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Sleeping sickness

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Serodiagnosis

Diagnosis of sleeping sickness currently depends on microscopic demonstration of trypanosomes in blood, cerebrospinal fluid (CSF) or lymph node aspirates. Parasitological methods are not easy to use in screening programmes. Development of serological tests for trypanosomiasis has been hindered by the ability of the parasite to keep changing its surface antigenic coat, which allows it to evade the host’s defense mechanisms. FIND is seeking to develop a serological method based on either antibody or antigen detection that will be specific and sensitive enough to guide treatment.

Antibody detection test

For passive case finding, parasitological examination is carried out only on individuals in at-risk populations who demonstrate anti-trypanosome antibodies with a screening test. The card agglutination test for trypanosomiasis (CATT), developed in 1978, is the primary screening tool used by control programs in areas where T.b. gambiense is endemic (Figure 1).

Performing the card agglutination test for trypanosomiasis (CATT) on human blood.

Figure 1: Performing the card agglutination test for trypanosomiasis (CATT) on human blood. The test is used as a first screen for the presence of antibodies against trypanosomes. A drop of blood or serum is mixed with reagent and placed on a rotor for a few minutes. People who are positive on the test (agglutination) are regarded as suspects and have to undergo further investigation to confirm them as cases. FIND is working with partners to develop an antibody or antigen detection test that is specific enough to guide treatment. (Photo courtesy of Veerle Lejon)

Detection of antibodies against trypanosomes using CATT is a sensitive indicator of infection. However, in populations undergoing screening, where the prevalence of disease is usually below 2% and specificity of the CATT test is around 95%, a large number of positive results turn out to be false-positives. Other problems with the CATT are:

it is not applicable for T.b. rhodesiense infections;

it is manufactured using whole T.b. gambiense organisms recovered from infected laboratory animals in a complex process;

it has inferior sensitivity in some disease foci;

it can only be performed by trained personnel.

Furthermore, the test is incapable of differentiating between active and cured infection, as antibodies tend to stay in the blood for prolonged periods after curative treatment. Finally, it is produced in 50-test packages, and is not available as individual tests, a factor that contributes to higher costs and wastage.

FIND is working with partners to determine the feasibility of developing another serological method that is simpler, more sensitive and more specific by using purified, recombinant or synthetic antigens, rather than whole organisms. As a by-product of fundamental research on African trypanosomes, many parasite-specific proteins with diagnostic potential have been described (published and unpublished data) but only minor attempts have been made to convert them into diagnostic formats.

The strategy FIND has adopted is to select candidate antigens amongst those that are currently available, rather than invest in a discovery phase. Scientists and laboratories with such antigens are collaborating with FIND in screening recombinant and synthetic peptides for their potential for diagnosis of both T.b. gambiense and T.b. rhodesiense, the two forms of sleeping sickness. In order to identify the most promising candidates, a panel of 32 different antigens was screened in a Dot Blot assay by Microcoat in Germany, using a collection of well defined sera from patients infected with T.b. gambiense and T.b. rhodesiense. The first screen led to selection of 14 antigens, which are undergoing a second round using dot-blot and ELISA, and a new collection of positive and negative serum samples, with greater emphasis on specificity.

Antigen detection test

Identification of antibodies that are suitable for antigen detection assays and antigens for use in developing antibody detection tests are running concurrently. FIND is working with the Institute of Biotechnology at the University of Brussels, Belgium, to determine the feasibility of using camel heavy-chain antibodies (nanobodies), and with the Department of Genetics at Darmstadt University of Technology in Germany and ITM, to explore the use of RNA aptamers in tests to detect parasite antigens.

FIND is also working with the Seattle Biomedical Research Institute (SBRI) to apply the single chain variable fragment (scFv) antibody engineering technology in development of optimized antibody probes for trypanosome antigens in blood. Using a technology called yeast display, high-affinity antibody fragments for a number of T. brucei proteins are being generated, and those that are best suited for diagnostic detection in human samples identified. The sensitivity and stability of the probes for the chosen antigens will be enhanced further by antibody engineering methods. The outcome will be a set of antibody probes with characteristics of sensitivity, stability, and manufacturability that are superior to probes generated by traditional methods. (Read more)