Environmental occurrence of Madurella mycetomatis, the major agent of human eumycetoma in Sudan

Abstract

Madurella mycetomatis is the main causative agent of human eumycetoma, a severe debilitating disease endemic in Sudan. It has been suggested that eumycetoma has a soil-borne or thorn prick-mediated origin. For this reason, efforts were undertaken to culture M. mycetomatis from soil samples (n = 43) and thorn collections (n = 35) derived from areas in which it is endemic. However, ribosomal sequencing data revealed that the black fungi obtained all belonged to other fungal species. In addition, we performed PCR-mediated detection followed by restriction fragment length polymorphism (RFLP) analysis for the identification of M. mycetomatis DNA from the environmental samples as well as biopsies from patients with mycetoma. In the case of the Sudanese soil samples, 17 out of 74 (23%) samples were positive for M. mycetomatis DNA. Among the thorn collections, 1 out of 22 (5%) was positive in the PCR. All PCR RFLP patterns clearly indicated the presence of M. mycetomatis. In contrast, 15 Dutch and English control soil samples were all negative. Clinically and environmentally obtained fungal PCR products share the same PCR RFLP patterns, suggesting identity, at least at the species level. These observations support the hypothesis that eumycetoma is primarily environmentally acquired and suggest that M. mycetomatis needs special conditions for growth, as direct isolation from the environment seems to be impossible.

FIG. 1.

Detailed map of the region in Sudan where the thorns were collected and the soil was sampled. Main sampling sites were in the encircled region comprising the region southeastern to Khartoum (Wad Madani and Sinnar). Other regions where mycetoma is highly endemic are highlighted by the elliptic red-dot regions. The red bars indicate the boundaries of the Sudanese mycetoma belt.

FIG. 2.

Molecular detection of M. mycetomatis in soils and thorn samples. (A) Results of the broad-spectrum ITS PCR employing primers ITS4 and ITS5. Note that amplicons can be observed in all lanes. (B) Result of the nested PCR using M. mycetomatis-specific primers. Lanes 1 to 6, amplicons derived from Dutch control samples: lanes 7 to 14, amplified material from Sudanese samples; lane 15, results obtained with purified M. mycetomatis DNA; lanes 16 and 17, negative controls; lanes M, 100-bp molecule sizing ladder, and the 600-bp fragment is indicated (arrow).

FIG. 3.

Molecular identification of M. mycetomatis from the environment and clinical biopsies. Shown are digests for the specific 26.1A/28.3A PCR products obtained for two clinical isolates (lanes 2 and 3, 5 and 6, and 8 and 9) and a single environmental isolate (lanes 1, 4, and 7). Note that per restriction digest, the patterns are identical. On the left (lane M), the 10-bp molecule sizing ladder is shown, and the 100- and 1,000-bp markers are indicated (arrow).