Detection of Mycobacterium ulcerans by the Loop Mediated Isothermal Amplification Method

Anthony Ablordey,Isaac Frimpong Aboagye,Diana Ackon Amissah,Harutaka Katano,Koichi Ishikawa,Ben Hatano,Tetsutaro Sata,Toshio Yamazaki,Christian Johnson

doi:10.1371/journal.pntd.0001590

Abstract

BackgroundBuruli ulcer (BU) caused by Mycobacterium ulcerans (M. ulcerans) has emerged as an important public health problem in several rural communities in sub-Saharan Africa. Early diagnosis and prompt treatment are important in preventing disfiguring complications associated with late stages of the disease progression. Presently there is no simple and rapid test that is appropriate for early diagnosis and use in the low-resource settings where M. ulcerans is most prevalent.MethodologyWe compared conventional and pocket warmer loop mediated isothermal amplification (LAMP) methods (using a heat block and a pocket warmer respectively as heat source for amplification reaction) for the detection of M. ulcerans in clinical specimens. The effect of purified and crude DNA preparations on the detection rate of the LAMP assays were also investigated and compared with that of IS2404 PCR, a reference assay for the detection of M. ulcerans. Thirty clinical specimens from suspected BU cases were examined by LAMP and IS2404 PCR.Principal FindingsThe lower detection limit of both LAMP methods at 60°C was 300 copies of IS2404 and 30 copies of IS2404 for the conventional LAMP at 65°C. When purified DNA extracts were used, both the conventional LAMP and IS2404 PCR concordantly detected 21 positive cases, while the pocket warmer LAMP detected 19 cases. Nine of 30 samples were positive by both the LAMP assays as well as IS2404 PCR when crude extracts of clinical specimens were used.Conclusion/SignificanceThe LAMP method can be used as a simple and rapid test for the detection of M. ulcerans in clinical specimens. However, obtaining purified DNA, as well as generating isothermal conditions, remains a major challenge for the use of the LAMP method under field conditions. With further improvement in DNA extraction and amplification conditions, the pwLAMP could be used as a point of care diagnostic test for BU

Highlights

Buruli ulcer (BU) caused by Mycobacterium ulcerans (M. ulcerans) is a necrotizing skin disease endemic mostly in rural wetland of tropical countries of Africa, America, Asia and Australia
In order to develop a simple and rapid test that can be used to diagnose Buruli ulcer under field conditions, we modified the conventional loop mediated isothermal amplification (LAMP) assay by using a disposable pocket warmer as a heating device for generating a constant temperature for the test reaction and employed the use of crude sample preparations consisting of boiled and unboiled extracts of the clinical specimen instead of using purified DNA as the diagnostic specimen
Thirty clinical specimens from suspected Buruli ulcer patients were investigated by the modified LAMP and the conventional LAMP, as well as IS2404 PCR, a reference method for the detection of Mycobacterium ulcerans

Summary

Introduction

Buruli ulcer (BU) caused by Mycobacterium ulcerans (M. ulcerans) is a necrotizing skin disease endemic mostly in rural wetland of tropical countries of Africa, America, Asia and Australia. Available data reveals an increase in BU incidence over the last several years in the west African countries of Ivory Coast, Ghana and Benin. In these countries BU has replaced leprosy as the second most prevalent mycobacterial disease [1], [3,4,5]. Buruli ulcer (BU) caused by Mycobacterium ulcerans (M. ulcerans) has emerged as an important public health problem in several rural communities in sub-Saharan Africa. There is no simple and rapid test that is appropriate for early diagnosis and use in the low-resource settings where M. ulcerans is most prevalent

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