Please cite this article in press as: Biéler, S., et al., Improved detection of Trypanosoma brucei by lysis of red blood cells, concentration and LED fluorescence microscopy. Acta Trop. (2011), doi:10.1016/j.actatropica.2011.10.016 ARTICLE IN PRESS G Model ACTROP-2751; No. of Pages 6 Acta Tropica xxx (2011) xxx–xxx Contents lists available at SciVerse ScienceDirect Acta Tropica journa l h o me pa g e: www.elsevier.com/locate/actatropica Improved detection of Trypanosoma brucei by lysis of red blood cells, concentration and LED fluorescence microscopy Sylvain Biéler a,* , Enock Matovu b , Patrick Mitashi c , Edward Ssewannyana d , Stomy Karhemere Bi Shamamba e , Paul Richard Bessell a , Joseph Mathu Ndung’u a a Foundation for Innovative New Diagnostics (FIND), 16 avenue de Budé, 1202 Geneva, Switzerland b Makerere University, Department of Veterinary Parasitology and Microbiology, Faculty of Veterinary Medicine, Makerere University, P.O. Box 7062, Kampala, Uganda c Tropical Medicine Department, Faculty of Medicine, University of Kinshasa, P.O. Box 747, Kinshasa 11, Democratic Republic of the Congo d National Livestock Resources Research Institute (NaLIRRI), P.O. Box 96, Tororo, Uganda e Institut National de Recherche Biomédicale (INRB), P.O. Box 1197, Kinshasa/Gombe, Democratic Republic of the Congo a r t i c l e i n f o Article history: Received 7 June 2011 Received in revised form 26 September 2011 Accepted 28 October 2011 Available online xxx Keywords: Human African trypanosomiasis Trypanosome LED fluorescence microscopy Red blood cell Lysis Diagnosis a b s t r a c t Confirmatory diagnosis of African trypanosomiasis relies on demonstration of parasites in body fluids by bright field microscopy. The parasitaemia in infected patients and animals is usually low, and con- centration methods are used to try and increase the chances of seeing parasites. Recently, fluorescence microscopes using light-emitting diodes (LED) have been developed. Since they emit strong light, their use does not require a dark room, making field application a possibility. We have combined LED fluores- cence microscopy with lysis of red blood cells (RBC) to improve the sensitivity and speed of detecting trypanosomes. In studies conducted at four centers in Uganda and the Democratic Republic of the Congo, parasitaemic blood was serially diluted and the RBCs lysed using commercial buffer. Samples were then concentrated by centrifugation, and different volumes of the sediment used to make thin and thick smears. Next, these were stained with acridine orange or Giemsa, and examined using an LED micro- scope under fluorescence or bright light, respectively. Detection of parasites was significantly improved by RBC lysis and concentration, regardless of the staining and microscopy method used. Further improve- ments were made when smears were prepared using larger volumes of sediment. The best results were obtained with thin smears prepared using 20 l of sediment and stained with acridine orange. The time taken to see the first parasite was dramatically reduced when smears were examined by LED fluorescence microscopy, compared to bright light. LED fluorescence microscopy was found to be easier and requiring less visual effort than bright field microscopy. These studies demonstrate the potential for incremental improvement in detection of Trypanosoma brucei by combining LED fluorescence microscopy with RBC lysis and concentration. The lysis and concentration method may also be useful in sample preparation for other diagnostic tests for trypanosomiasis. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Human African trypanosomiasis (HAT), also known as sleeping sickness, is a major cause of mortality in rural tsetse fly-infested Abbreviations: LED, light-emitting diodes; RBC, red blood cells; HAT, human African trypanosomiasis; CSF, cerebrospinal fluid; mHCT, micro haematocrit cen- trifugation technique; CTC, capillary centrifugation technique; mAECT, mini anion exchange centrifugation technique; DRC, Democratic Republic of the Congo; INRB, Institut National de Recherche Biomédicale; NaLIRRI, National Livestock Resources Research Institute; PBS, phosphate buffered saline. * Corresponding author. Tel.: +41 22 710 27 81; fax: +41 22 710 05 99. E-mail addresses: sylvain.bieler@finddiagnostics.org (S. Biéler), matovue04@yahoo.com (E. Matovu), drmitashi@yahoo.fr (P. Mitashi), edssewannyana@yahoo.com (E. Ssewannyana), stomy karhem@yahoo.fr (S.K. Bi Shamamba), paul.bessell@finddiagnostics.org (P.R. Bessell), joseph.ndungu@finddiagnostics.org (J.M. Ndung’u). sub-Saharan Africa. This debilitating disease is caused by Try- panosoma brucei gambiense in central and western Africa, and Trypanosoma brucei rhodesiense in eastern and southern Africa (World Health Organization, 1998). The former manifests as a chronic disease that claims its victims over a period of years, while the latter acute form can kill patients in just a few weeks. Accord- ing to annual incidence figures reported recently (World Health Organization, 2006; Simarro et al., 2008), HAT appears to be on the decline; however, a similar phenomenon of a diminishing number of cases was observed in the 1960s, yet was followed by devastat- ing epidemics that are now apparently being contained (Moore and Richer, 2001; Lutumba et al., 2005). Control of HAT largely relies on chemotherapy for which only a few drugs are available. Since they are associated with unac- ceptable toxicity, prescriptions based on clinical signs are never warranted (Brun et al., 2010). Thus, suspected individuals are 0001-706X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2011.10.016