The development of a novel diagnostic PCR for Madurella mycetomatis using a comparative genome approach

Abstract

Eumycetoma is a neglected tropical disease most commonly caused by the fungus Madurella mycetomatis. Identification of eumycetoma causative agents can only be reliably performed by molecular identification, most commonly by species-specific PCR. The current M. mycetomatis specific PCR primers were recently discovered to cross-react with Madurella pseudomycetomatis. Here, we used a comparative genome approach to develop a new M. mycetomatis specific PCR for species identification. Predicted-protein coding sequences unique to M. mycetomatis were first identified in BLASTCLUST based on E-value, size and presence of orthologues. Primers were then developed for 16 unique sequences and evaluated against 60 M. mycetomatis isolates and other eumycetoma causing agents including the Madurella sibling species. Out of the 16, only one was found to be specific to M. mycetomatis. We have discovered a predicted-protein coding sequence unique to M. mycetomatis and have developed a new species-specific PCR to be used as a novel diagnostic marker for M. mycetomatis. Mycetoma is a neglected tropical disease characterised by tumorous swellings and grain formation. This disease can be caused by more than 70 different micro-organisms and is categorised into actinomycetoma (caused by bacteria) and eumycetoma (caused by fungi). The most common causative agent of mycetoma is the fungus Madurella mycetomatis. Diagnosis of eumycetoma is often only done clinically or by histopathological examination and culturing of the grains. Unfortunately, that often leads to misidentifications. Molecular identification is currently the most reliable method to identify the causative agents. However, we have recently discovered that the only M. mycetomatis species-specific PCR primers cross-reacts to Madurella pseudomycetomatis. Since all Madurella species cause eumycetoma and have different susceptibilities to antifungal agents, it is important to be able to accurately identify them to the species level. Here we have used a comparative genome approach to identify and design new M. mycetomatis species-specific PCR primers. These primers can be used to identify M. mycetomatis directly from grains and do not cross-react with any of the other eumycetoma causative agents tested. We, therefore, recommended the use of these primers in reference centres and local laboratories to identify M. mycetomatis to the species level.