by Dr Andrew Lane
The emergence of SARS-CoV-2 and its global proliferation has spurred unprecedented efforts by academia and the in vitro diagnostics industry to develop rapid tests that can be used for point-of-care (POC) testing. At the time of writing, over 200 rapid test kits are under development or have already been commercialized for use. Yet, owing to relaxation of regulatory standards and the unprecedented pace at which tests have been developed, many have not been adequately assessed. In this article, Dr Andy Lane explains the role of POC diagnostics for COVID-19 and the improvements needed for their widespread use, including the importance of high-quality reagents.
Since its emergence in late 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread to nearly every country worldwide, infecting tens of millions of individuals and resulting in over one million deaths . With no specific treatments currently available, non-pharmaceutical interventions such as quarantine, personal protective equipment and social distancing have become the mainstay of disease control. Yet, despite these efforts, many countries have faced ongoing community and nosocomial transmission, with recent surges in cases. To reduce transmission and contain further outbreaks, diagnostic testing continues to play a crucial role. Not only does testing allow for the identification, isolation and management of cases, but also provides the epidemiological variables to inform ongoing changes to public health policy.
So far, the diagnosis of COVID-19 has largely relied on reverse transcriptase (RT)-PCR – an established technique that amplifies viral RNA from swab samples to detectable levels in order to confirm active infection. Since the outset of the pandemic, RT-PCR has been used to carry out the majority of testing and is considered the gold standard for acute-phase diagnosis . RT-PCR has shown its strengths in both speed and sensitivity, as swab samples can be sent to clinical laboratories to provide results within hours and only very small amounts of RNA are needed for initial amplification. However, RT-PCR also suffers from some well-recognized limitations. The processing of RNA samples requires specialized biocontainment laboratories, supported by complex and resource-costly sample collection and distribution systems. Given the unprecedented scale of the ongoing pandemic, this has severely limited the pace at which testing has been scaled and is typically extending turnaround times beyond 24 hours .
Furthermore, there are also inherent biological limitations to using RT-PCR: as virus is removed from the body during the convalescent phase, levels of RNA in the upper respiratory tract decrease rapidly, resulting in a limited time window for detection . This precludes the use of RT-PCR in confirming previous infection and makes asymptomatic or sub-clinical infections especially difficult to detect.
Antigen and antibody testing
Unlike molecular methodologies, immunoassays harness the interactions of immune proteins to indicate the presence of, or past exposure to, a pathogen. Traditionally, platforms such as the enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA) have formed the backbone of immunoassay testing and have largely been used to understand COVID-19 immunology and serostatus to-date (Fig. 1) .
Antigen immunoassays use specific antibodies to directly bind and detect the presence of viral antigens and produce a signal. Like RT-PCR, antigen testing is therefore limited to the acutephase of infection when virus is present and actively replicating in the respiratory tract. However, because immunoassay methods do not amplify proteins, antigen testing lacks the sensitivity of RT-PCR and is therefore not the preferred method of clinical diagnosis in medical facilities. Antibody immunoassays, on the other hand, indirectly detect pathogens via specific host antibodies present in the blood or sputum. The major advantage of antibody testing is that antibodies are long lasting and can typically be used to determine exposure to a pathogen years after the initial infection. Given its applications in immune status testing, vaccine development and epidemiology, widespread antibody testing is now considered an essential tool in fighting COVID-19 .
While there is a clear need for antibody diagnostics, ELISA and CLIA platforms suffer from the same facility, training and supply chain constraints of RT-PCR that limit their use to large hospital facilities with highly trained staff. Indeed, the sheer scale of the COVID-19 pandemic has exposed the shortcomings of traditional diagnostics and has spurred efforts to develop faster, simpler and more scalable platforms that can be decentralized for use at the point of care (POC).
Although various technologies have been investigated to meet these needs, POC immunoassays, such as the lateral flow assay (LFA), are the best-placed (Fig. 2). In short, LFAs are paper or polymer-based immunoassays that absorb a sample and run it along the surface of a pad, binding reporter antibodies and then detector antibodies to produce a confirmatory visual signal – usually within a matter of minutes . A well-known example of an LFA is the at-home pregnancy test, which uses antibodies to detect gonadotropin.
A particularly useful feature of the LFA is its design flexibility, which makes it well-suited for a variety of applications. For the detection of acute SARS-CoV-2 infection, virus or antigen can be collected from nasopharyngeal swab samples and detected by antibodies that are specific to the spike glycoprotein and nucleoproteins. Although rapid antigen tests tend to show lower performance than RT-PCR, their speed and portability has made them a viable contender to supplement existing methods for decentralized POC testing. For immune status testing, a range of different antibodies produced against SARS-CoV-2 can be measured from either sputum (IgA) or blood (IgM and IgG). Unlike antigens or RNA, antibodies appear unusually slowly in most COVID-19 patients, with a median time of 11 and 14 days for IgM and IgG, respectively . Therefore, the application of antibody tests for acute-phase diagnosis is not advised, with health authorities instead proposing that they are used in tandem with other diagnostic technologies, or in epidemiological studies .
Given their complementary features, the use of antigen and antibody LFAs in combination could help alleviate the pressures on public testing laboratories while providing much-needed serological data. Acutephase status could be used to inform isolation and treatment decisions – potentially in tandem with digital approaches to contract tracing – while the secure confirmation of antibody status may allow individuals to return work and guide policy-makers in their decision-making.
Quality over quantity
The emergence of SARS-CoV-2 and its global proliferation has spurred unprecedented efforts by academia and the diagnostics industry to provide timely and effective solutions. At the time of writing, over 200 rapid tests are in development or have already been commercialized for use, with many being employed in small- to medium-scale serological studies (Fig. 3) . However, although an abundance of tests are now available, there have been several hurdles to their effective deployment. Firstly, owing to the pace at which diagnostics have been developed, the performance characteristics of many kits have not been adequately assessed for use at the POC. Therefore, many kits are for research-use only and have not received full regulatory approval. In fact, at the time of writing, only one LFA has gained full U.S. Food & Drug Administration approval for use in clinical settings . This over-abundance of tests and under-abundance of product data has the potential to threaten not only public trust in healthcare, but patient safety too.
Complicating matters, the few studies assessing the performance of such tests have showed high risks of bias and heterogeneity in evaluation standards , with further clinical investigations tending to show less favourable performance, and some tests having even been identified to have “fraudulent documentation, incomplete technical files or unsubstantiated claims” . Finally, in the case of antibody tests, there is also still an incomplete understanding of antibody kinetics and correlates of immune protection, which limit the utility of LFAs in this application .
The ongoing research efforts and advances in complementary technologies will pave the way to new POC assays in the coming months. However, adapting and scale manufacturing such platforms for COVID-19 diagnosis is no small feat, and the performance of these assays needs to be thoroughly evaluated before they are employed for use. To remedy existing issues, further research and assay validation are a clear priority. Studies are needed in prospective cohorts for the intended use populations that include a range of ages, ethnicities and underlying conditions, with transparent reporting of data.
The right reagents
Central to the performance of any immunoassay is the development, selection and application of high-quality biological reagents. The majority of immunoassays use recombinant proteins expressed from cell culture, which offer the advantage of improved biosafety and batch-to-batch consistency . For COVID-19, there are two antigens that nearly all tests are based on: spike glycoprotein and nucleoprotein.
Spike glycoprotein (Fig. 4) is naturally found as a trimer that protrudes from the surface of the SARS-CoV-2 envelope and gives the coronaviruses their characteristic crown-like appearance. In addition to spike’s three polypeptide chains, each trimer contains up to 66 post-translationally added glycan sugars that mediate various functions during natural infection .
However, glycans also constitute many of the epitopes that antibodies recognize, and as a result, the use of unglycosylated spike proteins risks the binding of cross-reactive antibodies in assays. Consequently, reduced assay specificity may produce false-positive results. Developers must therefore take care in selecting and optimizing expression systems to ensure that recombinant spike protein is produced with full glycosylation patterns and proper conformational folding. Simpler organisms such as E. coli do not have the cell machinery to glycosylate antigens, requiring more advanced mammalian or insect systems. Using a mammalian cell expression system, such as The Native Antigen Company’s proprietary HEK293 cell-based platform for expression of recombinant proteins, allows for proper protein folding and full post-translational modifications, which are essential for full biological and antigenic activity. When scaling-up protein production, factors such as yield, and batch-to-batch consistency also require careful consideration.
To further improve specificity, manufacturers are investigating select regions of the spike glycoprotein which show greater SARS-CoV-2 sequence specificity and are therefore less able to bind cross-reacting antibodies. Popular choices are the spike protein S1 subunit and its receptor-binding domain, which is responsible for binding ACE2 to mediate viral entry to host cells. However, this in turn brings forth new challenges, as producing and presenting these truncated proteins in their native conformation is no easy feat.
For assays that use nucleoprotein, the challenges are somewhat different. Nucleoproteins are not present at the surface of the virus but are instead found within the viral capsid where they bind to genomic RNA. Unlike spike glycoprotein, nucleoprotein is unglycosylated and shows greater sequence conservation, especially at its N-terminus. The resulting structural similarity allows nucleoproteins to bind crossreactive antibodies that have the potential to produce false-positive results in assays. A common strategy to address this is to ‘ablate’ crossreactive epitopes by introducing point mutations in the nucleoprotein gene while minimizing structural changes to other regions. Alternatively, so-called ‘quenching antigens’ can be introduced in assays to ‘soak-up’ excess, cross-reacting antibodies from patient sera.
Andrew Lane PhD
The Native Antigen Company, Kidlington, Oxfordshire, OX5 1LH, UK