Antifungal resistance: why are we losing this battle?

The emergence of fungal pathogens and changes in the epidemiological landscape are prevalent issues in clinical mycology. Reports of resistance to antifungals have been reported. This review aims to evaluate molecular and nonmolecular mechanisms related to antifungal resistance. Mutations in the ERG genes and overexpression of the efflux pump (MDR1, CDR1 and CDR2 genes) were the most reported molecular mechanisms of resistance in clinical isolates, mainly related to Azoles. For echinocandins, a molecular mechanism described was mutation in the FSK genes. Furthermore, nonmolecular virulence factors contributed to therapeutic failure, such as biofilm formation and selective pressure due to previous exposure to antifungals. Thus, there are many public health challenges in treating fungal infections.

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Determinants of fluconazole resistance and echinocandin tolerance in C. parapsilosis isolates causing a large clonal candidemia outbreak among COVID-19 patients in a Brazilian ICU.

Patients presenting with severe COVID-19 are predisposed to acquire secondary fungal infections such as COVID-19-associated candidemia (CAC), which are associated with poor clinical outcomes despite antifungal treatment. The extreme burden imposed on clinical facilities during the COVID-19 pandemic has provided a permissive environment for the emergence of clonal outbreaks of multiple Candida species, including C. auris and C. parapsilosis. Here we report the largest clonal CAC outbreak to date caused by fluconazole resistant (FLZR) and echinocandin tolerant (ECT) C. parapsilosis. Sixty C. parapsilosis strains were obtained from 57 patients at a tertiary care hospital in Brazil, 90% of them were FLZR and ECT. Although only 35.8% of FLZR isolates contained an ERG11 mutation, all of them contained the TAC1L518F mutation and significantly overexpressed CDR1. Introduction of TAC1L518F into a susceptible background increased the MIC of fluconazole and voriconazole 8-fold and resulted in significant basal overexpression of CDR1. Additionally, FLZR isolates exclusively harboured E1939G outside of Fks1 hotspot-2, which did not confer echinocandin resistance, but significantly increased ECT. Multilocus microsatellite typing showed that 51/60 (85%) of the FLZR isolates belonged to the same cluster, while the susceptible isolates each represented a distinct lineage. Finally, biofilm production in FLZR isolates was significantly lower than in susceptible counterparts Suggesting that it may not be an outbreak determinant. In summary, we show that TAC1L518F and FKS1E1393G confer FLZR and ECT, respectively, in CAC-associated C. parapsilosis. Our study underscores the importance of antifungal stewardship and effective infection control strategies to mitigate clonal C. parapsilosis outbreaks.

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.

Examining Signatures of Natural Selection in Antifungal Resistance Genes Across Aspergillus Fungi.

Certain Aspergillus fungi cause aspergillosis, a set of diseases that typically affect immunocompromised individuals. Most cases of aspergillosis are caused by Aspergillus fumigatus, which infects millions of people annually. Some closely related so-called cryptic species, such as Aspergillus lentulus, can also cause aspergillosis, albeit at lower frequencies, and they are also clinically relevant. Few antifungal drugs are currently available for treating aspergillosis and there is increasing worldwide concern about the presence of antifungal drug resistance in Aspergillus species. Furthermore, isolates from both A. fumigatus and other Aspergillus pathogens exhibit substantial heterogeneity in their antifungal drug resistance profiles. To gain insights into the evolution of antifungal drug resistance genes in Aspergillus, we investigated signatures of positive selection in 41 genes known to be involved in drug resistance across 42 susceptible and resistant isolates from 12 Aspergillus section Fumigati species. Using codon-based site models of sequence evolution, we identified ten genes that contain 43 sites with signatures of ancient positive selection across our set of species. None of the sites that have experienced positive selection overlap with sites previously reported to be involved in drug resistance. These results identify sites that likely experienced ancient positive selection in Aspergillus genes involved in resistance to antifungal drugs and suggest that historical selective pressures on these genes likely differ from any current selective pressures imposed by antifungal drugs.

Biological Properties and Analytical Methods for Micafungin: A Critical Review.

Micafungin is characterized as one of the most active available drugs for candidemia treatment; however, their use is also associated in prophylaxis protocols in cases of invasive fungal infections. The use of this drug is widely appreciated in the medical field due to be the most active echinocandin available for invasive fungal infections. In order to provide important parameters related to the chemical, physical, biological and therapeutic characteristics, this review article gathers important research results that demonstrate the biological potential of this drug, as well as to present analytical methods that can be used to determine the antifungal potential and a monitoring of administered dosages. Important studies about the methods most commonly used in biological activity evaluation and determination/quantification by analytical methods are provided in this review article. With the data provided, the scientific community will have the possibility to choose the analytical methods and biological that can be employed in clinical and scientific research to provide greater safety and reliability of the results to be found.

Caspofungin and Polymyxin B Reduce the Cell Viability and Total Biomass of Mixed Biofilms of Carbapenem-Resistant Pseudomonas aeruginosa and Candida spp.

Pseudomonas aeruginosa and Candida spp. are biofilm-forming pathogens commonly found colonizing medical devices, being mainly associated with pneumonia and bloodstream infections. The coinfection by these pathogens presents higher mortality rates when compared to those caused by a single microbial species. This study aimed to evaluate the antibiofilm activity of echinocandins and polymyxin B (PMB) against polymicrobial biofilms of carbapenem-resistant (CR) Pseudomonas aeruginosa and Candida spp. (C. albicans, C. parapsilosis, C. tropicalis, and C. glabrata). In addition, we tested the antimicrobial effect on their planktonic and monomicrobial biofilm counterparties. Interestingly, beyond inhibition of planktonic [minimum inhibitory concentration (MIC) = 0.5 µg/ml] and biofilm [minimum biofilm inhibitory concentration (MBIC)50 ≤ 2-8 µg/ml] growth of P. aeruginosa, PMB was also effective against planktonic cells of C. tropicalis (MIC = 2 µg/ml), and polymicrobial biofilms of CR P. aeruginosa with C. tropicalis (MBIC50 ≤ 2 µg/ml), C. parapsilosis (MBIC50 = 4-16 µg/ml), C. glabrata (MBIC50 = 8-16 µg/ml), or C. albicans (MBIC50 = 8-64 µg/ml). On the other hand, while micafungin (MFG) showed highest inhibitory activity against planktonic (MIC ≤ 0.008-0.5 µg/ml) and biofilm (MBIC50 ≤ 2-16 µg/ml) growth of Candida spp.; caspofungin (CAS) displays inhibitory activity against planktonic cells (MIC = 0.03-0.25 µg/ml) and monomicrobial biofilms (MBIC50 ≤ 2-64 µg/ml) of Candida spp., and notably on planktonic and monomicrobial biofilms of CR P. aeruginosa (MIC or MBIC50 ≥ 64 µg/ml). Particularly, for mixed biofilms, while CAS reduced significantly viable cell counts of CR P. aeruginosa and Candida spp. at ≥32 and ≥ 2 µg/ml, respectively; PMB was effective in reducing viable cells of CR P. aeruginosa at ≥2 µg/ml and Candida spp. at ≥8 µg/ml. Similar reduction of viable cells was observed for CAS (32-64 µg/ml) combined with PMB (2 µg/ml). These findings highlight the potential of PMB and CAS for the treatment of polymicrobial infections caused by Candida spp. and critical priority CR P. aeruginosa.

Comparative study of Candida spp. isolates: Identification and echinocandin susceptibility in isolates obtained from blood cultures in 15 hospitals in Medellín, Colombia.

OBJECTIVES: Invasive candidiasis has a high impact on morbidity and mortality in hospitalised patients. Accurate and timely methods for identification of Candida spp. and determination of echinocandin susceptibility have become a priority for clinical microbiology laboratories. METHODS: This study was performed to compare matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) identification with sequencing of the D1/D2 region of the rRNA gene complex 28 subunit in 147 Candida spp. isolates obtained from patients with candidaemia. Antimicrobial susceptibility testing was performed by broth microdilution (BMD) and Etest. Sequencing of the FKS1 and FKS2 genes was performed. RESULTS: The most common species isolated were Candida albicans (40.8%), followed by Candida parapsilosis (23.1%) and Candida tropicalis (17.0%). Overall agreement between the results of identification by MALDI-TOF/MS and molecular identification was 99.3%. Anidulafungin and caspofungin susceptibility by the BMD method was 98.0% and 88.4%, respectively. Susceptibility to anidulafungin and caspofungin by Etest was 93.9% and 98.6%, respectively. Categorical agreement between Etest and BMD was 91.8% for anidulafungin and 89.8% for caspofungin, with lower agreements in C. parapsilosis for anidulafungin (76.5%) and C. glabrata for caspofungin (40.0%). No mutations related to resistance were found in the FKS genes, although 54 isolates presented synonymous polymorphisms in the hotspots sequenced. CONCLUSIONS: MALDI-TOF/MS is a good alternative for routine identification of Candida spp. isolates. DNA sequencing of the FKS genes suggested that the isolates analysed were susceptible to echinocandins; alternatively, unknown resistance mechanisms or limitations related to antifungal susceptibility tests may explain the resistance found in a few isolates.

Phenotypic and Molecular Evaluation of Echinocandin Susceptibility of Candida glabrata, Candida bracarensis, and Candida nivariensis Strains Isolated during 30 Years in Argentina.

The echinocandin susceptibilities of 122 Candida glabrata complex strains (including 5 Candida nivariensis and 3 Candida bracarensis strains) were evaluated by microdilution and compared with the results from a molecular tool able to detect FKS mutations. No echinocandin resistance was detected. The PCR results coincide with the MIC data in 99.25% of the cases (1 C. glabrata strain was misidentified as resistant) but were 20 h faster. C. nivariensis FKS genes were sequenced and showed differences with C. glabrataFKS genes.

Identification of Candida parapsilosis Sensu Lato in Pediatric Patients and Antifungal Susceptibility Testing.

A total of 59 Candida parapsilosis sensu stricto and 1 Candida orthopsilosis recovered from catheters and blood cultures of pediatric patients from the northeastern region of Argentina were studied. Susceptibility to azoles, amphotericin B, and echinocandins was tested by the broth microdilution method. According to CLSI clinical breakpoints, >91% of the strains were azole susceptible, whereas 15% showed high amphotericin B MICs.

Molecular Confirmation of the Relationship between Candida guilliermondii Fks1p Naturally Occurring Amino Acid Substitutions and Its Intrinsic Reduced Echinocandin Susceptibility.

Candida guilliermondii shows intrinsic reduced echinocandin susceptibility. It harbors two polymorphisms (L633M and T634A) in the Fks1p hot spot 1 region. Our objective was to confirm that the reduced echinocandin susceptibility of C. guilliermondii is due to those naturally occurring substitutions. We constructed a Saccharomyces cerevisiae mutant in which a region of the FKS1 gene (including hot spot 1) was replaced with that from C. guilliermondii The chimeric mutants showed 32-fold increases in echinocandin MIC values, confirming the hypothesis.

Antifungal activities of diphenyl diselenide and ebselen against echinocandin-susceptible and -resistant strains of Candida parapsilosis.

We evaluated the in vitro antifungal activity of diphenyl diselenide and ebselen against echinocandin-susceptible and -resistant strains of Candida parapsilosis using the broth microdilution method. Diphenyl diselenide (MIC range =1-8 µg/mL) and ebselen (MIC range =0.25-4 µg/mL) showed in vitro activity against echinocandin-susceptible isolates. However, ebselen also showed the highest antifungal activity against echinocandin-resistant strains (MIC range =0.06-4 µg/mL). This study demonstrated that the antifungal potential of diphenyl diselenide and ebselen deserves further investigation using in vivo experimental protocols.

Tratamiento exitoso de una mucormicosis rinocerebral persistente en un paciente pediátrico durante el debut de una leucemia aguda / Successful treatment of a persistent rhino-cerebral mucormycosis in a pediatric patient with a debut of acute lymphoblastic leukemia

The fungi of the order Mucorales cause mucormycosis, which usually presents as an invasive fungal disease with rapid angioinvasion in immunocompromised patients. Rhinocerebral is the most common presentation. The lipid formulations of amphotericin B are used as primary treatment in invasive mucormycosis; the combined use of posaconazole could allow a reduction in the dose of amphotericin B improving tolerance and adherence to treatment. Caspofungin and amphotericin B association has been shown to be synergistic in vitro and effective in murine models. We present the case of a preschool patient that during the debut of acute lymphoblastic leukemia developed a rhinocerebral mucormycosis successfully responding to antifungal treatment with the combination of liposomal amphotericin and caspofungin.

Los hongos del orden Mucorales causan la mucormicosis, que se presenta habitualmente como una enfermedad fúngica invasora con rápida angioinvasión en pacientes inmunocomprometidos. La presentación rino-cerebral es la más frecuente. Las formulaciones lipídicas de anfotericina B se usan como tratamiento primario en las mucormicosis invasoras; el uso combinado de posaconazol podría permitir reducir la dosis de anfotericina B generando una mejor tolerancia y adherencia al tratamiento. La asociación de caspofungina con anfotericina ha demostrado acción sinérgica in vitro y eficacia en modelos murinos. Se presenta el caso de una niña preescolar que durante el debut de una leucemia linfoblástica aguda evolucionó con una mucormicosis rino-cerebral persistente, que respondió en forma exitosa al tratamiento antifúngico combinado de anfotericina liposomal y caspofungina.

Advances in the treatment of invasive neonatal candidiasis.

INTRODUCTION: Invasive candidiasis is responsible for ∼ 10% of nosocomial sepsis in very-low-birth-weight infants and is associated with substantial morbidity and mortality. Over the last two decades, the antifungal armamentarium against Candida spp. has increased; however, efficacy and safety studies in this population are lacking. AREAS COVERED: We reviewed the medical literature and extracted information on clinical and observational studies evaluating the use of antifungal agents in neonates with invasive candidiasis. EXPERT OPINION: Efficacy and safety data for antifungals in neonates are lacking, and the majority of studies conducted to date have concentrated on pharmacokinetic/pharmacodynamic evaluations. Unlike other anti-infective agents, efficacy data in the setting of neonatal candidiasis cannot be extrapolated from adult studies due to differences in the pathophysiology of the disease in this population relative to older children and adults. Data for amphotericin B deoxycholate, fluconazole, and micafungin suggest that these are the current agents of choice for this disease in neonates until data for newer antifungal agents become available. For prophylaxis, data from fluconazole randomized controlled trials will be submitted to the regulatory agencies for labeling. Ultimately, the field of therapeutics for neonatal candidiasis will require multidisciplinary collaboration given the numerous challenges associated with conducting clinical trials in neonates.

Micafungin alone and in combination therapy with deferasirox against Pythium insidiosum.

This study evaluated the in vitro and in vivo activity of micafungin alone and in combination with the iron chelator deferasirox against Pythium insidiosum. Micafungin showed a poor in vitro activity when it was used alone, but synergistic interactions were observed for 88.2% of the strains when the drug was combined with deferasirox. Smaller lesions were observed in infected rabbits receiving the combination therapy, although it favored disease dissemination to the lungs. The present results show that micafungin alone is ineffective against P. insidiosum, and the combination micafungin-deferasirox might have deleterious effects for the host.