Caution in interpretation of SARS-CoV-2 quantification based on RT-PCR cycle threshold value

Abstract

RT-PCR is the reference method for diagnosis of a Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection. During the setting up of 6 SARS-CoV-2 RT-PCR assays in our laboratory, comparative evaluations were systematically undertaken and allowed to evidence major discrepancies on cycle threshold RT-PCR results between techniques. These tendencies were confirmed in routine application when analyzing sequential samples from the same patients. Our aim was to examine the impact of the technique among factors influencing RT-PCR result, a far surrogate of 'viral load' in the heterogeneous environment of respiratory specimens.

Fig. 1

SARS-CoV-2 RT-PCR cycle thresholds differences (delta Ct) between techniques on the same sample during comparative evaluation of 6 assays. A- in-house RT-PCR based on gene E amplification (Corman et al., January 23, 2020), B- Bosphore® v2 nCoV assay (Anatolia geneworks), C- Allplex^TM nCoV assay (Seegene), D- RealTime SARS-CoV-2 assay (Abbott), E- Xpert® Xpress SARS-CoV-2 assay (Cepheid) and F- Simplexa^TM COVID-19 Direct (Diasorin molecular). Techniques E and F are unitary rapid one-step extraction/RT-PCR assays. Open circles: clinical samples, selected in March and April 2020 for varied initial Ct value; open triangles: quality controls, composed of Qnostics (Randox laboratories) and/or QCMD 2020 panel for external quality assessment. Error bars show medians and interquartile ranges (GraphPad Prism v9 software).

Fig. 2

Bland-Altman representations of comparisons of RT-PCR cycle thresholds (Ct) obtained on the same sample by 2 comparative assays (GraphPad Prism v9 software). Technique A- in-house RT-PCR based on gene E amplification (Corman et al., January 23, 2020), B- Bosphore v2 nCoV assay (Anatolia geneworks), C- Allplex nCoV assay (Seegene), D- RealTime SARS-CoV-2 assay (Abbott), E- Xpert Xpress SARS-CoV-2 assay (Cepheid), F- Simplexa COVID-19 Direct (Diasorin molecular). Similar or higher numbers of negative specimens were also tested for evaluation and found negative by both techniques (n = 43, data not shown). Solid lines indicate bias and horizontal dotted lines indicate 95% limits of agreement. Black dotted lines indicate simple linear regression.

Fig. 3.

Temporal profiles of SARS-CoV-2 RT-PCR cycle thresholds (Ct) for six representative patients. Samples (nasopharyngeal swabs) were processed by 2 comparative assays: B- Bosphore v2 nCoV assay (Anatolia geneworks) or C- Allplex nCoV assay (Seegene), vs D- RealTime SARS-CoV-2 assay (Abbott) or F- Simplexa COVID-19 Direct (Diasorin molecular).