Trypanosomiasis: detection of parasites

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.

Category: Medicine Notes