LAMP for Human African Trypanosomiasis: A Comparative Study of Detection Formats

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Abstract
Loop-mediated isothermal amplification (LAMP) is at the forefront of the search for innovative diagnostics for human African trypanosomiasis (HAT). Several simple endpoint detection methods have been developed for LAMP and here we compare four of these: (i) visualization of turbidity; (ii) addition of hydroxynaphthol blue before incubation; (iii) addition of calcein with MnCl2 before incubation and (iv) addition of Quant-iT PicoGreen after incubation. These four methods were applied to four LAMP assays for the detection of human African trypanosomiasis, including two Trypanozoon specific and two Trypanosoma brucei rhodesiense specific reactions using DNA extracted from cryo-preserved procyclic form T. b. rhodesiense. A multi-observer study was performed to assess inter-observer reliability of two of these methods: hydroxynapthol blue and calcein with MnCl2, using DNA prepared from blood samples stored on Whatman FTA cards. Results showed that hydroxynaphthol blue was the best of the compared methods for easy, inexpensive, accurate and reliable interpretation of LAMP assays for HAT. Hydroxynapthol blue generates a violet to sky blue colour change that was easy to see and was consistently interpreted by independent observers. Visible turbidity detection is not possible for all currently available HAT LAMP reactions; Quant-iT PicoGreen is expensive and addition of calcein with MnCl2 adversely affects reaction sensitivity and was unpopular with several observers. Human African trypanosomiasis (HAT) is a disease of the rural poor in sub-Saharan Africa where diagnostic laboratories are scarce and often ill equipped. Specific LAMP (loop-mediated isothermal amplification) tests for HAT have been developed and represent a significant step forward in the search for simple, sensitive and reliable diagnosis. Easy, accurate and reliable methods to read the results of these tests are critical and several simple methods have been developed. In this study, four methods were compared including three different colour change methods, and one in which the reaction turned from clear to cloudy. The hydroxynaphthol blue method involving a colour change, from violet to sky blue, was easy to see, the test is cheap to use and the results were largely agreed upon by 33 independent observers.

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