Discovery and verification of osteopontin and beta-2-microglobulin as promising markers for staging human African trypanosomiasis

Human African trypanosomiasis (HAT), or sleeping sickness, is a parasitic disease endemic in sub-Saharan Africa, transmitted to humans through the bite of a tsetse fly. The first or haemolymphatic stage of the disease (S1) is associated with presence of parasites in the bloodstream, lymphatic system and body tissues. If patients are left untreated, parasites cross the blood-brain barrier (BBB) and invade the cerebrospinal fluid (CSF) and the brain parenchyma, giving rise to the second or meningoencephalitic stage (S2). Stage determination is a crucial step in guiding the choice of treatment, as drugs used for S2 are potentially dangerous. Current staging methods, based on counting white blood cells (WBC) and demonstrating trypanosomes in CSF, lack specificity and/or sensitivity. In the present study, we used a number of proteomic strategies to discover new markers with potential for staging HAT. CSF samples were collected from patients infected with Trypanosoma brucei gambiense in the Democratic Republic of Congo. The stage was determined following the guidelines of the national control program. The proteome of the samples was analysed by two-dimensional gel electrophoresis (n=9), and by sixplex tandem mass tag (TMT) isobaric labeling (n=6) quantitative mass spectrometry. Overall, 73 proteins were over-expressed in patients presenting the second stage of disease. Two of these, osteopontin and beta-2-microglobulin, were confirmed to be potential markers for staging HAT by Western blot and ELISA. The two proteins significantly discriminated between S1 and S2 patients with high sensitivity (68% and 78%, respectively) for 100% specificity, and a combination of both improved the sensitivity to 91%. The levels of osteopontin and beta-2-microglobulin in CSF of S2 patients (microg/ml range), as well as the fold increased concentration in S2 compared to S1 (3.8 and 5.5 respectively) make the two markers good candidates for the development of a test for staging HAT patients.