SARS-CoV-2 RNA identification in nasopharyngeal swabs: issues in pre-analytics

Abstract

<jats:title>Abstract</jats:title><jats:sec id="j_cclm-2020-0749_abs_001_w2aab3b7b1b1b6b1aab1c11b2Aa"><jats:title>Objectives</jats:title><jats:p>The direct identification of SARS-CoV-2 RNA in nasopharyngeal swabs is recommended for diagnosing the novel COVID-19 disease. Pre-analytical determinants, such as sampling procedures, time and temperature storage conditions, might impact on the end result. Our aim was to evaluate the effects of sampling procedures, time and temperature of the primary nasopharyngeal swabs storage on real-time reverse-transcription polymerase chain reaction (rRT-PCR) results.</jats:p></jats:sec><jats:sec id="j_cclm-2020-0749_abs_002_w2aab3b7b1b1b6b1aab1c11b3Aa"><jats:title>Methods</jats:title><jats:p>Each nasopharyngeal swab obtained from 10 hospitalized patients for COVID-19 was subdivided in 15 aliquots: five were kept at room temperature; five were refrigerated (+4 °C); five were immediately mixed with the extraction buffer and refrigerated at +4 °C. Every day and for 5 days, one aliquot per condition was analyzed (rRT-PCR) for SARS-CoV-2 gene E and RNaseP and threshold cycles (Ct) compared. To evaluate manual sampling, 70 nasopharyngeal swabs were sampled twice by two different operators and analyzed separately one from the other.</jats:p></jats:sec><jats:sec id="j_cclm-2020-0749_abs_003_w2aab3b7b1b1b6b1aab1c11b4Aa"><jats:title>Results</jats:title><jats:p>A total of 6/10 swabs were SARS-CoV-2 positive. No significant time or storage-dependent variations were observed in SARS-CoV-2 Ct. Re-sampling of swabs with SARS-CoV-2 Ct lower than 33 resulted in highly reproducible results (CV=2.9%), while a high variability was observed when Ct values were higher than 33 (CV=10.3%).</jats:p></jats:sec><jats:sec id="j_cclm-2020-0749_abs_004_w2aab3b7b1b1b6b1aab1c11b5Aa"><jats:title>Conclusions</jats:title><jats:p>This study demonstrates that time and temperature of nasopharyngeal swabs storage do not significantly impact on results reproducibility. However, swabs sampling is a critical step, and especially in case of low viral load, might be a potential source of diagnostic errors.</jats:p></jats:sec>

SARS-CoV-2 identification and IgA antibodies in saliva: One sample two tests approach for diagnosis

“…On admission, all patients were SARS-CoV-2 positive at naso-pharyngeal swab. During hospitalization they underwent repeat naso-pharyngeal swab collection for SARS-CoV-2 molecular testing, performed as described by us elsewhere [24] . Blood samples were also obtained to evaluate cell blood count, coagulation parameters (Prothrombin Time-PT, Partial Thrombplastin Time-PTT) and biochemical markers evidencing any systemic inflammation (C-reactive protein-CRP and fibrinogen), renal (creatinine) and liver (aspartate aminotransferase-AST, alanine aminotransferase- ALT, Bilirubin, alkaline phosphatase-ALP and gamma-glutamyl transferase-GGT) function, as well as heart (troponin I and brain natriuretic peptide-BNP) and pancreatic (amylase) involvement, measured using standard laboratory methods.…”

SARS-CoV-2 Subgenomic N (sgN) Transcripts in Oro-Nasopharyngeal Swabs Correlate with the Highest Viral Load, as Evaluated by Five Different Molecular Methods

“…However, since the market saturation did not make available large numbers of kits, reagents, chemicals and disposables [2][3][4], we were forced to use different tools in order not to stop our diagnostic activity, in compliance with the agreement with the regional task force for SARS-CoV-2 screening. However, many practical and technical issues frequently arise in laboratories performing COVID-19 molecular assays, particularly regarding the nucleic acid extraction, nucleic acid amplification reagents, and interpretation of test results [5,6].…”

Clinical performances of an ELISA for SARS-CoV-2 antibody assay and correlation with neutralization activity

“…A total of 171 leftover serum samples from 41 SARS-CoV-2 negative subjects (20 healthcare workers, 13 autoimmune patients, 8 pregnant women) and 130 COVID-19 patients (9 asymptomatic/mildly symptomatic recovered at home with supportive care and isolation, and 121 hospitalized, classified with moderate or severe disease following WHO interim guidance [5] ) were included in the study. All subjects underwent nasopharyngeal swab testing, analyzed by rRT-PCR as described elsewhere [6] . Healthcare workers were considered negative on the basis of at least three negative sequential rRT-PCR results obtained between February 26 th and May 29 th , 2020.…”