Fourier transform infrared (FTIR) spectroscopy provides a (bio)chemical snapshot of the sample, and was recently used in proof-of-concept cohort studies for COVID-19 saliva screening. However, the biological basis of the proposed technology has not been established. To investigate underlying pathophysiology, we conducted controlled infection experiments on Vero E6 cells in vitro and K18-hACE2 mice in vivo. Potentially infectious culture supernatant or mouse oral lavage samples were treated with ethanol or 75% (v/v) Trizol for attenuated total reflectance (ATR)-FTIR spectroscopy and proteomics, or RT-PCR, respectively. Controlled infection with UV-inactivated SARS-CoV-2 elicited strong biochemical changes in culture supernatant/oral lavage despite a lack of viral replication, determined by RT-PCR or a cell culture infectious dose 50% assay. Nevertheless, SARS-CoV-2 infection induced additional FTIR signals over UV-inactivated SARS-CoV-2 infection in both cell and mouse models, which correspond to aggregated proteins and RNA. Proteomics of mouse oral lavage revealed increased secretion of kallikreins and immune modulatory proteins. Next, we collected saliva from a cohort of human participants (n = 104) and developed a predictive model for COVID-19 using partial least squares discriminant analysis. While high sensitivity of 93.48% was achieved through leave-one-out cross-validation, COVID-19 patients testing negative on follow-up on the day of saliva sampling using RT-PCR was poorly predicted in this model. Importantly, COVID-19 vaccination did not lead to the misclassification of COVID-19 negatives. Finally, meta-analysis revealed that SARS-CoV-2 induced increases in the amide II band in all arms of this study and in recently published cohort studies, indicative of altered β-sheet structures in secreted proteins. In conclusion, this study reveals a consistent secretory pathophysiological response to SARS-CoV-2, as well as a simple, robust method for COVID-19 saliva screening using ATR-FTIR.

Fourier-transform infrared (FTIR) spectroscopy provides a (bio)chemical snapshot of the sample, and was recently proposed for COVID-19 saliva screening in proof-of-concept cohort studies. As a step towards translation of this technology, we conducted controlled validation experiments in multiple biological systems. SARS-CoV-2 or UV-inactivated SARS-CoV-2 were used to infect Vero E6 cells in vitro, and K18-hACE2 mice in vivo. Potentially infectious culture supernatant or mouse oral lavage samples were treated with ethanol or Trizol to 75% (v/v) for attenuated total reflectance (ATR)-FTIR spectroscopy, or RT-PCR, respectively. The control condition, UV-inactivated SARS-CoV-2 elicited strong biochemical changes in culture supernatant/oral lavage despite lack of replication determined by RT-PCR or cell culture infectious dose 50%. Crucially, we show that active SARS-CoV-2 infection induced additional FTIR signals over the UV-inactivated SARS-CoV-2 infection, which correspond to innate immune response, aggregated proteins, and RNA. For human patient cohort prediction, we achieved high sensitivity of 93.48% on leave-on-out cross validation (n=104 participants) for predicting COVID-19 positivity using a partial least squares discriminant analysis model, in agreement with recent studies. However, COVID-19 patients negative on follow-up (RT-PCR on day of saliva sampling) were poorly predicted in this model. Importantly, COVID-19 vaccination did not lead to mis-classification of COVID-19 negatives. Meta-analysis revealed SARS-CoV-2 induced increase in Amide II band in all arms of this study and recent studies, indicative of altered β-sheet structures in secreted proteins. In conclusion, ATR-FTIR is a robust, simple, portable method for COVID-19 saliva screening based on detection of pathophysiological responses to SARS-CoV-2.

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