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[Preprint]. 2021 Mar 1:2021.02.25.21252493.
doi: 10.1101/2021.02.25.21252493.

SARS-CoV-2 Total and Subgenomic RNA Viral Load in Hospitalized Patients

Derek E Dimcheff  1 , Andrew L Valesano  2   3 , Kalee E Rumfelt  2   3   4 , William J Fitzsimmons  2   3 , Christopher Blair  2   3 , Carmen Mirabelli  3 , Joshua G Petrie  4 , Emily T Martin  4 , Chandan Bhambhani  5 , Muneesh Tewari  5 , Adam S Lauring  2   3
Affiliations

SARS-CoV-2 Total and Subgenomic RNA Viral Load in Hospitalized Patients

Derek E Dimcheff et al. medRxiv. .

Update in

Abstract

Understanding viral load in patients infected with SARS-CoV-2 is critical to epidemiology and infection control. Previous studies have demonstrated that SARS-CoV-2 RNA can be detected for many weeks after symptom onset. The clinical significance of this finding is unclear and, in most patients, likely does not represent active infection. There are, however, patients who shed infectious virus for weeks. Detection of subgenomic RNA transcripts expressed by SARS-CoV-2 has been proposed to represent productive infection and may be a tractable marker for monitoring infectivity. Here, we use RT-PCR to quantify total and subgenomic nucleocapsid (N) and envelope (E) transcripts in 190 SARS-CoV-2 positive samples collected on hospital admission. We relate these findings to duration of symptoms. We find that all transcripts decline at the same rate; however, subgenomic E becomes undetectable before other transcripts. In Kaplan-Meier analysis the median duration of symptoms to a negative test is 14 days for sgE and 25 days for sgN. There is a linear decline in subgenomic RNA compared to total RNA suggesting subgenomic transcript copy number is highly dependent on copy number of total transcripts. The mean difference between total N and subgenomic N is 16-fold (4.0 cycles) and the mean difference between total E and sub-genomic E is 137-fold (7.1 cycles). This relationship is constant over duration of symptoms allowing prediction of subgenomic copy number from total copy number. Although Subgenomic E is undetectable at a time that may more closely reflect the duration of infectivity, its utility in determining active infection may be no more useful than a copy number threshold determined for total transcripts.

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Conflict of interest statement

CONFLICTS OF INTEREST

ASL reports receiving consulting fees from Sanofi on antiviral drugs and is a paid member of a steering committee for a clinical trial of baloxavir (Roche).

Figures

Figure 1
Figure 1
Comparison of cycle threshold versus day from symptom onset for clinical samples obtained from 185 inpatients. Total N (Panel A), subgenomic N (Panel B), total E (Panel C) and subgenomic E (Panel D). Red dots in panel A and C represent subgenomic negative samples and black dots represent subgenomic positive samples. Of the 185 patients, 56 were negative for sgE and 28 were negative for sgN (Shown on y-axis). Pearson correlation coefficients: N = −0.404 p<0.0001; SgN = −0.466, p<0.0001; E = −0.456 p<0.0001; sgE = −0.427 <0.0001. Linear regression line equations are indicated in each panel.
Figure 2
Figure 2
Kaplan-Meier analysis showing proportion of patients with positive RT-PCR for subgenomic transcripts versus day from symptom onset. The median duration from symptom onset to a negative sgN RT-PCR was 25 days (95% CI: 19.9–30.1) and 14 days for sgE (95% CI 9.6–18.4). This difference between the curves was significant P=0.001. Vertical hash marks indicate censored cases.
Figure 3
Figure 3
Box plots comparing difference in cycle threshold values, delta Ct (subgenomic Ct-genomic Ct) for cell culture and clinical samples. Total N is expressed at a 4.5-fold (2.2 cycles, STD 0.24) higher level than sgN and total E is expressed at a 13.9-fold (3.8 cycles STD 0.2) higher level compared to sgE in HuH-7 cells (Panel A). The difference was more dramatic in NP samples from inpatients where N was expressed at 16-fold (4.0 cycles, STD 1.1) greater than sgN and E was expressed at 137.2-fold (7.1 cycles, STD 1.3) greater than sgE expression (Panel B). This relationship was found at all times post symptom onset (n=185) for N (Panel C) and E (Panel D). Horizontal hash represents median value, if no IQM shown then N=1 for that sample. Moderate and extreme outliers noted by circles and stars respectively.
Figure 4
Figure 4
Relationship between total and subgenomic RNA in a persistently infectious patient. Total N was expressed at a 11-fold (3.5 cycles STD 0.75) higher level than subgenomic N, and total E was expressed at a 84-fold (6.4 cycles, STD 0.8) higher level than subgenomic E when all time points were combined (Panel A). Time course showing 45 minus cycle thresholds for total (black) and subgenomic RNA (red) over time. This patient showed persistently positive total and subgenomic RNA for 119 days. The ratio of total N and sgN of 3 to 4 cycles was maintained over the duration of infection (Panel B). This was also observed for E and sgE with the ratio of 6 to 7 cycles maintained thorough the course of infection (Panel C).
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