February 2010

Practical fluorescence microscopy for detection of trypanosomes

The most sensitive method for parasite detection has, until now, been performing mAECT, followed by bright field microscopy. We report the development and evaluation of a fluorescence microscope that could be applicable in rural settings where the disease occurs.

A simple and affordable LED-based fluorescence microscope, the Primo Star iLED, has been developed jointly by FIND and Carl Zeiss, with the following specifications:

  • high-grade optics
  • reflected rather than transmitted blue light for fluorescence applications
  • can be operated on battery power
  • permits easy switching between fluorescent and bright field viewing
  • no need for a dark room
  • bulb life of >10,000 hours 
  • inexpensive bulb
  • LED light source and filter sets that would use dyes for trypanosomiasis and other parasitologic examinations

The Primo Star iLED has been evaluated by a number of laboratories in Africa, including the Livestock Research Institute and Makerere University in Uganda, and the University of Kinshasa and Institut National de Recherche Biomédicale (INRB) in DRC.  Acridine Orange, a common fluorochrome stain, has been used to detect trypanosomes; figure 2 shows how easily parasites are visualized using this stain. The data that have been generated indicate that the speed of reading and sensitivity are higher with this method, when compared with common brightfield microscopy using Giemsa staining. The diagnostic capacity of the method is greatly enhanced when staining of samples is preceded by lysis of red blood cells using ammonium chloride or commercial lysis buffer (Figure 3).

Clinical evaluation of the LED microscope in the diagnosis of HAT using the red cell lysis and acridine orange staining methods on samples from sleeping sickness patients will be carried out at several hospitals in DRC and Uganda in early 2010 (Figure 1).

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Figure 1: Preparing slides for parasite detection using improved microscope developed with FIND partners. (Photo taken at National Livestock Resources Research Institute (NaLIRRI) in Uganda).
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Figure 2: A thin blood smear showing trypanosomes stained with acridine orange, as seen under the Primo Star iLED microscope. Parasites (orange) are easily recognizable alongside red blood cells (dark green). Clumps of platelets (cyan) are also visible adhering to red blood cells. This picture was taken using a simple, commercially-available digital camera (picture courtesy of Carl Zeiss).

Figure 3: Trypanosomes and white blood cells stained with acridine orange after lysis of red blood cells