-
Medical Mycology Nov 2023Mycetoma is a neglected tropical disease commonly caused by the fungus Madurella mycetomatis. Standard treatment consists of extensive treatment with itraconazole in...
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.
Topics: Animals; Itraconazole; Madurella; Mycetoma; Antifungal Agents
PubMed: 37960934
DOI: 10.1093/mmy/myad118 -
PLoS Neglected Tropical Diseases Sep 2023Filamentous fungi of the genus Madurella are the primary causative agents of mycetoma, a disease observed in tropical and subtropical regions. Since early diagnostics...
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.
Topics: Mycetoma; Madurella; Nucleic Acid Amplification Techniques
PubMed: 37721946
DOI: 10.1371/journal.pntd.0011644 -
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 -
PLoS Neglected Tropical Diseases Apr 2024Madurella mycetomatis is the main cause of mycetoma, a chronic granulomatous infection for which currently no adequate therapy is available. To improve therapy, more...
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.
Topics: Hygromycin B; Madurella; Protoplasts; Transformation, Genetic; Drug Resistance, Fungal; Mycetoma; Cinnamates
PubMed: 38578808
DOI: 10.1371/journal.pntd.0012092 -
Medical Mycology Aug 2023Eumycetoma is a subcutaneous implantation mycosis often found in the foot. One of the hallmarks of eumycetoma is the formation of grains. These grains are either black...
Eumycetoma is a subcutaneous implantation mycosis often found in the foot. One of the hallmarks of eumycetoma is the formation of grains. These grains are either black or white, and the consistency and morphology differs per causative agent. The two most common causative agents of black-grain eumycetoma are Madurella mycetomatis and Falciformispora senegalensis. Since grains cannot be formed in vitro, in vivo models are needed to study grain formation. Here, we used the invertebrate Galleria mellonella to establish an in vivo grain model for F. senegalensis. Three different F. senegalensis strains were selected, and four different inocula were used to infect G. mellonella larvae, ranging from 0.04 mg/larvae to 10 mg/larvae. Larval survival was monitored for 10 days. Grain formation was studied macroscopically and histologically. The efficacy of antifungal therapy was determined for itraconazole, amphotericin B, and terbinafine. A concentration of 10 mg F. senegalensis per larva was lethal for the majority of the larvae within 10 days. At this inoculum, grains were formed within 24 h after infection. The grains produced in the larvae resembled those formed in human patients. Amphotericin B given at 1 mg/kg 4 h, 28 h, and 52 h after infection prolonged larval survival. No enhanced survival was noted for itraconazole or terbinafine. In conclusion, we developed a F. senegalensis grain model in G. mellonella larvae in which grains were formed that were similar to those formed in patients. This model can be used to monitor grain formation over time and study antifungal efficacy.
Topics: Humans; Animals; Antifungal Agents; Larva; Amphotericin B; Terbinafine; Itraconazole; Mycetoma; Disease Models, Animal; Moths
PubMed: 37451815
DOI: 10.1093/mmy/myad070 -
Molecules (Basel, Switzerland) Apr 2024In the search for new bioactive agents against the infectious pathogen responsible for the neglected tropical disease (NTD) mycetoma, we tested a collection of 27...
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 , the primary pathogen responsible for the fungal form of mycetoma, termed eumycetoma. Among this series, the EO of (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 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 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 ()-α- and ()-β-santalol were isolated from the oil by column chromatography on AgNO-coated silica. They were tested as isolated compounds against the fungus, and ()-α-santalol was about two times more active than the β-isomer.
Topics: Madurella; Plant Oils; Oils, Volatile; Mycetoma; Santalum; Sesquiterpenes; Antifungal Agents; Microbial Sensitivity Tests
PubMed: 38675665
DOI: 10.3390/molecules29081846 -
Mycopathologia Jan 2024Madurella fahalii is a causative agent of the implantation mycosis mycetoma with decreased susceptibility to itraconazole, the preferred therapeutic drug to combat...
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.
Topics: Itraconazole; Mycetoma; Azoles; Madurella
PubMed: 38231295
DOI: 10.1007/s11046-023-00807-0 -
PLoS Neglected Tropical Diseases Nov 2023Mycetomas are endemic diseases in tropical and sub-tropical countries of Africa, Asia and America, mainly affecting rural populations living below the poverty line. We...
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.
Topics: Female; Humans; Pregnancy; Adult; Mycetoma; Antifungal Agents; Africa; Madurella; Asia
PubMed: 37956163
DOI: 10.1371/journal.pntd.0011372 -
Tropical Medicine and Health Dec 2023Mycetoma is a chronic disease affecting the skin and subcutaneous tissue endemic in the tropical and subtropical regions. Several bacteria and fungi can cause mycetoma,...
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.
PubMed: 38115141
DOI: 10.1186/s41182-023-00563-3