The global COVID-19 pandemic has resulted in a worldwide shortage of viral transport media and raised questions about specimen stability. The objective of this study was to determine the stability of SARS-CoV-2 virus RNA in specimen transport media under various storage conditions. Transport medium tested included: VCM, UTM®-RT, ESwab™, M4 and saline (0.9% NaCl). Specimen types tested included Nasopharyngeal/Oropharyngeal (NP/OP) swabs in the above transport media, bronchoalveolar lavage (BAL) and Sputum. A high-titer SARS-CoV-2 remnant patient specimen was spiked into pooled SARS-CoV-2 RNA-negative specimen remnants for the various media types. Aliquots of samples were stored at 18°C to 25°C, 2°C to 8°C and -10°C to -30°C and then tested at time points up to 14 days. Specimens consistently yielded amplifiable RNA with mean Ct differences of <3 over the various conditions assayed, thus supporting the use and transport of alternative collection media and specimen types under a variety of temperature storage conditions.

Key Words: Pim-1 Ⅲ mitochondria Ⅲ cardiomyocyte Ⅲ apoptosis C ardiovascular disease is the leading cause of death among men and women and affects approximately 33% of the US population. 1 A direct correlation between the decline in heart function and loss of cardiomyocytes via apoptosis involving the mitochondria occurs in cardiomyopathy, myocardial ischemia/reperfusion (I/R), and congestive heart failure. 2-9 Specifically, myocardial I/R injury generates calcium overload and oxidative stress, which initiate the intrinsic apoptotic pathway through activation of the mitochondrial permeability transition pore (mPTP). The ensuing chain of events result in dramatic changes to mitochondrial morphology associated with uncoupling of the electron transport chain, depolarization of the inner membrane, matrix swelling, unfolding of the cristae, and ultimately outer membrane rupture, with release of proapoptotic cytochrome c. 10 -15 Release of cytochrome c into the cytosol consequently activates apoptotic protease-activating factor, which mediates caspase cascade programmed cell death. 16 Thus, preservation of mitochondrial integrity is essential in designing molecular strategies to enhance cardiomyocyte cell survival by blunting injury attributed to cardiomyopathic insult.Cardioprotection mediated by survival kinase signal transduction acts through multiple mechanisms including preservation of mitochondrial integrity. 17 Numerous studies have documented antiapoptotic actions of the serine/threonine kinase AKT, which acts in part through protecting mitochon-

BackgroundGenomic surveillance efforts for SARS-CoV-2 are needed to understand the epidemiology of the COVID-19 pandemic. Viral variants may impact routine diagnostic testing, increase viral transmissibility, cause differences in disease severity, have decreased susceptibility to therapeutics, and/or confer the ability to evade host immunity. While viral whole-genome sequencing (WGS) has played a leading role in surveillance programs, many laboratories lack the expertise and resources for performing WGS. This study describes the performance of multiplexed real-time reverse transcription-PCR (RT-PCR) assays for identification of SARS-CoV-2 variants.MethodsSARS-CoV-2 specimens were tested for spike-gene variants using a combination of allele-specific primer and allele-specific detection technology (PlexPrime® and PlexZyme®). Targeted detection of spike gene mutations by RT-PCR was compared to variant detection in positive specimens by WGS, including the recently emerged SARS-CoV-2 Omicron variant.ResultsA total of 398 SAR-CoV-2 RT-PCR positive and 39 negative specimens previously tested by WGS were re-tested by RT-PCR genotyping. PCR detection of spike gene mutations N501Y, E484K, and S982A correlated 100% with WGS for the 29 lineages represented, including Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). Incorporating the P681R spike gene mutation also allowed screening for the SARS-CoV-2 Delta variant (B.1.617.2 and AY sublineages). Further sampling of 664 specimens that were screened by WGS between June and August 2021 and then re-tested by RT-PCR showed strong agreement for Delta variant positivity: 34.5% for WGS vs 32.9% for RT-PCR in June; 100% vs 97.8% in August. In a blinded panel of 16 Omicron and 16 Delta specimens, results of RT-PCR were 100% concordant with WGS results.ConclusionsThese data demonstrate that multiplexed real-time RT-PCR genotyping has strong agreement with WGS and may provide additional SARS-CoV-2 variant screening capabilities when WGS is unavailable or cost-prohibitive. RT-PCR genotyping assays may also supplement existing sequencing efforts while providing rapid results at or near the time of diagnosis to help guide patient management.

Background Nucleic acid amplification testing is a critical tool for addressing the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. Specimen pooling can increase throughput and conserve testing resources but requires validation to ensure that reduced sensitivity does not increase the false-negative rate. We evaluated the performance of a real time reverse transcription PCR (RT-PCR) test authorized by the U.S. Food and Drug Administration (FDA) for emergency use for pooled testing of upper respiratory specimens. Methods Positive specimens were selected from three prevalence groups, 1-3%, &gt;3-6%, and &gt;6-10%. Positive percent agreement (PPA) was assessed by pooling single-positive specimens with three negative specimens; performance was assessed using Passing-Bablok regression. Additionally, we assessed the distributions of RT-PCR cycle threshold (Ct) values for 3,091 positive specimens. Results PPA was 100% for the 101 pooled specimens. There was a linear relationship between Ct values for pooled and single-tested specimens (r: 0.96-0.99, slope ≈ 1). The mean pooled Ct shifts at 40 cycles were 2.38 and 1.90 respectively for the N1 and N3 targets. The median Cts for 3,091 positive specimens were 25.9 (N1) and 24.7 (N3). The percent of positive specimens with Cts between 40 and the shifted Ct was 1.42% (N1) and 0.0% (N3). Conclusions Pooled and individual testing of specimens positive for SARS-CoV-2 demonstrated 100% agreement and demonstrate the viability of pooled specimens for SARS-COV-2 testing using a dual-target RT-PCR system. Pooled specimen testing can help increase testing capacity for SARS-CoV-2, with a low risk of false-negative results.

Background The use of a remote specimen collection strategy employing a kit designed for unobserved self-collection for SARS-CoV-2 RT-PCR can decrease the use of PPE and exposure risk. To assess the impact of unobserved specimen self-collection on test performance, we examined results from a SARS-CoV-2 qualitative RT-PCR test for self-collected specimens from participants in a return-to-work screening program and assessed the impact of a pooled testing strategy in this cohort. Methods Self-collected anterior nasal swabs from employee return to work programs were tested using the Quest Diagnostics SARS-CoV-2 RT-PCR EUA. The Ct values for the N1 and N3 N-gene targets and a human RNase P (RP) gene control target were tabulated. For comparison, we utilized Ct values from a cohort of HCP-collected specimens from patients with and without COVID-19 symptoms. Results Among 47,923 participants, 1.8% were positive. RP failed to amplify for 13/115,435 (0.011%) specimens. The median (IQR) Cts were 32.7 (25.0-35.7) for N1 and 31.3 (23.8-34.2) for N3. Median Ct values in the self-collected cohort were significantly higher than those of symptomatic, but not asymptomatic patients. Based on Ct values, pooled testing with 4 specimens would have yielded inconclusive results in 67/1,268 (5.2%) specimens but only a single false-negative result. Conclusions Unobserved self-collection of nasal swabs provides adequate sampling for SARS-CoV-2 RT-PCR testing. These findings alleviate concerns of increased false negatives in this context. Specimen pooling could be used for this population as the likelihood of false negative results is very low due when using a sensitive, dual-target methodology.