Direct 16S/ITS rRNA Gene PCR followed by Sanger Sequencing for Detection of Mycetoma causative Agents in Dakar, Senegal: A pilot study among patients with mycetoma attending Aristide Le Dantec University Hospital

Introduction. A mycetoma is defined as “any pathological process in which fungal or actinomycotic agents of exogenous origin produce grains”. A precise identification of the causal agents is critical for the therapeutic outcome. Thus, the objective of this study was to identify the pathogens of mycetoma using direct 16S/ITS rRNA gene polymerase chain reaction (PCR) followed by Sanger sequencing directly on grains. Materials and Methods. In sum, 32 samples including 15 black grains, 12 red grains, and five white/yellow grains collected from patients with mycetoma at the Aristide Le Dantec University Hospital in Dakar, Senegal, between October 2014 and September 2020 were submitted to PCR/sequencing in IHU Méditerranée Infection in Marseille, France. For black grain eumycetoma, the ITS rRNA region was targeted. Similarly, the 16S rRNA gene was targeted for red grain actinomycetoma. These two regions were targeted in parallel for white/yellow grains, which could be of either bacterial or fungal origin. The obtained sequences were assembled and searched using BLAST against the NCBI GenBank nucleotide database with DNA sequence-based species identification defined by ≥99% sequence similarity. Results. The age of the patients ranged from 14 to 72 years with a mean age of 36±14 years. Thirteen (86%) of the 15 samples with black grains, were successfully sequenced with only one established eumycetoma pathogen, M. mycetomatis identified in 11 (73%). Cladosporium sphaerospermum was identified in one sample. For the 16S rRNA sequencing about red grains, a 58.3% (7/12) success rate was obtained with Actinomadura pelletieri identified in six (06) samples. Among the five samples sequenced twice, the ITS rRNA sequencing success rate was 60% (3/5); and no mycetoma causative agent was identified. The 16S rRNA sequencing success rate was 40% (2/5) with the established actinomycetoma causative organism, Actinomadura madurae , identified in one. In the second, A. geliboluensis was identified. Conclusion. Overall, direct 16S/ITS rRNA sequencing on grains for the detection and identification of mycetoma pathogens was successful in 59.4% of cases. The success rate depended on the colour of the grains. It was 80% for black, 50% for red and 40% for white/yellow grains. Fungi, led by Madurella mycetomatis , were the predominant pathogens identified. We identified two probable new causal agents, namely Cladosporium sphaerospermum , and Actinomadura geliboluensis . Yet, the involvement of both deserves confirmation.