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Journal of Clinical Microbiology, March 2009, p. 630-635, Vol. 47, No. 3
0095-1137/09/$08.00+0     doi:10.1128/JCM.01430-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Nucleic Acid Sequence-Based Amplification with Oligochromatography for Detection of Trypanosoma brucei in Clinical Samples{triangledown}

Claire M. Mugasa,1,2 Thierry Laurent,3 Gerard J. Schoone,2 Piet A. Kager,4 George W. Lubega,1 and Henk D. F. H. Schallig2*

Department of Veterinary Parasitology and Microbiology, Faculty of Veterinary Medicine, Makerere University, Kampala, Uganda,1 Koninklijk Instituut voor de Tropen (KIT)/Royal Tropical Institute, KIT Biomedical Research, Meibergdreef 39,2 Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands,4 Coris BioConcept, Gembloux, Belgium3

Received 25 July 2008/ Returned for modification 14 October 2008/ Accepted 22 December 2008

Molecular tools, such as real-time nucleic acid sequence-based amplification (NASBA) and PCR, have been developed to detect Trypanosoma brucei parasites in blood for the diagnosis of human African trypanosomiasis (HAT). Despite good sensitivity, these techniques are not implemented in HAT control programs due to the high cost of the equipment, which is unaffordable for laboratories in developing countries where HAT is endemic. In this study, a simplified technique, oligochromatography (OC), was developed for the detection of amplification products of T. brucei 18S rRNA by NASBA. The T. brucei NASBA-OC test has analytical sensitivities of 1 to 10 parasites/ml on nucleic acids extracted from parasite culture and 10 parasites/ml on spiked blood. The test showed no reaction with nontarget pathogens or with blood from healthy controls. Compared to the composite standard applied in the present study, i.e., parasitological confirmation of a HAT case by direct microscopy or by microscopy after concentration of parasites using either a microhematocrit centrifugation technique or a mini-anion-exchange centrifugation technique, NASBA-OC on blood samples had a sensitivity of 73.0% (95% confidence interval, 60 to 83%), while standard expert microscopy had a sensitivity of 57.1% (95% confidence interval, 44 to 69%). On cerebrospinal fluid samples, NASBA-OC had a sensitivity of 88.2% (95% confidence interval, 75 to 95%) and standard microscopy had a sensitivity of 86.2% (95% confidence interval, 64 to 88%). The T. brucei NASBA-OC test developed in this study can be employed in field laboratories, because it does not require a thermocycler; a simple heat block or a water bath maintained at two different temperatures is sufficient for amplification.

* Corresponding author. Mailing address: Koninklijk Instituut voor de Tropen (KIT)/Royal Tropical Institute, KIT Biomedical Research, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands. Phone: 31-20-5665447. Fax: 31-20-6971841. E-mail: h.schallig{at}kit.nl

{triangledown} Published ahead of print on 30 December 2008.

Journal of Clinical Microbiology, March 2009, p. 630-635, Vol. 47, No. 3
0095-1137/09/$08.00+0     doi:10.1128/JCM.01430-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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