2% (56.6-81.6) and 99.6% (98.9-99.9), respectively, and in asymptomatic children were 65.4% (55.6-74.4) and 99.0% (98.0-99.6), respectively. By cycle threshold (Ct) value cutoff, sensitivity in all subgroups combined (n=292 RT-PCR-positive individuals) was 99.3% with Ct [≤]25, 95.8% with [≤]30, and 81.2% with [≤]35. Twelve false positive BinaxNOW results (out of 2308 tests) were observed; in all twelve, the test bands were faint but otherwise normal, and were noted by both readers. One invalid BinaxNOW result was identified. Inter-operator agreement (positive versus negative BinaxNOW result) was 100% (n = 2230/2230 double reads). Each operator was able to process 20 RDTs per hour. In a separate set of 30 specimens (from individuals with symptoms [≤]7 days) run at temperatures below the manufacturers recommended range (46-58.5{degrees}F), sensitivity was 66.7% and specificity 95.2%. ConclusionsBinaxNOW had very high specificity in both adults and children and very high sensitivity in newly symptomatic adults. Overall, 95.8% sensitivity was observed with Ct [≤] 30. These data support public health recommendations for use of the BinaxNOW test in adults with symptoms for [≤]7 days without RT-PCR confirmation. Excellent inter-operator agreement indicates that an individual can perform and read the BinaxNOW test alone. A skilled laboratorian can perform and read 20 tests per hour. Careful attention to temperature is critical.

Evaluation of the accuracy and ease-of-use of Abbott PanBio - A WHO emergency use listed, rapid, antigen-detecting point-of-care diagnostic test for SARS-CoV-2 (preprint)

BackgroundDiagnostics are essential for controlling the pandemic. Identifying a reliable and fast diagnostic is needed to support testing. We assessed performance and ease-of-use of the Abbott PanBio antigen-detecting rapid diagnostic test (Ag-RDT). MethodsThis prospective, multi-centre diagnostic accuracy study enrolled at two sites in Germany. Following routine testing with RT-PCR, a second study-exclusive swab was performed for Ag-RDT testing. Routine swabs were nasopharyngeal (NP) or combined NP/oropharyngeal (OP) whereas the study-exclusive swabs were NP. To evaluate performance, sensitivity and specificity were assessed overall and in predefined sub analyses accordingly to cycle-threshold values, days of symptoms, disease severity and study site. Additionally, an ease-of-use assessment and System Usability Scale (SUS) were performed. Findings1108 participants were enrolled between Sept 28 and Oct 30, 2020. Of these, 106 (9{middle dot}6%) were PCR-positive. The Abbott PanBio detected 92/106 PCR-positive participants with a sensitivity of 86{middle dot}8% (95% CI: 79{middle dot}0% - 92{middle dot}0%) and a specificity of 99{middle dot}9% (95% CI: 99{middle dot}4%-100%). The sub analyses indicated that sensitivity was 95{middle dot}8% in CT-values <25 and within the first seven days from symptom onset. The test was characterized as easy to use (SUS: 86/100) and considered suitable for point-of- care settings. InterpretationThe Abbott PanBio Ag-RDT performs well for SARS-CoV-2 testing in this large manufacturer independent study, confirming its WHO recommendation for Emergency Use in settings with limited resources. FundingThe Foundation of Innovative New Diagnostics supplied the test kits for the study. The internal funds from the Heidelberg University as well as the Charite Berlin supported this study.

Performance of Saliva, Oropharyngeal Swabs, and Nasal Swabs for SARS-CoV-2 Molecular Detection: A Systematic Review and Meta-analysis (preprint)

BackgroundNasopharyngeal (NP) swabs are considered the highest-yield sample for diagnostic testing for respiratory viruses, including SARS-CoV-2. The need to increase capacity for SARS-CoV-2 testing in a variety of settings, combined with shortages of sample collection supplies, have motivated a search for alternative sample types with high sensitivity. We systematically reviewed the literature to understand the performance of alternative sample types compared to NP swabs. MethodsWe systematically searched PubMed, Google Scholar, medRxiv, and bioRxiv (last retrieval October 1st, 2020) for comparative studies of alternative specimen types [saliva, oropharyngeal (OP), and nasal (NS) swabs] versus NP swabs for SARS-CoV-2 diagnosis using nucleic acid amplification testing (NAAT). A logistic-normal random-effects meta-analysis was performed to calculate % positive alternative-specimen, % positive NP, and % dual positives overall and in sub-groups. The QUADAS 2 tool was used to assess bias. ResultsFrom 1,253 unique citations, we identified 25 saliva, 11 NS, 6 OP, and 4 OP/NS studies meeting inclusion criteria. Three specimen types captured lower % positives [NS (0.82, 95% CI: 0.73-0.90), OP (0.84, 95% CI: 0.57-1.0), saliva (0.88, 95% CI: 0.81 - 0.93)] than NP swabs, while combined OP/NS matched NP performance (0.97, 95% CI: 0.90-1.0). Absence of RNA extraction (saliva) and utilization of a more sensitive NAAT (NS) substantially decreased alternative-specimen yield. ConclusionsNP swabs remain the gold standard for diagnosis of SARS-CoV-2, although alternative specimens are promising. Much remains unknown about the impact of variations in specimen collection, processing protocols, and population (pediatric vs. adult, late vs. early in disease course) and head-to head studies of sampling strategies are urgently needed.

Correlation of SARS-CoV-2 nucleocapsid antigen and RNA concentrations in nasopharyngeal samples from children and adults using an ultrasensitive and quantitative antigen assay (preprint)

BackgroundDiagnosis of COVID-19 by PCR offers high sensitivity, but the utility of detecting samples with high cycle threshold (Ct) values remains controversial. Currently available rapid diagnostic tests (RDTs) for SARS-CoV-2 nucleocapsid antigens (Ag) have sensitivity well below PCR. The correlation of Ag and RNA quantities in clinical nasopharyngeal (NP) samples is unknown. MethodsAn ultrasensitive, quantitative electrochemiluminescence immunoassay for SARS-CoV-2 nucleocapsid (the MSD(R) S-PLEX(R) CoV-2 N assay) was used to measure Ag in clinical NP samples from adults and children previously tested by PCR. ResultsThe S-PLEX Ag assay had a limit of detection (LOD) of 0.16 pg/mL and a cutoff of 0.32 pg/mL. Ag concentrations measured in clinical NP samples (collected in 3.0 mL media) ranged from less than 160 fg/mL to 2.7 ug/mL. Log-transformed Ag concentrations correlated tightly with Ct values. In 35 adult and 101 pediatric PCR-positive samples, sensitivity was 91% (95% CI, 77-98%) and 79% (70-87%), respectively. In samples with Ct [≤] 35, sensitivity was 100% (88-100%) and 96% (88-99%), respectively. In 50 adult and 40 pediatric PCR-negative specimens, specificity was 100% (93-100%) and 98% (87-100%), respectively. ConclusionsNucleocapsid concentrations in clinical NP samples span 8 orders of magnitude and correlate closely with RNA concentrations (Ct values). The S-PLEX Ag assay had 96-100% sensitivity in samples from children and adults with Ct values [≤] 35, and 98-100% specificity. These results clarify Ag concentration distributions in clinical samples, providing insight into the performance of Ag RDTs and offering a new approach to diagnosis of COVID-19. Key pointsSARS-CoV-2 nucleocapsid concentrations in clinical nasopharyngeal samples, measured with an ultrasensitive assay, spanned an 8-log range and correlated closely with PCR Ct values. The assay was 96-100% sensitive in pediatric/adult samples with Ct values [≤] 35, and 98-100% specific.