Madurella real-time PCR, a novel approach for eumycetoma diagnosis

The genus Madurella comprising four species, M. fahalii, M. mycetomatis, M. pseudomycetomatis, and M. tropicana, represents the prevalent cause of eumycetoma worldwide. The four species are phenotypically similar and cause an invariable clinical picture, but differ markedly in their susceptibility to antifungal drugs, and epidemiological pattern. Therefore, specific identification is required for optimal management of Madurella infection and to reveal proper epidemiology of the species. In this study, a novel multiplex real-time PCR targeting the four Madurella species was developed and standardized. Evaluation of the assay using reference strains of the target and non-target species resulted in 100% specificity, high analytical reproducibility (R2 values >0.99) and a lowest detection limit of 3 pg target DNA. The accuracy of the real-time PCR was further assessed using biopsies from eumycetoma suspected patients. Unlike culture and DNA sequencing as gold standard diagnostic methods, the real-time PCR yielded accurate diagnosis with specific identification of the causative species in three hours compared to one or two weeks required for culture. The novel method reduces turnaround time as well as labor intensity and high costs associated with current reference methods.
Author summary Mycetoma, a progressive and disfiguring disease, is one of the neglected tropical diseases, caused by both bacteria and fungi. Eumycetoma is the fungal type and mainly caused by species of the genus Madurella. Madurella mycetomatis is the most prevalent species worldwide. However, other species such as M. fahalii, M. pseudomycetomatis, and M. tropicana can also cause mycetoma and have a different susceptibility towards the drug used for treating mycetoma patients. Currently, we lack a rapid and non-culture-based technique that can readily identify these four species from clinical samples. Due to its sensitivity, and specificity, real-time PCR is re-recognized by European Organization for Research and Treatment of Cancer (EORTC) to directly identify fungal agents from clinical samples. We developed and validated a multiplex real-time PCR-based technique using the least expensive chemistry to identify Madurella species within 3–4 hours. Development of such a technique will allow rapid diagnosis of eumycetoma and timely initiation of appropriate antifungal therapy.