Taking the Long View: Strategies for SARS-CoV-2 Surveillance

Although not the first virus to threaten the world in the 21st century, SARS-CoV-2’s emergence in late 2019 gave rise to the most severe pandemic in recent history. And although mass vaccination and widespread diagnostic testing have reduced COVID-19 case rates, the disease continues to pose a significant threat of infection and death. This is largely due to its ability to evolve new variants, which in turn is related to its high worldwide prevalence (1). SARS-CoV-2 therefore remains a real concern for public health systems and for the global economy (2) – so how do we manage the risks?

The consensus is that governments should focus on readiness; public health systems should be able to quickly and effectively respond to the appearance of easily transmissible SARS-CoV-2 variants, such as Omicron, while remaining attentive to outbreaks caused by other pathogens. This implies that we should adopt surveillance methods designed to detect new variants – but successful surveillance requires a robust combination of technology platforms, including flexible antibody detection platforms for seroprevalence studies, real-time quantitative PCR (qRT-PCR) to monitor the prevalence of variant sequences, next-generation sequencing (NGS) to identify the emergence of new variants, and droplet digital PCR (ddPCR) for community-based surveillance through wastewater testing.

Building the toolkit
 

PCR methods: good, but not sufficient

PCR testing is the gold standard for diagnosing COVID-19 in symptomatic individuals. qRT-PCR can detect viral RNA in nasal or salivary swabs with very high sensitivity and can therefore provide accurate diagnoses during early stages of infection. Relevant tests include assays specific for SARS-CoV-2 only and assays that test for a panel of respiratory viruses. The latter enables differentiation between COVID-19 and other infections – a useful capability during flu season.

But PCR-based tests are not foolproof; in particular, the rate of false negatives varies during the course of an infection due to changes in viral load (3). The probability of accurate PCR-based SARS-CoV-2 detection is high (77 percent) four days post-infection, when there is a high viral load in the nasal cavity, but significantly lower thereafter (~50 percent at 10 days post-infection and ~0 percent at 30 days post-infection). The effectiveness of routine screening of asymptomatic individuals is therefore constrained by testing frequency and viral load effects (3). Finally, a diagnostic assay may not be as useful for new variants as it is for the variants for which it was designed, depending on the nature and site of mutations; for this reason, commercial kit vendors must continually monitor the sensitivity and specificity of their assays against emerging variants.

Antibody methods: classic tests, still important

Antigen and antibody assays detect SARS-CoV-2 antibodies regardless of infection status and can therefore confirm either current or historic COVID-19. Seroprevalence studies using these kinds of tools allow us to better understand true exposure rates across a population and characterize the spread of new variants. This is important; because four in five patients with COVID-19 are asymptomatic or only mildly symptomatic, restricting tests to symptomatic individuals alone does not allow us to track the true incidence of infection (4). Some serology assays also enable us to measure IgA, IgG, and IgM antibody responses to nucleocapsid, receptor binding domain (RBD), spike 1, and spike 2 SARS-CoV-2 antigens, respectively, and therefore provide excellent data on natural infection and vaccine-induced immunity (5). In addition, serology assays give information on both population exposure rate and – because each isotype is associated with a different stage of infection – the profile and duration of humoral responses (6,7). Data from antibody-based population surveillance therefore helps us understand both the rate of immunity and its persistence. This has obvious utility in determining the longevity of vaccine effectiveness following mass vaccination programs (5,8). Such tests can also help us understand how symptom severity correlates with antibody response during disease progression, which may help plan new therapies to control disease severity and improve patient outcomes.

Using the toolkit
 

Define your task: monitoring variants

The rapid, global spread of SARS-CoV-2 has been accompanied by the emergence of numerous variants, categorized as variants of concern (VOCs) or variants of interest (VOIs) (see Table 1). We must monitor the circulation of these new subtypes, even within vaccinated populations, because accurate monitoring may identify variants that can evade immune responses. We need look no further than the Omicron variant, with its high transmissibility even among vaccinated individuals, to see that variant surveillance remains essential and may help prevent outbreaks caused by emerging VOCs (10).

Such surveillance should not be limited to measuring isotype-specific antibody responses; it must also assess antibody efficacy – that is, the extent to which vaccine-induced antibody responses remain effective against the new variant – by determining levels of neutralizing antibodies that protect against disease. Data from these kinds of tests are critical determinants of the level of concern public health systems attribute to arising variants.

^{Table 1. SARS-CoV-2 VOCs and VOIs. These variants contain mutations in the spike (S) protein, within the amino-terminal domain (NTD) or within the RBD. These alterations influence transmissibility, virulence, and/or evasion of immune responses (9).}

Combine your tools: Variant monitoring with qRT-PCR, NGS, and ddPCR

One of the most powerful ways to monitor changes in circulating SARS-CoV-2 sequences over time is to use ddPCR, qRT-PCR, and NGS technologies in combination, because each approach has different advantages. For example, qRT-PCR assays permit rapid, cost-effective assessment of the prevalence of known SARS-CoV-2 variants. In this context, commercial systems that assay multiple mutations in a single reaction are more cost-effective than single-mutation assays and provide more complete mutation profiles per sample. Furthermore, qRT-PCR assays are compatible with samples containing low levels of viral RNA (C_q values of 31–40), enabling mutation profiling of samples that cannot be reliably evaluated by NGS, which requires less challenging C_q values (11). NGS methods, however, can confirm variants indicated by qRT-PCR – and, indeed, qRT-PCR permits rapid prioritization of samples for subsequent NGS-mediated variant confirmation. Another advantage of qRT-PCR assays is their simplicity, which allows them to be rapidly deployed in laboratories around the world to enhance global surveillance measures (12).Finally, some qRT-PCR assays can be applied to ddPCR protocols. This is important because the ddPCR approach has several advantages, including lower rates of false positives, less variation between replicates across dilution levels, and maintenance of sensitivity and specificity even in very low abundance samples, such as wastewater (13).

In summary, continued surveillance using a combination of technologies will allow us to study new VOCs and VOIs, improve our understanding of variant transmission characteristics, and better monitor the effectiveness of vaccines. This knowledge will then guide key decisions, including whether and when to incorporate antigens from emerging VOCs into new vaccines or boosters.

Use tools wisely: variant surveillance by population and wastewater testing

Although PCR and antibody assays help determine whether an individual is or has been infected, they do not give a complete overview of a community’s susceptibility to SARS-CoV-2 outbreaks. A better approach is to track SARS-CoV-2 incidence at the community level in real time with wastewater-based epidemiology (WBE) using ddPCR(14). There is a clear correlation between the number of COVID-19 cases in a population and the SARS-CoV-2 gene concentration in that population’s wastewater (15) and, given that individuals shed SARS-CoV-2 virus into wastewater earlier and for longer periods than is suggested by respiratory samples, WBE permits more accurate monitoring of infections in a local population than conventional methods. Also, use of ddPCR protocols can mitigate the issues that make wastewater treatment plant influent notoriously difficult to work with: the large number of contaminants, the presence of PCR inhibitors, and low levels of virus. Furthermore, ddPCR offers levels of sensitivity and precision beyond those associated with qRT-PCR assays and is therefore particularly useful for low-abundance targets, for targets in complex backgrounds, or for monitoring subtle changes in analyte abundance. In particular, ddPCR’s high sensitivity makes it well suited for population screening through pooled testing (detecting one infected individual in 10,000) and for confirming negative results suggested by other methods. In addition, the ability of WBE-ddPCR to estimate the abundance of a specific variant in a sample and its independence of people’s propensity to opt for a PCR test allow indirect monitoring of variant incidence. Indeed, recent studies of WBE for early identification of infection hotspots indicate that it effectively identifies incidence peaks and predicts infection outbreaks (16). Finally, some ddPCR assays can accurately discriminate and quantify multiple variants in a sample using a single-well assay (17) and, in comparison with multiplexing approaches based on other technologies, ddPCR multiplexing provides data that are less prone to artifacts and easier to analyze.

Thus, ddPCR-based WBE is a powerful surveillance tool that accurately measures copy numbers of both wild-type and variant genomes. It is encouraging that fully quantitative WBE surveillance is now globally available; this approach should be recognized as complementary to clinical testing and academic research and as a key tool in the management of COVID-19 and other pandemics.

Looking ahead: surveillance for new variants
 

Now that vaccination programs have been rolled out, pandemic management has moved to a different phase; today’s priority is timely detection of emerging SARS-CoV-2 variants. This requires reliable, population-level assays that identify new variants and estimate their prevalence and transmission rates. WBE is now a key component of these surveillance strategies (18); nonetheless, other tools remain critically important for identifying novel VOCs or VOIs and determining their prevalence. Used in combination, these various assay methods employed in prevalence studies will not only help manage near-term SARS-CoV-2 outbreaks, but also ensure that we are prepared for future pandemics. In brief, development and intelligent use of a comprehensive surveillance toolkit enables us to detect SARS-CoV-2, track carriers, and inform public health strategies. This will help us to slow the spread of the virus and allow the public to live their lives in safety.