Raman spectroscopic analysis of skin as a diagnostic tool for Human African Trypanosomiasis

Abstract

Human African Trypanosomiasis (HAT) has been responsible for several deadly epidemics throughout the 20th century, but a renewed commitment to disease control has significantly reduced new cases and motivated a target for the elimination of Trypanosoma brucei gambiense-HAT by 2030. However, the recent identification of latent human infections, and the detection of trypanosomes in extravascular tissues hidden from current diagnostic tools, such as the skin, has added new complexity to identifying infected individuals. New and improved diagnostic tests to detect Trypanosoma brucei infection by interrogating the skin are therefore needed. Recent advances have improved the cost, sensitivity and portability of Raman spectroscopy technology for non-invasive medical diagnostics, making it an attractive tool for gambiense-HAT detection. The aim of this work was to assess and develop a new non-invasive diagnostic method for T. brucei through Raman spectroscopy of the skin. Infections were performed in an established murine disease model using the animal-infective Trypanosoma brucei brucei subspecies. The skin of infected and matched control mice was scrutinized ex vivo using a confocal Raman microscope with 532 nm excitation and in situ at 785 nm excitation with a portable field-compatible instrument. Spectral evaluation and Principal Component Analysis confirmed discrimination of T. brucei-infected from uninfected tissue, and a characterisation of biochemical changes in lipids and proteins in parasite-infected skin indicated by prominent Raman peak intensities was performed. This study is the first to demonstrate the application of Raman spectroscopy for the detection of T. brucei by targeting the skin of the host. The technique has significant potential to discriminate between infected and non-infected tissue and could represent a unique, non-invasive diagnostic tool in the goal for elimination of gambiense-HAT as well as for Animal African Trypanosomiasis (AAT).

Anti-trypanosomatid drug discovery: progress and challenges

“…Potentially further challenging elimination efforts, parasite reservoirs found in human skin in asymptomatic cases can contribute to transmission 38 . In this regard, the development of a portable and non-invasive human skin test could be particularly useful 41 .…”

Quantitative Assessment of Low-Dose Photodynamic Therapy Effects on Diabetic Wound Healing Using Raman Spectroscopy

“…The peak 1445 cm –1 is dominated by the vibrations of CH 2 bending, which indicate the total dermal matrix protein content [ 49 , 56 ]. In the 1050–1150 cm −1 spectral range, the peaks are mainly assigned to the ν(C–C) modes associated with lipid components [ 57 ]. Spectral features in the 450–1050 cm −1 spectral range were attributed to amino acids: 1003 cm −1 (phenylalanine), 936 cm −1 (proline), 877 cm –1 (hydroxyproline, tryptophan), 854 cm −1 (proline, tyrosine), and 755 cm −1 (tryptophan) [ 54 , 58 ].…”

“…In the 1020–1140 cm −1 spectral range, there are bands associated with phosphate groups, phospholipids, and the C–C stretch of proline [ 57 ]. On day 3, these bands’ intensities in the control group wounds are higher compared to the healthy skin, as shown in Figure 12 .…”

Spatially-resolved single cell transcriptomics reveal a critical role for γδ T cells in the control of skin inflammation and subcutaneous adipose wasting during chronicTrypanosoma bruceiinfection

“…and diluted to 4 ng/µl. Trypanosome load in the skin was determined using Taqman real-time PCR, using primers and probe specifically designed to detect the trypanosome Pfr2 gene, as previously reported 30 . Reactions were performed in a 25 µl reaction mix comprising 1X…”

“…and diluted to 4 ng/µl. Trypanosome load in the skin was determined using Taqman real-time PCR, using primers and probe specifically designed to detect the trypanosome Pfr2 gene, as previously reported30 . Reactions were performed in a 25 µl reaction mix comprising 1X Taqman Brilliant III master mix (Agilent, Stockport, UK), 0.2 pmol/µl forward primer using the ARIAMx qPCR software (Agilent, USA) as a proxy for estimated trypanosome load.…”