No correlation or association between SLPI and lipocalin 2 levels with other clinical (body mass index, somnolence, etc.) or laboratory parameters (number of trypanosomes in the CSF, intrathecal IgM titers, etc) was found. critical because early stage drugs do not efficiently cross the blood-brain barrier (BBB), and treating early-stage patients with the more toxic drugs should be avoided.3,4 According to current World Health Organization (WHO) criteria, HAT patients in late stage are those with trypanosomes present in the cerebrospinal fluid (CSF) and/or an elevated leukocyte count (> 5 cells/mm3) or an increase in protein TNFSF13B content of the CSF (> 37 mg/100 mL).5 However, diagnosis of late stage HAT based on these criteria is unsatisfactory because the O6BTG-octylglucoside number of white blood cells (WBCs) or parasites in the CSF may not be good indicators of passage of trypanosomes across the BBB.5C8 Thus, there is a critical need for biomarkers to efficiently and reliably stage HAT for treatment guidance. Recently, immunoglobulins,9 cytokines O6BTG-octylglucoside such as interleukin (IL)-10,9C11 and chemokines such as CXCL10 and CXCL1312C14 have been suggested as useful biomarkers. In the current study, aimed at the discovery of new staging markers for HAT, genes differentially expressed in the brain of mice at the early and late stage of infection were identified by transcriptome analysis. Because some molecules such as cytokines and chemokines, which may be secreted by WBC in the CSF, have been studied, molecules mainly secreted by WBCs were excluded from the selection. Instead, we focused on other molecules, e.g., those secreted from brain parenchymal cells, as potential novel markers for invasion of trypanosomes. We report that the level of lipocalin 2 and secretory leukocyte peptidase inhibitor (SLPI) transcripts is elevated in the brain of mice during the late phase of infection, when such infected mice cannot be cured with the early stage drug suramin. Levels of these molecules, together with CXCL10, were also increased in the CSF from late stage trypanosome-infected individuals. Thus, lipocalin 2 and SLPI might be considered as markers of the late stage of the disease to complement the use of chemokines or antibodies for better staging. Materials and Methods Patients and specimen. Early and late stage HAT patients with were recruited from the area around Dipumba Hospital, Mbuji Mayi, Democratic Republic of the Congo (DRC), where sleeping sickness caused by is endemic.15 Briefly, individuals who were seropositive in the card agglutination test for trypanosomiasis (CATT) or who presented suggestive clinical signs were examined for trypanosomes in the blood, lymph node aspirate, and CSF. The presence of trypanosomes in at least one of O6BTG-octylglucoside these body fluids O6BTG-octylglucoside was evidence of infection. The late stage disease was defined as either WBC count > 5 cells/L or detection of trypanosomes in CSF. The study protocol was approved by the Ministry of Health, Kinshasa, DRC, and the Ethical Committee of the University of Antwerp, Belgium. Patients were informed about the objectives and modalities of the study and were asked to provide consent. Patients younger than 12 years of age, moribund or with a blood-contaminated CSF were excluded from this study. In total, 180 patients (early stage [n = 90] and late stage [n = 90]) with HAT were considered for analyses reported in this study. Patients with infections (6 in early stage and 20 at late stage) were recruited in the Rumphi region of Malawi and were screened at the local hospitals after providing consent. Disease stage was determined as indicated previously, and the study protocol was approved by the Ministry of Health and Population, Lilongwe, Malawi and the Ethical Committee of the University of Antwerp, Belgium. Blood, CSF, saliva, and urine were collected from all consenting patients. The CSF and urine samples were spun briefly before the supernatant was snap frozen in O6BTG-octylglucoside liquid nitrogen, and remained at ?80C until testing. Blood samples were also spun and sera carefully pipetted out and snap frozen. Aliquots of all samples were kept at ?80C until testing. Body fluids from non-infected age and sex matched patients subjected to lumbar puncture for spinal anaesthesia in Heidelberg, Germany (n = 18), were used as control. Patients signed an informed consent form. The protocol was approved by the Ethical Committee of the Medical Faculty, University of Heidelberg, Germany. Mice, rats, parasites, and infection. The C57BL/6 mice (8C12 weeks old) and Sprague-Dawley rats (180C200 g), kept under specific pathogen-free conditions with food and water were used throughout. All experiments received institutional approval by the local animal ethical committees. Mice were infected by intraperitoneal (i.p.) injection with 2C3.