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 cut-off values for itraconazole and ravuconazole for Madurella mycetomatis, the most common causative agent of mycetoma.

BACKGROUND: Eumycetoma is a neglected tropical disease. It is a chronic inflammatory subcutaneous infection characterised by painless swellings which produce grains. It is currently treated with a combination of itraconazole and surgery. In an ongoing clinical study, the efficacy of fosravuconazole, the prodrug of ravuconazole, is being investigated. For both itraconazole and ravuconazole, no clinical breakpoints or epidemiological cut-off values (ECV) to guide treatment are currently available. OBJECTIVE: To determine tentative ECVs for itraconazole and ravuconazole in Madurella mycetomatis, the main causative agent of eumycetoma. MATERIALS AND METHODS: Minimal inhibitory concentrations (MICs) for itraconazole and ravuconazole were determined in 131 genetically diverse clinical M. mycetomatis isolates with the modified CLSI M38 broth microdilution method. The MIC distributions were established and used to determine ECVs with the ECOFFinder software. CYP51A sequences were sequenced to determine whether mutations occurred in this azole target gene, and comparisons were made between the different CYP51A variants and the MIC distributions. RESULTS: The MICs ranged from 0.008 to 1 mg/L for itraconazole and from 0.002 to 0.125 mg/L for ravuconazole. The M. mycetomatis ECV for itraconazole was 1 mg/L and for ravuconazole 0.064 mg/L. In the wild-type population, two CYP51A variants were found for M. mycetomatis, which differed in one amino acid at position 499 (S499G). The MIC distributions for itraconazole and ravuconazole were similar between the two variants. No mutations linked to decreased susceptibility were found. CONCLUSION: The proposed M. mycetomatis ECV for itraconazole is 1 mg/L and for ravuconazole 0.064 mg/L.

A Short-Tandem-Repeat Assay (MmySTR) for Studying Genetic Variation in Madurella mycetomatis.

Madurella mycetomatis is the major causative agent of eumycetoma, a neglected tropical infection characterized by painless subcutaneous lesions, inflammation, and grains draining from multiple sinuses. To study the epidemiology of mycetoma, a robust discriminatory typing technique is needed. We describe the use of a short-tandem-repeat assay (MmySTR) for genotyping of M. mycetomatis isolates predominantly from Sudan. Eleven microsatellite markers (3 dinucleotides, 4 trinucleotide repeats, and 4 tetranucleotide repeats) were selected from the M. mycetomatis MM55 genome using the Tandem Repeats Finder software. PCR amplification primers were designed for each microsatellite marker using primer3 software and amplified in a multicolor multiplex PCR approach. To establish the extent of genetic variation within the population, a collection of 120 clinical isolates from different regions was genotyped with this assay. The 11 selected MmySTR markers showed a large genotypic heterogeneity. From a collection of 120 isolates, 108 different genotypes were obtained. Simpson's diversity index (D) value for individual markers ranged from 0.081 to 0.881, and the combined panel displayed an overall D value of 0.997. The MmySTR assay demonstrated high stability, reproducibility, and specificity. The MmySTR assay is a promising new typing technique that can be used to genotype isolates of M. mycetomatis Apart from the possible contribution of host factors, the genetic diversity observed among this group of isolates might contribute to the different clinical manifestations of mycetoma. We recommend that the MmySTR assay be used to establish a global reference database for future study of M. mycetomatis isolates.

Diagnostic implications of mycetoma derived from Madurella pseudomycetomatis isolates from Mexico.

BACKGROUND: At the dermatology service of the General Hospital of Mexico City, Mexico, two patients, father and son, with black-grain mycetoma were seen. The grains were isolated, and the cultured fungi were identified as Madurella mycetomatis based on morphology. Using the M. mycetomatis specific PCR, amplicons of a different size than that of the M. mycetomatis type strain were obtained. OBJECTIVE: To determine the causative agent of the two black-grain mycetoma cases and develop non-culture-based diagnostic tools to identify them to the species level. METHODS: The M. mycetomatis specific, the internal transcribed spacer (ITS) region, ß-tubulin (BT) and ribosomal binding protein 2 (RBP2) PCRs were used to confirm the identity of the isolates. Genetic variation was established by amplification fragment length polymorphisms. To determine the antifungal susceptibility profile, the Sensititre™ YeastOne™ assay was used. To develop a species-specific PCR primers were designed on the sequenced PCR amplicon from the M. mycetomatis specific PCR. RESULTS: By analyzing the ITS, BT and RBP2 regions the isolates were identified as Madurella pseudomycetomatis. The isolates from father and son were similar but not identical to M. pseudomycetomatis from Venezuela and one from an unknown origin. Madurella pseudomycetomatis isolates were inhibited by itraconazole, posaconazole and voriconazole but showed increased MIC values for amphotericin B and fluconazole. They were not inhibited by the echinocandins and five flucytosine. The two patients were treated with itraconazole resulting in cure for the father while the son was lost to follow-up. The species-specific PCR developed for M. pseudomyceotmatis was discriminative and specific. CONCLUSION: Madurella pseudomycetomatis is genetically diverse with same susceptibility profile as M. mycetomatis and causes eumycetoma in Latin America. The M. pseudomycetomatis specific PCR can be used to identify this causative agent to the species level; however, this needs to be validated in an endemic setting.

Pyomelanin Secretion in Madurella mycetomatis Interferes with Spectrophotometric Endpoint Reading Using the Sensititre YeastOne alamarBlue Assay but Not with Visual Endpoint Reading.

The use of the Sensititre YeastOne YO10 alamarBlue assay for the in vitro susceptibility testing of Madurella mycetomatis was evaluated in M. mycetomatis isolates with and without pyomelanin secretion. Pyomelanin secretion did not influence visual endpoint reading; however, it caused a shift in peak absorbance from 570 nm to 620 nm when read spectrophotometrically. Therefore, when choosing the method for endpoint reading, the presence of pyomelanin should be considered.

VNTR confirms the heterogeneity of Madurella mycetomatis and is a promising typing tool for this mycetoma causing agent.

The neglected tropical disease mycetoma is a chronic granulomatous inflammatory and infectious disease affecting various body parts. The most common causative agent is the fungus Madurella mycetomatis. In order to study the genetic diversity of this fungus and to monitor any potential outbreaks, a good typing method that can be used in endemic settings is needed. Previous typing methods developed were not discriminative and not easy to perform in resource-limited laboratories. Variable-Number-Tandem-Repeat (VNTR) typing overcomes these difficulties and further enables interlaboratory data comparison. Therefore, in this study we developed a VNTR method for typing M. mycetomatis. Six tandem-repeats were identified in the genome of M. mycetomatis isolate MM55 using an online tandem repeats software. The variation in these repeats was determined by PCR and gel-electrophoresis on DNA obtained from 81 M. mycetomatis isolates obtained from patients. These patients originated from Sudan, Mali, Peru, and India. The 81 isolates were divided into 14 genotypes which separated into two main clusters with seven and five subdivisions, respectively. VNTR typing confirms the heterogeneity of M. mycetomatis strains and can be used to study the epidemiology of M. mycetomatis. The results presented in this article are made fully available to the scientific community on request from the Eumycetoma Working Group. We hope that this open resource approach will bridge scientific community working with mycetoma from all around the world and lead to a deeper understanding of M. mycetomatis.

Nigrograna mackinnonii, Not Trematosphaeria grisea (syn., Madurella grisea), Is the Main Agent of Black Grain Eumycetoma in Latin America.

Mycetoma, a chronic and mutilating subcutaneous infection recognized by the WHO as a neglected tropical disease, has been reported in >25 countries in Africa, Asia, and South America. In Latin America, Trematosphaeria grisea is assumed to be the prevalent fungal agent. Recent molecular studies have shown that this is an environmental saprobe in Europe, where it is rarely implicated in human diseases. The aim of the present paper is to establish the identity of Latin American cases ascribed to Trematosphaeria grisea Three cases analyzed were caused by Nigrograna mackinnonii Data on an additional 21 strains in the literature revealed that N. mackinnonii rather than T. grisea is responsible for most cases of black grain eumycetoma in Latin America.

Molecular identification of unusual Mycetoma agents isolated from patients in Venezuela.

Mycetoma is a chronic granulomatous, subcutaneous disease endemic in tropical and subtropical countries. It is currently a health problem in rural areas of Africa, Asia and South America. Nine cases of mycetoma were analysed in a retrospective study. All isolates were identified by morphological features. The level of species identification was reached by molecular tools. Definitive identification of fungi was performed using sequence analysis of the ITS of the ribosomal DNA region and the ribosomal large-subunit D1/D2. Identification of actinomycetes was accomplished by the 16S rRNA gene sequence. Six unusual clinical isolates were identified: Aspergillus ustus, Cyphellophora oxyspora, Exophiala oligosperma, Madurella pseudomycetomatis, Nocardia farcinica and Nocardia wallacei. The prevalence of mycetoma in Venezuela remains unknown. This study represents the first report in the literature of mycetoma caused by unusual pathogens identified by molecular techniques.

Protoplast-mediated transformation of Madurella mycetomatis using hygromycin resistance as a selection marker.

Madurella mycetomatis is the main cause of mycetoma, a chronic granulomatous infection for which currently no adequate therapy is available. To improve therapy, more knowledge on a molecular level is required to understand how M. mycetomatis is able to cause this disease. However, the genetic toolbox for M. mycetomatis is limited. To date, no method is available to genetically modify M. mycetomatis. In this paper, a protoplast-mediated transformation protocol was successfully developed for this fungal species, using hygromycin as a selection marker. Furthermore, using this method, a cytoplasmic-GFP-expressing M. mycetomatis strain was created. The reported methodology will be invaluable to explore the pathogenicity of M. mycetomatis and to develop reporter strains which can be useful in drug discovery as well as in genetic studies.

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.

New species of Madurella, causative agents of black-grain mycetoma.

A new species of nonsporulating fungus, isolated in a case of black-grain mycetoma in Sudan, is described as Madurella fahalii. The species is characterized by phenotypic and molecular criteria. Multigene phylogenies based on the ribosomal DNA (rDNA) internal transcribed spacer (ITS), the partial ß-tubulin gene (BT2), and the RNA polymerase II subunit 2 gene (RPB2) indicate that M. fahalii is closely related to Madurella mycetomatis and M. pseudomycetomatis; the latter name is validated according to the rules of botanical nomenclature. Madurella ikedae was found to be synonymous with M. mycetomatis. An isolate from Indonesia was found to be different from all known species based on multilocus analysis and is described as Madurella tropicana. Madurella is nested within the order Sordariales, with Chaetomium as its nearest neighbor. Madurella fahalii has a relatively low optimum growth temperature (30°C) and is less susceptible to the azoles than other Madurella species, with voriconazole and posaconazole MICs of 1 µg/ml, a ketoconazole MIC of 2 µg/ml, and an itraconazole MIC of >16 µg/ml. Since eumycetoma is still treated only with azoles, correct species identification is important for the optimal choice of antifungal therapy.

Fructose-bisphosphate aldolase and pyruvate kinase, two novel immunogens in Madurella mycetomatis.

Eumycetoma, a chronic granulomatous disease characterized by a subcutaneous mass, multiple sinuses and purulent discharge containing grains, remains difficult to diagnose and treat. Madurella mycetomatis is the most common causative agent of eumycetoma. Using a serum pool from patients with active mycetoma, we screened a M. mycetomatis-specific λgt11 cDNA library which was shown to contain 8% of cDNA inserts encoding proteins involved in glycolysis. Two of these enzymes, fructose-bisphosphate aldolase (FBA) and pyruvate kinase (PK), were produced in vitro and their antigenicity was studied with bead-based flow cytometry. It appeared that both FBA and PK IgG antibodies were present in eumycetoma patient sera. However, only FBA antibody levels were found to be significantly higher in eumycetoma patient sera when compared to healthy Sudanese controls. Furthermore, FBA and PK were also found to be expressed on the hyphae present in the mycetoma grain. In conclusion, this study presents two new antigenic proteins of M. mycetomatis next to the translationally controlled tumour protein (TCTP): the glycolytic enzymes FBA and PK. These antigens might be useful as vaccine-candidates in the prevention of mycetoma.

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.

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.

Optimisation of the Production and Bleaching Process for a New Laccase from Madurella mycetomatis, Expressed in Pichia pastoris: from Secretion to Yielding Prominent.

Laccases are polyphenol oxidoreductases used in a number of industrial applications. Due to the increasing demand for these "green catalysis" enzymes, the identification and biochemical characterisation of their novel properties is essential. In our study, cloned Madurella mycetomatis laccase (mmlac) genes were heterologously expressed in the methylotrophic yeast host Pichia pastoris. The high yield of the active recombinant protein in P. pastoris demonstrates the efficiency of a reliably constructed plasmid to express the laccase gene. The optimal biochemical conditions for the successfully expressed MmLac enzyme were identified. Detailed structural properties of the recombinant laccase were determined, and its utility in decolourisation and textile bleaching applications was examined. MmLac demonstrates good activity in an acidic pH range (4.0-6.0); is stable in the presence of cationic metals, organic solvents and under high temperatures (50-60 °C); and is stable for long-term storage at - 20 °C and - 80 °C for up to eight weeks. The structural analysis revealed that the catalytic residues are partially similar to other laccases. MmLac resulted in an increase in whiteness, whilst demonstrating high efficiency and stability and requiring the input of fewer chemicals. The performance of this enzyme makes it worthy of investigation for use in textile biotechnology applications, as well as within environmental and food technologies.

The synthetic synergistic cinnamon oil CIN-102 is active against Madurella mycetomatis, the most common causative agent of mycetoma.

Mycetoma is a devastating neglected tropical infection of the subcutaneous tissue and most commonly caused by the fungus Madurella mycetomatis. Treatment of mycetoma consists of a combination of a long term antifungal treatment with itraconazole and surgery. However, treatment is associated with low success rates. Therefore, there is a need to identify novel treatments for mycetoma. CIN-102 is a synthetic partial copy of cinnamon oils with activity against many pathogenic bacteria and fungi. In this study we determined the in vitro activity of CIN-102 against 21 M. mycetomatis isolates and its in vivo efficacy in a M. mycetomatis infected Galleria mellonella larval model. In vitro, CIN-102 was active against M. mycetomatis with MICs ranging from 32 µg/mL to 512 µg/mL. 128 µg/mL was needed to inhibit the growth in 50% of tested isolates. In vivo, concentrations below the MIC of 40 mg/kg and 80 mg/kg CIN-102 prolonged larval survival, but higher concentrations of CIN-102 did not.

Phenotypic and molecular characterization of Madurella pseudomycetomatis sp. nov., a novel opportunistic fungus possibly causing black-grain mycetoma.

A case of black-grain mycetoma occurring on the lower jaw with an odontogenic origin, which to our knowledge is the first case reported in China, is presented here. The clinical manifestation, histopathological morphology, and microbiological features are described. The new species, Madurella pseudomycetomatis, isolated from the black grains discharged by this patient, was analyzed using sequence data of the multiloci of ribosomal DNA (rDNA) and its ability to ferment carbohydrate as well as morphology. The analyses of the internal transcribed spacer (ITS) region and the D1/D2 hypervariable region of the 28S ribosomal gene sequences support a new species designation. Antifungal susceptibility testing was conducted, indicating that Madurella pseudomycetomatis was highly susceptible to itraconazole, voriconazole, and amphotericin B; moderately susceptible to terbinafine; and resistant to fluconazole and flucytosine.

Madurella real-time PCR, a novel approach for eumycetoma diagnosis.

The genus Madurella comprising four species, M. fahalii, M. mycetomatis, M. pseudomycetomatis, and M. tropicana, represents the prevalent cause of eumycetoma worldwide. The four species are phenotypically similar and cause an invariable clinical picture, but differ markedly in their susceptibility to antifungal drugs, and epidemiological pattern. Therefore, specific identification is required for optimal management of Madurella infection and to reveal proper epidemiology of the species. In this study, a novel multiplex real-time PCR targeting the four Madurella species was developed and standardized. Evaluation of the assay using reference strains of the target and non-target species resulted in 100% specificity, high analytical reproducibility (R2 values >0.99) and a lowest detection limit of 3 pg target DNA. The accuracy of the real-time PCR was further assessed using biopsies from eumycetoma suspected patients. Unlike culture and DNA sequencing as gold standard diagnostic methods, the real-time PCR yielded accurate diagnosis with specific identification of the causative species in three hours compared to one or two weeks required for culture. The novel method reduces turnaround time as well as labor intensity and high costs associated with current reference methods.