High-wavenumber Raman spectroscopy for the detection of Mycobacterium tuberculosis in saliva

Abstract

Despite the increased availability of low-cost and effective treatments for tuberculosis (TB), ~1 million people continue to die from TB-related symptoms annually. One major challenge limiting the effectiveness of TB treatment is delays in diagnosis, largely due to current detection methods requiring either weeks of culture time or complex processing steps that cannot be performed at the point-of-care. Thus, there is a need for alternative methods that are easier to use yet still effective in providing an accurate TB diagnosis. This work investigates the feasibility of using high-wavenumber Raman spectroscopy to detect the presence of the causative agent of TB, Mycobacterium tuberculosis, in human saliva. To accomplish this, raw saliva was collected from healthy participants and inoculated with a fixed, physiologically relevant, concentration of bacteria (10⁶ CFU/mL) and concentrated into a pellet. The samples were measured using Raman spectroscopy and analyzed with a spectral unmixing approach to determine the relative biochemical composition. The presence of M. tuberculosis resulted in a significant increase in the lipid signal of saliva pellets containing the spiked bacteria, with a median percent increase of 423.6% as compared to the control samples. Control experiments using Streptococcus mutans, a common oral bacterium, only resulted in a slight increase of 9.8%. Additionally, using linear regression analysis, a predictive relationship was found between the Raman lipid fractions of the raw saliva and the control saliva pellets. Using the 95% prediction interval of this relationship as a classification threshold, the presence of M. tuberculosis was accurately determined for all samples with an overall training accuracy of 98.5% and a cross-validation accuracy of 100%. These results showcase the potential of high-wavenumber Raman spectroscopy as a reagent-free method of detecting M. tuberculosis in saliva samples.