Antifungal susceptibility testing of Cryptococcus strains from Kinshasa (DRC), intronic substitution of the ERG11 gene in one of the fluconazole resistant strains

Background
The management of cryptococcal meningitis (CM) remains a real challenge specifically in the context of antifungal resistant strains emergence, mainly against fluconazole which is the most widely administered antifungal in poor world regions.
Objective
We focused here on the common used antifungal susceptibility testing of Cryptococcus spp. from people living with HIV (PLHIV) with CM. Moreover, the sterol 14-α-demethylase gene (ERG11) substitutions that would underlie the fluconazole high minimum inhibitory concentrations (MICs) were explored in the relevant strains.
Methods
Twenty-three strains of Cryptococcus neoformans (VNI), five strains of C. curvatus, and one strain of C. laurentii were analysed. MICs were determined according to the EUCAST E.Def 7.3.1 protocol. The ERG11 gene sequence was extracted from the cryptococcal genome NGS data and then analysed in comparison to the wild-type sequence of the C. neoformans ERG11 gene.
Results
Among the included strains, only C. laurentii was resistant to amphotericin B. Strains with high MICs values to 5-flucytosine were identified in 6.8% of cases (2/29), including the single C. laurentii and one C. neoformans isolates. Regarding the strains’ susceptibility to fluconazole, 13.8% (4/29) exhibited high MICs values (16-32 mg/L), among them, two of five (40%) C. curvatus strains and two of 23 (8.7%) C. neoformans strains. No statistical difference of mean MICs values was found comparing the major sequence type (ST)-MLST of C. neoformans strains (ST93) and the less common ST-MLST (ST53, ST31, ST5, ST4, ST659, and ST69). One of the two Cryptococcus neoformans strains showing high MICs to fluconazole wore three ERG11 gene substitutions, including two exonic silent point substitutions and one intronic point substitution located within a potential sequence involved in the splicing of the pre-mRNA (g.315C > T), which is suspected to be the underlying mechanism to this high MIC. The ERG11 gene sequence of Cryptococcus curvatus with high MIC to fluconazole was not analysed due to a lack of her wild-type gene sequence in NCBI database.
Conclusions
While slight disparities in antifungal susceptibility were found between Cryptococcus species, no significant differences were observed within C. neoformans strains with different ST-MLST. We suspect that an intronic point substitution in a sequence that may be involved in the pre-mRNA splicing of the ERG11 gene could be responsible for fluconazole resistance of C. neoformans. We consider that more elaborate and in-depth investigations to draw definitive conclusions are needed.