Quantitative cycle threshold (Ct) values of RdRp (RNA dependent RNA polymerase) RNA target sequence (IP4) of SARS-CoV-2 were obtained after serial ten-fold dilutions of synthetic SARS-CoV-2 RNA transcripts ranging from 108 to 103 copies. These positive controls for used for real-time RT-qPCR were synthetic RNA derived from cDNA plasmid of the BetaCoV_Wuhan_WIV04_2019 strain (EPI_ISL_402124). A significant correlation (R2 = 0.995, P < 10− 4) was displayed between Ct values of IP4 sequence of SARS-CoV-2 and log10 starting quantity of SARS-CoV-2 RNA copies: this curve was used in each RT-qPCR serial analyses to validate our quantitative assay to check that obtained Ct values inversely reflected viral load levels (Fig. 1A).
Among 17,911 study outpatients, 496 (2.8%) patients (298 women /198 men; mean age (± SD), 32.31 ± 17.79 years) were detected positive with low to high viral loads (median (± SD) Ct value of 23.57 ± 6.74) of which 180 (36.3%) were asymptomatic. We identified two distinct subsets of viral load values that were strongly segregated by a cut-off Ct value of 30 in both asymptomatic and symptomatic patients (P < 10− 4) and this result appeared to be not associated with significant differences in median age levels between these two subgroups (P = 0.85) (Fig. 1B). We found that the proportion of asymptomatic patients with low viral loads was significantly higher than that observed in symptomatic patients (Ct values ≥ 30: 25% versus 11.7%, P = 0.003, Fig. 1B). Remarkably 75% of asymptomatic patients had mean to high viral loads which median value was slightly higher than that observed in symptomatic patients (Ct values < 30: 21.60 ± 0.24 vs. 22.57 ± 0.38; P = 0.029) (Fig. 1B). As expected, viral RNA loads in upper respiratory tract of study outpatients appeared to be well correlated with the time delay between the respiratory sample and the onset symptoms (Fig. 1C). Only the viral loads of patients who were sampled between 8 and 28 days after symptom onset were significantly lower than those of asymptomatic patients (P = 0.041) (Fig. 1D).
Panel A. Cycle threshold (Ct) values of RNA dependent RNA polymerase (RdRp) target sequence (IP4) on SARS-CoV-2 RNA genome after serial ten-fold dilutions of synthetic SARS-CoV-2 RNA transcripts ranging from 108 to 103 copies. These positive controls for used for real-time RT-qPCR were synthetic RNA derived from cDNA plasmid of the BetaCoV_Wuhan_WIV04_2019 strain (EPI_ISL_402124). A significate correlation (R2 = 0.995, P < 10− 4) was displayed between Ct values of IP4 sequence of SARS-CoV-2 and log10 starting quantity of SARS-CoV-2 RNA copies: this curve was used in each RT-qPCR serial analyses to validate our quantitative assay and to check that obtained Ct values inversely reflected viral load levels. Panel B and D. Boxes denote interquartile ranges, and horizontal bars denote median CT values of IP4 sequence among patients asymptomatic or symptomatic. Whiskers IP4 Ct values denote the maximum and minimum values below or above the median. Panel B. The shaded boxes indicate IP4 Ct values < 30 among asymptomatic (n = 135) or symptomatic (n = 279) patients whereas white boxes indicate IP4 Ct values ≥ 30 among asymptomatic (n = 45) or symptomatic (n = 37) patients (P < 0.0001). Panel C. Correlation of means Ct values in COVID-19 positive patients and days after onset of symptoms. The outer dotted lines are 95% confidence interval. Data represent standard curve to regression sigmoidal with a goodness of fit (R2 = 0.997). Panel D. Comparison of IP4 Ct values between asymptomatic outpatients (n = 180) and symptomatic sub-groups of outpatients stratified according to the delay between the respiratory sample and the onset of symptoms; 0–4 (n = 220), 5–7 (n = 73) and 8–28 (n = 23) days after the apparition of clinical symptoms (P < 0.0001). Data represent the median ± SD. *: P < 0.05; according to ANOVA test. Data represent the median ± SD. *: P < 0.05; ****: P < 0.0001; according to ANOVA test.
To investigate whether asymptomatic patients with a mean to high viral load (Ct values < 30) were SARS-CoV-2 infectious shedders, we assessed the existence of a viral genomic RNA replication activity following infection of cultured cells by their upper respiratory tract specimens. Among the 135 asymptomatic patients with RT-PCR Ct values lower than 30, only 83 had frozen stored available samples that were tested by our culture assay protocol. Results were expressed as fold of RNA viral load level increase per ml of cell culture supernatants at 96 hours post-inoculation of Vero cells (Fig. 2). We observed a strong correlation (R2 = 0.591, P < 0.0001) between initial SARS-CoV-2 RT-PCR Ct values in respiratory specimens and fold increase of viral RNA levels following viral culture of the same samples (Fig. 2).
Linear regression curve between IP4 Ct values in COVID-19 asymptomatic patients and fold of viral SARS-CoV-2 RNA load level increase after 96 hours viral culture on Vero cells (log10 copies/mL) (R2 = 0.591, P < 10− 4) (n = 83). Fold values greater than 1 were considered as representative of a viral RNA replication activity related to an infection of cultured cells by viable viral particles. Linear regression was performed, and slopes were compared using Spearman test. P values < 0.05 were considered as statistically significant.
This correlation confirmed the reported association between RNA load levels and viral infectivity in upper respiratory tract of COVID-19 patients and demonstrated the reliability of our experimental approach 7–9. Among studied asymptomatic patients, 50.6% of them were characterized by the presence of viable virus (fold of viral RNA level increase > 1) associated with RT-PCR Ct values lower than 30 in their respiratory specimens, whereas 39.76% of them evidenced no viable viral particles (fold of viral RNA level increase < 1) despite RT-PCR Ct values lower than 30 in their respiratory specimens. None of the asymptomatic patients with RT-PCR Ct values upper than 30 in their upper respiratory tract demonstrated the presence of detectable replicative viral particles (Fig. 2).