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 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.

Madurella mycetomatis grains within a eumycetoma lesion are clonal.

Eumycetoma is a neglected tropical infection of the subcutaneous tissue, characterized by tumor-like lesions and most commonly caused by the fungus Madurella mycetomatis. In the tissue, M. mycetomatis organizes itself in grains, and within a single lesion, thousands of grains can be present. The current hypothesis is that all these grains originate from a single causative agent, however, this hypothesis was never proven. Here, we used our recently developed MmySTR assay, a highly discriminative typing method, to determine the genotypes of multiple grains within a single lesion. Multiple grains from surgical lesions obtained from 11 patients were isolated and genotyped using the MmySTR panel. Within a single lesion, all tested grains shared the same genotype. Only in one single grain from one patient, a difference of one repeat unit in one MmySTR marker was noted relative to the other grains from that patient. We conclude that within these lesions the grains originate from a single clone and that the inherent unstable nature of the microsatellite markers may lead to small genotypic differences. LAY ABSTRACT: In lesions of the implantation mycosis mycetoma many Madurella mycetomatis grains are noted. It was unknown if grains arose after implantation of a single isolate or a mixture of genetically diverse isolates. By typing the mycetoma grains we showed that all grains within a single lesion were clonal and originated from a single isolate.

Epidemiological observations and management challenges in extrapedal mycetoma: A three-decade review of 420 cases.

Mycetoma is a serious, destructive, disfiguring chronic granulomatous inflammatory disease affecting the subcutaneous tissues that spread to involve the skin, deep tissues and bone. The disease predominately affects the limbs, and extrapedal mycetoma is rarely reported. The reported extrapedal ones are characterised by high morbidity and mortality. This communication reports on 420 patients with extrapedal mycetoma seen and managed at the Mycetoma Research Centre (MRC), University of Khartoum, between January 1991 and December 2021. In this descriptive, cross-sectional, hospital-based study, the electronic records of all mycetoma-confirmed patients seen during the study period were carefully and meticulously reviewed. The confirmed patients with extrapedal mycetoma were included in this study. The study included 420 patients with extrapedal mycetoma, 298 (70.7%) had eumycetoma, and 122 (29.3%) had actinomycetoma. There were 343 male patients (81.7%) and 77 (18.3%) females, with a male-to-female ratio of 4:1. Their ages ranged between 1.5 and 95 years, with a median of 28 years. Most of the patients were students and farmers. The majority of patients were from El Gezira, North Kordofan, and the White Nile States. Mycetoma was painful in 21%, and a family history of mycetoma was recorded in 11.5% of patients. The buttocks (37.9%) and head and neck (16.9%) were affected most. Less frequently affected sites were the trunk and back (12%) each, abdominal and chest walls (4.5%) each and loin (1%). The prominent clinical presentation findings were multiple sinuses discharging grains (55%), massive swellings (46%), and lymphadenopathy (11.5%). Less commonly observed clinical findings were local hyperhidrosis (5.3%) and dilated tortuous veins close to mycetoma lesions (0.5%). The study showed that 204 patients (48.6%) had clinical improvement in terms of decreased lesion size and healing of sinuses following medical therapy. Sixty-six patients (15.7%) had no noticeable improvement. The lesion continued progressing despite treatment in 44 patients (10.5%). In the study, 118 patients were on regular follow-up, and in this group, a cure was documented in 25 patients (21.1%) with eumycetoma and 23 (19.4%) with actinomycetoma. Post-operative recurrence among eumycetoma patients was 40%, with a 1% mortality rate. The treatment outcome was unsatisfactory, characterised by a low cure rate, high recurrence (40%) and follow-up dropout (57%) rates. This emphasises the importance of early case detection and management, objective health education programmes and thorough patient counselling to urge people to seek treatment early and reduce dropouts.

The Mycetoma Research Center, University of Khartoum, Sudan's experience in community engagement initiatives spans 3 decades.

Mycetoma profoundly affects marginalised communities, especially in impoverished and remote areas with limited access to healthcare. This chronic and debilitating inflammatory disease highlights the typical issues of neglected tropical diseases (NTDs), such as insufficient attention, funding, and resources, which perpetuate neglect and suffering. Patients often delay seeking medical help, leading to advanced disease stages, severe complications, and lasting disabilities. The lack of medical infrastructure and skilled healthcare professionals worsens the situation, causing delays in diagnosis and inadequate treatment. Engaging affected communities in tailored interventions is essential to tackle these challenges, promote collaboration, raise awareness, and mobilise resources to improve healthcare access and enhance diagnostic and treatment capabilities. Since 1991, the Mycetoma Research Center (MRC) at the University of Khartoum, Sudan, has led community engagement initiatives aimed at improving the quality of life for mycetoma-affected individuals through education, advocacy, and local collaboration. In this communication, the MRC shares its extensive experience in community engagement to benefit mycetoma-affected communities.

Hope amidst neglect: Mycetoma Research Center, University of Khartoum. A holistic management approach to achieve the United Nations' Sustainable Development Goals.

Mycetoma is a debilitating neglected tropical disease that affects individuals worldwide, particularly in regions where there is poverty and limited health care access. The Mycetoma Research Center (MRC), based in Khartoum, Sudan, provides a sustainable, holistic approach to patient care as the only World Health Organization collaborating center for mycetoma. We describe MRC activities that align with the United Nations' Sustainable Development Goals to control mycetoma in Sudan and globally.

Emerging therapeutics: The imidazo[1,2-b]pyridazine scaffold as a novel drug candidate for eumycetoma, a neglected tropical disease.

Mycetoma is a neglected invasive infection endemic in tropical and subtropical regions, presenting as a chronic subcutaneous inflammatory mass that can spread to deeper structures, leading to deformities, disabilities, and potentially mortality. The current treatment of eumycetoma, the fungal form of mycetoma, involves antifungal agents, such as itraconazole, combined with surgical intervention. However, this approach has limited success, with low cure rates and a high risk of recurrence. This study addresses to the urgent need for more effective therapeutics by designing and synthesising 47 diversely pharmacomodulated imidazo [1,2-b]pyridazine derivatives using a simple synthetic pathway with good yields and purity. Of these, 17 showed promising in vitro activity against Madurella mycetomatis, the prime causative agent of eumycetoma, with IC50 ≤ 5 µM and demonstrated significantly lower cytotoxicity compared to standard treatments in NIH-3T3 fibroblasts. Notably, compound 14d exhibited an excellent activity with an IC50 of 0.9 µM, in the same order then itraconazole (IC50 = 1.1 µM), and achieved a favourable selectivity index of 16 compared to 0.8 for itraconazole. These promising results warrant further research to evaluate the clinical potential of these novel compounds as safer, more effective treatments for eumycetoma, thus addressing a profound gap in current therapeutic strategies.

The nutritional status of mycetoma affected patients seen at the Mycetoma Research Center, Sudan.

Nutrition plays a critical and crucial role in addressing neglected tropical diseases (NTDs) and their complications, as they often contribute to malnutrition, which can worsen the impact of these conditions. Therefore, it is necessary to investigate the nutritional status of mycetoma patients, which has not been explored previously. This descriptive cross-sectional hospital-based study was conducted at the Mycetoma Research Center (MRC), University of Khartoum, Sudan. The study included 179 confirmed mycetoma patients and an equal number of age- and sex-matched normal controls. The nutritional status of the mycetoma patients was assessed and compared with that of the control group. The majority of the patients were young adults with varying educational levels, predominantly from Central Sudan. The foot was the most commonly affected part; most patients had lesions more than 10 cm in diameter. The Body Mass Index (BMI) was calculated for both study groups, revealing that 43.5% of the patients and 53.6% of controls had a normal BMI. Furthermore, 36% of patients were underweight, contrasting with only 11% in the control group. Correlation analyses indicated no significant associations between BMI and age groups, educational levels, daily meals, food quantity, and appetite in the study population (p > 0.05). Similarly, no significant differences were observed in BMI concerning disease duration and affected sites (p = 0.0577). The Kruskal-Wallis test did not reveal significant differences in BMI means among the groups. The study revealed that most participants consumed three meals daily, and the control group showed a more robust appetite and consumed more food than the patient group (p = 0.005). Nevertheless, there were no significant differences in the consumption of different food types between the patient and control groups and among different BMI categories (p = 0.025 and 0.040, respectively).

Mycetoma and the environment.

Mycetoma is a chronic, incapacitating, destructive inflammatory disease with many serious damaging impacts. Currently, there is no control or prevention program as many of its epidemiological characteristics, such as the causative organisms' ecological niche, natural habitat, primary reservoir, transmission mode, geographical distribution, incidence, and prevalence, remain unclear. This may be due to a lack of research interest, as mycetoma is still a neglected disease and the scarcity of accurate molecular diagnostic techniques in disease-endemic regions for accurate causative microorganisms identification and mapping. With this background, this study set out to address this knowledge gap by considering the mycetoma environmental occurrence predictors. The medical literature obtained data showed a close association between mycetoma occurrence and its environment. The causative microorganisms are available in the environment in active or dormant forms. Animal dung may be a natural niche and reservoir for these organisms, and thorns may facilitate the subcutaneous inoculation. Some environmental factors, such as the soil type and consistency, temperature, water sources, aridity index, and thorny trees, may be risk factors. The population in endemic areas socioeconomic, hygiene, and health education status are contributory factors for mycetoma. The individual's genetic and immunological backgrounds may determine the disease's susceptibility and resistance. Environmental conditions and personal hygiene improvement are mandatory to reduce disease occurrence. Mycetoma spatial mapping can detect disease cluster areas and then develop public health strategies for early case detection and management to reduce the disease burden. More research interests and facilities are needed to understand disease pathogenesis and appropriate patient management better.

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.

Mycetoma management and clinical outcomes: the Mycetoma Research Center experience.

BACKGROUND: Mycetoma is a chronic granulomatous inflammatory disease that affects the cutaneous and subcutaneous tissues, leading to gruesome complications if not treated early. As a neglected disease, it has received scant attention in developing curable drugs. Mycetoma treatment is still based on expert opinions in the absence of guidelines. METHODS: This descriptive, cross-sectional, hospital-based study aimed to determine and assess the disease treatment outcomes observed at Mycetoma Research Center, Sudan. RESULTS: In this study, 75% of patients had eumycetoma, all of whom were treated with itraconazole and 37.4% underwent surgical excision, while 25% of the patients had actinomycetoma, 99.2% of whom were treated with a combination of cotrimoxazole and amoxicillin-clavulanate. The cure rate was 12.7% and 14.3% for patients with eumycetoma and actinomycetoma, respectively. Only 6.1% of eumycetoma patients underwent amputation. Remarkably, no patient with actinomycetoma underwent an amputation. Small lesions (OR=10.09, p<0.001) and good follow-up (OR=6.81, p=0.002) were positive predictors of complete cure. In terms of amputation, history of surgical recurrence at presentation (OR=3.67, p=0.020) and presence of grains (OR=7.13, p=0.012) were positive predictors, whereas small lesions were negative predictors (OR=0.06, p=0.009). CONCLUSIONS: Treatment of mycetoma was suboptimal, with a low cure rate despite a long treatment duration. Complete cure has a significant association with small lesions and good follow-up.

The First Case of Fusarium falciforme Eumycetoma in Sudan and an Extensive Literature Review about Treatment Worldwide.

Eumycetoma is an infectious disease caused by various fungal pathogens. The disease is characterised by black and pale-yellowish grain discharge. In this communication, we report a case of eumycetoma with a pale grain foot-eumycetoma caused by Fusarium falciforme. The patient presented at the outpatient clinic of the Mycetoma Research Centre in Sudan. The causative agent was initially misidentified as Aspergillus nidulans based on its seemingly similar histopathological appearance. However, sequencing the internally transcribed spacer region of the extracted grain confirmed infection with Fusarium falciforme. Although the patient received Itraconazole and underwent surgical excision, the disease was recurrent. To our knowledge, this is the first report on Fusarium falciforme causing eumycetoma in Sudan, indicating the expansion of the geographical distribution of this pathogen. This calls for raising the awareness of healthcare providers and improving the diagnostic and surveillance systems in at-risk areas to improve the case management and reduce the threat of further spread. Considering the potential impacts of F. falciforme infection including threatening the global health, food security, and ecosystem balance, as well as loss of biodiversity and negative socioeconomic changes in endemic countries, we recommend the implementation of an integrated transdisciplinary One Health strategy for the prevention and control of emerging infectious diseases including F. falciforme.

Environmental detection of eumycetoma pathogens using multiplex real-time PCR for soil DNA in Sennar State, Sudan.

BACKGROUND: Mycetoma is a chronic disease affecting the skin and subcutaneous tissue endemic in the tropical and subtropical regions. Several bacteria and fungi can cause mycetoma, but fungal mycetoma (eumycetoma) is challenging because the treatment requires a combination of a long-term antifungal agent and surgery. Although the transmission route has not yet been elucidated, infection from the soil is a leading hypothesis. However, there are few soil investigation studies, and the geographical distribution of mycetoma pathogens is not well documented. Here, we used multiplex real-time PCR technology to identify three fungal species from soil samples. METHODS: In total, 64 DNA samples were extracted from soil collected in seven villages in an endemic area in Sennar State, Sudan, in 2019. Primers and fluorescent probes specifically targeting the ribosomal DNA of Madurella mycetomatis, Falciformispora senegalensis, and F. tompkinsii were designed. RESULTS: Multiplex real-time PCR was performed and identified the major pathogen, M. mycetomatis that existed in most sites (95%). In addition, two other pathogens were identified from some sites. This is the first report on the use of this technique for identifying the eumycetoma causative microorganisms. CONCLUSIONS: This study demonstrated that soil DNA investigation can elucidate the risk area of mycetoma-causative agents. The results will contribute to the design of prevention measures, and further large-scale studies may be effective in understanding the natural habitats of mycetoma pathogens.

Tongue actinomycetoma due to Actinomadura madurae: a rare clinical presentation.

A 60-year-old male farmer presented with tongue swelling of 1-month duration. Local oral clinical examinations showed a painless firm mass in the anterolateral aspect of the anterior third of the tongue. Fine needle aspiration for cytology confirmed the diagnosis of tongue actinomycetoma due to Actinomadura madurae. The patient underwent wide local excision under general anesthesia and had an uneventful postoperative recovery. He was started on amikacin sulfate 15 mg/kg daily and cotrimoxazole 15 mg/kg twice per day for 6 months. The lesion healed completely, with no evidence of recurrence at 6-month follow-up. The route of infection in this patient is unclear; however, direct traumatic inoculation is the most likely route. To the authors' knowledge, this is the first report of tongue mycetoma in the medical literature.

The developed molecular biological identification tools for mycetoma causative agents: An update.

Mycetoma is a chronic granulomatous inflammatory disease that is caused either by bacteria or fungi. Bacterial mycetoma (actinomycetoma) can be caused by various causative agents of the genera Nocardia, Streptomyces and Actinomadura. On the other hand, fungal mycetoma (eumycetoma) is most commonly caused by causative agents belonging to the genera Madurella, Scedosporium and Falciformispora. Early and accurate diagnosis of the causative organisms can guide proper patient management and treatment. To allow rapid and accurate species identification, different molecular techniques were developed over the past decades. These techniques can be protein based (MALDI-TOF MS) as well as DNA based (Sequencing, PCR and isothermal amplification methods). In this review, we provide an overview of the different molecular techniques currently in use and identify knowledge gaps, which need to be addressed before we can implement molecular diagnostics for mycetoma in different clinical settings.

Individual Risk Factors of Mycetoma Occurrence in Eastern Sennar Locality, Sennar State, Sudan: A Case-Control Study.

Mycetoma is a serious chronic subcutaneous granulomatous inflammatory disease that is endemic in tropical and subtropical regions, where it impacts profoundly on patients, families, and communities. Individual-level risk factors for the disease are poorly understood. To address this, a case-control study was conducted based on data collected from 60 villages in Eastern Sennar Locality, Sennar State, Sudan. Based on the presence of swelling in any part of the body, or sinus formation with or without grain discharge evident from the lesion by ultrasound examination, we diagnosed 359 cases of mycetoma. For each case, we included three healthy sex-matched persons, with no evidence of mycetoma, from the same village as the control group (n = 1077). The odds for mycetoma were almost three times higher in individuals in the age group 16-30 years (Adjusted Odds Ratio (AOR) = 2.804, 95% CI = 1.424-5.523) compared to those in age group ≤ 15 years. Other factors contributing to the odds of mycetoma were history of local trauma (AOR = 1.892, 95% CI = 1.425-2.513), being unmarried (AOR = 3.179, 95% CI = 2.339-4.20) and owning livestock (AOR = 3.941, 95% CI = 2.874-5.405). In conclusion, certain factors found to be associated with mycetoma in this study could inform a high index of suspicion for mycetoma diagnosis, which would improve early case detection. Other factors found to be associated could inform the development of an interventional program for mycetoma control in Sudan, including education on healthy farming practices and the risks of puncture wounds for individuals residing in endemic areas. However, this work was conducted in one endemic state, while mycetoma cases occur in all states of Sudan. Replicating this study over a wider area would give a fuller picture of the situation, providing the control program with more comprehensive information on the risk factors for the disease.

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.

Role of socioeconomic factors in developing mycetoma: Results from a household survey in Sennar State, Sudan.

BACKGROUND: Mycetoma is a chronic, progressively destructive disease of subcutaneous tissues and bones caused by certain species of bacteria or fungi. We conducted a cross-sectional community-based study alongside mapping of mycetoma in five administrative units with high mycetoma endemicity in the Eastern Sennar Locality, Sennar State, Sudan. METHODS: A household survey was administered which included questions about the household members, household characteristics, economic activity and history of mycetoma. A clinical examination was conducted on all members of the household. If mycetoma was suspected, an individual questionnaire was completed collecting demographic, clinical and epidemiological data as well as information on the use of health care and associated costs. Geographical coordinates and photos of the lesions were taken, and the affected persons were referred to the medical centre for confirmation of the diagnosis and treatment. We compared the characteristics of households with confirmed cases of mycetoma with those without confirmed cases, and individuals with confirmed mycetoma with those in whom mycetoma was not confirmed. RESULTS: In total 7,798 households in 60 villages were surveyed; 515 suspected cases were identified and 359 cases of mycetoma were confirmed. Approximately 15% of households with mycetoma had more than one household member affected by this disease. Households with mycetoma were worse off with respect to water supply, toilet facilities, electricity and electrical appliances compared to the survey households. Only 23% of study participants with mycetoma had sought professional help. Of these, 77% of patients travelled an average of six hours to visit a medical facility. More than half of patients had to pay towards their treatment. The estimated average cost of treatment was 26,957 Sudanese pounds per year (566 US dollars, exchange rate 2018). CONCLUSIONS: Results of this survey suggest that agricultural practices and reduced access to sanitation and clean water can be risk factors in developing mycetoma. Poor access to health care and substantial financial costs were barriers to seeking treatment for mycetoma.

Detection of multiple mycetoma pathogens using fungal metabarcoding analysis of soil DNA in an endemic area of Sudan.

Mycetoma is a tropical disease caused by several fungi and bacteria present in the soil. Fungal mycetoma and eumycetoma are especially challenging to treat; therefore, prevention, early diagnosis, and early treatment are important, but it is also necessary to understand the geographic distribution of these pathogenic fungi. In this study, we used DNA metabarcoding methodology to identify fungal species from soil samples. Soil sampling was implemented at seven villages in an endemic area of Sennar State in Sudan in 2019, and ten sampling sites were selected in each village according to land-use conditions. In total, 70 soil samples were collected from ground surfaces, and DNA in the soil was extracted with a combined method of alkaline DNA extraction and a commercial soil DNA extraction kit. The region for universal primers was selected to be the ribosomal internal transcribed spacer one region for metabarcoding. After the second PCR for DNA library preparation, the amplicon-based DNA analysis was performed using next-generation sequencing with two sets of universal primers. A total of twelve mycetoma-causative fungal species were identified, including the prime agent, Madurella mycetomatis, and additional pathogens, Falciformispora senegalensis and Falciformispora tompkinsii, in 53 soil samples. This study demonstrated that soil DNA metabarcoding can elucidate the presence of multiple mycetoma-causative fungi, which may contribute to accurate diagnosis for patient treatment and geographical mapping.