. Significance: The primary method of COVID-19 detection is reverse transcription polymerase chain reaction (RT-PCR) testing. PCR test sensitivity may decrease as more variants of concern arise and reagents may become less specific to the virus. Aim: We aimed to develop a reagent-free way to detect COVID-19 in a real-world setting with minimal constraints on sample acquisition. The machine learning (ML) models involved could be frequently updated to include spectral information about variants without needing to develop new reagents. Approach: We present a workflow for collecting, preparing, and imaging dried saliva supernatant droplets using a non-invasive, label-free technique—Raman spectroscopy—to detect changes in the molecular profile of saliva associated with COVID-19 infection. Results: We used an innovative multiple instance learning-based ML approach and droplet segmentation to analyze droplets. Amongst all confounding factors, we discriminated between COVID-positive and COVID-negative individuals yielding receiver operating coefficient curves with an area under curve (AUC) of 0.8 in both males (79% sensitivity and 75% specificity) and females (84% sensitivity and 64% specificity). Taking the sex of the saliva donor into account increased the AUC by 5%. Conclusion: These findings may pave the way for new rapid Raman spectroscopic screening tools for COVID-19 and other infectious diseases.

Significance: The primary method of COVID-19 detection is reverse transcription polymerase chain reaction (RT-PCR) testing. PCR test sensitivity may decrease as more variants of concern arise. Aim: We aimed to develop a reagent-free way to detect COVID-19 in a real-world setting with minimal constraints on sample acquisition. Approach: We present a workflow for collecting, preparing and imaging dried saliva supernatant droplets using a non-invasive, label-free technique known as Raman spectroscopy to detect changes in the molecular profile of saliva associated with COVID-19 infection. Results: Using machine learning and droplet segmentation, amongst all confounding factors, we discriminated between COVID-positive and negative individuals yielding receiver operating coefficient (ROC) curves with an area under curve (AUC) of 0.8 in both males (79% sensitivity, 75% specificity) and females (84% sensitivity, 64% specificity). Taking the sex of the saliva donor into account increased the AUC by 5%. Conclusion:These findings may pave the way for new rapid Raman spectroscopic screening tools for COVID-19 and other infectious diseases.