Standardization and Comparison of Emergency Use Authorized COVID-19 Assays and Testing Laboratories (preprint)

SARS-CoV-2, the causal agent of the global COVID-19 pandemic, made its appearance at the end of 2019 and is still circulating in the population. The pandemic led to an urgent need for fast, reliable, and widely available testing. After December 2020, the emergence of new variants of SARS-CoV-2 led to additional challenges since new and existing tests had to detect variants to the same extent as the original Wuhan strain. When an antigen-based test is submitted to the Food and Drug Administration (FDA) for Emergency Use Authorization (EUA) consideration it is benchmarked against PCR comparator assays, which yield cycle threshold (CT) data as an indirect indicator of viral load - the lower the CT, the higher the viral load of the sample and the higher the CT, the lower the viral load. The FDA mandates that 10-20% of clinical samples used to evaluate the antigen test have to be low positive. Low positive, as defined by the FDA, are clinical samples in which the CT of the SARS-CoV-2 target gene is within 3 CT of the mean CT value of the approved comparator tests Limit of Detection (LOD). While all comparator tests are PCR-based, the results from different PCR assays used are not uniform. Results vary depending on assay platform, target gene, LOD and laboratory methodology. The emergence and dominance of the Omicron variant further challenged this approach as the fraction of low positive clinical samples dramatically increased as compared to earlier SARS-CoV-2 variants. This led to 20-40% of clinical samples having high CT values and therefore assays vying for an EUA were failing to achieve the 80% Percent Positive Agreement (PPA) threshold required. Here we describe the methods and statistical analyses used to establish a predefined cutoff, based on genome copies per ml (GE/ml) to classify samples as low positive (less than the cutoff GE/ml) or high positive (greater than the cutoff GE/mL). CT 30 for the E gene target using Cobas(R) SARS-CoV-2-FluA/B platform performed at TriCore Reference Laboratories, and this low positive cutoff value was used for two EUA authorizations. Using droplet digital PCR and methods described here, a value 49,447.72 was determined as the GE/ml equivalent for the low positive cutoff. The CT cutoff corresponding to 49447.72 GE/ml was determined across other platforms and laboratories. The methodology and statistical analysis described here can now be used for standardization of all comparators used for FDA submissions with a goal towards establishing uniform criteria for EUA authorization. MotivationThe motivation for this work was the need to establish a predefined cutoff based on genome copies per ml (GE/ml) rather than Ct, which can vary depending on the laboratory and assay used. A GE/ml-based threshold was necessary to define what constituted low positives" for samples that were included in data sets submitted to the FDA for emergency use approval for SARS-CoV-2 antigen tests.

Viral and host factors associated with SARS-CoV-2 disease severity in Georgia, USA (preprint)

While SARS-CoV-2 vaccines have shown strong efficacy, their suboptimal uptake combined with the continued emergence of new viral variants raises concerns about the ongoing and future public health impact of COVID-19. We investigated viral and host factors, including vaccination status, that were associated with SARS-CoV-2 disease severity in a setting with low vaccination rates. We analyzed clinical and demographic data from 1,957 individuals in the state of Georgia, USA, coupled with viral genome sequencing from 1,185 samples. We found no difference in disease severity between individuals infected with Delta and Omicron variants among the participants in this study, after controlling for other factors, and we found no specific mutations associated with disease severity. Compared to those who were unvaccinated, vaccinated individuals experienced less severe SARS-CoV-2 disease, and the effect was similar for both variants. Vaccination within 270 days before infection was associated with decreased odds of moderate and severe outcomes, with the strongest association observed at 91-270 days post-vaccination. Older age and underlying health conditions, especially immunosuppression and renal disease, were associated with increased disease severity. Overall, this study provides insights into the impact of vaccination status, variants/mutations, and clinical factors on disease severity in SARS-CoV-2 infection when vaccination rates are low. Understanding these associations will help refine and reinforce messaging around the crucial importance of vaccination in mitigating the severity of SARS-CoV-2 disease.

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.

Adequacy of Self-Collected Anterior Nares Swabs for SARS-CoV-2 Testing by Grade School Children (preprint)

Background. The goal of this study was to characterize the ability of school-aged children to self-collect adequate anterior nares (AN) swabs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing. Methods. From July to August 2021, 287 children, age 4-14 years-old, were prospectively enrolled in the Atlanta area. Symptomatic (n=197) and asymptomatic (n=90) children watched a short instructional video before providing a self-collected AN specimen. Health care workers (HCWs) then collected a second specimen, and useability was assessed by the child and HCW. Swabs were tested side-by-side for SARS-CoV-2. RNase P RNA detection was investigated as a measure of specimen adequacy. Results. Among symptomatic children, 87/196 (44.4%) tested positive for SARS-CoV-2 by both self- and HCW-swab. Two children each were positive by self- or HCW-swab; one child had an invalid HCW-swab. Compared to HCW-swabs, self-collected swabs had 97.8% and 98.1% positive and negative percent agreements, respectively, and SARS-CoV-2 Ct values did not differ significantly between groups. Participants [≤]8 years-old were less likely than those >8 to be rated as correctly completing self-collection, but SARS-CoV-2 detection did not differ. Based on RNase P RNA detection, 270/287 children (94.1%) provided adequate self-swabs versus 277/287 (96.5%) HCW-swabs (p=0.24) with no difference when stratified by age. Conclusions. Children, aged 4-14 years-old, can provide adequate AN specimens for SARS-CoV-2 detection when presented with age-appropriate instructional material, consisting of a video and a handout, at a single timepoint. These data support the use of self-collected AN swabs among school-age children for SARS-CoV-2 testing.

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

Nucleocapsid antigenemia is a marker of acute SARS-CoV-2 infection (preprint)

BackgroundReliable detection of SARS-CoV-2 infection is essential for diagnosis and treatment of disease as well as infection control and prevention during the ongoing COVID-19 pandemic. Existing nucleic acid tests do not reliably distinguish acute from resolved infection, as residual RNA is frequently detected in the absence of replication-competent virus. We hypothesized that viral nucleocapsid in serum or plasma may be a specific biomarker of acute infection that could enhance isolation and treatment strategies at an individualized level. MethodsSamples were obtained from a retrospective serological survey using a convenience sampling method from adult inpatient and outpatient encounters from January through March 2021. Samples were categorized along a timeline of infection (e.g. acute, late presenting, convalescent) based on timing of available SARS-CoV-2 testing and symptomatology. Nucleocapsid was quantified by digital immunoassay on the Quanterix HD-X platform. ResultsIn a large sample of 1860 specimens from 1607 patients, the highest level and frequency of antigenemia were observed in samples obtained during acute SARS-CoV-2 infection. Levels of antigenemia were highest in samples from seronegative individuals and in those with more severe disease. Using ROC analysis, we found that antigenemia exhibited up to 85.8% sensitivity and 98.6% specificity as a biomarker for acute COVID-19. ConclusionsNucleocapsid antigenemia is a sensitive and specific biomarker for acute SARS-CoV-2 infection and may aid in individualized assessment of SARS-CoV-2 infection resolution or persistence, although interpretation is limited by absence of a diagnostic gold standard for active infection.

Clinical evaluation of the Diagnostic Analyzer for Selective Hybridization (DASH): a point-of-care PCR test for rapid detection of SARS-CoV-2 infection (preprint)

Background: Rapid and accurate testing for SARS-CoV-2 is an essential tool in the medical and public health response to the COVID-19 pandemic. An ideal test for COVID-19 would combine the sensitivity of laboratory-based PCR combined with the speed and ease of use of point-of-care (POC) or home-based rapid antigen testing. Methods: To evaluate the performance of the Diagnostic Analyzer for Selective Hybridization (DASH) SARS-CoV-2 POC PCR (sample insertion to result time of 16 minutes), we conducted a cross-sectional study of adults with and without symptoms of COVID-19 at four clinical sites. We collected two bilateral anterior nasal swabs from each participant and information on COVID-19 symptoms, vaccination, and exposure. One swab was tested with the DASH SARS-CoV-2 POC PCR and the second in a central laboratory using Cepheid Xpert Xpress SARS-CoV-2 PCR. We assessed test concordance and calculated sensitivity, specificity, negative and positive predictive values using Xpert as the "gold standard." Results: We enrolled 315 and analyzed 313 participants with median age 42 years; 65% were female, 62% symptomatic, 75% had received [≥]2 doses of mRNA COVID-19 vaccine, and 16% currently COVID-19 positive. There were concordant results for 307 tests indicating an overall agreement for DASH of 0.98 [95% CI 0.96, 0.99] compared to Xpert. DASH performed at 0.96 [95% CI 0.86, 1.00] sensitivity and 0.98 [95% CI 0.96, 1.00] specificity, with a positive predictive value of 0.85 [95% CI 0.73, 0.96] and negative predictive value of 0.996 [95% CI 0.99, 1.00]. The six discordant tests between DASH and Xpert all had high Ct values (>30) on the respective positive assay. DASH and Xpert Ct values were highly correlated (R=0.89 [95% CI 0.81, 0.94]). Conclusions: DASH POC SARS-CoV-2 PCR was accurate, easy to use, and provided fast results in real-life clinical settings with an overall performance similar to an EUA-approved laboratory-based PCR. Its compact design and ease of use are optimal for a variety of healthcare, and potentially community settings, including areas with lack of access to central laboratory-based PCR testing.

Buzz about RT-qPCR: An RT-qPCR formulation for SARS-CoV-2 detection using reagents produced at Georgia Institute of Technology (preprint)

Widespread testing for the presence novel coronavirus SARS-CoV-2 in patients remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. The early testing shortfall in some parts of the US can be traced to an initial shortage of supplies, expertise and/or instrumentation necessary to detect the virus by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can produce the RT-qPCR assay and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. We compare the performance of our in-house kit to a commercial product used for diagnostic testing and describe implementation of environmental testing to monitor surfaces across various campus laboratories for the presence of SARS-CoV-2.