RT-PCR for SARS-CoV-2: quantitative versus qualitative
Authors: Mi Seon Han, Jung-Hyun Byun, Yonggeun Cho, John Hoon Rim
Journal: The Lancet Infectious Diseases (2020)

We read the Article by Lescure and colleagues1Lescure FX Bouadma L Nguyen D et al.Clinical and virological data of the first cases of COVID-19 in Europe: a case series.Lancet Infect Dis. 2020; (published online March 27.)https://doi.org/10.1016/S1473-3099(20)30200-0Summary Full Text Full Text PDF PubMed Scopus (803) Google Scholar with great interest. During the ongoing coronavirus disease 2019 (COVID-19) pandemic, monitoring patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using viral kinetics or viral loads in various sample types by real-time RT-PCR has become essential. However, understanding whether the RT-PCR test results are interpreted as quantitative, qualitative, or semi-quantitative is important. Since the cycle threshold (Ct) values from RT-PCR can be affected by batch effect, variations among different runs need to be closely monitored by laboratories—especially for quality control in quantitative RT-PCR. Unfortunately, several papers on COVID-19 use the naive Ct values from qualitative RT-PCR as a quantitation unit or use the ΔCt values with incorrect quantitation unit.2Zou L Ruan F Huang M et al.SARS-CoV-2 viral load in upper respiratory specimens of infected patients.N Engl J Med. 2020; 382: 1177-1179Crossref PubMed Scopus (3487) Google Scholar, 3Shen C Wang Z Zhao F et al.Treatment of 5 critically ill patients with COVID-19 with convalescent plasma.JAMA. 2020; (published online March 27.)DOI:10.1001/jama.2020.4783Crossref PubMed Scopus (1733) Google Scholar Quantitative RT-PCR is entirely different from qualitative RT-PCR. Ct value itself cannot be directly interpreted as viral load without a standard curve using reference materials. Thorough evaluation of the reliability and robustness of the standard curve is the key to accurately quantify the expected viral copy number. There is wide heterogeneity and inconsistency of the standard curves calculated from studies that provided Ct values from serial dilution samples and the estimated viral loads (figure).2Zou L Ruan F Huang M et al.SARS-CoV-2 viral load in upper respiratory specimens of infected patients.N Engl J Med. 2020; 382: 1177-1179Crossref PubMed Scopus (3487) Google Scholar, 4Chu DKW Pan Y Cheng SMS et al.Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia.Clin Chem. 2020; 66: 549-555Crossref PubMed Scopus (952) Google Scholar, 5Chan JF Yip CC To KK et al.Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel real-time reverse transcription-polymerase chain reaction assay validated in vitro and with clinical specimens.J Clin Microbiol. 2020; 58: e00310-e00320Crossref PubMed Scopus (727) Google Scholar An appropriate standard curve with adequate limit of detection is required for viral load quantification to correctly track the viral titre kinetics. A two-step approach using qualitative RT-PCR (for detection) and quantitative RT-PCR (for viral load quantification) is highly recommended for studies focusing on viral loads, as clearly presented by Lescure and colleagues.1Lescure FX Bouadma L Nguyen D et al.Clinical and virological data of the first cases of COVID-19 in Europe: a case series.Lancet Infect Dis. 2020; (published online March 27.)https://doi.org/10.1016/S1473-3099(20)30200-0Summary Full Text Full Text PDF PubMed Scopus (803) Google Scholar Furthermore, using appropriate quantification units according to different sample types—ie, copies per 1000 cells (for respiratory samples), copies per mL (for plasma), and copies per g (for stool)—should be followed by clinicians, as outstandingly shown by Lescure and colleagues.1Lescure FX Bouadma L Nguyen D et al.Clinical and virological data of the first cases of COVID-19 in Europe: a case series.Lancet Infect Dis. 2020; (published online March 27.)https://doi.org/10.1016/S1473-3099(20)30200-0Summary Full Text Full Text PDF PubMed Scopus (803) Google Scholar When interpreting the results of SARS-CoV-2 RT-PCR, the validity of the standard curve using reference materials or in-house plasmid controls with known viral copy numbers should be confirmed first to interpret Ct values as viral loads. In conclusion, precautions are needed when interpreting the Ct values of SARS-CoV-2 RT-PCR results shown in COVID-19 publications to avoid misunderstanding of viral load kinetics for comparison across different studies. We declare no competing interests. MSH and JHR contributed to study design, data collection, data analysis, data interpretation, literature search, and writing of the Correspondence. J-HB and YC contributed to data collection, data analysis, and data interpretation. All authors reviewed and approved the final version of the Correspondence. Clinical and virological data of the first cases of COVID-19 in Europe: a case seriesWe illustrated three different clinical and biological types of evolution in five patients infected with SARS-CoV-2 with detailed and comprehensive viral sampling strategy. We believe that these findings will contribute to a better understanding of the natural history of the disease and will contribute to advances in the implementation of more efficient infection control strategies. Full-Text PDF