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

Sally L Wastling,Abbas S L Kakembo,Susan C Welburn,Kim Picozzi,Daniel K Masiga

doi:10.1371/journal.pntd.0000865

Sally L Wastling, Abbas S L Kakembo + Show 3 more

Open Access

https://doi.org/10.1371/journal.pntd.0000865

Copy DOI

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.

Highlights

Loop-mediated isothermal amplification (LAMP) [1] is a DNA amplification technique whose advantages over traditional PCR have put it at the forefront of the search for innovative new diagnostics for infectious diseases such as human African trypanosomiasis (HAT) [2]
Since the performance of a subjective diagnostic method depends on sample variation in readers, as well as cases [13], we performed a multi-observer study to investigate the reliability of the two metal ion indicator methods
Turbidity Positive LAMP PfrA reactions were detectable under visible turbidity up to and including a 161024 dilution of the T. b. rhodesiense DNA

Summary

Introduction

Loop-mediated isothermal amplification (LAMP) [1] is a DNA amplification technique whose advantages over traditional PCR have put it at the forefront of the search for innovative new diagnostics for infectious diseases such as human African trypanosomiasis (HAT) [2]. Rapid and unambiguous visual discrimination of test results is essential for diagnostics and several simple endpoint detection methods have been developed for the LAMP method to allow visual discrimination of positive samples. These methods vary in cost and technological details. Gambiense which targets the 5.8S rRNA region [5]. This is controversial since the only widely accepted marker for specific identification of T. b. Rhodesiense specific LAMP assays which targets the serum resistance associated (SRA) gene, has been developed [8]. In addition a second LAMP assay for the SRA gene has been developed in our laboratory (Wastling and Picozzi, unpublished observations)

Methods

Results

Discussion

Conclusion