Loop-mediated isothermal amplification (LAMP)
Recommended procedures of DNA extraction
HAT LAMP SOPs: www.finddiagnostics.org/programs/hat/lamp/
sample_preparation/
For more information on the HAT LAMP project, please write to: hatlamp@finddiagnostics.org
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 and require confirmation by other methods. 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 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.
Furthermore, 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 that the LAMP technology has great potential for use 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.
Figure 1: Positive samples emit green fluorescence. PC: positive control, NC: negative control (Photo courtesy of Dr. Enock Matovu) |
Figure 2: The LoopampTM LF-160 incubator used to perform the HAT LAMP reaction. After extraction of DNA, the LAMP reaction is carried out in the reaction block (A), which is calibrated to maintain a constant temperature of 65°C, and to stop the reaction in 40 minutes. The results are visualized under LED light (B). |
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.
A technician displays the results of the LAMP test for HAT at Dipumba hospital in the ongoing evaluation studies. (click to enlarge) |
Performing the LAMP test for HAT at Katanda Hospital in East Kasaï province of the DRC during ongong evaluation studies. All equipment for preparing samples and performing LAMP reaction are operating using solar power. (click to enlarge) |
A technician at Bangumi Hospital applies blood on a filter paper from a person suspected of having HAT. After the sample dries, it is put in a plastic pouch and sent by motorcycle to the Bandundu LAMP centre, about 35km away, where the test is performed. (click to enlarge) |
Solar panels installed on the roof of Bangumi hosptital in North Bandundu province of the DRC. The power generated is used to operate equipment used for sample preparation in the ongoing evaluation of the LAMP kit for HAT. (click to enlarge) |
At some of the sites in the DRC, the whole procedure of preparing samples and performing LAMP is being carried out using solar power.
So far, we have generated enough evidence to confirm that the test is easy to perform in a basic laboratory, by staff with no training in molecular biology, and its performance is better than any test for HAT that is in clinical use.
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, are 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