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.

A Multi-Institutional Study Benchmarking Cycle Threshold Values for Major Clinical SARS-CoV-2 RT-PCR Assays (preprint)

Real-time, reverse transcriptase PCR assays are a pervasive technology used for diagnosis of SARS-CoV-2 infection. These assays produce a cycle threshold value (Ct) corresponding to the first amplification cycle in which reliable amplification is detected. Such Ct values have been used by clinicians and in public health settings to guide treatment, monitor disease progression, assess prognosis, and inform isolation practices. To understanding the risk of reporting out uncalibrated Ct values and potential for instead reporting out calibrated viral load values, we performed a multi-institutional study to benchmark major clinical platforms against a calibrated standard. We found that for any given Ct value, corresponding viral loads varied up to 1000-fold among the different tests. In contrast, when these different assays were calibrated against a common standard and then used to test unknown de-identified specimens at several dilutions, viral load values showed high precision between methods (standard deviation and range of 0.36 and 1.1 log10 genome copies) and high accuracy compared with droplet digital PCR (ddPCR) determinations (difference between mean CDC N2 and Sarbeco E ddPCR determinations and mean determinations by calibrated RT-PCR assays examined in our study of 0.044 log10 genome copies). We, therefore, find strong support for calibration of SARS-CoV-2 RT-PCR tests to allow conversion of cycle thresholds to accurate and precise viral load values that are reproducible across major clinical systems. Implementation of calibrated assays will provide more reliable information for clinical decision making and allow more rigorous interpretation of SARS-CoV-2 laboratory data in clinical and laboratory investigation.

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).

Limit of Detection for Rapid Antigen Testing of the SARS-CoV-2 Omicron Variant (preprint)

There has been debate in the literature about the ability of antigen tests to detect the SARS-CoV-2 Omicron variant including indication on the US Food and Drug administration website that antigen tests may have lower sensitivity for the Omicron variant without provision of data or the potential scale of the issue (see https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/sars-cov-2-viral-mutations-impact-covid-19-tests - omicronvariantimpact, accessed 1/27/2022). Here we determined the limit of detection (LoD) for the Omicron variant compared with the WA1 strain used for LoD studies described in the Instructions for Use for all Emergency Use Authorization (EUA)-approved antigen tests. Using live virus (to avoid artifactual findings potentially obtained with gamma-irradiated or heat-killed virus) quantified by plaque forming units (PFU), we examined the analytical sensitivity of three antigen tests widely used in the United States: the Abbott Binax Now, the AccessBio CareStart , and LumiraDx antigen tests. We found that the 95% detection threshold (LoD) for antigen tests was at least as good for Omicron as for the WA1 strain. Furthermore, the relationship of genome copies to plaque forming units for Omicron and WA1 overlap. Therefore, the LoD equivalency also applies if the quantitative comparator is genome copies determined from live virus preparations. Taken together, our data support the continued ability of the antigen tests examined to detect the Omicron variant.

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.

Large-scale, in-house production of viral transport media to support SARS-CoV-2 PCR testing in a multi-hospital healthcare network during the COVID-19 pandemic (preprint)

The COVID-19 pandemic has severely disrupted worldwide supplies of viral transport media (VTM) due to widespread demand for SARS-CoV-2 RT-PCR testing. In response to this ongoing shortage, we began production of VTM in-house in support of diagnostic testing in our hospital network. As our diagnostic laboratory was not equipped for reagent production, we took advantage of space and personnel that became available due to closure of the research division of our medical center. We utilized a formulation of VTM described by the CDC that was simple to produce, did not require filtration for sterilization, and used reagents that were available from commercial suppliers. Performance of VTM was evaluated by several quality assurance measures. Based on Ct values of spiking experiments, we found that our VTM supported highly consistent amplification of the SARS-CoV-2 target (coefficient of variation = 2.95%) using the Abbott RealTime SARS-CoV-2 EUA assay on the Abbott m2000 platform. VTM was also found to be compatible with multiple swab types and, based on accelerated stability studies, able to maintain functionality for at least four months at room temperature. We further discuss how we met logistical challenges associated with large-scale VTM production in a crisis setting including use of staged, assembly line for VTM transport tube production.

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.