SARS-CoV-2 RNA identification in nasopharyngeal swabs: issues in pre-analytics.
Author(s): Basso D, Aita A, Navaglia F, Franchin E, Fioretto P, Moz S, Bozzato D, Zambon CF, Martin B, Dal Prà C, Crisanti A, Plebani M
Publication: Clin Chem Lab Med, 2020, Vol. , Page
PubMed ID: 32573469 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to investigate the effects of storage duration, temperature, and condition on the detection of SARS-CoV-2, the virus that causes the novel 2019 coronavirus disease (COVID, COVID-19, COVID19) in nasopharyngeal swabs. The effects of resampling by another operator on the following day as well as vortexing prior to sampling and sampling volume on cycle threshold (CT) values and variation were also investigated.
Conclusion of Paper
SARS-CoV-2 was detected in the nasopharyngeal swabs of 6 of the 10 patients. Cycle threshold (CT) values for SARS-CoV-2 gene E were unaffected by storage duration (1-5 days), temperature (4°C versus room temperature), or condition (with extraction buffer or without), but ANOVA analysis of RNAseP CT values identified a significant effect of duration and condition. Significantly higher CT values were found for specimens stored at room temperature or 4°C versus those stored at 4°C with extraction buffer but significance depended on duration. Of the 57 patients with a second sampling, 27 tested negative in the second round and 30 tested positive. Swabs that tested negative in the second run were found to have had a higher CT in the first run than the 30 later found to be positive. The standard deviation of the CT values between replicate specimens was lower when the first CT values was below 33 than above 33 (CV 2.9% versus 10.3%); however, CT values in the second sampling of two patients were comparable between specimens obtained by the same operator and processed together regardless of volume used or vortexing prior to sampling.
Studies
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Study Purpose
The purpose of this study was to investigate the effects of storage duration, temperature, and condition on the detection of SARS-CoV-2 in nasopharyngeal swabs. The effects of resampling by another operator on the following day as well as vortexing prior to sampling and sampling volume on CT values and variation were also investigated. Two nasopharyngeal swabs were collected from each of 10 patients hospitalized with SARS-CoV-2 and stored viral transport media for up to 2 h at 4°C. The two specimens from each patient were then pooled, mixed, and aliquoted. Five aliquots were stored at room temperature, five were stored at 4°C, and five were mixed with Nuclisens easyMAG Extraction Buffer 1 and stored at 4°C. Specimens from each of the storage conditions were removed for testing each day for five days, mixed with Nuclisens easyMAG Extraction Buffer 1, and RNA was extracted using the Magna Pure 96 Instrument. SARS-CoV-2 was quantified by real-time RT-PCR amplification of the SARS-CoV-2 gene E and RNaseP. Specimens were considered positive if a CT value was below 45. Two specimens with high levels of SARS-CoV-2 were resampled (100 µL or 300 µL) 24 h after the first sampling without vortexing first (300 µL only) or after vortexing. To investigate operator dependent variability, replicate nasopharyngeal swab sampling was performed from 57 patients whose initial results were deemed doubtful based on the exponential curve by a different operator on a subsequent day.
Summary of Findings:
SARS-CoV-2 was detected in the nasopharyngeal swabs of 6 of the 10 patients. CT values for SARS-CoV-2 E-gene were unaffected by storage duration (1-5 days), temperature (4°C versus room temperature), or condition (with extraction buffer or without) but non-significant increases in CT values were noted for some patients when stored for 3 days or more. ANOVA analysis of RNAseP CT values identified a significant effect of duration (P=0.0018) and condition (P=0.0721) and an interaction between duration and condition (P=0.0205). Significantly higher CT values were found on days 4 and 5 of storage for specimens stored at room temperature versus those stored with extraction buffer at 4°C (P<0.05, both). Importantly, the coefficient of variance was not affected by storage. Further, analysis showed significantly lower CT values for specimens stored for 2 or more days at 4°C with extraction buffer than without (P<0.05, all). Of the 57 patients with a second nasopharyngeal swab, 27 tested negative in the second round and 30 tested positive. Swabs that tested negative in the second round were found to have had a higher CT in the first run than the 30 later found to be positive (38.44 versus 36.23, P<0.005). The standard deviation of the CT values between replicate specimens was lower when the first CT values was below 33 than above 33 (CV 2.9% versus 10.3%); however, CT values in the second sampling of two patients were comparable between specimens obtained by the same operator and processed together regardless of volume used or vortexing prior to sampling.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Pneumonia/Respiratory Infection
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Biospecimen mixing Vortexed before sampling
Not mixed
Biospecimen Aliquots and Components Aliquot size/volume 100µL
300µL
Storage Storage conditions 4°C with extraction buffer
4°C without extraction buffer
Storage Storage temperature 4°C
Room temperature
Storage Storage duration 1 day
2 days
3 days
4 days
5 days