Academic journal article Bulletin of the World Health Organization

Detection of Trypanosomes in Suspected Sleeping Sickness Patients in Uganda Using the Polymerase Chain Reaction

Academic journal article Bulletin of the World Health Organization

Detection of Trypanosomes in Suspected Sleeping Sickness Patients in Uganda Using the Polymerase Chain Reaction

Article excerpt

Voir page 123 le resume en francais. En la pagina 124 figura un resumen en espanol.

Introduction

In Uganda, human African trypanosomiasis (sleeping sickness) is caused by two different trypanosome subspecies -- Trypanosoma brucei rhodesiense (which causes the acute form of sleeping sickness in the south-east of the country), and T.b. gambiense (which causes the chronic form of sleeping sickness in the north-east and north-west). Epidemics of Gambian sleeping sickness, which have occurred periodically in north-east and north-west Uganda since 1983, have been attributed partly to population movements from the Sudan owing to the civil and political unrest there. The disease is fatal if untreated. Since most drugs currently available for treatment of sleeping sickness are very toxic (1), the diagnosis must be reliable. Various methods are at present used for diagnosis, including serological tests for specific antibodies, such as the card agglutination test for trypanosomiasis (CATT: Testryp-CATT; SmithKline-RIT, Antwerp, Belgium) (2). These tests are useful for mass screening, but do not discriminate between current and past infections (1). Parasitological diagnostic methods are more accurate, but have low sensitivity and may not give reproducible results, especially with the characteristically low parasitaemias in Gambian sleeping sickness which often remain undetected when they are used.

Techniques based on the amplification and characterization of nucleic acids from infectious organisms are sensitive and rapid (3, 4). Polymerase chain reaction (PCR) amplification has been advocated for the molecular diagnosis of several genetic diseases (5, 6) and for the identification of infectious disease agents, including trypanosomes (7, 8) and human immunodeficiency virus (9, 10).

In PCR diagnosis of trypanosome infections, the target DNA may be repetitive DNA, ribosomal RNA genes, or kinetoplast DNA minicircles. Since multiple copies of all these are present, the sensitivity is very high (3). In order to establish the stage of the disease, a PCR test with primers specific to T. brucei species (7, 8) was used in the present study to detect trypanosomes in the blood and cerebrospinal fluid from suspected sleeping sickness patients in Uganda.

Materials and methods

Field study and collection of samples

From November 1997 to January 1998, surveys were carried out in areas with endemic sleeping sickness in Arua district, north-west Uganda, by staff of the Livestock Health Research Institute (LIRI), Tororo. During active surveillance, persons were mobilized by sleeping sickness health aides and local chiefs to assemble at selected screening centres for examination using CATT as a primary screening test. Blood samples from suspected cases who were CATT-positive were then examined by thick blood smears (TBS) and the haematocrit centrifugation technique (HCT) for the presence of trypanosomes. The miniature anion-exchange centrifugation technique (MAECT) was used on suspected cases who were CATT-positive, but negative by TBS examination and HCT. If the clinical signs and a positive CATT indicated infection, but the blood was aparasitaemic, a lumbar puncture was performed. In each case the cerebrospinal fluid (CSF) was examined for the presence of trypanosomes after double centrifugation, and the cells were counted.

Blood samples from each suspected patient who gave a strong agglutination reaction (+++ or ++ in the CATT), or in whom parasites were detected, were collected into two microcentrifuge tubes with 10 mmol/l ethylenediaminetetraacetic acid (EDTA) for PCR (two 0.5-ml samples). The CSF samples from parasitologically confirmed cases and suspected cases, based on clinical signs, were also collected in microcentrifuge tubes for PCR (two 0.5-ml samples). In each case, the samples were stored in liquid nitrogen prior to transportation to the LIRI laboratory in Tororo. …

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