Perfil clínico-epidemiológico das infecções causadas por Trichosporon spp. em um hospital pediátrico de referência e busca de novas estratégias de controle do crescimento fúngico

Abstract

Invasive fungal infections (IFIs) involve contamination of the bloodstream and invasion of sterile tissues and/or organs and are considered highly relevant public health issues due to high morbidity and mortality rates. Trichosporon spp. are considered opportunistic fungi, as they take advantage of microbial dysbiosis and host immune imbalance to cause serious invasive infections. This study investigated the clinical and epidemiological profile of invasive infections by Trichosporon spp., as well as the impact of calcineurin and Hsp90 protein inhibition on the growth of this pathogen. The epidemiological study included 28 isolates of interest. Data from these patients were collected and analyzed regarding: age, sex, most prevalent species of the genus, distribution by hospital unit, reasons for care, use of invasive devices, clinical specimens from which pathogens were isolated, use of antimicrobials and other conditioning drugs, co-infection, and treatment measures – all of which were associated with the clinical outcome (discharge or death). The strains isolated and selected in the prospective phase (n=12) were studied for their ability to form biofilms and their sensitivity profile to amphotericin B (AMB), voriconazole (VRZ), and fluconazole (FLZ). The second phase of the work concerns the inhibition of calcineurin by the use of Cyclosporine A (CsA), against planktonic cells and biofilms of Trichosporon spp. strains in n=13 already contained in stock. These biofilms were formed in the presence of CsA at concentrations of 25 µg/mL, 50 µg/mL, and 100 µg/mL for the planktonic state and 50 µg/mL and 100 µg/mL for the biofilms. Subsequently, CsA at the same concentrations mentioned above were associated with the antifungals AMB (10 µg/mL), VRZ (50 µg/mL), and FLZ (64 µg/mL) and subjected to the same tests. The impact of CsA on the ultrastructure of the biofilms was evaluated by Scanning Electron Microscopy (SEM) both in isolation and in combination with the antifungals. Bioinformatics analyses were performed to investigate the antibiofilm potential caused by calcineurin inhibition in T. asahii. In the inhibition of Hsp90 using Radicicol (RAD), the strains were subjected to planktonic sensitivity testing and subsequently studied with mature biofilms (48h) exposed to RAD at concentrations of 6.25 µg/mL for T. asahii and 10 µg/mL for T. inkin. Then, the same concentrations of RAD in the mature biofilms were associated with AMB 4 µg/mL for T. asahii and 2 µg/mL for T. inkin and VRZ 0.125 µg/mL for an additional 24h in contact with the mature biofilm in vitro, the morphology of the biofilms was assessed by SEM, and virulence was evaluated using an experimental infection model with Galleria mellonella larvae. The epidemiological study showed that the most affected pediatric population over the years were boys, most of whom were admitted to Intensive Care Units (ICUs). Patients with neurological diseases and cancer; those using various invasive medical devices, such as feeding tubes; most samples isolated from urine; a significant portion used prophylactic antimicrobials; mortality rates are considerable; the most prevalent species is T. asahii; the strains obtained are capable of forming biofilms. CsA and RAD, isolated and in combination with antifungals, are able to inhibit the fungal growth of T. inkin and T. asahii in vitro and significantly alter the ultrastructure of biofilms. RAD is effective in treating infected G. mellonella larvae. These results suggest new and promising therapeutic targets for invasive trichosporonosis, considering the difficulty and resistance in treating these infections