Or specific detection of T. b. gambiense [32]. Product DNA was visualized by ethidium bromide staining of a 1.5 agarose gel. Results are included in Table 1.Methods Ethical IssuesWritten informed consent forms were obtained from patients and healthy individuals whose blood samples were collected and included in the present study. Blood samples were collected during a larger study for diagnostics development, within the framework of the World Health Organization control program for Trypanosomiases in West Africa (RPC 222/14.06.2007). Our study was also approved by the Heidelberg Ethical Commission (S-171/ 2012). All individuals who participated in the present study received an explanation of the scope of the study before they signed the consent forms.miRNA Expression ProfilingAnalysis of the differential expression of circulating miRNAs was done using the miRNA Microarray System with miRNA Epigenetics Complete Labeling and Hyb Kit (which represents 1205 human and 144 human viral miRNAs) (Agilent) following the manufacturer’s instructions. Briefly, after total RNA extraction and quality control using the Epigenetics Agilent Bioanalyzer, 100 ng of total RNA was dephosphorylated using calf intestinal alkaline phosphatase at 37uC for 30 min. The samples were then denatured in 100 DMSO at 100uC for 5 min and ligated to Cyanine3-pCp at 16uC in a circulating water bath for 2 h and purified on a micro bio spin column. The eluate was vacuum dried at 55uC. Samples were resuspended in 18 ml of nuclease-free water. 4.5 ml of the 10X GE Blocking Agent and 22.5 ml of 2x Hi-RPM hybridization buffer were added to each sample and mixed by vortexing. Samples were then heated at 100uC for 5 min and kept on ice. Hybridization was done in a SureHyb chamber at 55uC for 20 h in a hybridization oven. Slides were washed two times at room temperature and once at 37uC for 5 min and scanned using anBlood SamplesDuring routine field screening by teams of the WHO control program for Trypanosomiases in West Africa, people in the Boffa sleeping sickness focus (Guinea) were screened with the CATT for whole blood. Samples with a positive CATT result were screened using the CATT plasma dilution test; all individuals that were positive at a dilution of 1:4 or less were further examined for parasites using the buffy coat concentration technique [31], and by examination of lymph node aspirates if available, as well as a trypanolysis test [31]. Stage determination was done by white cellmiRNA in Human Sleeping SicknessTable 1. Sample classification based on multiple diagnostic tests.Patient Bo.470/6 Bo.471/6 Bo.472/6 Bo.475/6 Bo.480/6 Bo.481/6 Bo.484/6 Bo.487/6 Bo.502/6 Bo 482/6 Bo.473/6 Bo.474/6 Bo.476/6 Bo.477/6 Bo.478/6 Bo.479/6 Bo.
