Contrasting Effects of SARS-CoV-2 Vaccination vs. Infection on Antibody and TCR Repertoires (preprint)

Antibodies and helper T cells play important roles in SARS-CoV-2 infection and vaccination. We sequenced B- and T-cell receptor repertoires (BCR/TCR) from the blood of 251 infectees, vaccinees, and controls to investigate whether features of these repertoires could predict subjects' SARS-CoV-2 neutralizing antibody titer (NAbs), as measured by enzyme-linked immunosorbent assay (ELISA). We sequenced recombined immunoglobulin heavy-chain (IGH), TCRbeta (TRB), and TCRdelta (TRD) genes in parallel from all subjects, including select B- and T-cell subsets in most cases, with a focus on their hypervariable CDR3 regions, and correlated this AIRRseq data with demographics and clinical findings from subjects' electronic health records. We found that age affected NAb levels in vaccinees but not infectees. Intriguingly, we found that vaccination, but not infection, has a substantial effect on non-productively recombined IGHs, suggesting a vaccine effect that precedes clonal selection. We found that repertoires' binding capacity to known SARS-CoV-2-specific CD4+ TRBs performs as well as the best hand-tuned fuzzy matching at predicting a protective level of NAbs, while also being more robust to repertoire sample size and not requiring hand-tuning. The overall conclusion from this large, unbiased, clinically well annotated dataset is that B- and T-cell adaptive responses to SARS-CoV-2 infection and vaccination are surprising, subtle, and diffuse. We discuss methodological and statistical challenges faced in attempting to define and quantify such strong-but-diffuse repertoire signatures and present tools and strategies for addressing these challenges.

SARS-CoV-2 live virus culture and sample freeze-thaw stability (preprint)

The COVID-19 pandemic has presented unique diagnostic challenges including the need to store and test large number of samples for clinical and research studies. While SARS CoV-2 diagnosis relies on RT-qPCR and antigen testing, live virus culture remains an important surrogate for viral 'infectiousness', as we previously described in 'SARS-CoV-2 Antigen Tests Predict Infectivity Based on Viral Culture: Comparison of Antigen, PCR Viral Load and Viral Culture Testing on a Large Sample Cohort' (Clin Microbiol Infect, 2022, PMC9293398). Live virus isolation and characterization has also been important to the SARS CoV-2 research community, to assess viral fitness, cellular tropism, and live virus neutralization, particularly with the emergence of new variants. Many clinical and research studies make use of samples that are frozen in transport media and investigated at later dates. The effect of freezing on RT-qPCR results is well established. However, the effect of freeze-thaw on viral viability has not been. Here, we therefore examined the effect of freeze-thaw on viral culture isolation from a large number of clinical samples that were split, and then cultured either fresh or after being frozen for 7 or 17-18 days. Samples represented the range of viral loads (genome copies/mL) observed in our patient population. We found that freeze-thaw did not significantly affect viral culture isolation. Therefore, the ability to assess infectiousness of samples previously frozen in transport medium appears to be maintained.

The Coviral Portal: Multi-Cohort Viral Loads and Antigen-Test Virtual Trials for COVID-19 (preprint)

Background: Regulatory approval of new over-the-counter tests for infectious agents such as SARS-CoV-2 has historically required that clinical trials include diverse groups of specific patient populations, making the approval process slow and expensive. Showing that populations do not differ in their viral loads---the key factor determining test performance---could expedite the evaluation of new tests. Methods. 46,726 RT-qPCR-positive SARS-CoV-2 viral loads were annotated with patient demographics and health status. Real-world performance of two commercially available antigen tests was evaluated over a wide range of viral loads. An open-access web portal was created allowing comparisons of viral-load distributions across patient groups and application of antigen-test performance characteristics to patient distributions to predict antigen-test performance on these groups. Findings. In several cases distributions were surprisingly similar where a difference was expected (e.g. smokers vs. non-smokers); in other cases there was a difference that was the opposite direction from expectations (e.g. higher in patients who identified as White vs. Black). Sensitivity and specificity of antigen tests for detecting contagiousness were similar across most groups. The portal is at https://arnaoutlab.org/coviral/. Conclusions. In silico analyses of large-scale, real-world clinical data repositories can serve as a timely evidence-based proxy for dedicated trials of antigen tests for specific populations. Free availability of richly annotated data facilitates large-scale hypothesis generation and testing.

ct2vl: Converting Ct Values to Viral Loads for SARS-CoV-2 RT-qPCR Test Results (preprint)

RT-qPCR is the de facto reference method for detecting the presence of SARS-CoV-2 genomic material in infected individuals. Although RT-qPCR is inherently quantitative and despite SARS-CoV-2 viral loads varying by 10 orders of magnitude and therefore being potentially highly clinically informative, in practice SARS-CoV-2 RT-qPCR results are usually reported qualitatively as simply positive or negative. This is both because of the mathematical complexity of converting from Ct values to viral loads and because the same Ct value can correspond to orders-of-magnitude differences in viral load depending on the testing platform. To address this problem, here we present ct2vl, a Python package designed to help individual clinical laboratories, investigators, and test developers convert from Ct values to viral loads on their own platforms, using only the data generated during validation of those platforms. It allows any user to convert Ct values to viral loads and is readily applicable to other RT-qPCR tests. ct2vl is open source, has 100% code coverage, and is freely available via the Python Package Index (PyPI).

Visualizing Omicron: COVID-19 Deaths vs. Cases Over Time (preprint)

For most of the COVID-19 pandemic, the daily focus has been on the number of cases, and secondarily, deaths. The most recent wave is caused by the omicron variant, first identified at the end of 2021 and the dominant variant through the first part of 2022. South Africa, one of the first countries to experience and report data regarding omicron (variant 21.K), reported far fewer deaths, even as the number of reported cases rapidly eclipsed previous peaks. However, as the omicron wave has progressed, time series show that it has been markedly different from prior waves. To more readily visualize the dynamics of cases and deaths, it is natural to plot deaths per million against cases per million. Unlike the time-series plots of cases or deaths that have become daily features of pandemic updates during the pandemic, which have time as the x-axis, in a plot of deaths vs. cases, time is implicit, and is indicated in relation to the starting point. Here we present and briefly examine such plots from a number of countries and from the world as a whole, illustrating how they summarize features of the pandemic in ways that illustrate how, in most places, the omicron wave is very different from those that came before. Code for generating these plots for any country is provided on an automatically updating GitHub repository (https://github.com/rarnaout/Covidcycles).

SARS-CoV-2 Antigen Tests Predict Infectivity Based on Viral Culture: Comparison of Antigen, PCR Viral Load, and Viral Culture Testing on a Large Sample Cohort (preprint)

The relationship of SARS-CoV-2 antigen testing results, viral load, and viral culture detection remains to be fully defined. Presumptively, viral culture can provide a surrogate measure for infectivity of sampled individuals, and thereby inform how and where to most appropriately deploy available diagnostic testing modalities. We therefore determined the relationship of antigen testing results from three lateral flow and one microfluidics assay to viral culture performed in parallel in 181 nasopharyngeal swab samples positive for SARS-CoV-2. Sample viral loads, determined by RT-qPCR, were distributed across the range of viral load values observed in our testing population. We found that antigen tests were predictive of viral culture positivity, with the LumiraDx method showing enhanced sensitivity (90%; 95% confidence interval (95% CI) 83-94%) compared with the BD Veritor (74%, 95% CI 65-81%), CareStart (74%, 95% CI 65-81%) and Oscar Corona (74%, 95% CI 65-82%) lateral flow antigen tests. Antigen and viral culture positivity were also highly correlated with sample viral load, with areas under the receiver?operator characteristic curves (ROCs) of 0.94-0.97 and 0.92, respectively. In particular, a viral load threshold of 100,000 copies/mL was 95% sensitive (95% CI, 90-98%) and 72% specific (95% CI, 60-81%) for predicting viral culture positivity. Taken together, the detection of SARS-CoV-2 antigen identified highly infectious individuals, some of whom may harbor 10,000-fold more virus in their samples than those with any detectable infectious virus. As such, our data support use of antigen testing in defining infectivity status at the time of sampling.

Saliva Is Comparable to Nasopharyngeal Swabs for Molecular Detection of SARS-CoV-2 (preprint)

Abstract Background. The continued need for molecular testing for SARS-CoV-2 and promise of self-collected saliva as an alternative to nasopharyngeal (NP) swabs for sample acquisition led us to compare saliva to NP swabs in an outpatient setting, without special restrictions to avoid food, drink, smoking, or toothbrushing. Methods. A total of 385 pairs of NP and saliva specimens were obtained, the majority from individuals presenting for initial evaluation, and were tested on two high-sensitivity RT-PCR platforms: the Abbott m2000 and Abbott Alinity m, both with limits of detection (LoD) of 100 copies of viral RNA/mL. Results. Concordance between saliva and NP was excellent overall (Cohen's {kappa}=0.93) for initial as well as followup testing on both platforms, and for specimens treated with guanidinium transport medium as preservative and for untreated saliva ({kappa}=0.88-0.95). Viral loads were on average 16x higher in NP specimens than saliva specimens, suggesting that only the relatively small fraction of outpatients (~8% in this study) who present with very low viral loads (<1,600 copies/mL from NP swabs) would be missed by saliva testing relative to NP testing, for sensitive testing platforms. Special attention was necessary to ensure leak-resistant specimen collection and transport. Conclusions. The advantages of self-collection without additional restrictions will likely outweigh a minor potential decrease in clinical sensitivity in individuals less likely to pose an infectious risk to others for many real-world scenarios, especially for initial testing.

Verification of the Abbott Alinity m Resp-4-Plex Assay for detection of SARS-CoV-2, influenza A/B, and respiratory syncytial virus (preprint)

COVID-19 symptomology may overlap with those of other circulating respiratory virus. In the future, hospital epidemiology will be focused on viruses with the potential for causation of severe disease, and there will be additional need for detection of respiratory viruses with potential sequelae for which there are specific and potential life-saving treatments. Therefore, there will be increasing need for multiplex respiratory assays to detection such virus. The Abbott Alinity m Resp-4-Plex assay is a multiplex PCR assay that simultaneously detects and differentiates infection with SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus (RSV). Based on perceived assay utility, we characterized the new assay in a series of validation studies to characterize its accuracy, precision, and analytical sensitivity. All were found to be robust within the measures examined. In the context of sample-to-answer, near random access automation on the Alinity m platform, we believe that the Resp-4-Plex assay offers significant utility in addressing the needs of the current SARS-CoV-2 pandemic and especially in the future with anticipated endemic circulation of SARS-CoV-2 with other respiratory viruses.

Private Antibody Repertoires Are Public (preprint)

When faced with a given pathogen, the antibody response generally functions similarly across different people, 1–4 but the source of this similarity has been unclear. One hypothesis was that people share a high proportion of the same VDJ-recombined antibody genes, but this has been disproven. 5,6 An alternative is that people share a high proportion of functionally similar antibodies, 7,8 but testing this hypothesis requires a method for measuring functional similarity that scales to the millions of antibodies per repertoire and across multiple repertoires, which is impossible experimentally. We recently described a framework for doing so computationally, 9 which revealed that repertoires consist of loose overlapping functional classes of antibodies with similar antigen-binding capacities; 10–12 this framework allowed us to estimate a repertoire’s antigen-binding capacity, τ , for the ideal target of any given antibody. Here, we show that this framework supports the second hypothesis, and provide the first comprehensive demonstration of overwhelming functional overlap between repertoires from 20 different individuals directly from sequence, without need of binding studies. Overlap is highest among the young and falls with age, due to the selective loss of antibodies that represent a core set of shared or “public” antigen-binding capacities. We reveal considerable heterogeneity in antigen-binding capacities for antibodies against influenza, HIV, and SARS-CoV-2, and show that while some of these classes shrink with age, others persist across individuals. These discoveries change our understanding of repertoire diversity and have implications for vaccine and therapeutic-antibody development, especially for the aged.

Nasal-Swab Testing Misses Patients with Low SARS-CoV-2 Viral Loads (preprint)

The urgent need for large-scale diagnostic testing for SARS-CoV-2 has prompted pursuit of sample-collection methods of sufficient sensitivity to replace sampling of the nasopharynx (NP). Among these alternatives is collection of nasal-swab samples, which can be performed by the patient, avoiding the need for healthcare personnel and personal protective equipment. Previous studies have reached opposing conclusions regarding whether nasal sampling is concordant or discordant with NP. To resolve this disagreement, we compared nasal and NP specimens collected by healthcare workers in a cohort consisting of individuals clinically suspected of COVID-19 and outpatients known to be SARS-CoV-2 RT-PCR positive undergoing follow-up. We investigated three different transport conditions, including traditional viral transport media (VTM) and dry swabs, for each of two different nasal-swab collection protocols on a total of 308 study participants, and compared categorical results and Ct values to those from standard NP swabs collected at the same time from the same patients. All testing was performed by RT-PCR on the Abbott SARS-CoV-2 RealTime EUA (limit of detection [LoD], 100 copies viral genomic RNA/mL transport medium). We found high concordance (Cohen's kappa >0.8) only for patients with viral loads above 1,000 copies/mL. Those with viral loads below 1,000 copies/mL, the majority in our cohort, exhibited low concordance (Cohen's kappa = 0.49); most of these would have been missed by nasal testing alone. Previous reports of high concordance may have resulted from use of assays with higher LoD ([≥]1,000 copies/mL). These findings counsel caution in use of nasal testing in healthcare settings and contact-tracing efforts, as opposed to screening of asymptomatic, low-prevalence, low-risk populations. Nasal testing is an adjunct, not a replacement, for NP.

SARS-CoV2 Testing: The Limit of Detection Matters (preprint)

Resolving the COVID-19 pandemic requires diagnostic testing to determine which individuals are infected and which are not. The current gold standard is to perform RT-PCR on nasopharyngeal samples. Best-in-class assays demonstrate a limit of detection (LoD) of ~100 copies of viral RNA per milliliter of transport media. However, LoDs of currently approved assays vary over 10,000-fold. Assays with higher LoDs will miss more infected patients, resulting in more false negatives. However, the false-negative rate for a given LoD remains unknown. Here we address this question using over 27,500 test results for patients from across our healthcare network tested using the Abbott RealTime SARS-CoV-2 EUA. These results suggest that each 10-fold increase in LoD is expected to increase the false negative rate by 13%, missing an additional one in eight infected patients. The highest LoDs on the market will miss a majority of infected patients, with false negative rates as high as 70%. These results suggest that choice of assay has meaningful clinical and epidemiological consequences. The limit of detection matters.

Rapid Open Development and Clinical Validation of Multiple New 3D-Printed Nasopharyngeal Swabs in Response to the COVID-19 Pandemic (preprint)

Abstract The SARS-CoV-2 pandemic has caused a severe international shortage of the nasopharyngeal swabs that are required for collection of optimal specimens, creating a critical bottleneck in the way of high-sensitivity virological testing for COVID-19. To address this crisis, we designed and executed an innovative, radically cooperative, rapid-response translational-research program that brought together healthcare workers, manufacturers, and scientists to emergently develop and clinically validate new swabs for immediate mass production by 3D printing. We performed a rigorous multi-step preclinical evaluation on 160 swab designs and 48 materials from 24 companies, laboratories, and individuals, and shared results and other feedback via a public data repository (http://github.com/rarnaout/Covidswab/). We validated four prototypes through an institutional review board (IRB)-approved clinical trial that involved 276 outpatient volunteers who presented to our hospital's drive-through testing center with symptoms suspicious for COVID-19. Each participant was swabbed with a reference swab (the control) and a prototype, and SARS-CoV-2 reverse-transcriptase polymerase chain reaction (RT-PCR) results were compared. All prototypes displayed excellent concordance with the control ({kappa}=0.85-0.89). Cycle-threshold (Ct) values were not significantly different between each prototype and the control, supporting the new swabs' non-inferiority (Mann-Whitney U [MWU] p>0.05). Study staff preferred one of the prototypes over the others and the control swab overall. The total time elapsed between identification of the problem and validation of the first prototype was 22 days. The swabs are available to order (http://printedswabs.org). Our experience holds lessons for the rapid development, validation, and deployment of new technology for this pandemic and beyond.