Loop-mediated isothermal amplification (LAMP)
Recommended procedures for preparation of DNA template and performing the LAMP test
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The parasitologic tests in use for diagnosis of human African trypanosomiasis (HAT), have low sensitivity, and current serologic tests have inadequate specificity and require confirmation by other methods. Accurate detection of trypanosomal DNA from a patient’s blood, urine or saliva would be a significant improvement on parasitological examination. Loop-mediated isothermal amplification (LAMP) of DNA is a promising molecular technique that shows high sensitivity and specificity. The test amplifies target DNA at a constant temperature, meaning that it can be carried out with minimal equipment at low-level laboratories such as the ones in HAT endemic countries.
Positive samples are identified visually either through the formation of a white precipitate, a colour change or fluorescence (Figure 1). 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.
Feasibility studies carried out in a partnership between FIND, Murdoch and Obihiro Universities, as well as research institutes in endemic countries between 2007 and 2008, confirmed the great potential of the LAMP technology in the diagnosis of HAT. Sets of primers that are specific to the subgenus Trypanozoon (T.b. rhodesiense, T.b. gambiense, T. evansi, and T. equiperdum) were designed and tests optimized using DNA from various members of this subgenus. Primer sets were validated using samples from HAT patients, and reproducibility of the tests in laboratories based in the endemic regions was verified. The work gave sufficiently promising results to justify the need to develop a LAMP kit for diagnosis of HAT. See papers by Z.K. Njiru.
In October 2008, FIND and Eiken Chemical Company Ltd (Japan), the owner of the patent rights to LAMP, signed a development agreement for HAT. Prototype LAMP tests for HAT developed by Eiken were evaluated using blood samples from experimentally infected rodents at Makerere University in Uganda. This work included optimization of various methods of sample collection. The test can be performed on blood samples after they are freshly collected and when dried on microscopy slides or ordinary filter papers, and since the results are observed visually, no gel electrophoresis is required. The sensitivity of the test is greatly enhanced by initially lysing samples with SDS, and when blood is centrifuged and the buffy coat used in the LAMP reaction. The reagents for the LAMP kit developed by Eiken are dried down on the inside part of the cap of the reaction tube, which can be stored at room temperature for up to 12 months. When test samples are added, the tubes are heated at constant temperature in an incubator, and the results are read visually, using fluorescent light (Figure 2).
Development of the LAMP kit for HAT was completed by Eiken in July 2011, and the test launched at the ISCTRC conference in Bamako, Mali, in September 2011. Further optimization of sample preparation and clinical evaluation of the kit at multiple sites in the DRC and Uganda is at an advanced stage. Early results from clinical trials showed that while the LAMP method was highly sensitive and specific in diagnosing T.b. rhodesiense infections, detection of T.b. gambiense HAT at a sensitivity that is superior to that of routine parasitological methods would require additional work. Concentrating T.b. gambiense parasites by preparing a buffy coat prior to the LAMP reaction has been shown to improve sensitivity. This method is currently being evaluated in a field trial in the DRC.
At most of the sites where the LAMP method has been introduced, the whole procedure of preparing samples and performing LAMP is being carried out using solar energy, which is also used to power the equipment used to perform parasitology.
Whereas the LAMP test requires further evaluation before it can be considered as a confirmatory test to replace parasitological methods, it is already being introduced in a number of settings as a secondary screening tool to be used on serological suspects. Suspects who are found positive with the LAMP test, which can be performed on samples collected on filter paper, are referred for parasitology. Using this strategy, it is possible to only refer individuals who are strong HAT suspects, rather than all the individuals who are found positive with screening tools such as CATT or an RDT, which frequently result in false positives. This type of algorithm is currently being implemented [insert link to implementation strategies] in Uganda, Malawi, Guinea, Chad, South Sudan, Nigeria and DRC, with very encouraging results.
FIND and the Institute of Primate Research (IPR) in Nairobi, Makerere University in Uganda, the Swiss Tropical and Public Health Institute (Swiss TPH) in Switzerland, and the Institute of Neuroepidemiology and Tropical Neurology (INT) in France, have also been exploring 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 normally followed up for a period of 24 months to confirm that they have been cured. This exciting project is likely to dramatically reduce the follow up period, and could eliminate the need for a lumbar puncture. It would also make clinical trials and drug discovery studies to be carried out in shorter periods. Read more
Towards point-of-care diagnostic and staging tools for human African trypanosomiasis , Matovu E et al, Journal of Tropical Medicine, Volume 2012 (2012), Article ID 340538, 2012
Third World Biotech: Blood Test for African Sleeping Sickness, Aaron Rowe, WIRED