The development of a novel diagnostic PCR forMadurella mycetomatisusing a comparative genome approach

Abstract

14 15 16 17 18 19 Corresponding author 20 Wendy WJ van de Sande 21 w.vandesande@erasmusmc.nl 22 23 Keyword: Mycetoma, Madurella mycetomatis, Comparative genome, Neglected tropical disease 24 25 26 ABSTRACT: 27 28 Eumycetoma is a neglected tropical disease characterized by large tumorous lesions. It is most 29 commonly caused by the fungus Madurella mycetomatis which accounts for more than 70% of cases 30 in central Africa. Currently, identification of the causative agent can only be reliably performed by a 31 species-specific PCR. However, we recently demonstrated that our M. mycetomatis specific PCR can 32 cross-react with Madurella pseudomycetomatis. We therefore used a comparative genome approach 33 to develop a new M. mycetomatis specific PCR for species identification. For this we compared the 34 published M. mycetomatis genome to genomes of other organisms in BLASTCLUST to identify unique 35 M. mycetomatis predicted protein coding sequences. Based on 16 of these unique sequences, PCR36 primers were developed. The specificity of these primers was further evaluated in other 37 eumycetoma causing agents including the Madurella sibling species. Out of the 16 tested sequences, 38 only one was unique for M. mycetomatis and this should be used as a novel diagnostic marker for M.39 mycetomatis. 40 41 42 43 INTRODUCTION: 44 45The neglected tropical disease mycetoma presents itself as a subcutaneous chronic granulomatous 46 infectious and inflammatory disease and is characterized by tumorous lesions (1, 2). This disease can 47 be caused by more than 70 different micro-organisms and is categorized into actinomycetoma 48 (caused by bacteria) and eumycetoma (caused by fungi). Most cases occur in the mycetoma belt 49 between the latitudes 15° South and 30° North. Diagnosis of eumycetoma is often only made 50 clinically in endemic areas due to the scarcity of facilities, expertise and financial capacity. 51Identification of the causative agent is time consuming and often limited to culture and histology 52 which can leads to misidentifications (1, 3, 4). The only way to properly identify eumycetoma 53 causative agents to the species level is through molecular identification, commonly PCR. PCR 54 identification's reliability, high turnover and sensitivity has made it widely used in diagnosing fungal 55 disease, and in most cases, it has already replaced culturing of the microorganism as the primary 56 diagnostic method (5-11). 58Madurella mycetomatis is a fungus only recognised as a pathogen in mycetoma and is responsible for 59 more than 70% of all mycetoma infections in endemic areas (1, 12, 13). In 1999, specific PCR primers 60 based on the internal transcribed spacer (ITS) region were developed for M. mycetomatis by our 61 group (14), however, recently, this M. mycetomatis specific PCR primer pair was discovered to cross-62 react with Madurella pseudomycetomatis (15). Back then, M. pseudomycetomatis was not yet 63 discovered (16). Since new fungi causing eumycetoma are still being discovered, there is clearly a 64 need...