November 2012

Tests for parasite detection

Figure 1: A thin blood smear showing trypanosomes stained with Acridine Orange, as seen under the LED fluorescence microscope developed by FIND and Carl Zeiss (photo courtesy of Carl Zeiss).

Demonstration of parasites in body fluids is the most accurate way to confirm HAT, and is critical to avoid the risk of exposing healthy suspects to potentially dangerous therapy. Although parasitaemia is usually high enough in most T. b. rhodesiense patients for parasites to be seen by direct examination of blood under a light microscope, parasite numbers in T. b. gambiense infection are often below the detection limit of the most sensitive methods in use. In the cerebrospinal fluid (CSF), parasites exist in such low numbers that they have to be concentrated before viewing.

The most sensitive method for detection of trypanosomes in blood that is in clinical use is the mini Anion Exchange Centrifugation Technique (mAECT). The method is carried out in two stages, including chromatography of samples, then concentration by low speed centrifugation and viewing under low magnification microscopy. Similarly, in the modified single centrifugation technique (MSC), CSF is collected by lumbar puncture and centrifuged before viewing. Clinical use of this method has been limited by the high costs associated with it.

The mAECT has a number of limitations: it is expensive, time consuming, not easy to standardize, and it cannot be used in most field situations where electricity is often lacking. A number of previous attempts to produce the mAECT kit cheaply in Africa have been faced with problems of sustainability. Many programs therefore rely on the microhaematocrit centrifugation technique (MHCT or CTC), which involves collecting finger-prick blood in a capillary tube, centrifugation and examination of the interface between the buffy coat and plasma for motile trypanosomes. To achieve an acceptable sensitivity, six to eight tubes are usually prepared making the process lengthy and tiresome. An alternative approach is the quantitative buffy coat (QBC) method, where blood is collected in capillary tubes pre-coated with acridine orange and examined using a special microscope objective. Clinical use of this method has been impractical due to the high cost of the special microscope objective.

Figure 2: Malaria parasites (small orange structures) inside red blood cells (round, green) of a baboon experimentally infected with Plasmodium knowlesi and stained with acridine orange, as seen under the PrimoStar iLED microscope. White blood cells also stain orange. Blood smear courtesy of Dr. Maina Ngotho (IPR).

Figure 3: Cultured Leishmania promastigotes (orange with flagella) stained with acridine orange and viewed under the PrimoStar iLED microscope. Slide courtesy of Dr. Maina Ngotho (IPR).

The principle of fluorescence microscopy in trypanosomiasis is known to increase the sensitivity, speed and ease of demonstration of parasites, when compared to other methods. Field application of fluorescence microscopy has been constrained by lack of a simple, robust and affordable fluorescence microscope that can be applied in the rural settings where HAT occurs. A simple, dual-purpose LED-based microscope with facilities for both brightfield and fluorescence microscopy has recently been developed jointly by FIND and Carl Zeiss, which makes use of this technology in routine diagnosis a feasible option. Evaluation of the new microscope in experimental settings using acridine orange to stain parasites yielded very promising results (see Figure 1). (Abstract on fluorescence microscopy)

An added advantage of the microscope is the ability to disclose malaria and Leishmania parasites when samples are stained with acridine orange using a protocol that is similar to the one used to stain trypanosomes (Figures 2 & 3). This is especially important because sleeping sickness patients are frequently co-infected with malaria, which has to be treated first before the patients are put on HAT drugs.

It is possible to miss parasites in blood films, even when fluorescence microscopy is used, due to the low parasitaemia associated with T. b. gambiense infections. A maximum of 350 µl of blood is used on the mAECT technique, which is also inadequate. A parasite concentration procedure developed by FIND and partners has shown that it is possible to lyse red blood cells in large volumes (up to 3ml) of sample using ammonium chloride or commercial lysis buffer, without affecting the integrity of the parasites. Centrifugation of such samples and preparation of thick and thin smears using the sediment has significantly increased sensitivity, especially after staining with acridine orange. Studies carried out using this method by a number of laboratories in Africa, including the Livestock Research Institute and Makerere University in Uganda, University of Kinshasa and Institut National de Recherche Biomédicale (INRB) in DRC, have confirmed the reproducibility of this diagnostic approach.

Clinical evaluation of the LED microscope in the diagnosis of HAT using the red cell lysis and acridine orange staining methods has been completed at several sites in the DRC and Uganda. Widespread demonstration of the technique is planned to start in 2013.