Sandalwood Oils of Different Origins Are Active In Vitro against Madurella mycetomatis, the Major Fungal Pathogen Responsible for Eumycetoma.

In the search for new bioactive agents against the infectious pathogen responsible for the neglected tropical disease (NTD) mycetoma, we tested a collection of 27 essential oils (EOs) in vitro against Madurella mycetomatis, the primary pathogen responsible for the fungal form of mycetoma, termed eumycetoma. Among this series, the EO of Santalum album (Santalaceae), i.e., East Indian sandalwood oil, stood out prominently with the most potent inhibition in vitro. We, therefore, directed our research toward 15 EOs of Santalum species of different geographical origins, along with two samples of EOs from other plant species often commercialized as "sandalwood oils". Most of these EOs displayed similar strong activity against M. mycetomatis in vitro. All tested oils were thoroughly analyzed by GC-QTOF MS and most of their constituents were identified. Separation of the sandalwood oil into the fractions of sesquiterpene hydrocarbons and alcohols showed that its activity is associated with the sesquiterpene alcohols. The major constituents, the sesquiterpene alcohols (Z)-α- and (Z)-ß-santalol were isolated from the S. album oil by column chromatography on AgNO3-coated silica. They were tested as isolated compounds against the fungus, and (Z)-α-santalol was about two times more active than the ß-isomer.

Naphthylisoquinoline alkaloids: novel agents against the causative pathogens of eumycetoma and actinomycetoma-en route to broad-spectrum antimycetomal drugs.

Mycetoma is a devastating neglected tropical infection of the subcutaneous tissues. It is caused by fungal and bacterial pathogens recognized as eumycetoma and actinomycetoma, respectively. Mycetoma treatment involves diagnosing the causative microorganism as a prerequisite to prescribing a proper medication. Current therapy of fungal eumycetoma causative agents, such as Madurella mycetomatis, consists of long-term antifungal medication with itraconazole followed by surgery, yet with usually unsatisfactory clinical outcomes. Actinomycetoma, on the contrary, usually responds to treatment with co-trimoxazole and amikacin. Therefore, there is a pressing need to discover novel broad-spectrum antimicrobial agents to circumvent the time-consuming and costly diagnosis. Using the resazurin assay, a series of 23 naphthylisoquinoline (NIQ) alkaloids and related naphthoquinones were subjected to in vitro screening against two fungal strains of M. mycetomatis and three bacterial strains of Actinomadura madurae and A. syzygii. Seven NIQs, mostly dimers, showed promising in vitro activities against at least one strain of the mycetoma-causative pathogens, while the naphthoquinones did not show any activity. A synthetic NIQ dimer, 8,8'''-O,O-dimethylmichellamine A (18), inhibited all tested fungal and bacterial strains (IC50 = 2.81-12.07 µg/mL). One of the dimeric NIQs, michellamine B (14), inhibited a strain of M. mycetomatis and significantly enhanced the survival rate of Galleria mellonella larvae infected with M. mycetomatis at concentrations of 1 and 4 µg/mL, without being toxic to the uninfected larvae. As a result, broad-spectrum dimeric NIQs like 14 and 18 with antimicrobial activity are considered hit compounds that could be worth further optimization to develop novel lead antimycetomal agents.

Complete Genome Sequence of the Itraconazole Decreased Susceptible Madurella fahalii Type-Strain CBS 129176.

Madurella fahalii is a causative agent of the implantation mycosis mycetoma with decreased susceptibility to itraconazole, the preferred therapeutic drug to combat mycetoma. Here, we report the M. fahalii type-strain CBS 129176 genome assembly and annotation to identify a glutamic acid insert near the azole-binding pocket in the Cyp51A protein.

Using (1,3)-ß-D-glucan concentrations in serum to monitor the response of azole therapy in patients with eumycetoma caused by Madurella mycetomatis.

INTRODUCTION: (1,3)-ß-D-glucan is a panfungal biomarker secreted by many fungi, including Madurella mycetomatis, the main causative agent of eumycetoma. Previously we demonstrated that (1,3)-ß-D-glucan was present in serum of patients with eumycetoma. However, the use of (1,3)-ß-D-glucan to monitor treatment responses in patients with eumycetoma has not been evaluated. MATERIALS AND METHODS: In this study, we measured (1,3)-ß-D-glucan concentrations in serum with the WAKO (1,3)-ß-D-glucan assay in 104 patients with eumycetoma treated with either 400 mg itraconazole daily, or 200 mg or 300 mg fosravuconazole weekly. Serial serum (1,3)-ß-D-glucan concentrations were measured at seven different timepoints. Any correlation between initial and final (1,3)-ß-D-glucan concentrations and clinical outcome was evaluated. RESULTS: The concentration of (1,3)-ß-D-glucan was obtained in a total of 654 serum samples. Before treatment, the average (1,3)-ß-D-glucan concentration was 22.86 pg/mL. During the first 6 months of treatment, this concentration remained stable. (1,3)-ß-D-glucan concentrations significantly dropped after surgery to 8.56 pg/mL. After treatment was stopped, there was clinical evidence of recurrence in 18 patients. Seven of these 18 patients had a (1,3)-ß-D-glucan concentration above the 5.5 pg/mL cut-off value for positivity, while in the remaining 11 patients, (1,3)-ß-D-glucan concentrations were below the cut-off value. This resulted in a sensitivity of 38.9% and specificity of 75.0%. A correlation between lesion size and (1,3)-ß-D-glucan concentration was noted. CONCLUSION: Although in general (1,3)-ß-D-glucan concentrations can be measured in the serum of patients with eumycetoma during treatment, a sharp decrease in ß-glucan concentration was only noted after surgery and not during or after antimicrobial treatment. (1,3)-ß-D-glucan concentrations were not predictive for recurrence and seem to have no value in determining treatment response to azoles in patients with eumycetoma.

A Novel Report of mycetoma with Spinal Spread due to Madurella fahalli from India.

Eumycetoma caused by Madurella fahalii, a drug-resistant fungus, has never been reported in India. Here, we describe a fatal case of eumycetoma with spinal involvement due to M. fahalii for the first time in India.

Fungal mycetoma and pregnancy: An association with costly and difficult management, about a case.

Mycetomas are endemic diseases in tropical and sub-tropical countries of Africa, Asia and America, mainly affecting rural populations living below the poverty line. We report a particular case of a mycetoma associated with pregnancy whose evolution was good, but at the cost of significant financial expenses. This was a 39-year-old woman who developed a fungal mycetoma due to Madurella mycetomatis from the ingunocrural region. The patient had to develop several previous pregnancies on this site of mycetoma, the outcome of which was favorable. The last pregnancy was accompanied by an aggravation of the mycetoma in the form of polyfistulized inflammatory swelling of the right inguino-crural region emitting black grains. Magnetic Resonance Imaging (MRI) of the region showed invasion of the adductor muscles at the level of the root of the thigh on its antero-internal side with no sign of pelvic extension or underlying bone lesion. The patient was treated by surgery associated with antifungal treatment. The evolution was favorable for pregnancy and mycetoma.

The combination of manogepix and itraconazole is synergistic and inhibits the growth of Madurella mycetomatis in vitro but not in vivo.

Mycetoma is a neglected tropical disease commonly caused by the fungus Madurella mycetomatis. Standard treatment consists of extensive treatment with itraconazole in combination with surgical excision of the infected tissue, but has a low success rate. To improve treatment outcomes, novel treatment strategies are needed. Here, we determined the potential of manogepix, a novel antifungal agent that targets the GPI-anchor biosynthesis pathway by inhibition of the GWT1 enzyme. Manogepix was evaluated by determining the minimal inhibitory concentrations (MICs) according to the CLSI-based in vitro susceptibility assay for 22 M. mycetomatis strains and by in silico protein comparison of the target protein. The synergy between manogepix and itraconazole was determined using a checkerboard assay. The efficacy of clinically relevant dosages was assessed in an in vivo grain model in Galleria mellonella larvae. MICs for manogepix ranged from <0.008 to >8 mg/l and 16/22 M. mycetomatis strains had an MIC ≥4 mg/ml. Differences in MICs were not related to differences observed in the GWT1 protein sequence. For 70% of the tested isolates, synergism was found between manogepix and itraconazole in vitro. In vivo, enhanced survival was not observed upon admission of 8.6 mg/kg manogepix, nor in combination treatment with 5.7 mg/kg itraconazole. MICs of manogepix were high, but the in vitro antifungal activity of itraconazole was enhanced in combination therapy. However, no efficacy of manogepix was found in an in vivo grain model using clinically relevant dosages. Therefore, the therapeutic potential of manogepix in mycetoma caused by M. mycetomatis seems limited.

Treatment of Madurella mycetomatis-caused mycetoma consists of extensive exposure to antifungals and surgery. To improve therapy, we evaluated manogepix, a novel antifungal agent, as a therapeutic option against M. mycetomatis. Our findings suggest limited therapeutic potential for manogepix.

Specific and sensitive loop-mediated isothermal amplification (LAMP) method for Madurella strains, eumycetoma filamentous fungi causative agent.

BACKGROUND: Filamentous fungi of the genus Madurella are the primary causative agents of mycetoma, a disease observed in tropical and subtropical regions. Since early diagnostics based on a morphological approach are difficult and have many shortcomings, a molecular diagnostic method suitable for rural settings is required. In this study, we developed the loop-mediated isothermal amplification (LAMP) method to present a foundational technique of the diagnosis of Madurella spp. (M. mycetomatis, M. pseudomycetomatis, M. tropicana, and M. fahalii), the common causative organisms of eumycetoma. PRINCIPAL FINDINGS: We successfully designed a primer pair targeting the rDNAs of three Madurella spp. excluding M. fahalii, and detected up to 100 fg of genomic DNA extracted from isolates of M. mycetomatis and 1 pg of M. pseudomycetomatis and M. tropicana, within one hour. Second, a primer pair specific to M. mycetomatis, the most common causative species, or M. fahalii, a drug-resistant species, was constructed, and the detection limit of both primer pairs was 1 pg. The designed primers accurately distinguished 16 strains of the genus Madurella from various fungal species known to cause mycetomas. CONCLUSION: In summary, we established the first model of a LAMP detection method that rapidly and sensitively detects and identifies Madurella isolates for clinical diagnostics. Moreover, the combined designed primer sets could identify mycetoma-causing strains simultaneously.

First report on mycetoma in Turkana County-North-western Kenya.

Mycetoma is one of the six Neglected Tropical Diseases that are prevalent in Turkana County (northwest Kenya). The aim of the study was to estimate the prevalence of mycetoma in the county, as well as to describe the main causative agents involved in the disease using methods affordable locally. Based on the data collected by the team of cooperative medicine Cirugia en Turkana (Surgery in Turkana), a specific study for mycetoma was started during the 16th humanitarian medicine campaign in February 2019. Patients with suspected mycetoma were studied at the Lodwar County Referral Hospital (LCRH). After informing the patient and getting their consent, the lesions were examined and sampled (mainly by biopsy) and clinical data were recorded. Samples were washed in sterile saline solution and cut in fragments. Some of these were inoculated on Sabouraud Dextrose Agar, Malt Extract Agar, and diluted Nutrient Agar plates. One fragment of each sample was used for DNA extraction. The DNA and the rest of the fragments of samples were kept at -20°C. All cultures were incubated at room temperature at the LCRH laboratory. The DNA obtained from clinical samples was submitted to PCR amplification of the ITS-5.8S and the V4-V5 16S rRNA gene region, for the detection and identification of fungi and bacteria respectively. From February 2019 till February 2022, 60 patients were studied. Most of them were men (43, 74,1%) between 13 and 78 y.o. (mean age 37). Half of the patients were herdsmen but, among women 40% (6) were housewives and 26.7% (4) charcoal burners. Lesions were mainly located at the feet (87.9%) and most of the patients (54; 93.1%) reported discharge of grains in the exudate, being 27 (46.6%) yellow or pale colored and 19 (32.8%) of them dark grains. Culture of clinical samples yielded 35 fungal and bacterial putative causative agents. Culture and molecular methods allowed the identification of a total of 21 causative agents of mycetoma (39.6% of cases studied). Most of them (17) corresponded to fungi causing eumycetoma (80.9%) being the most prevalent the genus Madurella (7; 41.2%), with two species involved (M. mycetomatis and M. fahalii), followed by Aspergillus (2; 11.8%). Other minority genera detected were Cladosporium, Fusarium, Acremonium, Penicillium, and Trichophyton (5.9% each of them). Actinobacteria were detected in 19.1% of samples, but only Streptomyces somaliensis was identified as a known agent of mycetoma, the rest being actinobacteria not previously described as causative agents of the disease, such as Cellulosimicrobium cellulans detected in two of the patients. Although Kenya is geographically located in the mycetoma belt, to our knowledge this is the first report on mycetoma in this country from 1973, and the first one for Turkana County.

Antifungal Activity of Natural Naphthoquinones and Anthraquinones against Madurella mycetomatis.

Eumycetoma, the fungal form of the neglected tropical disease mycetoma, is a crippling infectious disease with low response rates to currently available antifungal drugs. In this study, a series of natural naphthoquinones and anthraquinones was evaluated for their activity against Madurella mycetomatis, which is the most common causative agent of eumycetoma. The metabolic activity of Madurella mycetomatis as well as the viability of Galleria mellonella larvae upon treatment with quinones was investigated. Several hydroxy-substituted naphthoquinones exhibited activity against Madurella mycetomatis. In particular, naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) was identified as a considerably active antifungal compound against Madurella mycetomatis (IC50 =1.4 µM), while it showed reduced toxicity to Galleria mellonella larvae, which is a well-established in vivo invertebrate model for mycetoma drug studies.

Using a Madurella mycetomatis-specific PCR on grains obtained via non-invasive fine-needle aspirated material is more accurate than cytology.

BACKGROUND: Eumycetoma is a chronic subcutaneous inflammatory fungal infection most often caused by the fungus Madurella mycetomatis. Using a species-specific PCR on DNA directly isolated from grains is currently the most reliable method for species identification. However, so far, PCR has been performed on grains obtained through deep-seated surgical biopsies, which are invasive procedures. Grains can also be obtained via ultrasound-guided fine-needle aspiration (US-FNA). Here we determined the diagnostic performance of species-specific PCRs performed on samples obtained through US-FNA. METHODS: From 63 patients, US-FNA was performed to obtain eumycetoma grains; 34 patients also underwent a deep-seated biopsy. From the grains, DNA was isolated, and one pan-fungal and two M. mycetomatis-specific PCRs were performed. The sensitivity and specificity were determined. RESULTS: Of the 63 patients who underwent US-FNA, 78% (49/63) had evidence of eumycetoma based on cytology and 93.7% (59/63) based on species-specific PCRs. In the 34 patients for whom surgical biopsies were performed as well, 31 patients had a positive PCR for M. mycetomatis when DNA was isolated from the deep-seated biopsy, and 30 had a positive PCR when DNA was obtained from the US-FNA material. This resulted in a 96.8% sensitivity, and 100% specificity with 97.1% diagnostic accuracy for PCR performed on US-FNA. CONCLUSION: PCR performed on the US-FNA material has a similar sensitivity and specificity as PCR performed on deep-seated biopsies. Therefore, when using PCR, a deep-seated biopsy may not be necessary to obtain grains.

Comparing the performance of the common used eumycetoma diagnostic tests.

OBJECTIVES: Mycetoma is a neglected tropical implantation disease caused by 70 different infectious agents. Identifying the causative organism to the species level is essential for appropriate patient management. Ultrasound, histopathology, culture and two species-specific PCRs are most the commonly used methods for species identification in endemic regions. The aim of this study was to compare the diagnostic performance of these commonly used assays using sequencing of barcoding genes as the gold standard. METHODS: This descriptive cross-sectional study was conducted at the Mycetoma Research Centre, University of Khartoum, Sudan. It included 222 patients suspected of fungal mycetoma caused by Madurella mycetomatis. RESULTS: 154 (69.3%) were correctly identified by ultrasound, histology, culture and both species-specific PCRs. In 60 patients, at least one of the diagnostic tests failed to identify M. mycetomatis. Five patients had no evidence of eumycetoma, and for three, only the ultrasound was indicative of mycetoma. The two species-specific PCRs were the most sensitive and specific methods, followed by culture and histology. Ultrasound was the least specific as it only allowed differentiation between actinomycetoma and eumycetoma. The time to result was 9.38 minutes for ultrasound, 3.76 hours for PCR, 8.5 days for histopathology and 21 days for grain culturing. CONCLUSION: Currently, PCR directly on DNA isolated from grains is the most rapid and reliable diagnostic tool to identify M. mycetomatis eumycetoma.

Clinical triad with multi-biopsy histopathology as a reliable diagnostic marker of mycetomas: a retrospective review from a tertiary care center.

BACKGROUND: Mycetoma is a neglected tropical infectious disease which runs a prolonged and protracted course. Microbiological confirmation is diagnostic yet unreliable due to poor sensitivity and variable availability of culture facilities in resource poor settings. METHODS: A retrospective review was performed on electronic records (histopathology, microbiology, and radiology) of all patients who underwent skin biopsies with mycetoma as one of the clinical differential diagnoses from year 2016 to 2020. RESULTS: Out of 73 patients biopsied with a differential of mycetoma, 42 fit the clinical triad of swelling-sinuses-granules. After clinical, microbiological, pathological, and radiological correlation, 31 cases were of eumycetoma and seven were of actinomycetoma. Mean patient age was 37.58 ± 13.8 years with a male to female ratio 2.45 : 1 and mean disease duration of 11.31 ± 10.9 years. Histopathological findings revealed fungal hyphae in 18 cases and gram-positive bacteria in six cases. Fungal culture was positive in 13 cases with the three commonest organisms being Madurella mycetomatis in five cases, Fusarium and Aspergillus nidulans in two cases each. X-ray changes of soft tissue, bones, and joints were seen in 25 cases, and "dot-in-circle" sign was seen in eight of nine MRIs. CONCLUSION: Eumycetoma was more common than actinomycetoma in our setup, ratio being 4.43 : 1. A clinical triad of swelling, multiple sinuses and grainy discharge with any one diagnostic support (histopathology/radiology) is sufficient to make a definitive diagnosis of mycetoma in the absence of microbiological identification.

Wako ß-D-glucan assay can be used to measure serum ß-D-glucan in Sudanese patients to aid with diagnosis of eumycetoma caused by Madurella mycetomatis.

BACKGROUND: Eumycetoma is a neglected tropical infection of the subcutaneous tissue commonly caused by the fungus Madurella mycetomatis. Previously, we demonstrated that ß-D-glucan was present in the serum of eumycetoma patients. OBJECTIVE: To compare the performance of the recently approved easy-to-use Wako ß-D-glucan assay to that of the Fungitell assay in eumycetoma patients. METHODS: Using sera obtained from 41 eumycetoma, 12 actinomycetoma and 29 healthy endemic controls, we measured the ß-glucan serum concentrations using the Wako assay and compared the performance to that of the Fungitell assay. RESULTS: With the Fungitell assay, median ß-glucan serum concentrations of 208, 70 and 27 pg/ml were obtained for the 41 eumycetoma patients, the 12 actinomycetoma patients and the 29 healthy endemic controls, respectively. With the Wako assay these concentrations were 14.45, 11.57 and 2.5 pg/ml, respectively. We demonstrated that when using the optimized cut-off value (5.5 pg/ml) for the Wako assay, the Wako and Fungitell assays had comparable performance in terms of sensitivity and specificity. CONCLUSION: The Wako assay is comparable to the Fungitell assay for measurement of serum ß-glucan in mycetoma patients and hence can be used in combination with current diagnostic tools. However, this test should be used in combination with other tests to differentiate actinomycetoma from eumycetoma.

Genomics and metagenomics of Madurella mycetomatis, a causative agent of black grain mycetoma in Sudan.

Madurella mycetomatis is one of the main causative agents of mycetoma, a debilitating neglected tropical disease. Improved understanding of the genomic diversity of the fungal and bacterial causes of mycetoma is essential to advances in diagnosis and treatment. Here, we describe a high-quality genome assembly of M. mycetomatis and results of the whole genome sequence analysis of 26 isolates from Sudan. We demonstrate evidence of at least seven genetically diverse lineages and extreme clonality among isolates within these lineages. We also performed shotgun metagenomic analysis of DNA extracted from mycetoma grains and showed that M. mycetomatis reads were detected in all sequenced samples with the average of 11,317 reads (s.d. +/- 21,269) per sample. In addition, 10 (12%) of the 81 tested grain samples contained bacterial reads including Streptococcus sp., Staphylococcus sp. and others.

Head and neck mycetoma: Clinical findings, investigations, and predictors for recurrence of the disease in Sudan: A retrospective study.

INTRODUCTION: Mycetoma is a unique neglected tropical disease which is found endemic in areas known as the "mycetoma belt". Head and neck mycetoma is a rarity and it has many devastating impacts on patients and communities. In this study, we assessed clinical findings, investigations, and predictors for recurrence of head and neck mycetoma in Sudan. METHODOLOGY: A retrospective study was conducted at Mycetoma Research Center in Khartoum between January 1999 and December 2020 for all patients with head and neck mycetoma. Data were analyzed using R software version 4.0.2. RESULTS: We included 107 patients with head and neck mycetoma. 65.4% were young adult males from mycetoma endemic areas in Sudan, and most of them were students (33.6%). Most of patients (64.4%) had actinomycetoma. Before presenting with head and neck mycetoma, majority (75.7%) had a long duration with mycetoma, and 30.8% had a history of trauma. The commonest invaded site was the parietal region (30.8%). The lesion started gradually in most of the patients (96.3%). 53.3% of the patients had large size lesions with no sweating, regional lymph nodes involvement, or distal vein involvement. CT scan was the most accurate diagnostic tool while 8.4% of patients were diagnosed by clinical examinations only. Laboratory investigations confirmed that 24/45 (44.4%) of actinomycetoma was caused by Streptomyces somaliensis while 13/28 (46.4%) of eumycetoma was caused by Madurella mycetomatis. All patients with recurrence of head and neck mycetoma underwent surgical excision of the lesion (n = 41/41 {100%}, p < 0.001). CONCLUSION: In head and neck mycetoma, the most common type was actinomycetes in Sudan. Majority had a long course of mycetoma and the commonest causative organism was Streptomyces somaliensis. The treatment outcome was poor and characterized by a low cure rate.

Metagenomic detection of eumycetoma causative agents from households of patients residing in two Sudanese endemic villages in White Nile State.

Eumycetoma is a chronic debilitating fungal disease endemic to tropical and subtropical regions, with Sudan featuring the highest eumycetoma incidence. Among the 50 species of fungi most commonly associated with eumycetoma Madurella mycetomatis (M. mycetomatis) is often referenced as the most common pathogen. However, there is an enormous knowledge gap related to this neglected disease and its pathogenesis, epidemiological features, and host-specific factors that could contribute to either the host susceptibility and resistance. In this study, we were able to utilize a metagenomic approach and samples collected from clinical black grains (BG) and familiar household environments aimed to assay both the habitat of eumycetoma-associated fungi and its possible connection with eumycetoma patients living in two different eumycetoma endemic villages within the White Nile State of Sudan. DNA sequencing targeting the fungal ITS2 domain was performed on soil, animal dung, housing walls and roofs, and Acacia-species thorn samples and compared with culture-dependent methods of fungal isolation. Additionally, we compared the soil samples obtained in the endemic zone with that from non-endemic zones, including Wagga village in Kassala State and Port Sudan suburb in Port Sudan State. Overall, a total of 392 Amplicon Sequence Variants (ASVs) were detected by ITS2 metagenomics Eumycetoma causative organisms accounted for 10% of total ASVs which included 11 genera: Exserohilum (2%), Aspergillus (1.7%), Curvularia (1%), Alternaria (0.9%), Madurella (0.5%), Fusarium (0.4%), Cladosporium (0.2%) Exophiala (0.15%), and, in a lesser extent, Microascus (0.05%) Bipolaris and Acremonium (0.01%) for each. Only five genera were identified by culture method, which included Fusarium (29%), Aspergillus (28%), Alternaria (2.5%), Bipolaris (1.6%), and Chaetomium (0.8%). M. mycetomatis was detected within all the studied patients' houses, accounting for 0.7% of total sequences. It was the first common eumycetoma-associated agent detected in soil samples and the third common in the dung and wall samples. In contrast, it was not detected in the roof or thorn samples nor in the soils from non-endemic regions. Exserohilum rostratum, Aspergillus spp and Cladosporium spp were detected in all samples. M. mycetomatis and other eumycetoma-associated fungal identified in the patients' black grains (BG) samples by metagenomics were identified in the environmental samples. Only Acremonium alternatum and Falciformispora senegalensis, responsible for eumycetoma in two patients were not detected, suggesting the infections in these patients happened outside these endemic areas. The soil, animal dung, and houses built from the same soil and dung are the main risk factors for M. mycetomatis infection in these endemic villages. Furthermore, the poor hygienic and environmental conditions, walking barefooted, and the presence of animals within the houses increase the risk of M. mycetomatis and other fungi causing eumycetoma.