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

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).Author summary: Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a disease caused by the parasite Trypanosoma brucei and has been responsible for the death of millions of people across Africa in the 20th century. It is also a major economic burden for countries endemic for trypanosomiasis, affecting livestock productivity in rural areas (Animal African Trypanosomiasis). A long-term international collaboration with the help of the World Health Organisation has resulted in the rate of human infection decreasing to less than 1000 new cases per year. However, the human disease continues to spread within remote villages. Current diagnosis is based on the detection of parasites in blood and serum samples, but this is challenging during chronic human infections with low or non-detectable parasitaemia. However, the recent discovery of extravascular skin-dwelling trypanosomes indicates that a reservoir of infection remains undetected, threatening the effort to eliminate the disease. In this study we have targeted the skin as a site for diagnosis using Raman spectroscopy and demonstrate that this method showed great promise in the laboratory, laying the foundation for field studies to examine its potential to strengthen current diagnostic strategies for detecting HAT cases.