Clinical Features

The signs and symptoms of East and West African trypanosomiasis are very similar. However, the former is a more acute illness, with overt clinical manifestations appearing within days to weeks of infection, and death supervening in weeks to months. West African trypanosomiasis runs a more indolent course, with an incubation period of months to years. Studies based on isoenzyme characterisation suggest that particular zymodemes may be associated with certain clinical features (Smith et al., 1997).

The haemolymphatic stage of trypanosomiasis is characterised by a chancre, which develops 2-3 days after the bite of an infecting tsetse fly as a tender, erythematous swelling. It is more common in T. brucei var. rhodesiense infection, and subsides within 3 weeks. Posterior cervical lymphadenopathy (Winterbottom's sign) often occurs with West African trypanosomiasis, lymphadenopathy being more generalised in East African disease. Fever, arthralgia, headache and myalgia are the commonest symptoms— unfortunately these symptoms occur in numerous febrile illnesses and are of little diagnostic value. The fever takes the form of recurrent febrile episodes coinciding with each wave of parasitae-mia, each bout lasting 1-3 days. The haemolymphatic stage of trypanosomiasis is also marked by anaemia, hepatosplenomegaly and characteristic cutaneous ring-like patches, with polycyclic contours called trypanids. Other manifestations that can develop in the haemolymphatic stage and progress in the meningoencephalitic stage include oedema, ascites, albuminuria, endocrine and cardiac dysfunction (including pericardial effusion) and intercurrent infection.

Although the clinical features of the haemolymphatic stage may persist, the meningo-encephalitic stage is characterised by the onset of neurological phenomena. These can manifest in a myriad of ways, the loss of the circadian sleep-wake rhythm being commonest. Daytime somnolence develops, sometimes alternating with nighttime insomnia. The progressive severity of the somnolence has resulted in the use of the term 'sleeping sickness'. Other manifestations include hyper-reflexia, presence of primitive reflexes, coordination disorders, sensory disorders, tremor and choreoathetosis, hyper- or hypotonia, convulsions, impairment of conscious level and alteration of the mental state, including confusion, disorientation, alteration of mood (e.g. depression or euphoria) and behavioural changes marked by progressive indifference.

LABORATORY DIAGNOSIS Parasite Detection

Diagnosis is most accurately made by demonstration of the parasite in body fluids. In early T. brucei var. rhodesiense infection, trypano-somes can be detected in serous fluid aspirates from the trypanosomal chancre, when present. In acute illness, trypanosomes can be detected in blood films. Wet blood films can be used for the visualisation of motile trypanosomes and have a detection limit of 25 parasites/ml of sample; thin (Figure 14a.6) and thick blood smears fixed in methanol and stained with Field's or Giemsa stain should also be made and have detection limits of 33 and 17 parasites/ml, respectively. The number of parasites in the blood is often very low, and multiple sampling and a variety of concentration techniques may be employed to facilitate detection:

• Capillary tube centrifugation (microhaemato-crit centrifugation) technique—microscopic examination of the buffy coat of blood spun in microhaematocrit tubes. Detection limit is about 16 parasites/ml (Anonymous, 1998).

• Quantitative buff coat technique—a glass haematocrit tube precoated with acridine orange and anticoagulant is centrifuged, with a float forcing the sedimentation of

Fig. 14a.6 Photomicrograph of a Giemsa-stained peripheral blood smear demonstrating African trypanosomes

erythrocytes. Motile fluorescent trypanosomes are concentrated in the buffy coat around the float. Detection limit is about 16 parasites/ml.

• Miniature anion-exchange centrifugation technique—the difference in electrical charge on the surface of trypanosomes from that in blood is used to effect a separation on an anion exchange (diethylaminoethyl cellulose) chromatography column. Trypanosomes are detected in the eluate after the passage of infected blood through the column, followed by centrifugation. This is the most sensitive of the blood concentration techniques, with a detection limit of 3-4 parasites/ml (Anonymous, 1998).

• Density gradients and differential haemolytic agentss can be employed to enable separation of trypanosomes from erythrocytes by centrifugation.

In early illness, lymph node aspiration is easily performed and microscopic examination of a wet preparation of aspirates often enables visualisation of trypanosomes. Examination of cerebrospinal fluid (CSF) is of particular use in demonstrating cerebral involvement. The double centrifugation technique substantially enhances sensitivity to a detection limit of 1 parasite/ml. In the absence of trypanosomes in CSF, a raised CSF leucocyte count (>5/mm3), the presence of morula cells and raised protein are all indicators of possible cerebral trypanosomiasis. In late disease, elevated IgM titres are also of diagnostic value, and can now be determined through a latex agglutination test (latex/IgM) which is sensitive, simple and stable (Lejon et al., 1998).

In vivo and in vitro culture systems may be used for the isolation of trypanosomes, but neither technique is currently practical for routine diagnosis. The in vivo technique is more sensitive for T. brucei var. rhodesiense (utilising mice and rats) than for T. brucei var. gambiense (utilising Mastomys natalensis, guinea-pigs and suckling rats) and achieves detection limits of 3-5 parasites/ml. A kit for in vitro isolation of trypanosomes (KIVI) from infected patients has been developed, but its diagnostic value is limited because detectable numbers of trypanosomes are produced only after several days (Aerts et al., 1992).

Indirect Diagnosis

As the density of trypanosomes in body fluids is often beyond the limits of even the most sensitive detection systems, indirect diagnostic techniques employing detection of antibodies, antigens or nucleic acids often need to be employed.

Antibody Tests

Various antibody detection tests have been developed, including ELISA, immunofluorescence, immune trypanolysis, direct agglutination, indirect haemagglutination, latex agglutination, Western blot and dot-blot. The card agglutination test for trypanosomiasis (CATT) uses a reagent made of fixed, stained intact trypanosomes of variable antigen type LiTat 1.3 (Buscher et al., 1999). This test has the advantages of high sensitivity and specificity, low costs, simplicity and speed, results being obtained within 5 minutes in the field. However, the LiTat 1.3 gene is not present in a small proportion of isolates, and non-expression of this gene could also produce a false-negative result. CATT is not equally effective in all geographical areas and is only currently available for T. brucei var. gambiense infection. Preliminary studies show that the Trypanosomiasis Agglutination Card Test (TACT) could be a promising development in the serological diagnosis of T. brucei var. rhodesiense infection (Akol et al., 1999).

Antigen Tests

Several direct, indirect and sandwich ELISA antigen-detection systems are being developed. The card indirect agglutination test for trypano-somiasis (TrypTect CIATT) uses specific antibodies coupled to latex beads to detect circulating trypanosomal antigens in patients' blood (Asonganyi et al, 1998). The antigens are invariant antigens expressed on the surface of procyclic forms of T. brucei, and are common to all T. brucei var. gambiense and T. brucei var. rhodesiense stocks. TrypTect CIATT has been shown to have high sensitivity and specificity (both >99%) and is simple and quick to perform. It is applicable for both T. brucei var. gambiense and T. brucei var. rhodesiense infection.

PCR techniques have been developed for trypanosome detection in both CSF and blood, and sensitivity thresholds of 1 parasite/ml have been reported. However, further evaluation is required before they are used for routine diagnosis.

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