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Antimicrobial Agents and Chemotherapy Aug 2021For many fungal infections, susceptibility testing is used to predict if an isolate is resistant or susceptible to the antifungal agent used to treat the infection. For...
For many fungal infections, susceptibility testing is used to predict if an isolate is resistant or susceptible to the antifungal agent used to treat the infection. For Madurella mycetomatis, the main causative agent of mycetoma, susceptibility testing currently is not performed on a routine basis. The current susceptibility testing method is labor-intensive, and sonication must be done to generate a hyphal inoculum. For endpoint visualization, expensive viability dyes are needed. Here, we investigated if the currently used susceptibility method could be adapted to make it amendable for use in a routine setting which can be used in low-income countries, where mycetoma is endemic. First, we developed a methodology in which hyphal fragments can be generated without the need for sonication, by comparing different bead beating methodologies. Next, susceptibility was assessed using standard broth microdilution assays as well as disc diffusion, Etest, and VIPcheck methodologies. We demonstrate that after a hyphal suspension is generated by glass bead beating, disc diffusion, Etest, and VIPcheck can be used to determine susceptibility of Madurella mycetomatis to itraconazole, posaconazole, and voriconazole. The MICs found with Etest were comparable to those obtained with our modified CLSI-based broth microdilution susceptibility assay for itraconazole and posaconazole. Furthermore, we found an inverse relationship between the zones of inhibition and MICs obtained with the Etest and those obtained by the modified CLSI broth microdilution technique.
Topics: Antifungal Agents; Disk Diffusion Antimicrobial Tests; Itraconazole; Madurella; Microbial Sensitivity Tests; Triazoles; Voriconazole
PubMed: 34181477
DOI: 10.1128/AAC.00433-21 -
Frontiers in Pharmacology 2023Eumycetoma, a chronic subcutaneous mycosis, responds poorly to the available antifungal treatments and patients often require extensive surgical resection or amputation... (Review)
Review
Eumycetoma, a chronic subcutaneous mycosis, responds poorly to the available antifungal treatments and patients often require extensive surgical resection or amputation of the affected limb. More effective treatments are needed for eumycetoma. This article will describe some of the approaches being used to develop and evaluate new treatments for eumycetoma, summarise the latest developments and discuss the challenges that lie ahead.
PubMed: 37274106
DOI: 10.3389/fphar.2023.1165273 -
PLoS Neglected Tropical Diseases Jul 2020Eumycetoma (mycotic mycetoma) is the fungal form of mycetoma, a subcutaneous infection occurring in individuals living in endemic areas of the disease. The Sudan is...
Eumycetoma (mycotic mycetoma) is the fungal form of mycetoma, a subcutaneous infection occurring in individuals living in endemic areas of the disease. The Sudan is hyperendemic for mycetoma, with the highest incidence being reported from Gezira State, Central Sudan. The present study was conducted at the Gezira Mycetoma Center and aimed to determine the cause of black-grain eumycetoma in the state and describe its epidemiology. Black-grain specimens were collected during the surgical operation and direct detection of the causative agent was performed using M. mycetomatis species-specific PCR and ITS PCR followed by sequencing. Black-grain was reported from 93.3% of all confirmed mycetoma cases (n = 111/119), with a prevalence in young males. Of the 91 samples subjected to direct PCR, 90.1% (n = 82) gave positive results. The predominant species (88.2%) was Madurella mycetomatis. One sample was identified as M. fahalii, one as M. tropicana, and one matched the phytopathogenic species Sphaerulina rhododendricola. The highest endemic zones were Southern Gezira (76.6%) and Northern Sinnar (23.4%). The study confirmed that direct molecular detection on grains provides rapid and specific diagnosis of agents of eumycetoma.
Topics: Adolescent; Adult; Aged; Child; Child, Preschool; Female; Humans; Infant; Madurella; Male; Middle Aged; Mycetoma; Phylogeny; Sudan; Young Adult
PubMed: 32730340
DOI: 10.1371/journal.pntd.0008420 -
International Journal of Molecular... Jun 2024Eumycetoma is a neglected tropical disease (NTD) characterized by subcutaneous lesions and the formation of grains. Attempts to treat eumycetoma involve a combination of...
OBJECTIVES
Eumycetoma is a neglected tropical disease (NTD) characterized by subcutaneous lesions and the formation of grains. Attempts to treat eumycetoma involve a combination of antifungal treatment and surgery, although the outcome is frequently disappointing. Therefore, there is a need to identify novel antifungal drugs to treat eumycetoma. In this respect, Medicines for Malaria Venture (MMV) has assembled libraries of compounds for researchers to use in drug discovery research against NTD. Therefore, we screened two MMVOpen compound libraries to identify novel leads for eumycetoma.
METHODS
A total of 400 compounds from the COVID Box and the Global Health Priority Box were screened in vitro at 100 µM and 25 µM against the most common causative agents of eumycetoma, namely and and the resulting IC and MIC values were obtained. Compounds with an IC < 8 µM were identified for possible in vivo efficacy studies using an grain model in larvae.
RESULTS
Out of the 400 compounds, 22 were able to inhibit both and growth at 100 µM and 25 µM, with compounds MMV1593278, MMV020335, and MMV1804559 being selected for in vivo testing. Of these three, only the pyrazolopyrimidine derivative MMV1804559 was able to prolong the survival of -infected larvae. Furthermore, the grains in MMV1804559-treated larvae were significantly smaller compared to the PBS-treated group.
CONCLUSION
MMV1804559 shows promising in vitro and in vivo activity against .
Topics: Madurella; Mycetoma; Antifungal Agents; Animals; Microbial Sensitivity Tests; Larva; Humans
PubMed: 38892422
DOI: 10.3390/ijms25116227 -
Colloids and Surfaces. B, Biointerfaces Jun 2021In this study, a laccase from Madurella mycetomatis (MmLac) was produced heterologously in Pichia pastoris; the initial immobilization in a metal-organic framework (MOF)...
In this study, a laccase from Madurella mycetomatis (MmLac) was produced heterologously in Pichia pastoris; the initial immobilization in a metal-organic framework (MOF) (MmLac/ZIF-8) was achieved using zinc nitrate and 2-methylimidazole. Due to the instability of MmLac/ZIF-8 in an acidic medium, a silica layer was created on the surface of MmLac/MOF-8. The immobilized laccase composite (silica@MmLac/ZIF-8) obtained was further treated with glutaraldehyde (silica@Glu-MmLac/ZIF-8) to increase stability of composite. Fourier-transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy techniques were used to confirm the immobilization of MmLac and to investigate the morphology of the immobilized laccase samples. The MmLac samples were also characterised in terms of optimum pH, temperature and thermal stability. The optimum pH of all the MmLac samples was determined to be 4.0. The free MmLac showed maximum activity at 55 °C, whereas both silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 were maximumly active at 65 °C. The silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 were 9.3- and 11.8-fold higher in stability, respectively, than the free MmLac at 65 °C. Furthermore, both silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 showed a higher bleaching performance than free MmLac on cotton woven fabric. According to these results, silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 may be promising candidates for biocatalysts in laccase-based biotechnological applications.
Topics: Enzyme Stability; Enzymes, Immobilized; Laccase; Madurella; Nanocomposites; Saccharomycetales; Silicon Dioxide
PubMed: 33690061
DOI: 10.1016/j.colsurfb.2021.111672 -
PLoS Neglected Tropical Diseases Aug 2022Eumycetoma is a chronic debilitating fungal disease endemic to tropical and subtropical regions, with Sudan featuring the highest eumycetoma incidence. Among the 50...
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.
Topics: Animals; Madurella; Metagenomics; Mycetoma; Neglected Diseases; Soil
PubMed: 36040926
DOI: 10.1371/journal.pntd.0010385 -
PLoS Neglected Tropical Diseases Apr 2020Mycetoma is a neglected chronic and granulomatous infection primarily associated with the fungal pathogen Madurella mycetomatis. Characteristic of this infection is the...
Mycetoma is a neglected chronic and granulomatous infection primarily associated with the fungal pathogen Madurella mycetomatis. Characteristic of this infection is the formation of grains. However, the processes leading to grain formation are not known. In this study, we employed a proteomic approach to characterise M. mycetomatis grain formation in Galleria mellonella larvae and map the processes leading to grain formation over time. For this, at 1 day, 3 days and 7 days post-inoculation, proteins from grains and hemolymph were extracted and analysed by label-free mass spectrometry. A total of 87, 51 and 48 M. mycetomatis proteins and 713, 997, 18 G. mellonella proteins were found in grains on day 1, 3 and 7 post-inoculation respectively. M. mycetomatis proteins were mainly involved in cellular metabolic processes and numerous enzymes were encountered. G. mellonella proteins were primarily involved in the nodulation process. The proteins identified were linked to nodulation and grain formation and four steps of grain formation were identified. The results of this proteomic approach could in the future be used to design novel strategies to interfere with mycetoma grain formation and to combat this difficult to treat infection.
Topics: Animals; Disease Models, Animal; Larva; Madurella; Moths; Mycetoma; Proteomics
PubMed: 32267851
DOI: 10.1371/journal.pntd.0008190 -
Molecular Biotechnology Jan 2021Laccases are polyphenol oxidoreductases used in a number of industrial applications. Due to the increasing demand for these "green catalysis" enzymes, the identification...
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.
Topics: Biotechnology; Bleaching Agents; Catalysis; Cloning, Molecular; Enzyme Stability; Gene Expression; Hydrogen-Ion Concentration; Kinetics; Laccase; Madurella; Recombinant Proteins; Saccharomycetales; Temperature
PubMed: 33058020
DOI: 10.1007/s12033-020-00281-9 -
PLoS Neglected Tropical Diseases Aug 2023Mycetoma 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 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.
Topics: Male; Humans; Female; Adult; Mycetoma; Kenya; Agar; RNA, Ribosomal, 16S; Polymerase Chain Reaction; Madurella
PubMed: 37578968
DOI: 10.1371/journal.pntd.0011327 -
Mycoses May 2023Mycetoma is a neglected tropical implantation disease caused by 70 different infectious agents. Identifying the causative organism to the species level is essential for...
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.
Topics: Humans; Mycetoma; Cross-Sectional Studies; Sudan; Polymerase Chain Reaction; Madurella; Diagnostic Tests, Routine
PubMed: 36583225
DOI: 10.1111/myc.13561