Objectives: 
Mycetoma is a poverty associated neglected tropical disease mainly affecting the foot. It is a chronic granulomatous infection that is characterised by grain formation. There are more than 70 different causative agents of mycetoma but Madurella mycetomatis is the most common. Currently, histology and/or culturing are used to identify these causative agents to the species level. However, misidentifications are common due to the lack of specific phenotypical characteristics. The only reliable method to identify these species is using molecular identification techniques such as sequencing or species-specific PCRs on isolates. However, in some laboratories, only histology is used to establish the nature of the causative agent. This may result in many misidentifications. Therefore, our aim is to develop a molecular identification technique which could be directly applied on histological sections. Here we report on the development of a specific in situ hybridization (ISH) method for staining mycetoma grains.

Methods: 
A probe specific for Madurella species was designed from the Large Subunit (LSU) region. Specificity of the probe was checked in silico with the database of Westerdijk Fungal Diversity Institute, Utrecht, The Netherlands using Bionumerics software (Applied-Maths). This probe was used in ISH staining performed on slides containing Madurella mycetomatis grains and isolates. The ISH method used in this study was optimised by comparing between different pre-treatment methods to obtain optimal staining range and intensity.

Results: Formalin fixation has a negative impact on ISH staining as staining can only be observed in samples fixed in formalin for a short period. Permeabilisation of the grains was crucial and it appeared to be most successful when slides were pre-treated with antigen retrieval. Grains stained with both the pan-fungal and Madurella specific probe. Staining was most intense on the rim of the grain, the center of the grains were not stained. 

Conclusion: 
This is the first study suggesting ISH method can be used to diagnose mycetoma. Antigen retrieval pre-treatment method is able to increase ISH staining intensity and range.

In Situ Hybridization for Madurella mycetomatis using Madurella specific probe