Genotyping of Madurella mycetomatis by Selective Amplification of Restriction Fragments (Amplified Fragment Length Polymorphism) and Subtype Correlation with Geographical Origin and Lesion Size

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Abstract
One of the causative organisms of mycetoma is the fungus Madurella mycetomatis . Previously, extensive molecular typing studies identified Sudanese isolates of this fungus as clonal, but polymorphic genetic markers have not yet been identified. Here, we report on the selective amplification of restriction fragment (AFLP) analysis of 37 Sudanese clinical isolates of M. mycetomatis . Of 93 AFLP fragments generated, 25 were polymorphic, and 12 of these 25 polymorphic fragments were found in a large fraction of the strains. Comparative analysis resulted into a tree, composed of two main (clusters I and II) and one minor cluster (cluster III). Seventy-five percent of the strains found in cluster I originated from central Sudan, while the origin of the strains in cluster II was more heterogeneous. Furthermore, the strains found in cluster I were generally obtained from lesions larger than those from which the strains found in cluster II were obtained (chi-square test for trend, P = 0.03). Among the 12 more commonly found polymorphisms, 4 showed sequence homology with known genes. Marker A7 was homologous to an endo-1,4-beta-glucanase from Aspergillus oryzae , 97% identical markers A12 and B3 matched a hypothetical protein from Gibberella zeae , and marker B4 was homologous to casein kinase I from Danio rerio . The last marker seemed to be associated with strains originating from central Sudan ( P = 0.001). This is the first report on a genotypic study where genetic markers which may be used to study pathogenicity in M. mycetomatis were obtained.