The development of a multiplex recombinase polymerase amplification reaction to detect the most common causative agents of eumycetoma

Purpose: Mycetoma is a neglected tropical disease that affects the subcutaneous tissue. The disease can be caused by over 90 different pathogens, including both bacteria (actinomycetoma) and fungi (eumycetoma). While diagnostic tools for eumycetoma causative agents are available, these are generally not well suited for use in endemic regions. This study aims to develop an isothermal based multiplex recombinase polymerase amplification reaction (RPA), that can be integrated in the diagnostic workflow of endemic regions. Methods: The RPA was designed targeting the Internal Transcribed Spacer (ITS) region to detect the presence of fungal DNA, and to differentiate between Madurella mycetomatis and Falciformispora senegalensis. The performance of the RPA was evaluated using 71 fungal isolates and five actinomycetes reference isolates. Furthermore, the limit of detection (LOD) was determined for the different probes in singleplex and multiplex. Results: The ITS probe was positive for all 71 fungal isolates with a mean detection time of 13.1 min. The M. mycetomatis and F. senegalensis probes were only positive for their respective targets, with a mean detection time of 9.3 and 7.6 min, respectively. No cross-reactivity was detected, and a limit of detection of 0.01 ng of fungal DNA was found. The costs of the RPA ranged from €1.56 to €10.03, depending on the workflow. Conclusion: We developed a field-friendly multiplex RPA, that successfully detects fungal DNA and discriminates between M. mycetomatis and F. senegalensis. This tool holds promise for enhancing diagnostic capabilities in eumycetoma endemic regions, paving the way for improved patient management and treatment outcomes.