Reliable Quantification of Sars-Cov-2 in Standard Clinical Samples is Not Possible

Reliable Quantification of Sars-Cov-2 in Standard Clinical Samples is Not Possible

Abstract
The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) kept the world in a stranglehold for more than two years. Infection with SARS-CoV-2 results in Covid-19, a disease that ranges from mild flu-like symptoms to respiratory failure from extensive lung injury and even death. To manage and harness the pandemic, people with clinical symptoms were tested to prove SARS-CoV-2 infection using RT-qPCR. This assay was used qualitatively rather than quantitatively. SARS-CoV-2 positive patients had to go in quarantine to prevent further spreading. Since RT-qPCR is a highly-sensitive quantitative technique which can detect as little as a single to many million copies in the reaction, researchers consider the possibility to stratify viral load, and thus clinical risk, of the individual patient.
The SARS-CoV-2 probe-based assay was standardized and is used world-wide. In an attempt to align the world-wide results, the WHO advocated a Cq cutoff of 25 cycles as an acceptable LOD for this point-of-care assay. Although the SARS-CoV-2 assay is standardized, the results of different laboratories cannot be compared and seem sometimes to be even contradictory. These differences do mostly reside from the use of different qPCR machines in different laboratories and the collection of the nasal and throat swaps.
To further investigate the quantitative nature of the SARS-CoV-2 RT-qPCR assay we retrieved the raw fluorescent data of the PCR analyses performed in one week on a single Roche Lightcycler 480 in the Academic Medical Center, Amsterdam. The included patients all suffered from SARS-CoV-2 clinical symptoms, were sampled and then PCR analyzed in the hospital. The delay between taking the swap and the PCR assay was at most overnight in the cooling and the entire procedure was performed in the same lab and using the same chemicals and equipment. For each of the assays we know the reported clinical diagnosis, but, due to privacy restrictions, we do not know the clinical outcome of the patient. The fluorescence data and amplification curve of each of the PCR assays were reanalyzed using LinRegPCR and the starting concentration of SARS-CoV-2 virus present in the reaction was determined using the PCR efficiency of the assay and those of the individual reactions.
The data show that, due to the properties of the amplification curves, even in a very standardized analysis pipeline reliable quantification of SARS-Cov-2 in standard clinical samples is not possible.