β-Thalassaemia Major in a Spanish Patient due to a Compound Heterozygosity for CD39 C → T/−28 A → C

A Spanish male patient with β-thalassaemia major was studied. Compound heterozygosity was found for one of the most common β-globin gene mutations in the Spanish population (codon 39 C → T) and for a mutation in the TATA box element of the β-globin gene promoter (−28 A → C mutation). To our knowledge this is the first report of a CD39 C → T and −28 A → C change association and the first report of the −28 A → C substitution in a Spanish patient

A novel combination of CYP51A mutations confers pan-azole resistance in aspergillus fumigatus

The treatment of invasive and chronic aspergillosis involves triazole drugs. Its intensive use has resulted in the selection of resistant isolates, and at present, azole resistance in Aspergillus fumigatus is considered an emerging threat to public health worldwide. The aim of this work is to uncover the molecular mechanism implicated in the azole resistance phenotype of three Aspergillus fumigatus clinical strains isolated from an Argentinian cystic fibrosis patient under long-term triazole treatment. Strain susceptibilities were assessed, and CYP51A gene sequences were analyzed. Two of the studied Aspergillus fumigatus strains harbored the TR34- L98H allele. These strains showed high MIC values for all tested triazoles (>16.00μg/ml, 1.00 μg/ml, 1.00 μg/ml, and 2.00 μg/ml for itraconazole, isavuconazole, posaconazole, and voriconazole, respectively). The third strain had a novel amino acid change (R65K) combined with the TR34-L98H mutations. This new mutation combination induces a pan-azole MIC augment compared with TR34-L98H mutants (>16 μg/ml, 4.00μg/ml, 4.00μg/ml, and 8.00μg/ml for itraconazole, isavuconazole, posaconazole, and voriconazole, respectively). The strain harboring the TR34-R65K-L98H allele showed no inhibition halo when voriconazole susceptibility was evaluated by disk diffusion. The effect of these mutations in the azole-resistant phenotype was confirmed by gene replacement experiments. Transformants harboring the TR34-L98H and TR34-R65KL98H alleles mimicked the azole-resistant phenotype of the clinical isolates, while the incorporation of the TR34-R65K and R65K alleles did not significantly increase azole MIC values. This is the first report of the TR34-L98H allele in Argentina. Moreover, a novel CYP51A allele (TR34-R65K-L98H) that induces a pan-azole MIC augment is described.Fil: Macedo, Daiana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Brito Devoto, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Pola, Santiago. Universidad de Buenos Aires; ArgentinaFil: Finquelievich, Jorge L.. Universidad de Buenos Aires; ArgentinaFil: Cuestas, María Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Garcia Effron, Guillermo. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentin

Requirement for Ergosterol in V-ATPase Function Underlies Antifungal Activity of Azole Drugs

Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H+-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma− phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca2+ and H+ surges triggered by the antimicrobial agent amiodarone, and impaired Ca2+ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections

Molecular Markers of Antifungal Resistance: Potential Uses in Routine Practice and Future Perspectives

Antifungal susceptibility testing (AST) has come to establish itself as a mandatory routine in clinical practice. At the same time, the mycological diagnosis seems to have headed in the direction of non-culture-based methodologies. The downside of these developments is that the strains that cause these infections are not able to be studied for their sensitivity to antifungals. Therefore, at present, the mycological diagnosis is correctly based on laboratory evidence, but the antifungal treatment is undergoing a growing tendency to revert back to being empirical, as it was in the last century. One of the explored options to circumvent these problems is to couple non-cultured based diagnostics with molecular-based detection of intrinsically resistant organisms and the identification of molecular mechanisms of resistance (secondary resistance). The aim of this work is to review the available molecular tools for antifungal resistance detection, their limitations, and their advantages. A comprehensive description of commercially available and in-house methods is included. In addition, gaps in the development of these molecular technologies are discussed

Improved Detection of Candida sp. fks Hot Spot Mutants by Using the Method of the CLSI M27-A3 Document with the Addition of Bovine Serum Albumin▿†

Echinocandins are highly bound to serum proteins, altering their antifungal properties. The addition of 50% human serum to the MIC assay improves the identification of echinocandin-resistant Candida spp. harboring fks hot spot mutations. However, this modification cannot readily be applied to the method of the CLSI M27-A3 document due to safety and standardization difficulties. The aim of this study was to evaluate commercial bovine serum albumin (BSA) as a safe and standardized alternative to human serum. A collection of 28 echinocandin-susceptible strains, 10 Candida parapsilosis sensu lato strains (with naturally reduced echinocandin susceptibility), and 40 FKS hot spot mutants was used in this work. When RPMI 1640 was used for susceptibility testing, wild-type strains and fks mutants showed MIC range overlaps (−2, −1, and −3 2-fold-dilution steps separated these populations for anidulafungin, caspofungin, and micafungin, respectively). On the other hand, the addition of BSA to RPMI 1640 differentially increased echinocandin MIC values for these groups of strains, allowing better separation between populations, with no MIC range overlaps for any of the echinocandin drugs tested. Moreover, the use of RPMI-BSA reduced the number of fks hot spot mutant isolates for which MIC values were less than or equal to the upper limit for the wild type (very major errors) from 9, 2, and 7 with RPMI alone to 3, 0, and 3 for anidulafungin, caspofungin, and micafungin, respectively. When RPMI-BSA was used to study the susceptibility of C. parapsilosis sensu lato species to echinocandins, the strains behaved as anidulafungin- and micafungin-resistant isolates (MIC, ≥8 μg/ml). These data support the need for a revision of the CLSI protocol for in vitro testing of echinocandin susceptibility in order to identify all or most of the fks hot spot mutants. Also, caspofungin could be used as a surrogate marker of reduced susceptibility to echinocandins

Emergence of Triazole Resistance in Aspergillus spp. in Latin America

Purpose of Review: Azole resistance in Aspergillus spp. is becoming a public health problem worldwide. However, data about this subject is lacking in Latin American countries. This review focuses in the epidemiology and molecular mechanisms of azole resistance in Aspergillus spp. emphasizing in Latin America. Data on Aspergillus fumigatus stands out because it is the most prevalent Aspergillus spp. pathogen. Recent Findings: Azole resistance in Aspergillus spp. emergence was linked with intensive use of these antifungals both in the clinical setting and in the environment (as pesticides). Reports on azole-resistant A. fumigatus strains are being constantly published in different countries. Molecular mechanisms of resistance mainly involve substitution in the azole target (CYP51A) and/or overexpression of this gene. However, several other non-CYP51A-related mechanisms were described. Moreover, intrinsically resistant cryptic Aspergillus species are starting to be reported as human pathogens. Summary: After a comprehensive literature review, it is clear that azole resistance in Aspergillus spp. is emerging in Latin America and perhaps it is underestimated. All the main molecular mechanisms of azole resistance were described in patients and/or environmental samples. Moreover, one of the molecular mechanisms was described only in South America. Cryptic intrinsic azole-resistant species are also described.Fil: Macedo, Daiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Leonardelli, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Gamarra, Soledad. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Garcia Effron, Guillermo. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentin

Correlating Echinocandin MIC and Kinetic Inhibition of fks1 Mutant Glucan Synthases for Candida albicans: Implications for Interpretive Breakpoints▿

A detailed kinetic characterization of echinocandin inhibition was performed for mutant 1,3-β-d-glucan synthase enzymes from clinical isolates of Candida albicans with nine different FKS1 mutations resulting in high MICs. Among 14 mutant Fks1p enzymes studied, the kinetic parameters 50% inhibitory concentration and Ki increased 50-fold to several thousandfold relative to those for the wild type. Enzymes with mutations at Ser645 (S645P, S645Y, and S645F) within hot spot 1 showed the most prominent decrease in sensitivity, while those with mutations at the N- and C-terminal ends of hot spot 1 generally retained greater sensitivity to all three drugs. Kinetic inhibitions by caspofungin, micafungin, and anidulafungin were comparable among the fks1 mutant enzymes, although absolute values did vary with specific mutations. Amino acid substitutions in Fks1p did not alter Km values, although some mutations decreased the Vmax. Given the association of FKS1 mutations with clinical resistance, an evaluation of the kinetic parameters for the inhibition of mutant 1,3-β-d-glucan synthase as a function of the MIC enabled an independent evaluation of the recently adopted susceptibility breakpoint for echinocandin drugs. Overall, a breakpoint MIC of ≥2 μg/ml for caspofungin captured nearly 100% of fks1 C. albicans strains when a kinetic inhibition rise threshold of ≤50-fold for the Ki was used as a measure of susceptibility. A similar MIC breakpoint for micafungin and anidulafungin was less inclusive, and a projected MIC of ≥0.5 μg/ml was required for >95% coverage of clinical isolates. However, when MIC determinations were performed in the presence of 50% serum, all fks1 mutants showed MIC values of ≥2 μg/ml for the three echinocandin drugs. The 1,3-β-d-glucan synthase kinetic inhibition data support the proposed susceptibility breakpoint for caspofungin in C. albicans, but a lower susceptibility breakpoint (≤0.5 μg/ml) may be more appropriate for anidulafungin and micafungin. Overall, the data indicate that MIC testing with caspofungin may serve as a surrogate marker for resistance among the class of echinocandin drugs

Black grain eumycetoma due to Diaporthe ueckerae. Taxonomical update of previous agents of infections due to Diaporthe spp.

A black-grain eumycetoma due to Diaporthe uekerae in a kidney transplant recipient is presented. The isolate was identified by using the newly available NCBI's curated database (rRNA_typestrains/ITS_RefSeq_Fungi) and the NCBI's GenBank + EMBL + DDBI + PDB + RefSeq database. The isolate's antifungal susceptibility was evaluated. The studied isolate showed low MIC values to the eight tested antifungals. Using this updated database, the identities of previous agents of Diaporthe spp. infections were revised

A Ser678Pro Substitution in Fks1p Confers Resistance to Echinocandin Drugs in Aspergillus fumigatus▿

An S678P substitution in Fks1p, the major subunit of glucan synthase, was sufficient to confer echinocandin resistance in Aspergillus fumigatus. The equivalent mutation in Candida spp. has been implicated in echinocandin resistance. This work demonstrates that modification of Fks1p is a conserved mechanism for echinocandin resistance in pathogenic fungi

Reduced Candida glabrata Susceptibility Secondary to an FKS1 Mutation Developed during Candidemia Treatment▿

We describe a case of recurring Candida glabrata infection in a 68-year-old African-American female on caspofungin therapy. The initial isolate was susceptible, but isolates recovered during following relapses were not. All isolates were clonal, and high-MIC strains contained a mutation in the highly conserved hot spot 1 region of Fks1p