Activities > Sleeping sickness > Molecular Diagnosis

Sleeping sickness

Parasite detection

The number of parasites in the blood and cerebrospinal fluid (CSF) of Trypanosoma brucei rhodesiense patients is usually high enough for them to be seen by direct examination of blood or CSF under a light microscope. However, T.b. gambiense exists in such low numbers that they have to be concentrated before viewing.

The most sensitive method for detection of trypanosomes in blood is the mini Anion Exchange Centrifugation Technique (mAECT) (Figure 1). This method involves separating the trypanosomes from venous blood by anion exchange chromatography and concentrating them at the bottom of a sealed glass tube by low speed centrifugation (3000rpm). After centrifugation, the tip of the glass tube is examined under low magnification microscopy for the presence of motile trypanosomes (Figure 2). The large volume of blood (300μl) enables detection of parasites in samples containing less than 100 parasites/ml, resulting in high sensitivity. The mAECT, which still requires some improvements, has a number of limitations: it is expensive, time consuming, not easy to standardize, and cannot be used in most field situations where electricity is most often lacking.

Figure 1	Figure 2
Performing the miniature anion exchange centrifugation test (mAECT). Parasites are captured in the filtrate and after centrifugation are visualized under a microscope (Figure 2 shows an old model of collector tube and viewing chamber). Both pictures are courtesy of the Unit of Parasite Diagnostics, ITM

Although several attempts to produce the mAECT kit in Africa have been made, they have always been met with problems of sustainability. FIND has been working with other partners to:

a) Improvement and production of mAECT

FIND has been working with the Institute of Tropical Medicine (ITM) in Antwerp and a number of other companies to improve some of the components of mAECT, including the collector tube, gel column, holding racks and viewing chambers (Figures 3, 4 & 5). A mAECT production unit at the Institut National de Recherche Biomédicale (INRB) in Kinshasa, DRC, has been upgraded and staff trained to produce and market the kit in disease-endemic countries. The upgrading included training, implementation of a rigorous quality control system and in-depth analysis of costs to guarantee sustainability of production.

Figure 3: New model of collector tube (2nd from right) and gel column (2nd from left), whose improvement have been carried out by ITM, with FIND support.

Figure 4: A mAECT set-up, including the gel column, collector tube and holding rack, developed by ITM, with FIND support.

Figure 5: Visualization of trypanosomes under a microscope using the new collector tube and holder, developed by ITM, with FIND support.

b) Development of a new test to replace mAECT

For reasons cited above, the mAECT kit is problematic for use in low income countries and in many field conditions. FIND is working closely with several partners to determine the feasibility of developing an alternative technique to disclose parasites, which will be more sensitive and simpler to perform than mAECT, including membrane filtration and fluorescence microscopy. A simple iLED fluorescence microscope, developed jointly by FIND and Carl Zeiss, is currently under evaluation for trypanosomiasis. In this evaluation, a common fluorochrome stain, Acridine Orange, is being used to detect trypanosomes. FIND is also working with partners to develop protocols for parasite detection using fluorescent trypanosome-specific probes, such as aptamers, nanobodies and single chain variable fragment antibodies (scFVs).

Print this page  Add to favorites  Send to a friend

---

© Foundation for Innovative New Diagnostics 2008. Click here to see the copyright notice