S4.5c Using serum beta-glucan measurements and sequencing of the Madurella mycetomatis azole target gene to predict therapeutic outcome during azole treatment in human mycetoma
Authors: Nyuykonge Bertrand , Siddig Emmanuel , Mhmoud Najwa , Nyaoke Borna , Zijlstra Ed , Verbon Annelies , Bakhiet Sahar , Fahal Ahmed , Van de Sande Wendy 
Published: September 2022

Abstract

S4.5 Mycetoma Clinical Trial on fosravuconazole treatment in eumycetoma– Top Line Results, September 22, 2022, 10:30 AM - 12:00 PM

Objectives
Eumycetoma is a neglected tropical disease characterized by large subcutaneous swellings and the formation of grains and most commonly caused by Madurella mycetomatis. The currently recommended therapy is a combination of antifungal therapy with an azole and surgery. Itraconazole is the current recommended drug and fosravuconazole, the pro-drug of ravuconzole, is currently clinically investigated. At the moment, there are no epidemiological cut-off values (ECV) for M. mycetomatis for either of these drugs or rapid diagnostic tests which can predict the therapeutic outcome of these treatments. Therefore, in this study, we determined the ECV for these drugs and determined whether there was a correlation between minimal inhibitory concentration (MIC) and the DNA sequence of the azole target gene CYP51A. We also assessed beta-glucan concentrations in the serum of mycetoma patients during treatment to establish whether any of these values were predictive for therapeutic outcomes.

Methods
In order to determine the ECV for M. mycetomatis, MIC distributions for itraconazole and ravuconazole were determined in genetically diverse clinical M. mycetomatis isolates using the ECOFFinder software. CYP51A sequences were sequenced and comparisons were made between the different CYP51A variants and the MIC distributions. Beta-glucan concentrations were measured in serum with the WAKO beta-glucan assay. Time points analyzed were 0, 22, 85, 176, 267, 358, and 455 days after the start of treatment.

Results
For M. mycetomatis 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. The MIC distributions for itraconazole and ravuconazole were similar between the two variants. No mutations linked to decreased susceptibility were found. Before the start of treatment, beta-glucan concentrations ranged from below the detection limit to 217.9 pg/ml. Of these patients, 61.2% had a beta-glucan concentration above 7 pg/ml, the recommended cut-off value for positivity by the manufacturer, 72.8% had a beta-glucan concentration above 5.5 pg/ml, the recommended cut-off value for M. mycetomatis. During the first months of azole treatment, the beta-glucan concentrations remained relatively stable. After surgery, a sharp decrease in beta-glucan concentration in serum was noted. At the end of the observation period, only 13 patients had a beta-glucan concentration above 7 pg/ml and 14 above 5.5 pg/ml. Of these patients, for only 3, there was clinical evidence of a recurrence. For the remaining 4 patients with clinical evidence of a recurrence, the beta-glucan concentration was below the cut-off value for positivity.

Conclusion
In conclusion, so far there was no link established with the initial in vitro susceptibility and failure or success of the treatment therapy. Beta-glucan levels, in general, remained high during azole treatment, and a sharp drop in beta-glucan concentration in serum was only noted after surgery. A positive beta-glucan concentration at the end of the treatment was not indicative of a recurrence.