Rapid identification of black grain eumycetoma causative agents using rolling circle amplification.

Joseph M. Vinetz,Sarah A. Ahmed,Wendy W. J. van de Sande,Bert H. G. Gerrits van den Ende,G. S. de Hoog,Ahmed H. Fahal

doi:10.1371/journal.pntd.0003368

Abstract

Accurate identification of mycetoma causative agent is a priority for treatment. However, current identification tools are far from being satisfactory for both reliable diagnosis and epidemiological investigations. A rapid, simple, and highly efficient molecular based method for identification of agents of black grain eumycetoma is introduced, aiming to improve diagnostic in endemic areas. Rolling Circle Amplification (RCA) uses species-specific padlock probes and isothermal DNA amplification. The tests were based on ITS sequences and developed for Falciformispora senegalensis, F. tompkinsii, Madurella fahalii, M. mycetomatis, M. pseudomycetomatis, M. tropicana, Medicopsis romeroi, and Trematosphaeria grisea. With the isothermal RCA assay, 62 isolates were successfully identified with 100% specificity and no cross reactivity or false results. The main advantage of this technique is the low-cost, high specificity, and simplicity. In addition, it is highly reproducible and can be performed within a single day.

Highlights

Black grain eumycetoma represents the most common fungal mycetoma worldwide
We describe rolling circle amplification method for identification of black grain eumycetoma using species-specific padlock probes
Rolling circle amplification (RCA) provides a simple, reproducible, and costeffective method for rapid identification of mycetoma agent that can be used in low-resource clinical settings

Summary

Introduction

Black grain eumycetoma represents the most common fungal mycetoma worldwide This chronic, erosive infection of subcutaneous tissues affects the lower extremities and leads to severe disability [1]. A fully developed mycetoma lesion is identified clinically, whereas in early stages with the absence of grains, the infection may be confused with phaeomycosis or soft tissue tumors [1]. In such cases fine needle aspiration cytology or deep surgical biopsy for histological examination are useful [1,5]. There is a need for a fast, simple and reliable method for identification

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Conclusion