Thermal treatment of nasopharyngeal samples before cobas SARS-CoV-2 testing

Pryce, Todd M.; Boan, Peter; Kay, Ian; Flexman, James

doi:10.1016/j.cmi.2020.07.042

Cited by 7 publications

(14 citation statements)

References 7 publications

(13 reference statements)

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“…We found no significant differences in the qualitative outcomes (detected, presumptive or negative) for thermally treated samples compared to room-temperature samples in this study. In the previous study ( Pryce et al, 2021 ) we observed additional positive results ( n= 3) and presumptive results ( n= 3) for thermal treatment compared to room-temperature (17.6%; 6/34 samples in the positive group), compared to only one additional positive result for thermal treatment (0.4%; 1/238 samples in the positive group) in this study. In the previous study we performed 10-fold serial dilutions ( n= 34) for each patient sample ( n= 8) compared to a single dilution for each patient this study ( n= 238).…”

Section: Discussionsupporting

confidence: 64%

“…We implemented thermal treatment of patient samples to improve laboratory safety and reduce additional PPE use ( Pryce et al, 2021 ). Preliminary results with a limited number of positive samples ( n= 34) showed increased C t values for thermal treatment compared to room-temperature despite a potential improvement in the qualitative detection of SARS-CoV-2 for thermally treated samples.…”

Section: Discussionmentioning

confidence: 99%

“…The effects of temperature on the viability of SARS-CoV-2 and other coronaviruses have been reported ( Chin et al, 2020 , Pagat et al, 2007 , Rabenau et al, 2005 , Yunoki et al, 2004 ). Our initial investigations suggested thermal treatment of nasopharyngeal samples resulted in statistically significant higher cycle threshold ( C t ) values for E-gene (p 0.040), suggesting a reduction in detectable virus RNA ( Pryce et al, 2021 ). Despite marginally higher cobas C t values for thermally treated samples, conflicting findings were observed for the qualitative detection of SARS-CoV-2, specifically detection of ORF1a and E-gene targets at the limit of detection.…”

Section: Introductionmentioning

confidence: 96%

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Qualitative and quantitative effects of thermal treatment of naso-oropharyngeal samples before cobas SARS-CoV-2 testing

Pryce

Boan

Kay

et al. 2021

Diagnostic Microbiology and Infectious Disease

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To improve laboratory safety we thermally treated naso-oropharyngeal samples before testing with the cobas SARS-CoV-2 assay. This study aimed to determine if thermal treatment significantly affects the qualitative detection of SARS-CoV-2 and the quantitative measurement of cobas SARS-CoV-2 ORF1a and E-gene target copy number using an in-house quantitative method. A collection of positive ( n= 238) and negative samples ( n= 196) was tested in parallel comparing thermal treatment (75°C for 15 minutes) to room-temperature. There were no significant differences in the final qualitative outcomes for thermal treatment versus room-temperature (99.8% agreement) despite a statistically significant reduction (p<0.05) in target copy number following thermal treatment. The median ORF1a and E-gene reduction in target copy number was 0.07 (1.6%) and 0.22 (4.2%) log 10 copies/mL respectively. The standard curves for both ORF1a and E-gene targets were highly linear (r 2 =0.99). Good correlation was observed for ORF1a (r 2 =0.96) and E-gene (r 2 =0.98) comparing thermal treatment to room-temperature control.

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Section: Discussionsupporting

confidence: 64%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 96%

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Qualitative and quantitative effects of thermal treatment of naso-oropharyngeal samples before cobas SARS-CoV-2 testing

Pryce

Boan

Kay

et al. 2021

Diagnostic Microbiology and Infectious Disease

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“…This is the rst report of a high-throughput HI protocol for SARS-CoV-2 testing that is performed on UN733 packaged OP/NP swab samples in bulk. Although the use of heat is an effective means of SARS-CoV-2 inactivation [7,[10][11][12][22][23][24], COVID-19 testing laboratories have relied on chemical lysis during RNA extraction for viral inactivation. Chemical lysis is highly effective mechanism of virus inactivation [25,26], but requires handling of potentially viable SARS-CoV-2 samples that represents a risk to operators.…”

Section: Discussionmentioning

confidence: 99%

“…Insu cient exposure at the required temperature could result in incomplete viral inactivation [12,27] which is especially important for samples with high viral load [10,11,28]. In addition our bulk HI protocol employs wet heat as opposed to dry heat as previously used [29], which can lead to evaporation and incomplete viral inactivation [27] This study is the rst to demonstrate successful SARS-CoV-2 detection following a bulk HI of clinical OP/NP swab samples at scale [22,25]. We tested approximately 1200 OP/NP swab samples in concordance studies, in addition to the use of known positive OP/NP swab samples for the supporting studies.…”

Section: Discussionmentioning

confidence: 99%

Heat Inactivation of Clinical COVID-19 Samples on an Industrial Scale for Low Risk and Efficient High-throughput qRT-PCR Diagnostic Testing

Delpuech

Douthwaite

Hill

et al. 2021

Preprint

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We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 120 million confirmed cases, over 3.8 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as ‘direct to PCR’ assays that do not involve RNA extraction or chemical neutralisation methods.

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Clinical associations of SARS-CoV-2 viral load using the first WHO International Standard for SARS-CoV-2 RNA

2022

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SARS-CoV-2 viral load declines from the time of symptom onset; in some studies viral load is higher or persists longer in more severe COVID-19 infection, and viral load correlates with culture positivity. This was a retrospective cohort study of inpatients and outpatients during the first wave of COVID-19 infection in Western Australia, March to May 2020, of the relationship of SARS-CoV-2 viral load (using the First WHO International Standard for SARS-CoV-2 RNA) from symptom onset, by clinical subgroups determined from the public health database and hospital records, using regression analysis. We studied 320 samples from 201 COVID-19 cases: 181 mild, seven severe, 11 critical, and four cases who died (two were also critical cases). At symptom onset the mean viral load was 4.34 log 10 IU/mL (3.92–4.77 log 10 IU/mL 95% CI, cobas SARS-CoV-2 assay ORF1a Ct 28.9 cycles). The mean viral load change was –0.09 log 10 IU/mL/day (–0.12 to –0.06 95% CI). R 2 was 0.08 and residual standard deviation 2.68 log 10 IU/mL. Viral load at symptom onset was higher for those reporting fever compared to those not reporting fever. Viral load kinetics were not different for gender, age, shortness of breath, or those requiring oxygen. Mean viral load at usual release from isolation at 14 days was 2.5 log 10 IU/mL or day 20 was 1.8 log 10 IU/mL. Variability in respiratory sample SARS-CoV-2 viral load kinetics suggests viral loads will only have a role supporting clinical decision making, and an uncertain role for prognostication.

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Thermal treatment of nasopharyngeal samples before cobas SARS-CoV-2 testing

Cited by 7 publications

References 7 publications

Qualitative and quantitative effects of thermal treatment of naso-oropharyngeal samples before cobas SARS-CoV-2 testing

Qualitative and quantitative effects of thermal treatment of naso-oropharyngeal samples before cobas SARS-CoV-2 testing

Heat Inactivation of Clinical COVID-19 Samples on an Industrial Scale for Low Risk and Efficient High-throughput qRT-PCR Diagnostic Testing

Clinical associations of SARS-CoV-2 viral load using the first WHO International Standard for SARS-CoV-2 RNA

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