The COVID-19 pandemic, now classified by the WHO as an epidemic, has swept across the globe, transforming public health paradigms and highlighting the crucial need for rapid and accurate diagnostic testing. Since SARS-CoV-2 was identified as the causative agent of COVID-19 in December 2019 (Wu et al. 2020), diagnostic methods have been at the forefront of pandemic control measures.

The real-time reverse transcription-polymerase chain reaction (RT-PCR) is the gold standard for SARS-CoV-2 detection because of its high sensitivity and specificity (Behera et al. 2021). However, RT-PCR testing is costly and time-consuming, requiring specialised laboratory set-ups and technical expertise (Alhamid et al. 2022), limiting its accessibility in remote or resource-constrained regions. In the later stages of the pandemic, antigen rapid diagnostic tests (AgRDTs) became an attractive alternative for population-level screening due to their ease of use, low cost and quick results turnaround time (Peeling and Heymann 2021).

Most antigen-detection rapid diagnostic tests (Ag-RDTs) rely on viral nucleocapsid recognition to detect SARS-CoV-2 infection because it is the most abundant viral protein (Dinnes et al. 2022, Lippi et al. 2023, Aboagye et al. 2024a). These tests offer numerous advantages, particularly in settings that demand quick results, such as airports, schools and healthcare facilities, where large-scale screening is essential to curb viral transmission. However, the rapid evolution of SARS-CoV-2, characterised by mutations in the viral genome, has raised significant concerns about the reliability of these tests, particularly as new variants of concern (VOCs) continue to emerge. Variants like Alpha (B.1.1.7), Delta (B.1.617.2) and Omicron (B.1.1.529) have presented unique challenges for AgRDTs, as mutations in key structural proteins could potentially alter antigenicity (Wijayanti et al. 2023) and, by extension, test performance.

The sensitivity of AgRDTs, defined as their ability to identify infected individuals correctly, has shown variability across different SARS-CoV-2 variants. This variability can have serious public health consequences, leading to missed diagnoses (false negatives) and subsequent uncontrolled virus transmission. As a result, understanding the molecular underpinnings of this variability is critical. Specifically, it is necessary to elucidate how mutations in the S and N proteins affect antigen-antibody binding interactions, a key determinant of AgRDT performance.

This study aims to address these critical gaps by investigating the molecular mechanisms underlying the variable sensitivity of AgRDTs across SARS-CoV-2 variants. A comprehensive understanding of the structural and functional implications of these mutations will provide information for the development of more robust diagnostic tools, ensuring accurate detection regardless of viral evolution. Furthermore, this study could have far-reaching implications for the design of diagnostic tests for other rapidly mutating viral pathogens, thus contributing to global preparedness for future pandemics.

The development of diagnostic tests is fundamental to controlling infectious disease outbreaks. During the early stages of the COVID-19 pandemic, the World Health Organisation (WHO) recommended diagnostic testing as one of the primary strategies to mitigate the spread of SARS-CoV-2 (WHO 2020). Amongst the array of diagnostic tools developed, antigen rapid diagnostic tests (AgRDTs) have been crucial in facilitating large-scale COVID-19 testing (Joji and Shahid 2021). Unlike RT-PCR, which detects viral RNA, AgRDTs target viral proteins, specifically the spike (S) or nucleocapsid (N) proteins, using antigen-antibody interactions on lateral flow assay platforms (Alhabbab 2022). These tests are designed to deliver results within 15–30 minutes, making them invaluable for prompt preliminary real-time decision-making in clinical and public health settings (Amadi 2024).

Despite their advantages, AgRDTs have been scrutinised for their variable performance, particularly their reduced sensitivity compared to RT-PCR (Ghasemi et al. 2022). The sensitivity of AgRDTs has been reported to range between 50% and 80%, depending on factors such as the viral load, stage of infection and the specific test used (Abdul-Mumin et al. 2021, Karon et al. 2021, Dong et al. 2022). Several studies have shown that AgRDT sensitivity is highest when viral loads are high, typically during the early phase of infection when viral replication peaks (Diao et al. 2020, Kahn et al. 2021, Liotti et al. 2021). However, evolving mutations in SARS-CoV-2 variants affecting viral gene expression have resulted in alterations in viral proteins targeted by these tests, complicating the detection process (Harvey et al. 2021, Khan et al. 2022, Thakur et al. 2022). The emergence of SARS-CoV-2 variants has added a new layer of complexity to diagnostic testing. Variants such as Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529) harbour mutations in the viral genome, particularly in the genes encoding the S and N proteins (Andre et al. 2023). The S protein, which facilitates viral entry into host cells via the angiotensin-converting enzyme 2 (ACE2) receptor, is the most rapidly evolving region of the virus due to the selection pressures exerted by the host immune system and vaccine interventions (Lu et al. 2023, Yao et al. 2024). Mutations in the S protein, particularly in the receptor-binding domain (RBD), can alter the structural conformation of the protein, potentially affecting the binding of antibodies used in AgRDTs (Xue et al. 2024). Similarly, mutations in the N protein, a highly conserved and abundant structural protein responsible for packaging the viral RNA (Miller et al. 2021), may also impact AgRDT sensitivity. Although the N protein is considered a more stable target for antigen detection due to its lower mutation rate compared to the S protein, recent variants have exhibited changes in key epitopes of the N protein that could reduce the binding efficiency of antibodies in certain AgRDTs (Vecchio et al. 2021, Rodrigues-da-Silva et al. 2023). Bekliz et al. (2022) showed that some commercially available AgRDTs had diminished performance when detecting the Delta variant, which harbours mutations in both the S and N proteins.

The molecular mechanisms by which these mutations impact AgRDT sensitivity remain poorly understood. It is hypothesised that changes in protein conformation, antigenicity or protein stability may alter epitope recognition, reducing the affinity of AgRDT antibodies for their target antigens (Alexander et al. 1992, Liang et al. 2016). Structural biology techniques, such as X-ray crystallography and cryo-electron microscopy (cryo-EM), can provide insights into how specific mutations affect protein structure and antibody binding (Bodakuntla et al. 2023). In addition, molecular dynamics simulations and immunoassays, such as surface plasmon resonance (SPR), can be used to quantify changes in antigen-antibody interactions caused by these mutations.

This study will combine some techniques and concepts in virology, structural biology and immunology to investigate how mutations in the S and N proteins of SARS-CoV-2 variants affect the diagnostic sensitivity of AgRDTs. By systematically characterising the structural and functional impacts of these mutations, this study aims to uncover the molecular mechanisms that drive variability in AgRDT performance. The results will provide crucial information for improving current diagnostic tools and developing new tests that are resilient to viral evolution, as well as identifying new and/or more conserved or stable diagnostic targets.

Antigen rapid diagnostic tests (AgRDTs) have been pivotal in COVID-19 control efforts globally, including in Ghana, due to their accessibility and speed. AgRDTs were initially developed to detect the original Wuhan strain of SARS-CoV-2 (Goux et al. 2024). With the rise of numerous mutations in different variants of concern (VOCs), there is a growing concern that these tests may now have either reduced or compromised antigen recognition capabilities (Osterman et al. 2022, Raïch-Regué et al. 2022). Numerous studies have demonstrated that diagnostic sensitivity varies across different emerging SARS-CoV-2 variants (Raïch-Regué et al. 2022, Aboagye et al. 2024b), with strains like Delta and Omicron exhibiting reduced detection rates in some AgRDTs (Bayart et al. 2022, Cocherie et al. 2022, Krutova et al. 2022, Soni et al. 2022). This inconsistency raises concerns about false-negative results, which can exacerbate viral transmission, especially in low-resource settings like Ghana, where testing infrastructure is already limited. Globally, the issue poses a broader threat to public health, as these undetected cases may fuel new outbreaks. Structural mutations in the spike (S) and nucleocapsid (N) proteins are suspected to disrupt antigen-antibody interactions, thereby reducing test sensitivity (Springer et al. 2022). Despite these concerns, the molecular mechanisms driving this variability remain poorly understood. As the virus continues to evolve, a lack of insight into these interactions could undermine future diagnostic efforts, both in Ghana and worldwide. This study aims to fill this critical knowledge gap by investigating how mutations in these viral proteins affect AgRDT performance, providing data essential for enhancing diagnostic reliability across diverse settings.

Rapid and accurate diagnosis is fundamental to controlling the spread of infectious diseases like COVID-19. AgRDTs offer a practical solution for large-scale testing, particularly in regions where access to PCR testing is limited. However, the reduced sensitivity of AgRDTs against certain SARS-CoV-2 variants poses a significant challenge to public health efforts. By uncovering the molecular mechanisms behind the variability in AgRDT sensitivity, this study will provide critical insights that could lead to the development of more reliable diagnostic tools. The study underscores the hypothesis of Raïch-Regué et al. (2022) that “the performance of AgRDTs for various VOCs depends on the specific antibodies used by each test and viral mutations alone cannot accurately predict their performance”. As a result, understanding the viral epitopes recognised by the capture antibodies in each commercial test is essential for ensuring the efficacy of AgRDTs in detecting different SARS-CoV-2 variants. The findings from this study will contribute to overcoming current diagnostic limitations, providing important information for improving test performance in varied public health settings.

The findings from this study on SARS-CoV-2 antigen rapid diagnostic tests (AgRDTs) hold significant potential for advancing diagnostics for other rapidly evolving viruses, such as influenza, HIV and various respiratory pathogens. Similar to SARS-CoV-2, these viruses experience frequent mutations that can alter antigen-antibody interactions which, in turn, can reduce diagnostic sensitivity. By elucidating the molecular mechanisms that lead to variability in AgRDT performance for SARS-CoV-2, this research establishes a foundation for improving diagnostic accuracy across other viruses.

The study seeks to investigate the molecular mechanisms responsible for the variability in diagnostic sensitivity of antigen rapid diagnostic tests (AgRDTs) across different SARS-CoV-2 variants.

How does the diagnostic sensitivity of AgRDTs vary across different SARS-CoV-2 variants?

Variability in the diagnostic sensitivity of AgRDTs across SARS-CoV-2 variants is driven by specific mutations in the viral nucleocapsid and spike proteins, which alter antigen-antibody interactions and reduce binding affinity in some variants.

This study will use a combination of virological, structural and immunological techniques to investigate the impact of SARS-CoV-2 variants on the sensitivity of AgRDTs. AgRDTs will be tested against clinical samples containing different SARS-CoV-2 variants to determine sensitivity thresholds. Structural biology techniques, such as X-ray crystallography and cryo-EM, will be employed to examine the conformational changes in viral proteins. Molecular dynamics simulations and immunoassays, including surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA), will quantify the impact of mutations on antigen-antibody binding affinity.

The findings from this study on SARS-CoV-2 AgRDTs have significant potential to advance diagnostic strategies for other rapidly evolving viruses, such as influenza, HIV and various respiratory pathogens. Like SARS-CoV-2, these viruses undergo frequent mutations that can alter antigen-antibody interactions, which can reduce diagnostic sensitivity. By elucidating the molecular mechanisms behind the variability in AgRDT performance for SARS-CoV-2, this research can serve as a foundation for improving diagnostic accuracy for other viruses.

In the case of influenza viruses, which frequently undergo antigenic drift and shift leading to mutations in hemagglutinin (HA) and neuraminidase (NA) (Luczo and Spackman 2024, Perofsky et al. 2024) that may result in false negatives in rapid diagnostic tests. The insights from this study could help design influenza diagnostics that are more resistant to such mutations. In the case of HIV, where the envelope glycoprotein (gp120) mutates rapidly and affects antigen-based detection (Li et al. 2022), the methods used in this research can be adapted to optimise AgRDT performance by predicting how these mutations impact antigen-antibody interactions. Additionally, the structural and computational strategies applied here could support the development of more reliable diagnostics for respiratory viruses, such as respiratory syncytial virus (RSV) and adenoviruses, by focusing on conserved antigenic regions that are less susceptible to mutation, thereby maintaining consistent diagnostic accuracy across viral strains.

In the context of global health, the study’s findings will be especially relevant in low-resource settings, where access to PCR-based diagnostics may be limited and rapid tests are the mainstay for controlling infectious disease outbreaks. By developing more mutation-resistant diagnostics for viruses like influenza and HIV, the findings could help ensure that high-sensitivity tests remain available and effective in regions that experience high viral mutation rates and strain diversity. This would strengthen diagnostic capacity for future pandemics, ensuring rapid identification of cases even as viruses continue to evolve.