Assessment of inactivation procedures for SARS-CoV-2

Auerswald, Heidi; Yann, Sokhoun; Dul, Sokha; In, Saraden; Dussart, Philippe; Martin, Nicholas J.; Karlsson, Erik A.; Garcia-Rivera, Jose A.

doi:10.1099/jgv.0.001539

Cited by 51 publications

(49 citation statements)

References 35 publications

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“…Searching for a solution capable of stabilizing SARS-CoV-2 RNA in saliva, we initially screened six formulations containing Proteinase K (namely PK1-6). AVL, a commercial guanidine-based buffer recommended for SARS-CoV-2 inactivation [10], was used as the experimental control, without any heat treatment. Since unprotected viral RNA is rapidly degraded in crude saliva, we simulated samples by first mixing saliva from a healthy donor with the solutions (1:1, v/v) and heating at 65 • C for 15 min followed by a step at 95 • C for 2 min, before spiking in SARS-CoV-2 RNA.…”

Section: Stabilization Of Sars-cov-2 Rna In Salivamentioning

confidence: 99%

A Novel Saliva RT-LAMP Workflow for Rapid Identification of COVID-19 Cases and Restraining Viral Spread

et al. 2021

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Rapid diagnostics is pivotal to curb SARS-CoV-2 transmission, and saliva has emerged as a practical alternative to naso/oropharyngeal (NOP) specimens. We aimed to develop a direct RT-LAMP (reverse transcription loop-mediated isothermal amplification) workflow for viral detection in saliva, and to provide more information regarding its potential in curbing COVID-19 transmission. Clinical and contrived specimens were used to optimize formulations and sample processing protocols. Salivary viral load was determined in symptomatic patients to evaluate the clinical performance of the test and to characterize saliva based on age, gender and time from onset of symptoms. Our workflow achieved an overall sensitivity of 77.2% (n = 90), with 93.2% sensitivity, 97% specificity, and 0.895 Kappa for specimens containing >102 copies/μL (n = 77). Further analyses in saliva showed that viral load peaks in the first days of symptoms and decreases afterwards, and that viral load is ~10 times lower in females compared to males, and declines following symptom onset. NOP RT-PCR data did not yield relevant associations. This work suggests that saliva reflects the transmission dynamics better than NOP specimens, and reveals gender differences that may reflect higher transmission by males. This saliva RT-LAMP workflow can be applied to track viral spread and, to maximize detection, testing should be performed immediately after symptoms are presented, especially in females.

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Section: Stabilization Of Sars-cov-2 Rna In Salivamentioning

confidence: 99%

A Novel Saliva RT-LAMP Workflow for Rapid Identification of COVID-19 Cases and Restraining Viral Spread

et al. 2021

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“…To control for extensive variation in qPCR test and target (each of which reported varying thresholds for positivity), we carried out in vitro experiments using SARS-CoV-2 isolates from infected patients reporting similar Ct values on the TaqPath platform at the time of sampling. Briefly, three SARS-CoV-2 isolates (designated hCoV-19/Madagascar/IPM-00754/2021, hCoV-19/Madagascar/IPM-01263/2021 and hCoV-19/Madagascar/IPM-01315/2021) were obtained and cultured in Vero cells as previously described [34]. Upon infection with SARS-CoV-2, the culture medium was replaced by infection medium containing DMEM, 5 % FBS, antibiotics, 2.5 μg/ml Amphotericin B (Gibco) and 16 μg/ml TPCK-trypsin (Gibco).…”

Section: Standardizing Ct Values Across Tests and Targetsmentioning

confidence: 99%

Cross-sectional cycle threshold values reflect epidemic dynamics of COVID-19 in Madagascar

Andriamandimby

Brook

Razanazatovo

et al. 2021

Preprint

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As the national reference laboratory for febrile illness in Madagascar, we processed samples from the first epidemic wave of COVID-19, between March and September 2020. We fit generalized additive models to cycle threshold (Ct) value data from our RT-qPCR platform, demonstrating a peak in high viral load, low-Ct value infections temporally coincident with peak epidemic growth rates estimated in real time from publicly-reported incidence data and retrospectively from our own laboratory testing data across three administrative regions. We additionally demonstrate a statistically significant effect of duration of time since infection onset on Ct value, suggesting that Ct value can be used as a biomarker of the stage at which an individual is sampled in the course of an infection trajectory. As an extension, the population-level Ct distribution at a given timepoint can be used to estimate population-level epidemiological dynamics. We illustrate this concept by adopting a recently-developed, nested modeling approach, embedding a within-host viral kinetics model within a population-level Susceptible-Exposed-Infectious-Recovered (SEIR) framework, to mechanistically estimate epidemic growth rates from cross-sectional Ct distributions across three regions in Madagascar. We find that Ct-derived epidemic growth estimates slightly precede those derived from incidence data across the first epidemic wave, suggesting delays in surveillance and case reporting. Our findings indicate that public reporting of Ct values could offer an important resource for epidemiological inference in low surveillance settings, enabling forecasts of impending incidence peaks in regions with limited case reporting.

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“…Moreover, samples with high viral load pose a risk of infection for the handler. Heat treatment of viral particles has been shown effective in inactivating SARS-CoV-2 with 70˚C 5min treatment rendering viral infectivity undetectable employing Vero E6 cells (limit of detection of TCID50 assay was 100 TCID50/mL) [18][19][20]. Other heat treatment protocols have also been demonstrated, with variable effects on PCR sensitivity [21].…”

Section: Heat Inactivation Comparisonmentioning

confidence: 99%

Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation

et al. 2021

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There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP. RNA extraction methods provided similar results, with Beckman performing better with our primer-probe combinations. Luna proved most sensitive although overall the three reagents did not show significant differences. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrated that heat treatment of nasopharyngeal swabs at 70°C for 10 or 30 min, or 90°C for 10 or 30 min (both original variant and B 1.1.7) inactivated SARS-CoV-2 employing plaque assays, and had minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable in settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/.

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Assessment of inactivation procedures for SARS-CoV-2

Cited by 51 publications

References 35 publications

A Novel Saliva RT-LAMP Workflow for Rapid Identification of COVID-19 Cases and Restraining Viral Spread

A Novel Saliva RT-LAMP Workflow for Rapid Identification of COVID-19 Cases and Restraining Viral Spread

Cross-sectional cycle threshold values reflect epidemic dynamics of COVID-19 in Madagascar

Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation

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