In the peripheral blood there is usually a normal white blood cell count (no leukocytosis or leukopaenia), a normal blood platelet count and a slight normocytic anaemia. The erythrocyte sedimentation rate is quite high. The diagnosis is best made by detection of the parasite. The sensitivity of the conventional parasitological techniques is, however, quite low. The parasite can be found in fluid from the inoculation chancre, blood (direct examination, thin smear, thick smear, buffy coat), lymph node fluid (needle aspiration) or cerebrospinal fluid (lumbar puncture). In a wet blood smear, the motility of the parasites attracts the eye, but the sensitivity of the technique is too low. A Giemsa-stained thick blood smear is more sensitive, but parasites are frequently deformed in this preparation and are therefore easily missed. Lymph node aspiration is done with a dry needle. After puncture the needle is left in place for a while and the node is massaged. A syringe is then fitted to the needle and after aspiration the fluid is put on a microscope slide for direct examination (the motile trypanosomes can then be observed). Several samples will often be needed, as the parasites are not present in large numbers and appear in the blood in intermittent waves. Concentration techniques (buffy coat from a centrifuged microhaematocrit tube or quantitative buffy coat test (QBC) can facilitate the diagnosis. In better equipped laboratories a mini-anion exchange column technique (mAECT) is used (Lanham or Lumsden method). Such a column contains diethylaminoethyl-cellulose (DEAE-52). The separation of blood cells from trypanosomes depends on a difference in surface charge of the blood cells and the parasites. This charge is pH-dependent (importance of iso-electric point). When blood is mixed with a particular buffer (PSG = Phosphate-Saline-Glucose) added to column, the red and white blood cells adhere to the DEAE gel particles. In this buffer, the trypanosomes are at their isoelectric point (= neither positive nor negative charge) so flow through the column. The eluate containing the parasites is collected and centrifuged. The sediment is examined microscopically to determine if parasites are present. The type of buffer and the temperature at which the test is carried out are of very great importance. The more the disease advances, the less frequently are trypanosomes found in the blood, though they are then found more often in the cerebrospinal fluid. The parasites can be cultured in vitro in a specific medium (KIVI; Kit for in Vitro Isolation). In theory as few as 1 trypanosome can be detected in 5 ml, though in 50% of the tested cases the culture remains sterile. The blood is initially mixed with the toxic anticoagulant polyanethol sulphate. The latter also has anti-complement activity. An antigen-capture ELISA (Enzyme Linked ImmunoSorbent Assay) also exists, but there are at present too many false positive results with this.

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Antibodies can be detected serologically. Several techniques (immunofluorescence etc.) have been developed. There are also methods for use in primitive rural conditions. A cheap and practical method is a direct agglutination reaction of trypanosomes on a plastic card, with macroscopic read-off (CATT = Card Agglutination Test for Trypanosomiasis). This is a good screening method in population studies for T. b. gambiense. A drop of blood (finger prick) and a drop of reagent that contains blue-coloured parasites of a known serotype are mixed on a white plastic card. The card is mechanically shaken for 5 minutes and then immediately read. When the test is positive (presence of antibodies) the trypanosomes agglutinate and form a blue clot. CATT must not be confused with the CIATT (Card Indirect Agglutination Test for Trypanosomiasis). Another method is to take a blood drop on very small filter papers (confetti) and examine this later in a laboratory. The patient should be called back later if the result is positive. Antigen-detection methods (ELISA) have also been developed, but are not yet in routine use. A problem arises in persons who have a positive serology, but who are asymptomatic and in whom no parasites are found (wait and see with follow-up or treatment with suramin or pentamidine?). After successful treatment the antibodies remain for years. Antibody detection therefore cannot be used for detecting relapse or reinfection. It is hoped that in the future we shall be able to prove a cure by monitoring reductions in the levels of circulating antigens, either directly (e.g. ELISA) or by PCR or variants of these. Such tests remain to be validated by further clinical investigation. Further invention of genetic detection methods will possibly also facilitate the detection of resistant parasites, which may then of course influence treatment.

Mr Eddy Magnus, Unit of Applied Technology and Production

Institute of Tropical Medicine, Nationalestraat 155, B-2000 Belgium

Tel : 32.3.247.63.68

A correct diagnosis can sometimes be reached even though parasites cannot be detected. These "clinical cases" are patients from an endemic area, with clinical symptoms of late stage trypanosomiasis and lymphocytes in the cerebrospinal fluid. Such "clinical cases" may amount to no more than 5% of the total number of trypanosomiasis patients.

Antibodies should if possible be detected in the cerebrospinal fluid (technically difficult). Determining the IgM content in the cerebrospinal fluid can be very difficult or even impossible to carry out in endemic areas and under field conditions. An experimental latex agglutination test for detection of IgM was developed at the Institute of Tropical Medicine, Antwerp, Belgium. Blood-CSF barrier dysfunction is usually absent or mild and occurs in very advanced late-stage disease. It is possible to calculate and plot diagrams of the quotients CSF/serum concentration for IgG, IgA and IgM (demonstration of intrathecal production of antibodies). Especially intrathecal IgM production will be present in late-stage sleeping disease (occurs in 98% of people with leukocyte counts higher than 20/µl). Similar patterns do occasionally occur in Lyme neuroborreliosis, neurosyphilis, mumps meningoencephalitis and in non-Hodgkin lymphoma involving the central nervous system.

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A lumbar puncture is important :

• sometimes in order to make a diagnosis
• in order to determine the stage (main purpose)
• in order to monitor therapy.

In the 2nd stage the cerebrospinal fluid is characterised by:

• white blood cell count (WBC) > 5 per mm^3, (normally <3)
• protein > 45 mg%,      (normally 15-45 mg%)
• IgM increase, (difficult to carry out; Latex IgM)
• Sometimes trypanosomes and/or Mott cells    

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In African trypanosomiasis, the cerebrospinal fluid can contain numerous lymphocytes. Some of them will have developed into plasma cells containing cytoplasmic vacuoles filled with accumulated immunoglobulines. These cells are also known as morula cells or Mott cells.

Control lumbar punctures should be carried out for late stage West African trypanosomiasis every 6 months for 2 to 3 years after diagnosis and therapy. In East African trypanosomiasis they should be carried out more frequently (every 3 months, certainly in the first year). Good follow-up is essential. Recurrence often presents as a deterioration in results obtained with the cerebrospinal fluid (increased lymphocyte count). Parasitological proof of recurrence is often lacking. Immediately after treatment the lymphocyte count in the cerebrospinal fluid increases (so-called "cerebrospinal fluid storm"). This should not be regarded as a recurrence. There is no permanent protective immunity. Reinfection can occur.