A qPCR identification scheme to detect the most common causative agents of actinomycetoma in Africa

Mycetoma is a neglected tropical disease characterized by mutilating tumorous lesions in the subcutaneous tissue. The causative agents are found embedded in granules called grains. Mycetoma is either caused by bacteria (actinomycetoma) or fungi (eumycetoma). To initiate the appropriate treatment, it is important to identify the causative agent rapidly and molecular identification for eumycetoma revolutionized the time to identification. For actinomycetoma this was not possible yet. Here we developed a multiplex qPCR identification scheme for the most common causative agents of actinomycetoma in Africa. Whole genome sequencing was used to identify species-specific gene families for Actinomadura madurae, Actinomadura pelletieri, Streptomyces somaliensis and Streptomyces sudanensis. qPCR primers and probes were developed on these species and validated against DNA isolated from mycetoma strains and grains. Each probe was unique with no cross-reactivity with other tested species. The limit of detection ranged from 0.000013 to 0.00067 ng bacterial DNA. When the qPCRs were validated against 28 grain samples, all fungal grains remained negative and 11 out of 12 Actinomadura grains were correctly identified. This resulted in a sensitivity of 85.7% for the A. pelletieri probe and a specificity of 100%. For the A. madurae probe, a sensitivity and specificity of 100% was obtained. The actinomycetoma qPCR developed in this study can be used to identify the most common causative agents of actinomycetoma in Africa.