SARS-CoV-2 viral load and shedding kinetics

Abstract

SARS-CoV-2 viral load and detection of infectious virus in the respiratory tract are the two key parameters for estimating infectiousness. As shedding of infectious virus is required for onward transmission, understanding shedding characteristics is relevant for public health interventions. Viral shedding is influenced by biological characteristics of the virus, host factors and pre-existing immunity (previous infection or vaccination) of the infected individual. Although the process of human-to-human transmission is multifactorial, viral load substantially contributed to human-to-human transmission, with higher viral load posing a greater risk for onward transmission. Emerging SARS-CoV-2 variants of concern have further complicated the picture of virus shedding. As underlying immunity in the population through previous infection, vaccination or a combination of both has rapidly increased on a global scale after almost 3 years of the pandemic, viral shedding patterns have become more distinct from those of ancestral SARS-CoV-2. Understanding the factors and mechanisms that influence infectious virus shedding and the period during which individuals infected with SARS-CoV-2 are contagious is crucial to guide public health measures and limit transmission. Furthermore, diagnostic tools to demonstrate the presence of infectious virus from routine diagnostic specimens are needed.

Fig. 1. Methods to measure infectious virus and RNA viral load.

Swab specimens from the nasopharynx or oropharynx are used for detection of SARS-CoV-2 viral loads. Detection of viral nucleic acids (RNA) is performed by quantitative real-time PCR (qRT-PCR). Viral RNA is extracted from lysed virus, reverse transcribed and amplified by qPCR using primers specific for one or more target regions in the viral genome. The amplification cycle at which samples cross the threshold (cycle threshold) defines the amount of viral RNA. RNA viral load can be expressed as the number of viral RNA copies per millilitre, or by the arbitrary test-specific cycle threshold value. Lateral flow assays detect the presence of specific viral proteins in the lysed viral particles. SARS-CoV-2 nucleocapsid is used in most antigen-detecting (rapid) diagnostic tests. The presence of infectious (replication-competent) virus in respiratory specimens can only be determined by the recovery of virus in cell culture by isolation or by quantification of infectious virus titres using 50% tissue culture infectious dose (TCID₅₀), focus-forming assays or plaque-forming assays. Virus isolation is performed by applying infectious medium on the monolayer of cells; isolation success is determined by the presence of a cytopathic effect approximately 3–5 days post-infection. White colour indicates the presence of a cytopathic effect in cells. For quantification of infectious virus titres, serial dilutions of respiratory samples are performed and used for inoculation on the monolayer of cells. In TCID₅₀, 3–5 days post-infection, viral-induced cytopathic effect is classically defined using microscopy. In focus-forming assays, cells are fixed 1 day post-infection and immunostaining with virus-specific antibodies is performed to detect groups of infected cells (foci). The foci, indicating the presence of infectious virus, are displayed in blue. In plaque-forming assays, plates are fixed 2–3 days post-infection and stained with crystal violet; wells with individual plaques are used to determine viral titres. The plaques, indicating the presence of infectious virus, are displayed in white.

Fig. 2. Kinetics of RNA viral loads and infectious virus for ancestral SARS-CoV-2 in patients with mild-to-moderate disease.

According to different studies, the incubation period for ancestral SARS-CoV-2 was estimated to lie between 4.6 and 6.4 days. On average, symptoms continue to persist for 10 days. RNA can already be detected before the onset of symptoms; RNA levels peak around the onset of symptoms and then gradually decline. Median clearance for RNA viral load is 16 days post-onset of symptoms. Infectious virus titres are highest around symptom onset, and infectious virus can be isolated up to 8 or 10 days post-onset of symptoms. RNA can be detected for prolonged periods by real-time PCR, when infectious virus is no longer detectable, whereas virus detection by antigen-detecting (rapid) diagnostic tests (Ag-RDTs) was shown to be a better correlate for infectiousness. Gradients reflect variability between individuals (lighter shade towards the end of infection shows that viral loads continue to be detected in some but not all individuals). The grey dashed line marks the initial infection, the blue dashed lines mark the PCR-positive period and the red dashed lines mark Ag-RDT positivity. Details of the underlying studies used to generate Fig. 2 can be found in Supplementary Table 1.

Fig. 3. Infectious viral load and symptom onset in SARS-CoV-2 Delta and Omicron BA.1 variants of concern.

Overall patterns of shedding dynamics are conserved between SARS-CoV-2 variants. In comparison to ancestral SARS-CoV-2, Delta and Omicron BA.1 have shorter incubation periods, estimated as approximately 3.7–4 days for Delta and approximately 3–3.4 days for Omicron BA.1. Higher infectious viral loads were detected in patients infected with Delta than in patients infected with Omicron BA.1 or ancestral SARS-CoV-2. Only a limited number of studies have determined when virus shedding for Delta and Omicron BA.1 ends, so this time point is not well defined. Owing to the low number of studies comparing the end of the infectious period between different SARS-CoV-2 variants of concern, the end point of infectivity is not well defined (shown as a colour gradient). Details of the underlying studies used to generate Fig. 3 can be found in Supplementary Table 2.

Fig. 4. Influence of vaccination on viral load.

Similar RNA viral loads were detected in vaccinated and unvaccinated patients infected with the Delta variant of concern during the first 5 days post-onset of symptoms. However, faster clearance of viral RNA was shown in vaccinated patients. Infectious viral loads (IVLs) were significantly lower in vaccinated individuals and declined faster than in unvaccinated individuals infected with Delta. Dynamics of viral loads in vaccinated individuals may vary widely in case of infection with another variant. Details of the underlying studies used to generate Fig. 4 can be found in Supplementary Table 3.