Every single conidium in Aspergillus fumigatus caspofungin tolerant strains are intrinsically caspofungin tolerant

Aspergillus fumigatus is a human fungal pathogen that causes a disease named aspergillosis. Echinocandins, such as the fungistatic drug caspofungin (CAS) are used as second-line therapy. Some A. fumigatus clinical isolates can survive and grow in higher CAS concentrations, a phenomenon known as “caspofungin paradoxical effect” (CPE). Here we investigate if CPE is due to a subpopulation of conidia produced by a CAS tolerant strain, indicative of a persistence phenotype or is caused by all the conidia which would be consistent with a tolerance phenotype. We evaluated 67 A. fumigatus clinical isolates for CPE growth and used a novel CPE Index (CPEI) classified them as CPE⁺ (CPEI ≥ 0.40) or CPE⁻ (CPEI ≤ 0.20). Conidia produced by three CPE⁺ clinical isolates, CEA17 (CPEI=0.52), Af293 (CPEI=0.64), CM7555 (CPEI=0.58) all showed the ability to grow in high levels of CAS while all conidia produced by the CPE⁻ isolate IFM61407 (CPEI=0.12) strain showed no evidence of tolerance. Given the importance of calcium/calcineurin/transcription factor CrzA pathway in CPE regulation, we also evaluated ΔcrzA^Af293 (CPE⁻) and ΔcrzA^CEA17 (CPE⁺) conidia tolerance to CAS. All ΔcrzA^CEA17 conidia showed CPE⁺ while 100 % of ΔcrzA^Af293 spores are CPE⁻. As all spores derived from an individual strain are phenotypically indistinct with respect to CPE it is likely that CPE is a genetically encoded adaptive trait that should be considered an antifungal tolerant phenotype. As the CPEI shows that the strength of the CPE is not uniform between strains we propose that the mechanisms that govern this phenomenon are multi-factorial.