The Salivary Raman COVID-19 Fingerprint

BACKGROUND/RATIONALE: The outbreak of coronavirus disease 2019 (COVID-19), caused by
infection of SARS-CoV-2, has rapidly spread to become a worldwide pandemic. Global
research focused on the understanding of the biochemical infective mechanism and on the
discovery of a fast, sensitive and cheap diagnostic tool, able to discriminate the
current and past SARS-CoV-2 infections from a minimal invasive biofluid. The fast
diagnosis of COVID-19 is fundamental in order to limit and isolate the positive cases,
decreasing with a prompt intervention the infection spreading. Moreover, the prediction
of the respiratory infection severity could be of crucial importance for the fast
identification and discrimination between mild clinical course, severe illness, and Acute
Respiratory Distress Syndrome (ARDS). One of the first infection sites of SARS-CoV-2 is
the oral cavity where the virus is able to bind and penetrate through the ACE2 receptors
present on the epithelial cells of the salivary glands. Thus, a high concentration of
virus particles could be found in saliva in the preliminary phases of the infection.
Saliva is a complex biofluid composed of bioactive molecules that can be collected with a
really minimal-invasive procedure. Raman spectroscopy is a non-invasive, fast and
label-free vibrational technique, able to provide information regarding presence,
concentration, environment, modifications and interactions of all the biochemical species
present in a specific biofluid. Using the Raman spectroscopy, the investiators will
analyze saliva collected from healthy subjects, patients affected by COVID-19 and
subjects with a past infection by COVID-19. The data collected will be analyzed and used
to create a Raman database able to provide a classification model based on machine
learning. The possibility to monitor and characterize a potential salivary COVID-19
fingerprint could be of crucial importance for the monitoring and discrimination of
COVID-19 subjects with a current and past infection from the healthy subjects.

OBJECTIVES: The aim of the project is to characterize and validate the salivary Raman
fingerprint of COVID-19, understanding the principal biomolecules involved in the
differences between the three experimental groups: 1) healthy subjects, 2) COVID-19
patients and 3) subjects with a past infection by COVID-19. The large amount of Raman
data will be used to create a salivary Raman database, associating each data with the
relative clinical data collected. The Raman database will be used for the creation of a
classification model through the application of multivariate analysis in terms of
principal component analysis and linear discriminant analysis. This classification model
will provide a fast tool for the discrimination of the COVID-19 condition, potentially
providing also information on the respiratory clinical course of the patient. The model
will be translated for the application to a portable Raman spectrometer, leading to the
creation of a Raman Point of Care METHODS: Starting from the preliminary results and
protocols of the Laboratory of Nanomedicine and Clinical Biophotonics (LABION) - IRCCS
Fondazione Don Gnocchi Milano, the saliva collected from each experimental group will be
analysed using Raman spectroscopy. All the data will be processed for the baseline, shift
and normalization in order to homogenize the signals collected and creating in this way
the Raman database. The average spectrum calculated from each group will be
characterized, identifying the principal families of biological molecules responsible for
the spectral differences. Consecutively, all the spectra will be processed through
multivariate analysis (principal component analysis and linear discriminant analysis)
obtaining in this way the classification model. LOOCV will be used for the training of
the classification model, which will be questioned using the subset validation analysis.
The partial correlation coefficient (Pearson's and Spearman's correlation) will be used
for the Raman correlation with the clinical parameter (e.g. COVID-19 clinical course)
using as control covariates the age and sex of the subjects. The classification model
will be then translated and used as point of care using a portable Raman equipped with a
laser emitting at 785 nm, with a comparable spectral resolution.

- SAMPLE COLLECTION: Saliva will be collected with Salivette (SARSTEDT, Germany),
following the manufacturer's instructions. The cotton swab will be inserted in the
subject mouth and chewed for 60 seconds. The saliva collection will be achieved
through centrifugation of the swab (1000 g x 2 min), recording all the related
parameters (storage temperature and the time between collection and analysis). All
the collection procedures will be performed at least two hours after the last meal
and teeth brushing.

- SAMPLE PROCESSING: Before the analysis, saliva (3 ul) will be deposited on aluminum
foil and dried overnight. The aluminum foil is fundamental to achieve the Surface
Enhanced Raman Scattering, increasing the saliva Raman signal.

- DATA COLLECTION: Raman spectra will be acquired using an Aramis Raman microscope
(Horiba Jobin-Yvon, France) equipped with a laser light source operating at 785 nm
with 100% (512mW) laser power. Acquisition time will be set at 30 seconds with
double acquisition and 2 seconds delay time to prevent the formation of artifact
spectra. Before each analysis, the instrument will be calibrated using the reference
band of silicon. All the signals will be acquired in the region between 400 and 1600
cm-1 with a resolution of 0.8cm-1, acquiring at least 25 spectra following a
square-map. The software package LabSpec 6 (Horiba Jobin-Yvon) will be used for map
design and the acquisition of spectra.

- DATA ANALYSIS: All the data will be fit using a fourth-degree polynomial curve to
set the baseline and consecutively normalized using unit vector. The contribution of
aluminum will be removed from each spectrum. The statistical analysis will be
performed using the multivariate approach. Briefly, principal component analysis and
linear discriminant analysis will be applied to extract the principal components and
the canonical variables. These features will be used for the leave one out
cross-validation (LOOCV), subset validation and correlation with the clinical
parameters. Mann-Whitney will be performed on PCs scores to verify the differences
statistically relevant between the analysed groups. The analysis will be performed
using Origin software (OriginLab, USA)

- CORRELATION: Partial correlation with Pearson's and Spearman's coefficients will be
performed on the variables extracted and the clinical parameters, using as control
covariates the age and sex of the subjects. Only values with p < 0.001 will be
considered as statistically relevant.

- TRANSLATION: The data and the classification model will be applied with a portable
Raman equipped with a laser emitting at 785 nm and with a spectral resolution
comparable with the one used for the previous analysis.