Clinical performance and sample freeze-thaw stability of the cobas®6800 SARS-CoV-2 assay for the detection of SARS-CoV-2 in oro-/nasopharyngeal swabs and lower respiratory specimens

The events of the last year have highlighted the complexity of implementing large-scale molecular diagnostic testing for novel pathogens. The purpose of this study was to determine the chemical influences of sample collection media and storage on the stability and detection of viral nucleic acids by qRT-PCR. We studied the mechanism(s) through which viral transport media (VTM) and number of freeze-thaw cycles influenced the analytical sensitivity of qRT-PCR detection of SARS-CoV-2. Our goal is to reinforce testing capabilities and identify weaknesses that could arise in resource-limited environments that do not have well-controlled cold chains. The sensitivity of qRT-PCR analysis was studied in four VTM for synthetic single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) simulants of the SARS-CoV-2 genome. The sensitivity and reproducibility of qRT-PCR for the synthetic ssRNA and dsDNA were found to be highly sensitive to VTM with the best results observed for ssRNA in HBSS and PBS-G. Surprisingly, the presence of epithelial cellular material with the ssRNA increased the sensitivity of the qRT-PCR assay. Repeated freeze-thaw cycling decreased the sensitivity of the qRT-PCR with two noted exceptions. The choice of VTM is critically important to defining the sensitivity of COVID-19 molecular diagnostics assays and this study suggests they can impact upon the stability of the SARS-CoV-2 viral genome. This becomes increasingly important if the virus structure is destabilised before analysis, which can occur due to poor storage conditions. This study suggests that COVID-19 testing performed with glycerol-containing PBS will produce a high level of stability and sensitivity. These results are in agreement with clinical studies reported for patient-derived samples.

show abstract

“…When analysing qRT-PCR results, a cut-off point of cycle threshold ( Ct ) ≥ 35 was set for a negative sample. 34,35,28…”

Section: Resultsmentioning

confidence: 99%

Influence of viral transport media and freeze-thaw cycling on the sensitivity of qRT-PCR detection of SARS-CoV-2 nucleic acids

Holohan

Hanrahan

Feely

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…However, the majority of samples from the positive sample group were obtained from symptomatic individuals (96.6%) and also contained all of the discordant results. Finally, our retrospective study required freezing and thawing of specimens, potentially resulting in degradation of viral nucleic acids and rupturing of virions causing initially positive specimens to provide false negative results [26] . This could especially impact samples with viral target levels at or below the LOD.…”

Section: Discussionmentioning

confidence: 99%

Comparison of two nucleic acid amplification tests (NAATs) and two antigen tests for detection of SARS-CoV-2 from upper respiratory specimens

Kuo¹,

Realegeno²,

Pride³

2021

Journal of Clinical Virology Plus

View full text Add to dashboard Cite

There are numerous tests available for acute diagnosis of SARS-CoV-2, the virus that causes the disease COVID-19. These tests fall into two main groups: nucleic acid amplification tests (NAATs) and antigen-based assays. We evaluated the clinical performance of two rapid antigen assays (BD Veritor System for Rapid Detection of SARS CoV-2 and Abbott BinaxNOW COVID-19 Ag Card) and one NAAT (Hologic Aptima SARS CoV-2 Assay) by comparing them with the initial test of record, the Roche cobas SARS-CoV-2 assay; the antigen tests were also compared to Aptima. We tested remnant frozen specimens from patients suspected of SARS-CoV-2 infections (either due to symptoms or exposure) on the comparator platforms to evaluate assay performance across a wide range of positive results, including cobas cycle threshold (Ct) values ranging between 12 and 35. We tested 250 previous positive and 50 previous negative specimens and found 95.6% positive percent agreement (PPA) with the Aptima assay. The few discrepancies between the NAATs occurred only when Ct values were >32. Agreement was much lower for the rapid antigen tests, with 45.2%/47.3% PPA for the Veritor and 47.0%/47.0% PPA for the Binax compared to cobas/Aptima. Discrepancies occurred when cobas Ct values were >20 for Veritor and >25 for Binax. The negative percent agreement (NPA) was 100% for all assay comparisons. These data indicate similar performance between the cobas and Aptima NAATs but demonstrate that antigen-based assays may be insufficient to diagnose SARS-CoV-2 infection when lower levels of the virus are shed.

show abstract

“…We think this is unlikely (1) because prolonged storage at −80°C with a single freeze-thaw cycle has generally not been considered deleterious to detection of enveloped RNA viruses from nasopharyngeal specimens 16 and (2) because a recent evaluation of a single freeze-thaw cycle on SARS-CoV-2 RNA detection using the same assay resulted in 0 of 66 positive samples converting to negative and only 4 of 66 (6%) converting to inconclusive. 17 Finally, household exposure risk may vary over time, as may adherence to infection control recommendations from public health authorities. The NPV was estimated from a sample of HCWs studied during a period when schools and businesses were closed, possibly lowering the risk of household exposure during self-isolation.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of a home-based 7-day infection control strategy for healthcare workers following high-risk exposure to severe acute respiratory coronavirus virus 2 (SARS-CoV-2): A cohort study

Benea

Rendon

Papenburg

et al. 2020

Infect. Control Hosp. Epidemiol.

View full text Add to dashboard Cite

Objective: Evidence-based infection control strategies are needed for healthcare workers (HCWs) following high-risk exposure to severe acute respiratory coronavirus virus 2 (SARS-CoV-2). In this study, we evaluated the negative predictive value (NPV) of a home-based 7-day infection control strategy. Methods: HCWs advised by their infection control or occupational health officer to self-isolate due to a high-risk SARS-CoV-2 exposure were enrolled between May and October 2020. The strategy consisted of symptom-triggered nasopharyngeal SARS-CoV-2 RNA testing from day 0 to day 7 after exposure and standardized home-based nasopharyngeal swab and saliva testing on day 7. The NPV of this strategy was calculated for (1) clinical coronavirus disease 2019 (COVID-19) diagnosis from day 8–14 after exposure, and for (2) asymptomatic SARS-CoV-2 detected by standardized nasopharyngeal swab and saliva specimens collected at days 9, 10, and 14 after exposure. Interim results are reported in the context of a second wave threatening this essential workforce. Results: Among 30 HCWs enrolled, the mean age was 31 years (SD, ±9), and 24 (80%) were female. Moreover, 3 were diagnosed with COVID-19 by day 14 after exposure (secondary attack rate, 10.0%), and all cases were detected using the 7-day infection control strategy: the NPV for subsequent clinical COVID-19 or asymptomatic SARS-CoV-2 detection by day 14 was 100.0% (95% CI, 93.1%–100.0%). Conclusions: Among HCWs with high-risk exposure to SARS-CoV-2, a home-based 7-day infection control strategy may have a high NPV for subsequent COVID-19 and asymptomatic SARS-CoV-2 detection. Ongoing data collection and data sharing are needed to improve the precision of the estimated NPV, and here we report interim results to inform infection control strategies in light of a second wave threatening this essential workforce.

show abstract

Clinical performance and sample freeze-thaw stability of the cobas®6800 SARS-CoV-2 assay for the detection of SARS-CoV-2 in oro-/nasopharyngeal swabs and lower respiratory specimens

Cited by 15 publications

References 16 publications

Influence of viral transport media and freeze-thaw cycling on the sensitivity of qRT-PCR detection of SARS-CoV-2 nucleic acids

Influence of viral transport media and freeze-thaw cycling on the sensitivity of qRT-PCR detection of SARS-CoV-2 nucleic acids

Comparison of two nucleic acid amplification tests (NAATs) and two antigen tests for detection of SARS-CoV-2 from upper respiratory specimens

Evaluation of a home-based 7-day infection control strategy for healthcare workers following high-risk exposure to severe acute respiratory coronavirus virus 2 (SARS-CoV-2): A cohort study

Contact Info

Product

Resources

About