New biomarkers for stage determination in Trypanosoma brucei rhodesiense sleeping sickness patients

Natalia Tiberti 0 Enock Matovu 2 Alexandre Hainard 0 John Charles Enyaru 1 Veerle Lejon 7 Xavier Robin 0 Natacha Turck 0 Dieudonn Mumba Ngoyi 6 Sanjeev Krishna 5 Sylvie Bisser 4 9 Bertrand Courtioux 4 9 Philippe Bscher 7 Krister Kristensson 8 Joseph Mathu Ndung'u 3 Jean-Charles Sanchez 0 0 Department of Human Protein Sciences, University of Geneva , Geneva, Switzerland 1 Department of Biochemistry, College of Natural Sciences, Makerere University , Kampala, Uganda 2 Department of Biotechnical and Diagnostics Sciences, College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University , Kampala, Uganda 3 Foundation for Innovative New Diagnostics (FIND) , Geneva, Switzerland 4 INSERM UMR1094, Tropical Neuroepidemiology , Limoges, France 5 Division of Cellular and Molecular Medicine , St. George's, University of London , London , Great Britain, UK 6 Department of Parasitology, Institut National de Recherche Biomdicale , Kinshasa, D. R. Congo 7 Department of Biomedical Sciences, Institute of Tropical Medicine , Antwerp, Belgium 8 Department of Neuroscience, Karolinska Institutet , Stockholm, Sweden 9 Institute of Neuroepidemiology and Tropical Neurology, University of Limoges, School of Medicine , CNRS FR 3503 GEIST, Limoges, France Accurate stage determination is crucial in the choice of treatment for patients suffering from sleeping sickness, also known as human African trypanosomiasis (HAT). Current staging methods, based on the counting of white blood cells (WBC) and the detection of parasites in the cerebrospinal fluid (CSF) have limited accuracy. We hypothesized that immune mediators reliable for staging T. b. gambiense HAT could also be used to stratify T. b. rhodesiense patients, the less common form of HAT. A population comprising 85 T. b. rhodesiense patients, 14 stage 1 (S1) and 71 stage 2 (S2) enrolled in Malawi and Uganda, was investigated. The CSF levels of IgM, MMP-9, CXCL13, CXCL10, ICAM-1, VCAM-1, neopterin and B2MG were measured and their staging performances evaluated using receiver operating characteristic (ROC) analyses. IgM, MMP-9 and CXCL13 were the most accurate markers for stage determination (partial AUC 88%, 86% and 85%, respectively). The combination in panels of three molecules comprising CXCL13-CXCL10-MMP-9 or CXCL13-CXCL10-IgM significantly increased their staging ability to partial AUC 94% (p value < 0.01). The present study highlighted new potential markers for stage determination of T. b. rhodesiense patients. Further investigations are needed to better evaluate these molecules, alone or in panels, as alternatives to WBC to make reliable choice of treatment. - Background Human African trypanosomiasis (HAT), commonly known as sleeping sickness, is a neglected tropical disease caused by the Trypanosoma brucei parasite and transmitted to humans through the bite of the tsetse fly [1]. Two morphologically identical subspecies of parasites are responsible for the disease: Trypanosoma brucei gambiense and T. b. rhodesiense [2]. In both cases, the disease progresses from a haemolymphatic first stage (S1), to a meningo-encephalitic second stage (S2). The latter reflects invasion of the central nervous system (CNS) by the parasites across the bloodbrain barrier (BBB) with severe neurological complications, which can ultimately lead to coma and death, when untreated [3]. The two forms of HAT differ in their clinical presentations and geographic distribution. The gambiense form is widespread in Central and Western Africa and is commonly considered to be a chronic infection, which slowly progresses from the first to the second stage. The rhodesiense form of sleeping sickness, that affects communities in Eastern Africa, is a more aggressive illness, which rapidly progresses to the meningo-encephalitic stage [3] and accounts for less than 5% of all HAT cases [4]. Contrary to T. b. gambiense, for which a relatively safe drug combination has recently been introduced for treatment of S2 patients [4-6], treatment of S2 T. b. rhodesiense patients still relies on melarsoprol [7-9]. Melarsoprol has been reported to cause reactive encephalopathies in 8% of T. b. rhodesiense treated patients, which are fatal in 57% of them [8]. As a drug to safely treat both stage 1 and stage 2 patients is yet to be identified, and as S2 treatment is associated with severe side effects and toxicity [8], stage determination remains a key step in the management of patients suffering from T. b. rhodesiense HAT. Staging is based on the examination of the cerebrospinal fluid (CSF) by microscopy. According to WHO, patients having 5 white blood cells (WBC) per microliter of CSF and absence of parasites are considered to be in the first stage of the disease, while patients having more than 5 WBC/L and/or presence of parasites in the CSF are considered as S2 [10]. These methods suffer from limited specificity and reproducibility of the counting of WBC and lack of sensitivity in finding of parasites in CSF [11,12] (Dieudonn Mumba Ngoyi, personal communication). The discovery of surrogate markers to complement or replace the counting of WBC in the staging of HAT is highly desired [11,13,14]. Many studies have focused on the staging in T. b. gambiense HAT [13,15-20], while less attention has been paid to T. b. rhodesiense, with a paucity of data on staging markers [13,21,22]. Some proand anti-inflammatory factors have been shown to be associated with the late stage of T. b. rhodesiense sleeping sickness, including IL-10, IL-6, CXCL10 and neopterin [13,21,22]. The aim of the present study was to investigate eight immune-related factors, shown to be powerful markers for stratification of T. b. gambiense HAT patients [13,1520,23], as staging markers for T. b. rhodesiense sleeping sickness. Methods Patients Eighty five patients (14 stage 1 and 71 stage 2) with evidence of parasites in blood, lymph or CSF were investigated in the present study (Table 1). Patients were enrolled by active or passive case finding in Malawi Stage 1 (n=14) Stage 2 (n=71) Age, years [mean SD] Trypanosome positive, n * Fishers exact test, no significant differences. MannWhitney U test, no significant differences. (NEUROTRYP study [13]) and Uganda (FINDTRYP study), in regions endemic for T. b. rhodesiense HAT. The studies were approved by the Ministry of Health and Population, Lilongwe, Malawi and by the Uganda National Council for Science and Technology (UNCST). All patients signed a written informed consent before inclusion into the study. Children (< 18 years old) or patients with altered mental status were only included in the studies after written consent of a parent or a guardian. All enrolled patients had the possibility to withdraw at any moment. Details on sample collection, inclusion and exclusion criteria of the two cohorts are reported in Additional file 1: Table 1. CSF samples were collected by lumbar puncture and the number of WBC counted. The presence of parasites was de (...truncated)