Investigação da heterorresistência ao fluconazol de isolados clínicos e ambientais do Complexo de Espécies Cryptococcus neoformans e Complexo de Espécies Cryptococcus gattii do Estado do Amazonas

Abstract

Heteroresistance is a phenomenon that has been described when an apparently homogeneous population has different degrees of susceptibility to a particular antifungal. The aim of this study was to investigate the heteroresistance to fluconazole of clinical and environmental isolates of theCryptococcus neoformans / Cryptococcus gattii species complex in the State of Amazonas/Brazil. Were analyzed 45 isolates and 09 standard strains. The clinical isolates came from the Dr Heitor Vieira Dourado Tropical Medicine Foundation (FMT-HVD), obtained between the years 2017 to 2019. The isolates of environmental origin belong to the collection of fungi of medical interest of the National Research Institute of the Amazon that were isolated from samples in the city of Manaus/Amazonas/Brazil.The tests performed were the determination of the minimum inhibitory concentration (MIC), test to determine the level of resistance to fluconazole (LHF), test of adaptation to FCL levels above NHF (ADP) and the heteroresistance stability test. The clinical isolates of the Cryptococcus gattii complex showed MIC higher than the environmental isolates. All clinical and environmental isolates showed heteroresistance to fluconazole and a significant part presented NHF values above 16 µg / mL. In the adaptation experiment, a significant part of the isolates were able to show growth in concentrations of fluconazole even higher than NHF. After nine replications in culture media without fluconazole, the isolate submitted to the adaptation experiment had their NHF reduced, however, in the period, no NHF returned to the original NHF value. The experiments showed that, on average, the NHF value of the isolates was 15,9 times greater than the original MIC. The experiments also demonstrated that after the adaptation experiment, the NHF value was 59,3 times greater than the original MIC value. Therefore, most of the isolates had NHF values well above 16 µg / mL. These results call into question the clinical relevance of the MIC test and motivate the development of in vivo studies to measure the impact of heteroresistance in the course of cryptococcosis.