Metagenomics of Black Grains: New Highlights in the Understanding Eumycetoma

Background: Eumycetoma is a debilitating chronic subcutaneous granulomatous disease, endemic in Sudan and many other tropical and subtropical countries. It can be caused by eight different fungal orders and Madurella mycetomatis as the main causative species. The gold standard diagnostic test is culture, however culture-independent methods as imaging, histopathological and molecular techniques can support diagnosis, especially in case of negative cultures. Methods: Amplicon-based Internal Transcribed Spacer 2 (ITS2) metagenomic technique was used to study black grains isolated from 14 tissue biopsies from patients with mycetoma. Furthermore, grains mycological culture and surgical biopsies histopathological examination were done. Results: M. mycetomatis (n=5) and Falciformispora spp. (n=4) organisms were identified by culture and confirmed by metagenomics. Metagenomics recognised, at the species level, Falciformispora as F. tompkinsii (n=3) and F. senegalensis (n=1), while in culture-negative cases (n=5), M. mycetomatis (n=3), F. senegalensis (n=1) and Fusarium spp. (n=1) were identified. Interestingly, the metagenomics results showed a “consortium” of different fungi in each sample, mainly Ascomycota phylum, including various species associated with eumycetoma. The microbial co-occurrence in eumycetoma showed the co-presence of Madurella with Trichoderma, Chaetomium, Malasseziales and Sordariales spp. While, Falciformispora was co-presented with Inocybe and Alternaria, and mutual exclusion with Subramaniula, Aspergillus and Trichothecium. Interpretation: Metagenomics had provided new insight into the aetiology of eumycetoma in samples with negative culture. Furthermore, into diversity and complexity of grains mycobiota, and it questioned the accuracy of traditional culture for the identification of causative agents. Funding Information: This study was supported by the Italian Agency for Development 62 Cooperation (AICS) (Grants AID10821 and AID10861). Declaration of Interests: The research work of Dr. Aleksandra Barac was supported by the Ministry of Education, Science and Technology, the Republic of Serbia (No. III45005). Ethical Approval Statement: The study was approved by the Mycetoma Research Center IRB, No. 12/2018. Informed consent was obtained from each adult patient, while an assent was taken from minors (age less than 18 years) with approval of their guardian.