Correlation between cycle threshold and viral load through comparison of RT-PCR qualitative <em>versus</em> quantitative assay for SARS-CoV-2

Abstract

Background and aims. Real-time reverse transcription polymerase chain reaction (RT-PCR) is the gold-standard assay to detect SARS-CoV-2, but it has limitations compared to viral load analysis. Quantitative detection improves surveillance, diagnosis, and prevention. We performed a comparative study of qualitative and quantitative tests for the diagnosis of COVID-19 on respiratory samples from patients screened for SARS-CoV-2 infection, and explored the correlation between viral load compared to the threshold cycle (Ct) value obtained in RT-PCR.Materials and methods. Sixty respiratory samples from patients affected by SARS-CoV-2 were subjected to both the qualitative (Allplex ™ 2019-nCoV Seegene) and the quantitative (Clonit® Quanty COVID-19) assays, and the relationship between viral load and Ct value was assessed by Spearman correlation analysis (ρ). In addition, the viral load of samples collected from a patient with symptomatic cancer was monitored. Results. The results show 100% agreement between the results obtained with quantitative assay and the reference standards, whereas 99.2% agreement was found for the qualitative test. A strong negative Spearman’s correlation between the Ct values of the N genes and RdRP gene was observed from qualitative assay values and viral loads.Conclusions. Quantitative assay has a higher sensitivity than qualitative assay, and viral load testing allows the clinicians to better orient themself in the choice of therapeutic treatment to be adopted. The constantly higher viral load of clinical cases considered, irrespective of the different therapies used, confirms that viral load monitoring could represent a great advantage in clinical practice.

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“…Independent evaluations [ 36 , 37 ] have reported an optimal agreement between Allplex 2019-nCoV and Quanty COVID-19v2 assays. However, in the case of inconclusive or discordant results, both qualitative and quantitative RT-PCRs were repeated.…”

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“…Figure 5 shows the estimated range of Ct values at a viral load of 100,000 Copies/mL from 42 identified standard curves. [12][13][14]20,22,23,[27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43] The median Ct value at this viral load was 25.82 (interquartile range 23.03 to 29.27), with 46% of Ct values below 25, and 83% below 30. [12][13][14]20,22,23,[27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43] The median Ct value at this viral load was 25.82 (interquartile range 23.03 to 29.27).…”

“…Figure 5. Ct Values at a Viral Load of 100,000 Copies/mL Estimated From 42 Standard Curves Box and Whisker plot of Ct values at a viral load of 100,000 Copies/mL from 42 identified PCR standard curves reported in 23 original articles [12][13][14]20,22,23,[27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43]. The median Ct value at this viral load was 25.82 (interquartile range 23.03 to 29.27).…”

“…Figure 6. Cumulative Distribution of Viral Loads at a Ct Value of 30 as Estimated From 42 Standard Curves Cumulative distribution curve derived from 42 PCR standard curves reported in 23 original articles [12][13][14]20,22,23,[27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43]. At a Ct value of 30, the median viral load was 5,400 copies/mL, and an estimated 83% of assays reported viral loads below 100,000 copies/mL at this threshold.…”