. 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.

.SignificanceStandardized data processing approaches are required in the field of bio-Raman spectroscopy to ensure information associated with spectral data acquired by different research groups, and with different systems, can be compared on an equal footing.AimAn open-sourced data processing software package was developed, implementing algorithms associated with all steps required to isolate the inelastic scattering component from signals acquired using Raman spectroscopy devices. The package includes a novel morphological baseline removal technique (BubbleFill) that provides increased adaptability to complex baseline shapes compared to current gold standard techniques. Also incorporated in the package is a versatile tool simulating spectroscopic data with varying levels of Raman signal-to-background ratios, baselines with different morphologies, and varying levels of stochastic noise.ResultsApplication of the BubbleFill technique to simulated data demonstrated superior baseline removal performance compared to standard algorithms, including iModPoly and MorphBR. The data processing workflow of the open-sourced package was validated in four independent in-human datasets, demonstrating it leads to inter-systems data compatibility.ConclusionsA new open-sourced spectroscopic data pre-processing package was validated on simulated and real-world in-human data and is now available to researchers and clinicians for the development of new clinical applications using Raman spectroscopy.

. Significance: The diagnosis of prostate cancer (PCa) and focal treatment by brachytherapy are limited by the lack of precise intraoperative information to target tumors during biopsy collection and radiation seed placement. Image-guidance techniques could improve the safety and diagnostic yield of biopsy collection as well as increase the efficacy of radiotherapy. Aim: To estimate the accuracy of PCa detection using in situ Raman spectroscopy (RS) in a pilot in-human clinical study and assess biochemical differences between in vivo and ex vivo measurements. Approach: A new miniature RS fiber-optics system equipped with an electromagnetic (EM) tracker was guided by trans-rectal ultrasound-guided imaging, fused with preoperative magnetic resonance imaging to acquire 49 spectra in situ ( in vivo ) from 18 PCa patients. In addition, 179 spectra were acquired ex vivo in fresh prostate samples from 14 patients who underwent radical prostatectomy. Two machine-learning models were trained to discriminate cancer from normal prostate tissue from both in situ and ex vivo datasets. Results: A support vector machine (SVM) model was trained on the in situ dataset and its performance was evaluated using leave-one-patient-out cross validation from 28 normal prostate measurements and 21 in-tumor measurements. The model performed at 86% sensitivity and 72% specificity. Similarly, an SVM model was trained with the ex vivo dataset from 152 normal prostate measurements and 27 tumor measurements showing reduced cancer detection performance mostly attributable to spatial registration inaccuracies between probe measurements and histology assessment. A qualitative comparison between in situ and ex vivo measurements demonstrated a one-to-one correspondence and similar ratios between the main Raman bands (e.g., amide I-II bands, phenylalanine). Conclusions: PCa detection can be achieved using RS and machine learning models for image-guidance applications using in situ measurements during prostate biopsy procedures.

Significance: Ensuring spectral quality is prerequisite to Raman spectroscopy applied to surgery. This is because the inclusion of poor-quality spectra in the training phase of Raman-based pathology detection models can compromise prediction robustness and generalizability to new data. Currently, there exists no quantitative spectral quality assessment technique that can be used to either reject low-quality data points in existing Raman datasets based on spectral morphology or, perhaps more importantly, to optimize the in vivo data acquisition process to ensure minimal spectral quality standards are met.Aim: To develop a quantitative method evaluating Raman signal quality based on the variance associated with stochastic noise in important tissue bands, including C─C stretch, CH 2 ∕CH 3 deformation, and the amide bands.Approach: A single-point hand-held Raman spectroscopy probe system was used to acquire 315 spectra from 44 brain cancer patients. All measurements were classified as either high or low quality based on visual assessment (qualitative) and using a quantitative quality factor (QF) metric. Receiver-operator-characteristic (ROC) analyses were performed to evaluate the performance of the quantitative metric to assess spectral quality and improve cancer detection accuracy. Results:The method can separate high-and low-quality spectra with a sensitivity of 89% and a specificity of 90% which is shown to increase cancer detection sensitivity and specificity by up to 20% and 12%, respectively. Conclusions:The QF threshold is effective in stratifying spectra in terms of spectral quality and the observed false negatives and false positives can be linked to limitations of qualitative spectral quality assessment.

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