Synergistic Antifungal Effect and In Vivo Toxicity of a Monoterpene Isoespintanol Obtained from Oxandra xylopioides Diels.

Candida sp. infections are a threat to global health, with high morbidity and mortality rates due to drug resistance, especially in immunocompromised people. For this reason, the search for new alternatives is urgent, and in recent years, a combined therapy with natural compounds has been proposed. Considering the biological potential of isoespintanol (ISO) and continuing its study, the objective of this research was to assess the effect of ISO in combination with the antifungals fluconazole (FLZ), amphotericin B (AFB) and caspofungin (CASP) against clinical isolates of C. tropicalis and to evaluate the cytotoxic effect of this compound in the acute phase (days 0 and 14) and chronic phase (days 0, 14, 28, 42, 56, 70 and 84) in female mice (Mus musculus) of the Balb/c lineage. The results show that ISO can potentiate the effect of FLZ, AFB and CASP, showing synergism with these antifungals. An evaluation of the mice via direct observation showed no behavioral changes or variations in weight during treatment; furthermore, an analysis of the cytokines IFN-γ and TNF in plasma, peritoneal cavity lavage (PCL) and bronchoalveolar lavage (BAL) indicated that there was no inflammation process. In addition, histopathological studies of the lungs, liver and kidneys showed no signs of toxicity caused by ISO. This was consistent with an analysis of oxaloacetic transaminases (GOT) and pyruvic transaminases (GPT), which remained in the standard range. These findings indicate that ISO does not have a cytotoxic effect at the doses evaluated, placing it as a monoterpene of interest in the search for compounds with pharmacological potential.

Brilacidin, a novel antifungal agent against Cryptococcus neoformans.

Cryptococcus neoformans causes cryptococcosis, one of the most prevalent fungal diseases, generally characterized by meningitis. There is a limited and not very effective number of drugs available to combat this disease. In this manuscript, we show the host defense peptide mimetic brilacidin (BRI) as a promising antifungal drug against C. neoformans. BRI can affect the organization of the cell membrane, increasing the fungal cell permeability. We also investigated the effects of BRI against the model system Saccharomyces cerevisiae by analyzing libraries of mutants grown in the presence of BRI. In S. cerevisiae, BRI also affects the cell membrane organization, but in addition the cell wall integrity pathway and calcium metabolism. In vivo experiments show BRI significantly reduces C. neoformans survival inside macrophages and partially clears C. neoformans lung infection in an immunocompetent murine model of invasive pulmonary cryptococcosis. We also observed that BRI interacts with caspofungin (CAS) and amphotericin (AmB), potentiating their mechanism of action against C. neoformans. BRI + CAS affects endocytic movement, calcineurin, and mitogen-activated protein kinases. Our results indicate that BRI is a novel antifungal drug against cryptococcosis. IMPORTANCE: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Cryptococcosis, one of the most prevalent fungal diseases, is generally characterized by meningitis and is mainly caused by two closely related species of basidiomycetous yeasts, Cryptococcus neoformans and Cryptococcus gattii. There are few therapeutic options for treating cryptococcosis, and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a potential antifungal agent against C. neoformans. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. BRI alone was shown to inhibit the growth of C. neoformans, acting as a fungicidal drug, but surprisingly also potentiated the activity of caspofungin (CAS) against this species. We investigated the mechanism of action of BRI and BRI + CAS against C. neoformans. We propose BRI as a new antifungal agent against cryptococcosis.

Microevolution of Candida glabrata (Nakaseomyces glabrata) during an infection.

Candida glabrata (Nakaseomyces glabrata) is an emergent and opportunistic fungal pathogen that colonizes and persists in different niches within its human host. In this work, we studied five clinical isolates from one patient (P7), that have a clonal origin, and all of which come from blood cultures except one, P7-3, obtained from a urine culture. We found phenotypic variation such as sensitivity to high temperature, oxidative stress, susceptibility to two classes of antifungal agents, and cell wall porosity. Only isolate P7-3 is highly resistant to the echinocandin caspofungin while the other four isolates from P7 are sensitive. However, this same isolate P7-3, is the only one that displays susceptibility to fluconazole (FLC), while the rest of the isolates are resistant to this antifungal. We sequenced the PDR1 gene which encodes a transcription factor required to induce the expression of several genes involved in the resistance to FLC and found that all the isolates encode for the same Pdr1 amino acid sequence except for the last isolate P7-5, which contains a single amino acid change, G1099C in the putative Pdr1 transactivation domain. Consistent with the resistance to FLC, we found that the CDR1 gene, encoding the main drug efflux pump in C. glabrata, is highly overexpressed in the FLC-resistant isolates, but not in the FLC-sensitive P7-3. In addition, the resistance to FLC observed in these isolates is dependent on the PDR1 gene. Additionally, we found that all P7 isolates have a different proportion of cell wall carbohydrates compared to our standard strains CBS138 and BG14. In P7 isolates, mannan is the most abundant cell wall component, whereas ß-glucan is the most abundant component in our standard strains. Consistently, all P7 isolates have a relatively low cell wall porosity compared to our standard strains. These data show phenotypic and genotypic variability between clonal isolates from different niches within a single host, suggesting microevolution of C. glabrata during an infection.

Rise and fall of Caspofungin: the current status of Caspofungin as a treatment for Cryptococcus neoformans infection.

Antifungal infections are becoming a major concern to human health due to antimicrobial resistance. Echinocandins have been promising agents against resistant fungal infections, primarily caspofungin, which has a more effective mechanism of action than azoles and polyenes. However, fungi such as Cryptococcus neoformans appear to be inheritably resistant to these drugs, which is concerning due to the high clinical importance of C. neoformans. In this review, we review the history of C. neoformans and the treatments used to treat antifungals over the years, focusing on caspofungin, while highlighting the C. neoformans problem and possible explanations for its inherent resistance.

Caspofungin is a drug used to treat several types of fungal infections. Resistance to caspofungin is a huge problem, especially in those that are immunocompromised. It is important to understand the history of caspofungin discovery, its clinical applications and its mechanism of action, as well as if a new drug target could be used overcome resistance. This review may perform guide new studies combining caspofungin with other drugs and indicate new potential targets for caspofungin.

Helium Cold Atmospheric Plasma Causes Morphological and Biochemical Alterations in Candida albicans Cells.

(1) Background: Previous studies reported the promising inhibitory effect of cold atmospheric plasma (CAP) on Candida albicans. However, the exact mechanisms of CAP's action on the fungal cell are still poorly understood. This study aims to elucidate the CAP effect on C. albicans cell wall, by evaluating the alterations on its structure and biochemical composition; (2) Methods: C. albicans cells treated with Helium-CAP were analyzed by atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) in order to detect morphological, topographic and biochemical changes in the fungal cell wall. Cells treated with caspofungin were also analyzed for comparative purposes; (3) Results: Expressive morphological and topographic changes, such as increased roughness and shape modification, were observed in the cells after CAP exposure. The alterations detected were similar to those observed after the treatment with caspofungin. The main biochemical changes occurred in polysaccharides content, and an overall decrease in glucans and an increase in chitin synthesis were detected; (4) Conclusions: Helium-CAP caused morphological and topographic alterations in C. albicans cells and affected the cell wall polysaccharide content.

A host defense peptide mimetic, brilacidin, potentiates caspofungin antifungal activity against human pathogenic fungi.

Fungal infections cause more than 1.5 million deaths a year. Due to emerging antifungal drug resistance, novel strategies are urgently needed to combat life-threatening fungal diseases. Here, we identify the host defense peptide mimetic, brilacidin (BRI) as a synergizer with caspofungin (CAS) against CAS-sensitive and CAS-resistant isolates of Aspergillus fumigatus, Candida albicans, C. auris, and CAS-intrinsically resistant Cryptococcus neoformans. BRI also potentiates azoles against A. fumigatus and several Mucorales fungi. BRI acts in A. fumigatus by affecting cell wall integrity pathway and cell membrane potential. BRI combined with CAS significantly clears A. fumigatus lung infection in an immunosuppressed murine model of invasive pulmonary aspergillosis. BRI alone also decreases A. fumigatus fungal burden and ablates disease development in a murine model of fungal keratitis. Our results indicate that combinations of BRI and antifungal drugs in clinical use are likely to improve the treatment outcome of aspergillosis and other fungal infections.

Pandemic Response Box® library as a source of antifungal drugs against Scedosporium and Lomentospora species.

Scedosporium and Lomentospora species are opportunistic filamentous fungi that cause localized and disseminated infections in immunocompetent and immunocompromised patients. These species are considered resistant fungi due to their low susceptibility to most current antifungal agents used in healthcare settings. The search for new compounds that could work as promising candidate antifungal drugs is an increasing field of interest. In this context, in the present study we screened the Pandemic Response Box® library (Medicines for Malaria Venture [MMV], Switzerland) to identify compounds with antifungal activity against Scedosporium and Lomentospora species. An initial screening of the drugs from this collection at 5 µM was performed using a clinical Scedosporium aurantiacum isolate according to the EUCAST protocol. Compounds with activity against this fungus were also tested against four other species (S. boydii¸ S. dehoogii, S. apiospermum and L. prolificans) at concentrations ranging from 0.078 to 10 µM. Seven compounds inhibited more than 80% of S. aurantiacum growth, three of them (alexidine, amorolfine and olorofim) were selected due to their differences in mechanism of action, especially when compared to drugs from the azole class. These compounds were more active against biofilm formation than against preformed biofilm in Scedosporium and Lomentospora species, except alexidine, which was able to decrease preformed biofilm about 50%. Analysis of the potential synergism of these compounds with voriconazole and caspofungin was performed by the checkerboard method for S. aurantiacum. The analysis by Bliss methodology revealed synergistic effects among selected drugs with caspofungin. When these drugs were combined with voriconazole, only alexidine and amorolfine showed a synergistic effect, whereas olorofim showed an antagonistic effect. Scanning electron microscopy revealed that alexidine induces morphology alterations in S. aurantiacum biofilm grown on a catheter surface. Reactive oxygen species production, mitochondrial activity and surface components were analyzed by fluorescent probes when S. aurantiacum was treated with selected drugs and revealed that some cell parameters are altered by these compounds. In conclusion, alexidine, amorolfine and olorofim were identified as promising compounds to be studied against scedosporiosis and lomentosporiosis.

Aspects related to biofilm production and antifungal susceptibility of clinically relevant yeasts of the genus Trichosporon.

Trichosporonosis corresponds to a systemic fungal disease that leads to high mortality rates and is frequently associated with medical devices. It affects immunosuppressed patients in particular and is strongly linked to acquired human immunodeficiency, organ and tissue transplants, and malignant hematologic diseases such as leukemia and lymphomas. Trichosporon infections have been increasingly reported worldwide; however, little information is available either about their characteristics or the causative microorganism. Thus, the aims of the present study were: to investigate 59 yeasts of the genus Trichosporon by verifying the biofilm formation capacity of isolates; to analyze the susceptibility patterns of planktonic cells against the antifungals fluconazole, itraconazole, amphotericin-B, voriconazole, and caspofungin by comparing European Committee for Antimicrobial Susceptibility Testing (EUCAST) broth microdilution technique with the commercial method Etest; and to assess the susceptibility patterns of biofilm cells (sessile) against the same antifungals through broth microdilution. The ability to form biofilm on the surface of polystyrene plates was noted for all isolates, and 54.3% of samples were considered strong producers. Comparison between the antifungal susceptibility techniques evidenced that Etest showed higher and discordant minimum inhibitory concentrations (MICs) from those obtained by the microdilution method, especially for fluconazole, itraconazole, and caspofungin. Considering the susceptibility of biofilms, most species had high MIC50 and MIC90 against the tested antifungals, showing 4-to-66-fold higher concentrations for amphotericin B and 2-to-33-fold greater concentrations for caspofungin. These results highlight the importance of further studies with Trichosporon spp. for comparison between laboratory findings and in vivo response, considering both the susceptibility tests and the behavior of biofilm cells against drugs.

This study investigated 59 isolates of the medically important yeast Trichosporon in relation to their ability to form biofilms and the susceptibility of biofilms to antifungal agents. All isolates were able to produce biofilms and biofilms showed lower antifungal susceptibility.

Infecciones fúngicas invasivas en niños hospitalizados en un centro de referencia de Uruguay / Invasive fungal infections in children hospitalized at a pediatric reference hospital in Uruguay / Infecções fúngicas invasivas em crianças internadas em um centro hospitalar de referência no Uruguai

Introducción: las infecciones fúngicas invasivas (IFI) son un problema de salud en creciente aumento. Objetivo: describir las características epidemiológicas, microbiológicas y clínicas de los menores de 15 años con IFI hospitalizados en el Hospital Pediátrico, Centro Hospitalario Pereira Rossell entre 2010- 2019. Metodología: estudio retrospectivo, mediante revisión de historias clínicas. Variables: edad, sexo, comorbilidades, factores de riesgo, clínica, patógenos, tratamiento y evolución. Resultados: se registraron 26 casos de IFI en 23 niños. La mediana de edad fue 8 años, de sexo femenino 17, con comorbilidades 17: infección por VIH 5, enfermedad hematooncológica 4. Todos presentaban factores de riesgo para IFI. Las manifestaciones clínicas de sospecha fueron: fiebre en 19, síntomas neurológicos 11, respiratorios 9, gastrointestinales 6, urinarios 2, sepsis/shock en 3. Los agentes identificados fueron: Candida spp en 14, Cryptococcus neoformans complex 8 y Aspergillus fumigatus complex 4. Tratamiento: se indicó fluconazol en 15, asociado a anfotericina B 11. Todas las infecciones por candida fueron sensibles a los azoles. Fallecieron 7 niños, la mediana de edad fue 1 año. En 4 se identificó Candida spp, Aspergillus fumigatus complex 2 y Cryptococcus neoformans complex 1. Conclusiones: las IFI son poco frecuentes, afectan en su mayoría a niños inmunocomprometidos asociando elevada mortalidad. El diagnóstico requiere alto índice de sospecha. Candida spp y Cryptococcus spp fueron los agentes más involucrados. El inicio precoz del tratamiento acorde a la susceptibilidad disponible se asocia a menor mortalidad.

Summary: Introduction: invasive fungal infections (IFI) are an increasing health problem. Objective: describe the epidemiological, microbiological and clinical characteristics of children under 15 years of age with IFI hospitalized at the Pereira Rossell Hospital Center between 2010-2019. Methodology: retrospective study, review of medical records. Variables: age, sex, comorbidities, risk factors, symptoms, pathogens, treatment and evolution. Results: 26 cases of IFI were recorded involving 23 children. Median age 8 years, female 17, comorbidities 17, HIV infection 5, hematological-oncological disease 4. All with risk factors. Suspicion symptoms: fever 19, neurological symptoms 11, respiratory 9, gastrointestinal 6, urinary 2, sepsis / shock 3. Identified agents: Candida spp 14, Cryptococcus neoformans complex 8 and Aspergillus fumigatus complex 4. Treatment: fluconazole 15, associated with amphotericin B 11. All candida infections were sensitive to azoles. 7 died, median age 1 year. In 4, Candida spp was isolated, Aspergillus fumigatus complex in 2 and Cryptococcus neoformans complex in 1. Conclusions: IFI are rare, mostly affecting immunocompromised children, associated with high mortality. The diagnosis requires a high index of suspicion. Candida spp and Cryptococcus spp were the most involved agents. Early treatment according to available susceptibility is associated with lower mortality.

Introdução: as infecções fúngicas invasivas (IFI) são um problema de saúde crescente. Objetivo: descrever as características epidemiológicas, microbiológicas e clínicas de crianças menores de 15 anos com IFI internadas no Centro Hospitalar Pereira Rossell entre 2010 e 2019. Metodologia: estudo retrospectivo, revisão de prontuários. Variáveis: idade, sexo, comorbidades, fatores de risco, sintomas, patógenos, tratamento e evolução. Resultados: foram registrados 26 casos de IFI em 23 crianças. Idade mediana 8 anos, sexo feminino 17, comorbidades 17, infecção por HIV 5, doença hemato-oncológica 4. Todos com fatores de risco. Suspeita clínica: febre 19, sintomas neurológicos 11, respiratórios 9, gastrointestinais 6, urinários 2, sepse/choque 3. Agentes identificados: Candida spp 14, Cryptococcus neoformans complexo 8 e Aspergillus fumigatus complexo 4. Tratamento: fluconazol 15, associado à anfotericina B 11. Todas as infecções por cândida foram sensíveis aos azóis. 7 morreram, idade média de 1 ano. Em 4 das crianças Cândida spp foi isolada, Aspergillus fumigatus complexo em 2 e Cryptococcus neoformans complexo em 1. Conclusões: IFIs são raras, afetando principalmente crianças imunocomprometidas, associadas a alta mortalidade. O diagnóstico requer alto índice de suspeita. Cândida spp e Cryptococcus spp são os agentes mais envolvidos. O tratamento precoce de acordo com a suscetibilidade disponível está associado a menor mortalidade.

Aspergillus fumigatus FhdA Transcription Factor Is Important for Mitochondrial Activity and Codon Usage Regulation during the Caspofungin Paradoxical Effect.

Aspergillus fumigatus is the main etiological agent of aspergillosis. The antifungal drug caspofungin (CSP) can be used against A. fumigatus, and CSP tolerance is observed. We have previously shown that the transcription factor FhdA is important for mitochondrial activity. Here, we show that FhdA regulates genes transcribed by RNA polymerase II and III. FhdA influences the expression of tRNAs that are important for mitochondrial function upon CSP. Our results show a completely novel mechanism that is impacted by CSP.

The Caspofungin Paradoxical Effect is a Tolerant "Eagle Effect" in the Filamentous Fungal Pathogen Aspergillus fumigatus.

Cell responses against antifungals other than resistance have rarely been studied in filamentous fungi, while terms such as tolerance and persistence are well-described for bacteria and increasingly examined in yeast-like organisms. Aspergillus fumigatus is a filamentous fungal pathogen that causes a disease named aspergillosis, for which caspofungin (CAS), a fungistatic drug, is used as a second-line therapy. Some A. fumigatus clinical isolates can survive and grow in CAS concentrations above the minimum effective concentration (MEC), a phenomenon known as "caspofungin paradoxical effect" (CPE). Here, we evaluated the CPE in 67 A. fumigatus clinical isolates by calculating recovery rate (RR) values, where isolates with an RR of ≥0.1 were considered CPE+ while isolates with an RR of <0.1 were classified as CPE-. Conidia produced by three CPE+ clinical isolates, CEA17 (RR = 0.42), Af293 (0.59), and CM7555 (0.38), all showed the ability to grow in high levels of CAS, while all conidia produced by the CPE- isolate IFM61407 (RR = 0.00) showed no evidence of paradoxical growth. Given the importance of the calcium/calcineurin/transcription factor-CrzA pathway in CPE regulation, we also demonstrated that all ΔcrzACEA17 (CPE+) conidia exhibited CPE while 100% of ΔcrzAAf293 (CPE-) did not exhibit CPE. Because all spores derived from an individual strain were 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. Because the RR parameter showed that the strength of the CPE was not uniform between strains, we propose that the mechanisms which govern this phenomenon are multifactorial. IMPORTANCE The "Eagle effect," initially described for bacterial species, which reflects the capacity of some strains to growth above the minimum inhibitory concentration (MIC) of specific antimicrobial agents, has been known for more than 70 years. However, its underlying mechanism of action in fungi is not fully understood and its connection with other phenomena such as tolerance or persistence is not clear yet. Here, based on the characterization of the "caspofungin paradoxical effect" in several Aspergillus fumigatus clinical isolates, we demonstrate that all conidia from A. fumigatus CPE+ strains are able to grow in high levels of the drug while all conidia produced by CPE- strains show no evidence of paradoxical growth. This work fills a gap in the understanding of this multifactorial phenomenon by proposing that CPE in A. fumigatus should be considered a tolerant but not persistent phenotype.

Antifungal susceptibility of Malassezia pachydermatis isolated from the external auditive conduct from dogs, in central Chile.

Background: External otitis is common in dogs, and one of the main agents involved is Malassezia pachydermatis, a yeast belonging to the otic microbiota. Empirical treatment can fail; therefore, it is essential to know the antifungal susceptibility profile to prescribe appropriate treatment, a fact scarcely reported in Chile. Aim: This study aimed to determine the antifungal sensitivity of M. pachydermatis isolated from the external auditory canal of dogs in central Chile. Methods: Ear swabs from 30 dogs (15 healthy and 15 with external otitis) were used. Samples were subjected to cytology and fungal culture. The antifungal susceptibility was determined according to clinical and laboratory standards institute (CLSI) document M44A-2 using the disk diffusion test from amphotericin B, Caspofungin, fluconazole, nystatin, clotrimazole, and voriconazole were used. Results: The prevalence of M. pachydermatis was 66.7% from 8 healthy dogs and 12 with otitis. While fungal culture was not associated with the patient's clinical condition (p = 0.24), the yeast count by cytology was significantly higher in dogs with otitis (p = 0.003). The strains were sensitive to all antifungals except for Caspofungin, where 55% of the strains were resistant. Conclusion: Malassezia pachydermatis is isolated more frequently in dogs with otitis, and the ear cytological examination is useful to differentiate colonized dogs versus dogs with otitis. In addition, most antifungals in vitro are active against this yeast, except Caspofungin, an antifungal used in human medicine. This situation should be further monitored in epidemiological programs to evaluate the possible impact on Chilean public health.

Caspofungin resistance in Candida albicans: genetic factors and synergistic compounds for combination therapies.

Caspofungin and other echinocandins have been used for the treatment of human infections by the opportunistic yeast pathogen, Candida albicans. There has been an increase in infections by non-albicans Candida species such as Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, and Candida auris in clinical or hospital settings. This is problematic to public health due to the increasing prevalence of echinocandin resistant species/strains. This review will present a summary on various studies that investigated the inhibitory action of caspofungin on 1,3-ß-D-glucan synthesis, on cell wall structure, and biofilm formation of C. albicans. It will highlight some of the issues linked to caspofungin resistance or reduced caspofungin sensitivity in various Candida species and the potential benefits of antimicrobial peptides and other compounds in synergy with caspofungin.

Prevalence of biofilms in Candida spp. bloodstream infections: A meta-analysis.

CONTEXT: Candida-related infections are nowadays a serious Public Health Problem emerging multidrug-resistant strains. Candida biofilm also leads bloodstream infections to invasive systemic infections. OBJECTIVE: The present meta-analysis aimed to analyze Candida biofilm rate, type, and antifungal resistance among hospitalized patients between 1995 and 2020. DATA SOURCES: Web of Science, Scopus, PubMed, and Google Scholar databases were searched for English papers using the following medical subject heading terms (MESH): "invasive candidiasis"; "bloodstream infections"; "biofilm formation"; "biofilm-related infections"; "mortality"; and "prevalence". STUDY SELECTION: The major inclusion criteria included reporting the rate of biofilm formation and the prevalence of biofilm-related to Candida species, including observational studies (more exactly, cohort, retrospective, and case-control studies). Furthermore, data regarding the mortality rate, the geographical location of the study set, and the use of anti-fungal agents in clinical isolates were also extracted from the studies. DATA EXTRACTION: Independent extraction of articles by 2 authors using predefined data fields, including study quality indicators. DATA SYNTHESIS: A total of 31 studies from publicly available databases met our inclusion criteria. The biofilm formation in the data set varied greatly from 16 to 100% in blood samples. Most of the studies belonged to Europe (17/31) and Asia (9/31). Forest plot showed a pooled rate of biofilm formation of 80.0% (CI: 67-90), with high heterogeneity (Q = 2567.45, I2 = 98.83, τ2 = 0.150) in random effects model (p < 0.001). The funnel plot and Egger's linear regression test failed to find publication bias (p = 0.896). The mortality rate in Candida-related bloodstream infections was 37.9% of which 70.0% were from biofilm-associated infections. Furthermore, Candida isolates were also characterized in low, intermediate, or high biofilm formers through their level of biofilm mass (crystal violet staining or XTT assays) after a 24h growth. When comparing between countries, statistical differences were obtained (p = 0.0074), showing the lower and higher biofilm prevalence values in Italy and Spain, respectively. The prevalence of low, intermediate, and high biofilms were 36.2, 18.9, and 35.0% (p < 0.0001), respectively. C. tropicalis was the prevalent species in high biofilm formation (67.5%) showing statistically significant differences when compared to other Candida species, except for C. krusei and C. glabrata. Finally, the rates of antifungal resistance to fluconazole, voriconazole, and caspofungin related to biofilm were 70.5, 67.9 and 72.8% (p < 0.001), respectively. CONCLUSIONS: Early detection of biofilms and a better characterization of Candida spp. bloodstream infections should be considered, which eventually will help preserve public health resources and ultimately diminish mortality among patients.

Genotypic, proteomic, and phenotypic approaches to decipher the response to caspofungin and calcineurin inhibitors in clinical isolates of echinocandin-resistant Candida glabrata.

BACKGROUND: Echinocandin resistance represents a great concern, as these drugs are recommended as first-line therapy for invasive candidiasis. Echinocandin resistance is conferred by mutations in FKS genes. Nevertheless, pathways are crucial for enabling tolerance, evolution, and maintenance of resistance. Therefore, understanding the biological processes and proteins involved in the response to caspofungin may provide clues indicating new therapeutic targets. OBJECTIVES: We determined the resistance mechanism and assessed the proteome response to caspofungin exposure. We then evaluated the phenotypic impact of calcineurin inhibition by FK506 and cephalosporine A (CsA) on caspofungin-resistant Candida glabrata isolates. METHODS: Twenty-five genes associated with caspofungin resistance were analysed by NGS, followed by studies of the quantitative proteomic response to caspofungin exposure. Then, susceptibility testing of caspofungin in presence of FK506 and CsA was performed. The effects of calcineurin inhibitor/caspofungin combinations on heat stress (40°C), oxidative stress (0.2 and 0.4 mM menadione) and on biofilm formation (polyurethane catheter) were analysed. Finally, a Galleria mellonella model using blastospores (1 × 109 cfu/mL) was developed to evaluate the impact of the combinations on larval survival. RESULTS: F659-del was found in the FKS2 gene of resistant strains. Proteomics data showed some up-regulated proteins are involved in cell-wall biosynthesis, response to stress and pathogenesis, some of them being members of calmodulin-calcineurin pathway. Therefore, the impact of calmodulin inhibition was explored. Calmodulin inhibition restored caspofungin susceptibility, decreased capacity to respond to stress conditions, and reduced biofilm formation and in vivo pathogenicity. CONCLUSIONS: Our findings confirm that calmodulin-calcineurin-Crz1 could provide a relevant target in life-threatening invasive candidiasis.

Caspofungin Inhibits Mixed Biofilms of Candida albicans and Methicillin-Resistant Staphylococcus aureus and Displays Effectiveness in Coinfected Galleria mellonella Larvae.

Candida albicans and Staphylococcus aureus are pathogens commonly isolated from bloodstream infections worldwide. While coinfection by both pathogens is associated with mixed biofilms and more severe clinical manifestations, due to the combined expression of virulence and resistance factors, effective treatments remain a challenge. In this study, we evaluated the activity of echinocandins, especially caspofungin, against mixed biofilms of C. albicans and methicillin-resistant (MRSA) or methicillin-susceptible S. aureus (MSSA) and their effectiveness in vivo using the Galleria mellonella coinfection model. Although caspofungin (CAS) and micafungin (MFG) inhibited the mixed biofilm formation, with CAS exhibiting inhibitory activity at lower concentrations, only CAS was active against preformed mixed biofilms. CAS significantly decreased the total biomass of mixed biofilms at concentrations of ≥2 µg/ml, whereas the microbial viability was reduced at high concentrations (32 to 128 µg/ml), leading to fungus and bacterium cell wall disruption and fungal cell enlargement. Notably, CAS (20 or 50 mg/kg of body weight) treatment led to an increased survival and improved outcomes of G. mellonella larvae coinfected with C. albicans and MRSA, since a significant reduction of fungal and bacterial burden in larval tissues was achieved with induction of granuloma formation. Our results reveal that CAS can be a therapeutic option for the treatment of mixed infections caused by C. albicans and S. aureus, supporting additional investigation. IMPORTANCE Infections by microorganisms resistant to antimicrobials is a major challenge that leads to high morbidity and mortality rates and increased time and cost with hospitalization. It was estimated that 27 to 56% of bloodstream infections by C. albicans are polymicrobial, with S. aureus being one of the microorganisms commonly coisolated worldwide. About 80% of infections are associated with biofilms by single or mixed species that can be formed on invasive medical devices, e.g., catheter, and are considered a dissemination source. The increased resistance to antimicrobials in bacterial and fungal cells when they are in biofilms is the most medically relevant behavior that frequently results in therapeutic failure. Although there are several studies evaluating treatments for polymicrobial infections associated or not with biofilms, there is still no consensus on an effective antimicrobial therapy to combat the coinfection by bacteria and fungi.

Caspofungin Effects on Electrocardiogram of Mice: An Evaluation of Cardiac Safety.

Caspofungin is an echinocandin, exhibiting efficacy against most Candida species invasive infection. Its cardiotoxicity was reported in isolated rat heart and ventricular myocytes, but in vivo and clinical studies are insufficient. Our objective was to evaluate caspofungin in vivo cardiac effects using an efficacious dose against Candida albicans. Female Swiss mice were infected with C. albicans, and treated with caspofungin, 5 or 10 mg/kg, intraperitoneal along 5 days. Survival rate and colony-forming units (CFU) into vital organs were determined. For cardiac effects study, mice were treated with caspofungin 10 mg/kg, and electrocardiogram (ECG) signal was obtained on C. albicans-infected mice, single dose-treated, and uninfected mice treated along 5 days, both groups to measure ECG intervals. Besides, ECG was also obtained by telemetry on uninfected mice to evaluate heart rate variability (HRV) parameters. The MIC for caspofungin on the wild-type C. albicans SC5314 strain was 0.3 µg/ml, indicating the susceptible. Survival rate increased significantly in infected mice treated with caspofungin compared to mice treated with vehicle. None of the survived infected mice presented positive CFU after treatment with 10 mg/kg. C. albicans infection induced prolongation of QRS, QT, and QTc intervals; caspofungin did not alter this effect. Caspofungin induced increase of PR and an additional increase of QRS after 24 h of a single dose in infected mice. No significant alterations occurred in ECG intervals and HRV parameters of uninfected mice, after caspofungin treatment. Caspofungin showed in vivo cardiac relative safety maintaining its antifungal efficacy against C. albicans.

Eficacia y seguridad de las terapias antifúngicas intravenosas para la aspergilosis invasiva, en pacientes adultos con nefrotoxicidad o intolerancia a la anfotericina B deoxicolato y enfermedad aguda o progresiva (segunda línea de tratamiento) / Efficacy and safety of intravenous antifungal therapies for invasive aspergillosis in adult patients with nephrotoxicity or intolerance to amphotericin B deoxycholate and acute or progressive disease (second-line treatment)

INTRODUCCIÓN: El presente dictamen preliminar expone la evaluación de la eficacia y seguridad de las terapias antifúngicas intravenosas para la aspergilosis invasiva, en pacientes adultos con nefrotoxicidad o intolerancia a la anfotericina B deoxicolato y enfermedad aguda o progresiva. La aspergilosis invasiva (AI) es una infección fúngica altamente letal en hospedadores inmunodeprimidos. Se estima que la tasa de mortalidad es de aproximadamente 30 a 80 %. La anfotericina B deoxicolato es el agente antifúngico (intravenoso) con mayor experiencia de uso para tratar la AI; sin embargo, se asocia con importantes toxicidades que limitan su uso, principalmente la nefrotoxicidad. En EsSalud, los pacientes con AI, nefrotoxicidad o intolerancia a anfotericina B deoxicolato y enfermedad aguda, disponen de caspofungina. Sin embargo, el IETSI recibió una solicitud de uso de isavuconazol intravenoso bajo la argumentación de que este fármaco representa la mejor opción terapéutica para este tipo de pacientes, basado en un mejor perfil de eficacia y seguridad. Considerando que, además del isavuconazol, existen otras alternativas antifúngicas intravenosas indicadas para este tipo de pacientes, algunas de las cuales tienen experiencia de uso en EsSalud, el equipo evaluador del IETSI optó por re-evaluar el problema de decisión y realizar una evaluación de múltiples tecnologías sanitarias con el fin de identificar la opción de tratamiento más efectiva, segura y costo-efectiva para la población de interés. METODOLOGÍA: Se realizó una búsqueda sistemática de literatura con el objetivo de identificar evidencia sobre la eficacia y seguridad de las terapias antifúngicas intravenosas para la aspergilosis invasiva, en pacientes adultos con nefrotoxicidad o intolerancia a la anfotericina B deoxicolato y enfermedad aguda o progresiva. Se utilizaron las bases de datos PubMed, Cochrane Library y LILACS, priorizándose la evidencia proveniente de ensayos clínicos controlados aleatorizados. Asimismo, se realizó una búsqueda dentro de bases de datos pertenecientes a grupos que realizan evaluación de tecnologías sanitarias (ETS) y guías de práctica clínica (GPC), incluyendo el Healthcare Improvement Scotland, el National Institute for Health and Care Excellence (NICE), la Canadian Agency for Drugs and Technologies in Health (CADTH), la Haute Autorité de Santé (HAS), el Institut für Qualität und Wirtschaftlichkeit im Gesundheitswesen (IQWiG), además de la Base Regional de Informes de Evaluación de Tecnologías en Salud de las Américas (BRISA) y páginas web de sociedades especializadas en aspergilosis como American Thoracic Society (ATS), Infectious Diseases Society of America (IDSA), European Society for Clinical Microbiology and Infectious Diseases (ESCMID) y European Conference on Infections in Leukaemia (ECIL). Se hizo una búsqueda adicional en la página web del Registro administrado por la Biblioteca Nacional de Medicina de los Estados Unidos (https://clinicaltrials.gov/) e International Clinical Trial Registry Platform (ICTRP) (https://apps.who.int/trialsearch/), para poder identificar ensayos clínicos en curso o que no hayan sido publicados para, de este modo, disminuir el riesgo de sesgo de publicación. Para que la búsqueda de información pueda ser empleada para responder a la pregunta PICO se utilizaron estrategias de búsqueda que incluyeron términos relacionados a la población de interés, la intervención, y el comparador. Se emplearon términos MeSH1 y términos libre junto con operadores booleanos acordes a cada una de las bases de datos elegidas para la búsqueda. Con la estrategia de búsqueda diseñada para PubMed, se generaron alertas diarias vía correo electrónico con el objetivo de identificar estudios publicados luego del 09 de octubre de 2020. La búsqueda bibliográfica se limitó a GPC, ETS, revisiones sistemáticas con meta-análisis, y ECA que hayan evaluado la pregunta PICO de interés del presente dictamen. Ante la ausencia de ECA, también se buscaron estudios observacionales comparativos. La búsqueda se limitó a estudios en inglés y español. Se excluyeron las series de casos, los reportes de casos, las cartas al editor, los comentarios, las editoriales, los resúmenes de congresos y los estudios in vitro. La selección de los estudios fue llevada a cabo en dos fases. La primera fase consistió en la revisión de los títulos o los resúmenes a través del aplicativo web Rayyan (https://rayyan.qcri.org), que permitió pre-seleccionar los estudios a incluir y/o los que requerían más información para decidir. En la segunda fase se aplicaron de nuevo los criterios de elegibilidad empleando el texto completo de los estudios que fueron pre-seleccionados. RESULTADOS: Se realizó una búsqueda de la literatura con respecto a la eficacia y seguridad de las terapias antifúngicas intravenosas para la aspergilosis invasiva, en pacientes adultos con nefrotoxicidad o intolerancia a la anfotericina B deoxicolato y enfermedad aguda o progresiva. A continuación, se describe la evidencia disponible según el orden jerárquico del nivel de evidencia o pirámide de Haynes 6S. CONCLUSIONES: El presente dictamen preliminar tuvo como objetivo evaluar la mejor evidencia sobre la eficacia y seguridad de las terapias antifúngicas intravenosas para la aspergilosis invasiva, en pacientes adultos con nefrotoxicidad o intolerancia a la anfotericina B deoxicolato y enfermedad aguda o progresiva (segunda línea de tratamiento). Las intervenciones de interés fueron el isavuconazol intravenoso, el voriconazol intravenoso y la L-AmB (intravenosa), y el comparador de interés fue la caspofungina. Los desenlaces de interés fueron la respuesta clínica, microbiológica y radiológica, la sobrevida global, la mortalidad, la calidad de vida y los eventos adversos. La evidencia disponible sobre la mejor alternativa antifúngica para pacientes con AI y nefrotoxicidad o intolerancia a anfotericina B deoxicolato (o tratamientos antifúngicos convencionales en general) es de baja calidad metodológica. La evidencia disponible sugiere, aunque con una gran incertidumbre, que los antifúngicos de interés para la presente evaluación (isavuconazol, voriconazol, L-AmB y caspofungina) tendrían similar eficacia, en términos de respuesta antifúngica, pero con diferentes perfiles de seguridad. De estos, caspofungina es el que tiene más evidencia en un contexto de intolerancia y sus estudios incluyen a la población de interés del presente dictamen preliminar. Caspofungina es mejor tolerado que otras clases de antifúngicos sistémicos, no habiéndose reportado eventos de nefrotoxicidad. En ese sentido, caspofungina sería el fármaco más adecuado para tratar a la población de interés del presente dictamen. De manera adicional, es importante resaltar que optar por el uso de caspofungina, en lugar de los otros antifúngicos evaluados, es una decisión con mejor perfil de costo-oportunidad; ya que este fármaco tiene los costos más asequibles a nivel institucional. Por lo expuesto, el IETSI no aprueba el uso de isavuconazol intravenoso, voriconazol intravenoso y L-AmB (intravenosa) en pacientes adultos con AI, nefrotoxicidad o intolerancia a la anfotericina B deoxicolato y enfermedad aguda o progresiva.

Infección invasiva por Candida spp. En pacientes inmunocomprometidos: Descripción de curso clínico experiencia diagnóstica, manejo y seguimiento en centro de alta complejidad / Invasive infection by Candida spp. in immunocompromised patients: A description of clinical, diagnostics, therapeutic and follow up experience in high complexity healthcare center

Introducción: Candida spp. Es la principal causa de fungemia, cuya incidencia ha aumentado en los últimos años. Existen datos locales insuficientes sobre este tipo de infecciones. Materiales y métodos: Este fue un estudio observacional retrospectivo de 44 pacientes diagnosticados con candidiasis invasiva hospitalizados en la Fundación Valle del Lili, el cual es un centro de cuarto nivel afiliado a la Universidad Icesi en el Suroccidente Colombiano, entre los años 2012 a 2017. Resultados: Se identificaron 44 pacientes con candidiasis invasiva, 27 de ellos mujeres (61%). La mediana de edad fue de 56 años (36 - 70). Más del 50% tenían una enfermedad crónica subyacente, uso de antibióticos (84%), catéter venoso central (80%), ventilación mecánica (68%) y nutrición enteral (66%) El 80% requirió manejo en unidad de cuidados intensivos (UCI) donde debutaron con sepsis (68%) y falla respiratoria (61%). En el 90% de los casos se aisló alguna especie de Candida spp. A partir de hemocultivo y sólo al 22% se le realizó prueba de sensibilidad. El tratamiento de elección fue con fluconazol (80%), asociado a caspofungina (70%). La tasa de mortalidad fue del 49%, con una mediana de 33 (22-49,5) días desde el ingreso hasta el fallecimiento. C. albicans fue el principal microorganismo aislado. La resistencia a azoles en especies no albicans existe en nuestro medio. Conclusión: La candidiasis se presenta como candidemia asociada a infección bacteriana concomitante, que cobra mayor importancia en el contexto del paciente inmunosuprimido asociado a elevadas tasas de mortalidad.

Introduction: Candida spp. is the main cause of fungemia, whose incidence has increased in recent years. There are insufficient local data about this pathology. Materials and methods: This was an observational, retrospective chart review of 44 patients diagnosed with invasive candida who were hospitalized at Fundación Valle del Lili, which is a fourth level center affiliated to Icesi university between 2012 and 2017. Results: We identified 44 patients with invasive candidiasis, 27 of them women (61%). The median age was 56 years (36 - 70). More than 50% had an underlying chronic disease, use of antibiotics (84%), central venous catheter (80%), mechanical ventilation (68%) and enteral nutrition (66%). 80% required management in an intensive care unit. Sepsis (68%) and respiratory failure (61%) were the most common clinical presentation. Almost 90% of the cases, had positive blood cultures, but only 22% presented susceptibility tests. The treatment was mainly fluconazole (80%), associated with caspofungin (70%). The mortality rate was 49%, median of 33 (22-49.5) days from admission to death. Candida albicans was the main isolated organism. Azole resistance in non-albicans species was observed. Conclusion: Candidiasis presents as bacterial infection associated candidemia, which becomes more important in the context of the immunosuppressed patient with high mortality rates.

Aspergillus fumigatus Transcription Factors Involved in the Caspofungin Paradoxical Effect.

Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, in the last 10 years there have been several reports of azole resistance in A. fumigatus and new strategies are needed to combat invasive aspergillosis. Caspofungin is effective against other human-pathogenic fungal species, but it is fungistatic only against A. fumigatus Resistance to caspofungin in A. fumigatus has been linked to mutations in the fksA gene that encodes the target enzyme of the drug ß-1,3-glucan synthase. However, tolerance of high caspofungin concentrations, a phenomenon known as the caspofungin paradoxical effect (CPE), is also important for subsequent adaptation and drug resistance evolution. Here, we identified and characterized the transcription factors involved in the response to CPE by screening an A. fumigatus library of 484 null transcription factors (TFs) in CPE drug concentrations. We identified 11 TFs that had reduced CPE and that encoded proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism, and cell wall remodeling. One of these TFs, FhdA, was important for mitochondrial respiratory function and iron metabolism. The ΔfhdA mutant showed decreased growth when exposed to Congo red or to high temperature. Transcriptome sequencing (RNA-seq) analysis and further experimental validation indicated that the ΔfhdA mutant showed diminished respiratory capacity, probably affecting several pathways related to the caspofungin tolerance and resistance. Our results provide the foundation to understand signaling pathways that are important for caspofungin tolerance and resistance.IMPORTANCEAspergillus fumigatus, one of the most important human-pathogenic fungal species, is able to cause aspergillosis, a heterogeneous group of diseases that presents a wide range of clinical manifestations. Invasive pulmonary aspergillosis is the most serious pathology in terms of patient outcome and treatment, with a high mortality rate ranging from 50% to 95% primarily affecting immunocompromised patients. Azoles have been used for many years as the main antifungal agents to treat and prevent invasive aspergillosis. However, there were several reports of evolution of clinical azole resistance in the last decade. Caspofungin, a noncompetitive ß-1,3-glucan synthase inhibitor, has been used against A. fumigatus, but it is fungistatic and is recommended as second-line therapy for invasive aspergillosis. More information about caspofungin tolerance and resistance is necessary in order to refine antifungal strategies that target the fungal cell wall. Here, we screened a transcription factor (TF) deletion library for TFs that can mediate caspofungin tolerance and resistance. We have identified 11 TFs that are important for caspofungin sensitivity and/or for the caspofungin paradoxical effect (CPE). These TFs encode proteins involved in the basal modulation of the RNA polymerase II initiation sites, calcium metabolism or cell wall remodeling, and mitochondrial respiratory function. The study of those genes regulated by TFs identified in this work will provide a better understanding of the signaling pathways that are important for caspofungin tolerance and resistance.