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

Koller, G.; Morrell, A. P.; Galao, R.; Pickering, S.; MacMahon, E.; Johnson, J.; Ignatyev, K.; Neil, S. J.; Elsharkawy, S.; Fleck, R.; Machado, P.; Addison, O.

Containing the global SARS-CoV-2 pandemic has been an unprecedented challenge due to high rates of both horizontal transmissivity and asymptomatic carriage. Lateral Flow Device (LFD) immunoassays were introduced in late 2020 to rapidly detect SARS-CoV-2 infection in asymptomatic or pre-symptomatic individuals in the population. Although 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. In particular, the low sensitivity (<50%) shown in several studies is a critical public health concern, given that asymptomatic or pre-symptomatic carriers may wrongly be assumed to be non-infectious, and 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. Using high sensitivity synchrotron X-ray fluorescence imaging we were able to quantify significant immobilized antigen-antibody-label conjugates within the LFDs visually scored as negative. 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). This is of significant concern should these tests be used to control community spread. 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. Our 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.