Molecular diagnostic tools
The parasitologic tests in use for diagnosis of human African trypanosomiasis (HAT), or sleeping sickness, have low sensitivity, and current serologic tests have inadequate specificity. Detection of trypanosomal DNA sequences from a patient’s blood, urine or saliva could be a significant improvement on parasitological examination. Loop-mediated isothermal amplification (LAMP) of DNA is a promising new molecular technique that shows high sensitivity and specificity, amplifies target DNA under isothermal conditions, meaning that the test can be carried out with minimal equipment.
Furthermore, positive samples are identified visually either through the formation of a white precipitate, a color change (see example with SYBR Green in Figure 1), or fluorescence. LAMP can also be used for the simultaneous analysis of large numbers of samples, and can be performed by staff with minimal experience in molecular biology. This test may also be useful for confirming cure in treatment follow-up.
Figure 1: Example of a loop-mediated isothermal amplification (LAMP) test carried out using purified trypanosome DNA. Positive samples turn green (Tubes 1-3) and negative ones remain orange (Tube 4 and NC-negative control). LAMP is highly sensitive and specific, works under isothermal conditions, needing minimal instrumentation. (Photo courtesy of Z.K.Njiru)
FIND has been working with Murdoch and Obihiro Universities, as well as research institutes in endemic countries, to develop and evaluate the potential for HAT diagnostics based on LAMP technology. Sets of primers that are specific to the subgenus Trypanozoon T.b. rhodesiense and T.b. gambiense were designed and tests optimized using DNA from various members of this subgenus. Primer sets were validated using samples from HAT patients. Reproducibility of the tests in laboratories based in the endemic regions was verified. The work gave sufficiently promising results to support adaptation of the LAMP technique for diagnosis of HAT. See papers by Z.K. Njiru, et al:
- African trypanosomiasis: Sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA [pdf 360kb]
- Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Trypanosoma brucei rhodesiense [pdf 420kb]
In 2005, FIND partnered with Eiken Chemical Company Ltd (Japan), the owner of the patent rights to LAMP, to work together in developing and supplying a diagnostic test for Mycobacterium tuberculosis for the developing world. Eiken has already developed and marketed other infectious disease tests, including assays for Salmonella detection, various E. coli detection kits, Listeria monocytogenes and Campylobacter detection kits based on LAMP. FIND has a close and ongoing collaboration with Eiken to assess opportunities to use LAMP for diagnosing other infectious diseases, and in October 27, 2008, the two signed a development agreement for HAT.
More recently, FIND has partnered with the BPRG in Geneva and the Institute of Primate Research (IPR) in Nairobi to explore the feasibility of using LAMP as a test of cure, and for predicting relapses when treatment is not successful. After treatment is completed, patients are followed up for a period of 24 months to confirm that they have been cured. This exciting new project is likely to dramatically reduce the follow up period. It would also make clinical trials and drug discovery studies to be carried out in shorter periods. More
Issue 6 of FIND Newsletter, 2007 New strides towards a simple molecular test for diagnosis of sleeping sickness