In a recent article published in Nature Reviews Microbiology, researchers attempted to establish an association between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load, indicating its ribonucleic acid (RNA) levels, and the presence of infectious virions.
Additionally, they examined host and other biological factors that affect infectious virion(s) shedding by SARS-CoV-2. They also evaluated the strengths and limitations of diagnostic tools that might help precisely estimate viral load, a robust substitute for characterizing the shedding of infectious virions by SARS-CoV-2.
Background
SARS-CoV-2 shedding patterns have become unique and distinguished from that of its ancestral strain in three years of the coronavirus disease 2019 (COVID-19) pandemic. The emergence of SARS-CoV-2 variants of concern (VOCs) has likely further complicated this pattern.
A detailed understanding of viral shedding patterns is crucial for designing public health interventions to contain human-to-human SARS-CoV-2 transmission. Although multifactorial, biological characteristics of SARS-CoV-2 and its variants, host factors, and pre-existing immunity of the diseased individual affect the shedding of its infectious virions, which, in turn, determines its onward transmission.
Nevertheless, viral load is a key parameter for estimating SARS-CoV-2 infectiousness, and a higher viral load in an infected individual’s upper respiratory tract (URT) implies a greater risk for onward transmission. To date, no diagnostic test precisely identifies or distinguishes between replication-competent SARS-CoV-2 and its residual RNA.
Factors at play in SARS-CoV-2 viral load and infectious virion(s) shedding dynamics
Reverse transcription-polymerase chain reaction (RT-PCR) assay, for instance, only detects viral RNA with high sensitivity in the respiratory tract of an infected individual, which remains detectable even in the absence of infectious virions. It presents results as viral RNA copies per milliliter of swab sample or by the discretionary test-specific cycle threshold (CT) value. Then, there could be SARS-CoV-2 RNA in peripheral blood, urine, ocular secretions, and the patient’s stool. Yet again, diagnostic tests are unable to detect infectious viruses in these non-respiratory specimens.
Contrastingly, RT-PCR estimates infectiousness qualitatively or quantitatively by viral replication in cell culture. Clinical samples with lower viral load often show delayed development of a cytopathic effect (CPE). Likewise, there are other methods for infectious virion quantification, such as plaque assays and focus-forming assays, to name a few. If swab samples are not immediately submerged in a viral transport medium and stored at −80 °C after collection, it loses viability resulting in complete loss of infectious virions. Other factors influencing successful virus isolation include cell lines used for isolation. Most importantly, working with SARS-CoV-2 infectious virions requires adherence to biosafety level 3 conditions, and detection of the viable virus is restricted to research only, deemed unsuitable for diagnostics. Likewise, CT values are only a weak predictor of infectious virions in the first five days post-onset of symptoms (dpos).
Antigen-detecting (rapid) diagnostic tests (Ag-RDT) fetch faster results and are inexpensive. They show good harmony with RT-PCR positivity when CT values are below 25 to 30, a viral load suggesting the presence of infectious virions; however, they fetch unreliable results at higher CT values. Further, it remains unclear whether Ag-RDT positivity beyond 10 dpos correlates with infectious virion(s) shedding. With the increasing presence of mucosal antibodies and rising hybrid immunity, Ag-RDTs may further lose sensitivity.
It is worth noting that it is difficult to estimate the time for which a SARS-CoV-2-infected individual remains infectious. A patient’s age, gender, immunity, and infecting variant; all these factors and more influence viral shedding dynamics. Studies showing the difference in the infectious virus titers between Alpha and Delta VOCs and ancestral SARS-CoV-2 have consistently fetched conflicting results. Nevertheless, both VOCs generate higher viral RNA loads than the parental strain, with an increased probability of cell culture isolation.
Despite its high transmissibility, Omicron led to much lower RNA viral loads and cell culture isolation probability. Studies have observed different behaviors of Omicron sublineages concerning infectious virus titers, with Omicron BA.2 leading to higher RNA viral loads and taking more time to clear infection than Omicron BA.1.
Infectious virion shedding by the ancestral SARS-CoV-2 strain resolved faster in those under 18 year-olds compared to individuals >50 years of age. A study showed its higher RNA loads for prolonged times in men infected with Alpha or Delta VOCs than in women. Furthermore, population-scale analysis of viral RNA loads in different age groups showed minimal differences in the dissemination of SARS-CoV-2 RNA load between adults and children; however, it had diminished in those below five years.
Studies have estimated the average incubation period of ancestral SARS-CoV-2 between 4.6 and 6.4 days. However, as the time of infection is often unknown, researchers use dpos when analyzing viral load and infectious virions. However, findings concerning viral shedding patterns have shown considerable heterogeneity in symptomatic and asymptomatic COVID-19 patients. Regardless of clinical symptoms, studies have detected high viral loads in the URT of infected individuals, which makes clinical symptoms – a not-so-robust indicator of infectiousness.
Likewise, the viral load of an index case is an unreliable proxy for SARS-CoV-2 transmission. Viral load can substantially vary between two individuals due to susceptibility and immunity from previous infections or vaccination, which affects their disposition to transmit SARS-CoV-2. In the same way, some sites, in particular, represent a higher risk of transmission; accordingly, many SARS-CoV-2 superspreading events typically occurred indoors in crowded places, such as music auditoriums, cruise ships, care centers, and hospitals.
Researchers have been pursuing several approaches to find a proxy for infectiousness to guide SARS-CoV-2 isolation techniques. In this context, single guide RNAs, transcribed in SARS-CoV-2-infected cells but not packaged in their infectious virions, could serve as a good proxy of infectious virions. However, the assays devised to detect sgRNAs with specific RT-PCR assays have yet to be successfully incorporated into routine diagnostic tools owing to their lower sensitivity. Also, the absence of sgRNA does not necessarily indicate infectiousness but the absence of active SARS-CoV-2 replication.
Conclusions
To summarize, all the currently available diagnostic tools have limitations concerning infectious SARS-CoV-2 virion detection. Though they have not been able to contain every SARS-CoV-2 infection, they are indispensable components of the anti-COVID-19 public health arsenal that have helped reduce the number of infectious individuals in the community and, subsequently, the number of secondary transmissions. Concerted and continuous efforts to evaluate viral-shedding characteristics amid changing biological properties of emerging SARS-CoV-2 variants would remain highly significant and help inform global future public health policies.
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