More than the eye can see – shedding new light on SARS-CoV-2 Lateral Flow Device-based immunoassays

Containing the global SARS-CoV-2 pandemic has been an unprecedented challenge due to high horizontal transmissivity and asymptomatic carriage rates. Lateral Flow Device (LFD) immunoassays were introduced in late 2020 to detect SARS-CoV-2 infection in asymptomatic or pre-symptomatic individuals rapidly. Whilst LFD technologies have been used for over 60 years, their widespread use as a public health tool during a pandemic is unprecedented. By the end of 2020, data from studies into the efficacy of the LFDs emerged and showed these point-of-care devices to have very high specificity (ability to identify true negatives) but inadequate sensitivity with high false-negative rates. The low sensitivity (<50%) shown in several studies is a critical public health concern, as asymptomatic or pre-symptomatic carriers may wrongly be assumed to be non-infectious, posing a significant risk of further spread in the community. Here we show that the direct visual readout of SARS-CoV-2 LFDs is an inadequate approach to discriminate a potentially infective viral concentration in a bio-sample. We quantified significant immobilized antigen-antibody-label conjugate complexes within the LFDs visually scored as negative using high-sensitivity synchrotron X-ray fluorescence imaging. Correlating quantitative X-ray fluorescence measurements and qRT-PCR determined numbers of viral copies, we identified that negatively scored samples could contain up to 100 PFU (equivalent here to ∼10,000 RNA copies/test). The study demonstrates where the shortcomings arise in many of the current direct-readout SARS-CoV-2 LFDs, namely being a deficiency in the readout as opposed to the potential level of detection of the test, which is orders of magnitude higher. The present findings are of importance, both to public health monitoring during the COVID-19 pandemic and to the rapid refinement of these tools for immediate and future applications. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The authors gratefully acknowledge the rapid access beamtime session SP28216 awarded at the Diamond Light Source. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Not applicable All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Associated data is contained within the document.