Sofia Perea

Prevalence of Molecular Mechanisms of Resistance to Azole Antifungal Agents in Candida albicans Strains Displaying High-Level Fluconazole Resistance Isolated from Human Immunodeficiency Virus-Infected Patients

ABSTRACT Molecular mechanisms of azole resistance in Candida albicans, including alterations in the target enzyme and increased efflux of drug, have been described, but the epidemiology of the resistance mechanisms has not been established. We have investigated the molecular mechanisms of resistance to azoles inC. albicans strains displaying high-level fluconazole resistance (MICs, ≥64 μg/ml) isolated from human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis. The levels of expression of genes encoding lanosterol 14α-demethylase (ERG11) and efflux transporters (MDR1 and CDR) implicated in azole resistance were monitored in matched sets of susceptible and resistant isolates. In addition,ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. The analysis confirmed the multifactorial nature of azole resistance and the prevalence of these mechanisms of resistance in C. albicans clinical isolates exhibiting frank fluconazole resistance, with a predominance of overexpression of genes encoding efflux pumps, detected in 85% of all resistant isolates, being found. Alterations in the target enzyme, including functional amino acid substitutions and overexpression of the gene that encodes the enzyme, were detected in 65 and 35% of the isolates, respectively. Overall, multiple mechanisms of resistance were combined in 75% of the isolates displaying high-level fluconazole resistance. These results may help in the development of new strategies to overcome the problem of resistance as well as new treatments for this condition.

Efficacy of Caspofungin Alone and in Combination with Voriconazole in a Guinea Pig Model of Invasive Aspergillosis

ABSTRACT The antifungal activity of caspofungin acetate (CAS) alone and in combination with voriconazole (VRC) was evaluated in an immunosuppressed transiently neutropenic guinea pig model of invasive aspergillosis. Guinea pigs were immunosuppressed with triamcinolone at 20 mg/kg of body weight/day subcutaneously beginning 4 days prior to lethal intravenous challenge with Aspergillus fumigatus and were made temporarily neutropenic with cyclophosphamide administered at 150 mg/kg intraperitoneally (i.p.) 1 day prior to challenge. Therapy with i.p. CAS at 1 and 2.5 mg/kg/day (with and without oral VRC at 5 mg/kg/day), oral VRC at 5 mg/kg/day, or i.p. amphotericin B (AMB) at 1.25 mg/kg/day was begun 24 h after challenge and was continued for 5 days. Mortality occurred in 12 of 12 untreated controls, whereas mortality occurred in 4 of 12 and 6 of 12 guinea pigs treated with CAS at 1 and 2.5 mg/kg/day, respectively, and in 3 of 12 guinea pigs treated with AMB. No mortality occurred among animals treated with CAS at 1 mg/kg/day plus VRC at 5 mg/kg/day, CAS at 2.5 mg/kg/day plus VRC at 5 mg/kg/day, or VRC at 5 mg/kg/day alone. Both CAS regimens increased the survival times and reduced the colony counts in tissue compared with those for the controls. Treatment with VRC and AMB significantly reduced the colony counts in the tissues of selected animals compared with those in the tissues of the controls. Treatment with VRC and AMB also resulted in reductions in colony counts in tissues compared with those in the tissues of animals treated with CAS (the difference was not statistically significant) and improved the survival times but did not sterilize tissues. Combination therapies with CAS plus VRC at either dose reduced colony counts in tissues 1,000-fold over those for the controls and were the only regimens that significantly reduced the numbers of positive cultures. The combinations of CAS plus VRC were highly effective in this model and should be further evaluated for use against invasive aspergillosis.

Antifungal Resistance in Pathogenic Fungi

Pathogenic fungi are the cause of life-threatening infections in an increasing number of immunocompromised patients. The intrinsic resistance to antifungal therapy observed in some genera, along with the development of resistance during treatment in others, is becoming a major problem in the management of these diseases. We reviewed the epidemiology of the most common systemic fungal infections for which antifungal resistance is a potential problem, the mechanisms of antifungal resistance, the correlation between in vitro susceptibility testing and clinical outcome, and the clinical implications of antifungal resistance.

In Vitro Interaction of Caspofungin Acetate with Voriconazole against Clinical Isolates of Aspergillus spp.

ABSTRACT The interaction between caspofungin acetate and voriconazole was studied in vitro by using 48 clinical Aspergillus spp. isolates obtained from patients with invasive aspergillosis. MICs were determined by the NCCLS broth microdilution method. Synergy, defined as a fractional inhibitory concentration (FIC) index of <1, was detected in 87.5% of the interactions; an additive effect, defined as an FIC index of 1.0, was observed in 4.2% of the interactions; and a subadditive effect, defined as an FIC index of 1.0 to 2.0, was found in 8.3% of the interactions. No antagonism was observed. Animal models are required to validate the in vivo significance of these in vitro data presented for the combination of caspofungin and voriconazole.

Comparison of High-Performance Liquid Chromatographic and Microbiological Methods for Determination of Voriconazole Levels in Plasma

ABSTRACT A new selective high-performance liquid chromatography (HPLC) method with UV detection for the determination of the investigational triazole voriconazole in human plasma by using acetonitrile precipitation followed by reverse-phase HPLC on a C18column was compared with a simple agar well diffusion bioassay method with Candida kefyr ATCC 46764 as the assay organism. Pooled plasma was used to prepare standard and control samples for both methods. The results of analyses with spiked serum samples (run as unknowns) were concordant by the bioassay and HPLC methods, with expected values being obtained. HPLC demonstrated an improved precision (3.47 versus 12.12%) and accuracy (0.81 versus 1.28%) compared to those of the bioassay method. The range of linearity obtained by both methods (from 0.2 to 10 μg/ml for HPLC and from 0.25 to 20 μg/ml for the bioassay) includes the range of concentrations of voriconazole (from 1.2 to 4.7 μg/ml) which are considered clinically relevant. Although either methodology could be used for the monitoring of patient therapy, the smaller variability observed with HPLC compared to that observed with the bioassay favors the use of HPLC for pharmacokinetic studies.

Molecular Mechanisms of Fluconazole Resistance in Candida dubliniensis Isolates from Human Immunodeficiency Virus-Infected Patients with Oropharyngeal Candidiasis

ABSTRACT Candida dubliniensis is a newly identified species of Candida that is phenotypically similar to but genetically distinct from C. albicans. This organism has been recovered with increasing frequency from the oral cavities of human immunodeficiency virus (HIV)-infected and AIDS patients and has been implicated as a causative agent of oral candidiasis and systemic disease. In the present study we characterized the molecular mechanisms of resistance to fluconazole (FLC) in C. dubliniensis clinical isolates from two different HIV-infected patients with oropharyngeal candidiasis. Isolates were identified to the species level by phenotypic and genotypic tests. DNA-typing techniques were used to assess strain identity. Antifungal susceptibility testing was performed by NCCLS techniques. Northern blotting analysis was used to monitor the expression of genes encoding lanosterol demethylase (ERG11) and efflux transporters (CDR and MDR1) in matched sets of C. dubliniensis-susceptible and -resistant isolates by using probes generated from their homologous C. albicans sequences. In addition, ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. Decreasing susceptibilities to FLC were detected in C. dubliniensis isolates recovered from both patients during the course of treatment. FLC-resistant C. dubliniensis isolates from one patient demonstrated combined upregulation of the MDR1, CDR1, and ERG11 genes. Among the isolates from the second patient, all isolates showing decreased susceptibility to FLC demonstrated upregulation of MDR1, whereas the levels of mRNA for the ERG11 genes remained constant and the expression of CDR genes was negligible. Fourteen point mutations were found in the ERG11 genes of the isolates with decreased susceptibility to FLC. These data demonstrate that the development of azole resistance in C. dublinensis clinical isolates from HIV-infected patients treated with FLC is mediated by multiple molecular mechanisms of resistance, similar to the observations found in the case of C. albicans.

Outpatient therapy with oral ofloxacin for patients with low risk neutropenia and fever

Hospitalization and treatment with broad‐spectrum intravenous antibiotics is the standard care for patients with neutropenia and fever. This randomized clinical trial evaluated the feasibility and efficacy of ambulatory care with oral ofloxacin for patients with low risk, chemotherapy‐induced neutropenia and fever.

Comparison of In Vitro Activities of Voriconazole and Five Established Antifungal Agents against Different Species of Dermatophytes Using a Broth Macrodilution Method

ABSTRACT The in vitro activities of voriconazole against 19 different species of dermatophytes were compared with those of terbinafine, itraconazole, ketoconazole, griseofulvin, and fluconazole. MICs were determined according to a National Committee for Clinical Laboratory Standards broth macrodilution method. Voriconazole appeared more active than ketoconazole, griseofulvin, and fluconazole and less active than itraconazole and terbinafine. Based on these results, voriconazole merits further investigation as a potentially useful agent for the treatment of dermatophytosis.

In vitro activities of terbinafine in combination with fluconazole, itraconazole, voriconazole, and posaconazole against clinical isolates of Candida glabrata with decreased susceptibility to azoles.

ABSTRACT A checkerboard microdilution method, performed according to the recommendations of the National Committee for Clinical Laboratory Standards, was used to study the in vitro interaction of terbinafine (TRB) with fluconazole (FLU), itraconazole (ITRA), voriconazole (VRC), and posaconazole (PSZ) in 24 isolates of Candida glabrata with decreased susceptibility to azoles isolated from the oral cavities of human immunodeficiency virus patients. Synergy, defined as a fractional inhibitory concentration index of ≤0.5, was observed in 17% of TRB-FLU interactions, 21% of TRB-ITRA interactions, 33% of TRB-VRC interactions, and 12% of TRB-PSZ interactions. Where synergy was not achieved, there was still a decrease in the MIC of one or both drugs when used in combination. Antagonism was not observed in any drug combination. Clinical studies are warranted to elucidate the potential utility of these combination therapies.

Identification of Continuous B-Cell Epitopes on the Protein Moiety of the 58-Kilodalton Cell Wall Mannoprotein of Candida albicans Belonging to a Family of Immunodominant Fungal Antigens

ABSTRACT The 58-kiloDalton mannoprotein (mp58) on the surface ofCandida albicans is highly immunogenic, is expressed by allC. albicans isolates tested, and elicits strong antibody responses during candidiasis. It belongs to a family of immunodominant fungal antigens with representatives also in different species ofAspergillus. The amino acid sequence of the protein portion of mp58 as deduced from the DNA sequence of its encoding gene (FBP1/PRA1) was used to synthesize a complete set of overlapping dodecapeptides (overlap, 7; offset, 5) covalently attached to the surface of derivatized polyethylene pins. The pin-coupled peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by a number of antiserum preparations containing anti-mp58 antibodies. This comprehensive epitope-scanning study revealed the presence of multiple immunoreactive continuous B-cell epitopes within the protein sequence. Regions of increased reactivity included both the amino and carboxy termini of the mature protein (encompassing amino acid residues 16 to 50 and 286 to 299, respectively) and four internal regions spanning amino acids at positions 66 to 92, 121 to 142, 148 to 192, and 211 to 232. Further delineation of epitopic regions and identification of the boundaries of the antigenic sites was performed upon ELISA testing with a second Pepset consisting of completely overlapping 8-mer peptides spanning these reactive regions in the protein moiety of mp58. The highly reactive epitopic region at the C terminus of the protein was further evaluated using both window net and replacement net analyses. A synthetic peptide corresponding to the last 10 amino acid residues at the C terminus of the protein was immunogenic when injected into mice after being coupled to a carrier protein. Moreover, antibodies in the resulting sera specifically recognized the homologus mp58 in ELISAs and immunoblot assays. Delineation of the antibody responses to mp58 could provide the basis for the development of novel immunity-based prophylactic, therapeutic, and diagnostic techniques for the management of candidiasis.