Determining the impact of vaccination on SARS-CoV-2 RT-PCR cycle threshold values and infectious viral titres

Abstract

Background: As the COVID-19 pandemic continues, efforts to better understand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral shedding and transmission in both unvaccinated and vaccinated populations remain critical to informing public health policies and vaccine development. The utility of using real time RT-PCR cycle threshold values (C_T values) as a proxy for infectious viral litres from individuals infected with SARS-CoV-2 is yet to be fully understood. This retrospective observational cohort study compares quantitative infectious viral litres derived from a focus-forming viral titre assay with SARS-CoV-2 RT-PCR C_T values in both unvaccinated and vaccinated individuals infected with the Delta strain.

Methods: Nasopharyngeal swabs positive for SARS-CoV-2 by RT-PCR with a C_T value <27 collected from 26 June to 17 October 2021 at the University of Vermont Medical Center Clinical Laboratory for which vaccination records were available were included. Partially vaccinated and individuals <18 years of age were excluded. Infectious viral litres were determined using a micro-focus forming assay under BSL-3 containment.

Results: In total, 119 specimens from 22 unvaccinated and 97 vaccinated individuals met all inclusion criteria and had sufficient residual volume to undergo viral titring. A negative correlation between RT-PCR C_T values and viral litres was observed in both unvaccinated and vaccinated groups. No difference in mean C_T value or viral titre was detected between vaccinated and unvaccinated groups. Viral litres did not change as a function of time since vaccination.

Conclusions: Our results add to the growing body of knowledge regarding the correlation of SARS-CoV-2 RNA levels and levels of infectious virus. At similar C_T values, vaccination does not appear to impact an individual's potential infectivity when infected with the Delta variant.

Keywords: SARS-CoV-2; COVID-19; CT; RT-qPCR; micro-focus forming assay; viral titre.

Fig. 1.

Infectious viral titres and C_T values for unvaccinated versus vaccinated individuals infected with Delta variant. (a) Viral RNA (C_T) versus vaccination status. Data are summarized by boxplots and overlaid with points representing individual subjects. The y axis is flipped for visualization as C_T values are inversely proportional to the amount of viral RNA. (b) Viral titre (f.f.u. ml^–1) by vaccination status. Data are summarized by boxplots and overlaid with points representing individual subjects. Dashed line indicates the limit of detection for infectious titer (10 f.f.u. ml^–1). (c) Viral RNA (C_T) on the x axis plotted against viral titre (f.f.u. ml^–1) on the y axis. Separate linear regression lines (ŷ = β0 + β1x̄ + Ɛ) were fit to unvaccinated and vaccinated individuals. Shading indicates confidence interval (0.95) for each line. (a−c) Red symbols and lines indicate unvaccinated individuals (N = 12), blue symbols and lines indicate vaccinated individuals (N = 76). Thermocycler method is indicated by shape (Cobas circles, non-Cobas triangles). f.f.u. stands for focus forming unit.

Fig. 2.

Infectious viral titres and C_T values as a function of time since vaccination. Clinical specimens from vaccinated individuals (N = 97) infected with SARS-CoV-2 Delta variant were used to visualize the relationship between viral titer, viral RNA (C_T), and time since full vaccination. (a) Days since fully vaccinated (≥14 days since completion of a primary COVID-19 vaccine series) on the x axis plotted against viral titer (f.f.u. ml^–1) on the y axis. (b) Viral RNA (C_T) on the x axis plotted against viral titre (f.f.u. ml^–1) on the y axis. Data point fill color corresponds with days post vaccination. Samples were grouped by <100 days (purple) or >100 days (green) post vaccination, and separate linear regression lines (ŷ = β0 + β1x̄ + Ɛ) were fit to each group. Shading indicates confidence interval (0.95) for each line. (a, b) Dashed line indicates the limit of detection for infectious titer (10 f.f.u. ml^–1). f.f.u. stands for focus forming unit.

Fig. 3.

CONSORT diagram. Flow chart of clinical specimens included for analysis.