Development and Validation of an
            <i>In Vitro</i>
            Resazurin-Based Susceptibility Assay against
            <i>Madurella mycetomatis</i>

Algaffar, S. O. Abd; Verbon, Annelies; Sande, Wendy W. J. van de; Khalid, Sami A.

doi:10.1128/aac.01338-20

Cited by 15 publications

(17 citation statements)

References 13 publications

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“…For in vitro susceptibility testing, experiments were performed as previously described 29 . Briefly, approximately 3 cm fungal mycelia were transferred to 15 ml RMPI 1640 medium (Lonza) supplemented with 0.35 g/L L‐glutamine and 1.98 mM 4‐morpholinepropanesulfonic acid (MOPS).…”

Section: Methodsmentioning

confidence: 99%

“…For in vitro susceptibility testing, experiments were performed as previously described. 29 Briefly, approximately 3 cm fungal mycelia were transferred to 15 ml RMPI 1640 medium (Lonza) supplemented with 0.35 g/L L-glutamine and 1.98 mM 4-morpholinepropanesulfonic acid (MOPS). This was followed by sonication of the fungal mycelium for 10 s at 10 microns (Beun de Ronde), after which 10 ml of RMPI medium was added.…”

Section: In Vitro Susceptibility Testingmentioning

confidence: 99%

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Eumycetoma causative agents are inhibited in vitro by luliconazole, lanoconazole and ravuconazole

Nyuykonge

Lim

Amelsvoort

et al. 2022

Mycoses

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Introduction Eumycetoma is a subcutaneous mutilating disease that can be caused by many different fungi. Current treatment consists of prolonged itraconazole administration in combination with surgery. In many centres, due to their slow growth rate, the treatment for eumycetoma is often started before the causative agent is identified. This harbours the risk that the causative fungus is not susceptible to the given empirical therapy. In the open‐source drug program MycetOS, ravuconazole and luliconazole were promising antifungal agents that were able to inhibit the growth of Madurella mycetomatis, the most common causative agent of mycetoma. However, it is currently not known whether these drugs inhibit the growth of other eumycetoma causative agents. Materials and methods Here, we determined the in vitro activity of luliconazole, lanoconazole and ravuconazole against commonly encountered eumycetoma causative agents. MICs were determined for lanoconazole, luliconazole and ravuconazole against 37 fungal isolates which included Madurella species, Falciformispora senegalensis, Medicopsis romeroi and Trematosphaeria grisea and compared to those of itraconazole. Results Ravuconazole, luliconazole and lanoconazole showed high activity against all eumycetoma causative agents tested with median minimal inhibitory concentrations (MICs) ranging from 0.008–2 µg/ml, 0.001–0.064 µg/ml and 0.001–0.064 µg/ml, respectively. Even Ma. fahalii and Me. romeroi, which are not inhibited in growth by itraconazole at a concentration of 4 µg/ml, were inhibited by these azoles. Conclusion The commonly encountered eumycetoma causative agents are inhibited by lanoconazole, luliconazole and ravuconazole. These drugs are promising candidates for further evaluation as potential treatment for eumycetoma.

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Section: Methodsmentioning

confidence: 99%

Section: In Vitro Susceptibility Testingmentioning

confidence: 99%

Eumycetoma causative agents are inhibited in vitro by luliconazole, lanoconazole and ravuconazole

Nyuykonge

Lim

Amelsvoort

et al. 2022

Mycoses

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“…The final inoculum was prepared from the homogenized fungal suspension mixed with RPMI medium to obtain a transmission of 70% at 660 nm (Novaspec II; Pharmacia Biotech) [15,16]. The screening procedure was performed in 96 well microplates and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) or resazurin was used to facilitate end-point reading [10,17]. Initial screening of the compounds was performed at the concentration of 25 μM.…”

Section: Screening the Pandemic Response Boxmentioning

confidence: 99%

Screening the pandemic response box identified benzimidazole carbamates, Olorofim and ravuconazole as promising drug candidates for the treatment of eumycetoma

et al. 2022

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Eumycetoma is a chronic subcutaneous neglected tropical disease that can be caused by more than 40 different fungal causative agents. The most common causative agents produce black grains and belong to the fungal orders Sordariales and Pleosporales. The current antifungal agents used to treat eumycetoma are itraconazole or terbinafine, however, their cure rates are low. To find novel drugs for eumycetoma, we screened 400 diverse drug-like molecules from the Pandemic Response Box against common eumycetoma causative agents as part of the Open Source Mycetoma initiative (MycetOS). 26 compounds were able to inhibit the growth of Madurella mycetomatis, Madurella pseudomycetomatis and Madurella tropicana, 26 compounds inhibited Falciformispora senegalensis and seven inhibited growth of Medicopsis romeroi in vitro. Four compounds were able to inhibit the growth of all five species of fungi tested. They are the benzimidazole carbamates fenbendazole and carbendazim, the 8-aminoquinolone derivative tafenoquine and MMV1578570. Minimal inhibitory concentrations were then determined for the compounds active against M. mycetomatis. Compounds showing potent activity in vitro were further tested in vivo. Fenbendazole, MMV1782387, ravuconazole and olorofim were able to significantly prolong Galleria mellonella larvae survival and are promising candidates to explore in mycetoma treatment and to also serve as scaffolds for medicinal chemistry optimisation in the search for novel antifungals to treat eumycetoma.

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“…Itraconazole was included as a reference. All molecules were tested in serial dilution against M. mycetomatis cultures [ 12 ]. The minimal inhibitory concentration (MIC) was defined as the lowest concentration that, after 7 days of incubation, had inhibited the growth by at least 80% compared to untreated cultures.…”

Section: Resultsmentioning

confidence: 99%

“…Exactly 100 µL of adjusted fungal suspension was added to each well along with 1 µL of the drug. Next, 1 µL of resazurin was added to a final concentration of 0.15 mg/mL [ 12 ]. The plates were sealed and incubated at 37 °C for 7 days.…”

Section: Methodsmentioning

confidence: 99%

Niclosamide Is Active In Vitro against Mycetoma Pathogens

et al. 2021

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Redox-active drugs are the mainstay of parasite chemotherapy. To assess their repurposing potential for eumycetoma, we have tested a set of nitroheterocycles and peroxides in vitro against two isolates of Madurella mycetomatis, the main causative agent of eumycetoma in Sudan. All the tested compounds were inactive except for niclosamide, which had minimal inhibitory concentrations of around 1 µg/mL. Further tests with niclosamide and niclosamide ethanolamine demonstrated in vitro activity not only against M. mycetomatis but also against Actinomadura spp., causative agents of actinomycetoma, with minimal inhibitory concentrations below 1 µg/mL. The experimental compound MMV665807, a related salicylanilide without a nitro group, was as active as niclosamide, indicating that the antimycetomal action of niclosamide is independent of its redox chemistry (which is in agreement with the complete lack of activity in all other nitroheterocyclic drugs tested). Based on these results, we propose to further evaluate the salicylanilides, niclosamidein particular, as drug repurposing candidates for mycetoma.

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Development and Validation of an In Vitro Resazurin-Based Susceptibility Assay against Madurella mycetomatis

Cited by 15 publications

References 13 publications

Eumycetoma causative agents are inhibited in vitro by luliconazole, lanoconazole and ravuconazole

Eumycetoma causative agents are inhibited in vitro by luliconazole, lanoconazole and ravuconazole

Screening the pandemic response box identified benzimidazole carbamates, Olorofim and ravuconazole as promising drug candidates for the treatment of eumycetoma

Niclosamide Is Active In Vitro against Mycetoma Pathogens

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