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

Abstract

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.

Keywords: CYP51A; Madurella mycetomatis; azoles; epidemiological cut-off values; eumycetoma; mycetoma.

FIGURE 1

Minimum spanning tree showing 131 M. mycetomatis isolates obtained from unique patients. Each circle represents a genotype, and the size is directly proportional to the number of isolates within a genotype. The number and thickness of the lines between the circles are directly proportional to the differences in STR markers between genotypes. In total, 56 different genotypes are noted. With blue coloured circles indicate isolates which harbour the CYP51A‐499S variant. The yellow coloured circles indicate isolates which harbour the CYP51A‐499G genotype.

FIGURE 2

Two CYP51A variants are available in the M. mycetomatis wild‐type (WT) population: The CYP51A (eburicol 14‐alpha‐demethylase) DNA sequence of Madurella mycetomatis. It is 1726 bp long and contains two introns of 62 and 74 bp, which are underlined and highlighted in yellow, with three exons of 246, 198 and 1146 bp. Based on sequence analysis, two variants of CYP51A are present in the population.

FIGURE 3

Mutations in the M. mycetomatis wild‐type (WT) population: (A) There are five synonymous mutations in the population analysed (white bars) with one nonsynonymous mutation at position 1631 (dark‐grey bar). (B) Based on this mutation, sequence analysis reveals two variants of CYP51A present in the population; CYP51A‐499S, which is dominant in the population and CYP51A‐499G. Serine is present at position 499 in the CYP51A‐499S genotype, while glycine is present in the CYP51A‐499G genotype.

FIGURE 4

CYP51A variants do not alter susceptibility towards azoles. (A &B) Depiction of the protein structures of the two variants of the CYP51A of M. mycetomatis. The main protein structure in the two variants is represented by ribbons, while the haem group is shown as ball‐and‐stick. The position where the two variants differ (499) is highlighted by arrows; this is further from the haem group and hence unlikely to affect drug interaction. The CYP51A variants did not alter the susceptibility to either itraconazole (C) or ravuconazole (D). There was no significant difference (Mann–Whitney) between the two variants, with a median MIC of 0.064 mg/L and 0.032 mg/L for itraconazole (p = .82) and a similar median MIC (0.008 mg/L) for ravuconazole (p = .87).