Managing uncertainty in antifungal dosing: antibiograms, therapeutic drug monitoring and drug-drug interactions.

PURPOSE OF REVIEW: A number of pharmacokinetic and pharmacodynamic factors in critically ill or severely immunosuppressed patients influence the effectiveness of antifungal therapy making dosing less certain. Recent position papers from infectious diseases societies and working groups have proposed methods for dosage individualization of antibiotics in critically ill patients using a combination of population pharmacokinetic models, Monte-Carlo simulation and therapeutic drug monitoring (TDM) to guide dosing. In this review, we examine the current limitations and practical issues of adapting a pharmacometrics-guided dosing approaches to dosing of antifungals in critically ill or severely immunosuppressed populations. RECENT FINDINGS: We review the current status of antifungal susceptibility testing and challenges in incorporating TDM into Bayesian dose prediction models. We also discuss issues facing pharmacometrics dosage adjustment of newer targeted chemotherapies that exhibit severe pharmacokinetic drug-drug interactions with triazole antifungals. SUMMARY: Although knowledge of antifungal pharmacokinetic/pharmacodynamic is maturing, the practical application of these concepts towards point-of-care dosage individualization is still limited. User-friendly pharmacometric models are needed to improve the utility of TDM and management of a growing number of severe pharmacokinetic antifungal drug-drug interactions with targeted chemotherapies.

Saprochaete clavata infections in patients undergoing treatment for haematological malignancies: A report of a monocentric outbreak and review of the literature.

Saprochaete clavata is a rare cause of fungaemia with deep organ involvement in patients with haematological malignancies with reported mortality rates of 60%-80%. We describe four cases of S clavata infection in a haematology unit over several months that were treated with voriconazole-based regimens. We also review the literature on factors that could contribute to earlier recognition and effective treatment of S clavata. We included all cases of culture-positive S clavata from sterile sites with associated signs of infection in patients undergoing treatment for a haematological malignancy. Isolates were identified by MALDI-TOF MS, and spectrum profiles were used to prepare clustering analysis of isolates. Susceptibility testing was performed using a commercial microtitre methods. Saprochaete clavata was isolated from the bloodstream in three cases and bronchial alveolar lavage (BAL) fluid in one case. Clustering analysis suggested strains of S clavata were clonal without evidence of divergence although a common source was not identified. Susceptibility testing yielded elevated MICs to fluconazole (8 mg/L) and echinocandins (>1-8 mg/L). All patients were treated with voriconazole-based regimens resulting in survival of 3/4 patients, who continued chemotherapy for their underlying malignancy without evidence of relapse. Saprochaete clavata is a rare but aggressive cause of breakthrough yeast infection in patients undergoing treatment for haematological malignancies, particularly patients with a prior history of echinocandin treatment. Timely initiation of appropriate treatment, aided by more rapid identification in microbiology laboratory, can reduce the risk of deep organ dissemination and patient death.

In vivo evolution of resistant subpopulations of KPC-producing Klebsiella pneumoniae during ceftazidime/avibactam treatment.

Objectives: KPC-producing Klebsiella pneumoniae (KPC-Kp) represent a serious problem worldwide. Herein, we describe the evolution of ceftazidime/avibactam resistance by sequencing longitudinal clinical isolates from a patient with KPC-Kp bloodstream infection undergoing ceftazidime/avibactam treatment. Methods: WGS was performed on one ceftazidime/avibactam-susceptible KPC-Kp (BOT-CA-S) and two phenotypically different ceftazidime/avibactam-resistant KPC-Kp with low (BOT-CA-R) and high (BOT-EMO) carbapenem MICs. The population diversity was assessed by the frequency of allele mutations and population analysis profiles (PAPs). Results: Phylogenetic analysis demonstrated clonal relatedness of the KPC-Kp isolates, all belonging to the clone ST1519. The D179Y mutation in blaKPC-3 was detected in both of the ceftazidime/avibactam-resistant KPC-Kp, whereas it was absent in the ceftazidime/avibactam-susceptible isolate. The mutation emerged independently in the two ceftazidime/avibactam-resistant isolates and was associated with a significant reduction in carbapenem MICs in BOT-CA-R, but not in BOT-EMO. WGS analysis revealed that the frequency of the D179Y mutation was 96.32% and 51.05% in BOT-CA-R and BOT-EMO, respectively. PAP results demonstrated that carbapenem resistance in BOT-EMO was due to the coexistence of mixed subpopulations harbouring WT and mutated blaKPC-3. A bacterial subpopulation with high ceftazidime/avibactam resistance for BOT-EMO KPC-Kp showed low carbapenem MICs, whereas a subpopulation with high meropenem resistance had a low MIC of ceftazidime/avibactam. Conclusions: Our analysis indicates that mixed subpopulations of ceftazidime/avibactam-resistant KPC-Kp emerge after ceftazidime/avibactam treatment. The evolution of different subpopulations that are highly resistant to ceftazidime/avibactam likely contributes to treatment failure, thereby highlighting the need for combination treatment strategies to limit selection of ceftazidime/avibactam-resistant KPC-Kp subpopulations.

Liver transplantation is associated with good clinical outcome in patients with active tuberculosis and acute liver failure due to anti-tubercular treatment.

BACKGROUND: Active tuberculosis (TB) is commonly considered a contraindication for liver transplantation (LT). However, in patients with TB who develop acute liver failure (ALF) due to toxicity induced by anti-tubercular treatment (ATT), LT could be the only opportunity for treatment. The aim of this study was to evaluate the feasibility of LT in this scenario. METHODS: We described 2 cases and comprehensively reviewed the literature finding 26 cases of LT performed in patients having a concomitant active TB and liver failure secondary to ATT toxicity. RESULTS: TB was classified as pulmonary in 18/26 (69%), nodal in 3/26 (11%) TB cases, while the remaining 5/26 cases included disseminated, pleural, renal, ovarian, and vertebral TB localization (1 case each). ATT following LT consisted mainly of isoniazid or rifampin (RIF)-sparing regimens and included primarily fluoroquinolones and ethambutol. Rejection episodes and liver toxicity were reported in 19% and 8% of patients respectively. Graft rejection was more frequent among patients treated with RIF-containing regimens (P<.001). Mortality rate was 15% after a median follow up of 12 months. In only one case was death attributed to uncontrolled TB infection. CONCLUSION: Our findings suggest that LT is an effective therapeutic option for patients with active TB developing ALF following ATT and should be considered for patients failing medical treatment.

Statin Concentrations Below the Minimum Inhibitory Concentration Attenuate the Virulence of Rhizopus oryzae.

BACKGROUND: Mucormycosis is a destructive invasive mold infection afflicting patients with diabetes and hematologic malignancies. Patients with diabetes are often treated with statins, which have been shown to have antifungal properties. We sought to examine the effects of statins on Rhizopus oryzae, a common cause of mucormycosis. METHODS: Clinical strains of R. oryzae were exposed to lovastatin, atorvastatin, and simvastatin and the minimum inhibitory concentrations (MICs) were determined. R. oryzae germination, DNA fragmentation, susceptibility to oxidative stress, and ability to damage endothelial cells were assessed. We further investigated the impact of exposure to lovastatin on the virulence of R. oryzae RESULTS: All statins had MICs of >64 µg/mL against R. oryzae Exposure of R. oryzae to statins decreased germling formation, induced DNA fragmentation, and attenuated damage to endothelial cells independently of the expression of GRP78 and CotH. Additionally, R. oryzae exposed to lovastatin showed macroscopic loss of melanin, yielded increased susceptibility to the oxidative agent peroxide, and had attenuated virulence in both fly and mouse models of mucormycosis. CONCLUSIONS: Exposure of R. oryzae to statins at concentrations below their MICs decreased virulence both in vitro and in vivo. Further investigation is warranted into the use of statins as adjunctive therapy in mucormycosis.

Comparative pharmacodynamics of posaconazole in neutropenic murine models of invasive pulmonary aspergillosis and mucormycosis.

We used two established neutropenic murine models of pulmonary aspergillosis and mucormycosis to explore the association between the posaconazole area under the concentration-time curve (AUC)-to-MIC ratio (AUC/MIC) and treatment outcome. Posaconazole serum pharmacokinetics were verified in infected mice to ensure that the studied doses reflected human exposures with the oral suspension, delayed-release tablet, and intravenous formulations of posaconazole. Sinopulmonary infections were then induced in groups of neutropenic mice with Aspergillus fumigatus strain 293 (posaconazole MIC, 0.5 mg/liter) or Rhizopus oryzae strain 969 (posaconazole MIC, 2 mg/liter) and treated with escalating daily dosages of oral posaconazole, which was designed to achieve AUCs ranging from 1.10 to 392 mg · h/liter. After 5 days of treatment, lung fungal burden was analyzed by quantitative real-time PCR. The relationships of the total drug AUC/MIC and the treatment response were similar in both models, with 90% effective concentrations (EC90s) corresponding to an AUC/MIC threshold of 76 (95% confidence interval [CI], 46 to 102) for strain 293 versus 87 (95% CI, 66 to 101) for strain 969. Using a provisional AUC/MIC target of >100, these exposures correlated with minimum serum posaconazole concentrations (Cmins) of 1.25 mg/liter for strain 293 and 4.0 mg/liter for strain 969. The addition of deferasirox, but not liposomal amphotericin or caspofungin, improved the activity of a suboptimal posaconazole regimen (AUC/MIC, 33) in animals with pulmonary mucormycosis. However, no combination was as effective as the high-dose posaconazole monotherapy regimen (AUC/MIC, 184). Our analysis suggests that posaconazole pharmacodynamics are similar for A. fumigatus and R. oryzae when indexed to pathogen MICs.

Proangiogenic growth factors potentiate in situ angiogenesis and enhance antifungal drug activity in murine invasive aspergillosis.

In invasive pulmonary aspergillosis, direct invasion and occlusion of pulmonary vasculature by Aspergillus hyphae causes tissue hypoxia, which is enhanced by secreted fungal metabolites that downregulate compensatory angiogenic signaling pathways. We assessed the effects of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) on survival rates, fungal burden, and in situ angiogenesis in a murine invasive pulmonary aspergillosis model. bFGF and VEGF monotherapy significantly increased survival rates and potentiated the activity of amphotericin B. bFGF-containing regimens were associated with reduced tissue fungal burdens. bFGF and VEGF reversed the antiangiogenic activity of Aspergillus fumigatus; however, VEGF induced the formation of immature neovessels, providing an explanation for its lesser efficacy. Treatment with bFGF plus amphotericin B was associated with neutrophil influx into Aspergillus-infected pulmonary tissue, suggesting that this combination limits fungal growth through neutrophil trafficking. Vasculogenic pathways are unexplored targets for the treatment of invasive pulmonary aspergillosis and may potentiate both innate immunity and antifungal drug activity against A. fumigatus.

Echinocandin resistance in Candida species: mechanisms of reduced susceptibility and therapeutic approaches.

OBJECTIVE: To summarize published data regarding mechanisms of reduced echinocandin susceptibility in Candida spp., the impact of echinocandin resistance on the fitness and virulence of Candida isolates, and current and future treatment approaches. DATA SOURCES: A search of MEDLINE databases (1966-September 2011) was conducted. STUDY SELECTION AND DATA EXTRACTION: Databases were searched using the terms echinocandin, resistance, and Candida. Citations from publications were reviewed for additional references. DATA SYNTHESIS: Echinocandins have in vitro activity against most Candida spp. and are first-line agents in the treatment of candidemia. However, case reports describing echinocandin treatment failure due to resistant isolates have been published. Reduced echinocandin susceptibility has been shown to occur via 3 main mechanisms: (1) adaptive stress responses, which result in elevated cell wall chitin content and paradoxical growth in vitro at supra minimum inhibitory concentrations (MICs); (2) acquired FKS mutations, which confer reduced glucan synthase sensitivity, elevated MICs, and are associated with clinical failure; and (3) intrinsic FKS mutations, which are naturally occurring mutations in C. parapsilosis and C. guilliermondii, which confer elevated MIC levels but a lower level of reduced glucan synthase sensitivity compared with acquired FKS mutations. Some FKS mutants have been shown to have significantly reduced fitness and virulence versus wild type isolates and may contribute to the low incidence of echinocandin resistance reported in large surveillance studies. Treatment strategies evaluated for FKS mutants include echinocandin dose escalation and combination with agents such as calcineurin inhibitors, HSP90 inhibitors, and chitin synthase inhibitors. CONCLUSIONS: While the incidence of echinocandin resistance in Candida spp. is low, it can present a significant therapeutic challenge, especially in multidrug-resistant Candida isolates. Dose escalation is unlikely to be effective in treating FKS mutant isolates, and significant adverse effects limit the clinical use of agents evaluated as combination therapy. Patients with infections failing to respond to echinocandin therapy should undergo susceptibility testing and be treated with an alternative antifungal agent if possible.

Comparative pharmacodynamics of amphotericin B lipid complex and liposomal amphotericin B in a murine model of pulmonary mucormycosis.

We compared the kinetics of amphotericin B (AMB) lung accumulation and fungal clearance by liposomal amphotericin B (L-AMB) and amphotericin B lipid complex (ABLC) in a neutropenic murine model of invasive pulmonary mucormycosis (IPM). Immunosuppressed BALB/c mice were inoculated with 1 x 10(6) Rhizopus oryzae spores and administered L-AMB or ABLC at daily intravenous doses of 1, 5, or 10 mg/kg of body weight for 5 days starting 12 h after infection. At a dose of 10 mg/kg/day, both L-AMB and ABLC were effective at reducing the R. oryzae lung fungal burden and achieved lung tissue concentrations exceeding the isolate mean fungicidal concentration (MFC) of 8 microg/ml by 72 h. When ABLC was dosed at 5 mg/kg/day, the ABLC-treated animals had significantly higher AMB lung concentrations than the L-AMB treated animals at 24 h (6.64 and 1.44 microg/g, respectively; P = 0.013) and 72 h (7.49 and 1.03 microg/g, respectively; P = 0.005), and these higher concentrations were associated with improved fungal clearance, as determined by quantitative real-time PCR (mean conidial equivalent of R. oryzae DNA per lung, 4.44 +/- 0.44 and 6.57 +/- 0.74 log(10), respectively; P < 0.001). Analysis of the AMB tissue concentration-response relationships revealed that the suppression of R. oryzae growth in the lung required tissue concentrations that approached the MFC for the infecting isolate (50% effective concentration, 8.19 microg/g [95% confidence interval, 2.81 to 18.1 microg/g]). The rates of survival were similar in the animals treated with L-AMB and ABLC at 10 mg/kg/day. These data suggest that higher initial doses may be required during L-AMB treatment than during ABLC treatment of experimental IPM.

Monotherapy with caspofungin for candidaemia in adult patients with cancer: a retrospective, single institution study.

This study was performed to evaluate retrospectively the efficacy and safety of caspofungin monotherapy for candidaemia in patients with cancer. The medical records were reviewed of 63 consecutive adult patients with cancer who had candidaemia treated with caspofungin alone for at least 3 consecutive days at The University of Texas M.D. Anderson Cancer Center (March 2001-February 2007). Twenty patients (32%) had haematological malignancies. Most of the candidaemia cases (54%) were caused by non-albicans Candida spp. The most frequent isolates were C. albicans (38%) followed by Candidaparapsilosis (21%), Candidatropicalis (16%) and Candidaglabrata (10%). In vitro susceptibility studies showed that of 30 Candida isolates tested, only one C. parapsilosis isolate had a caspofungin minimum inhibitory concentration > 1 microg/mL. The clinical and mycological response rates after 7 days of treatment with caspofungin were 78% and 77%, respectively. The overall mortality rate was 5% on Day 7, 12% on Day 14 and 21% on Day 30. The 30-day Candida-attributable mortality rate was 11% and was significantly higher in patients with fluconazole-resistant or susceptible-dose-dependent Candida isolates. Caspofungin was well tolerated in all patients. Although selection of candidaemic patients with cancer who received caspofungin monotherapy may have accounted for the favourable outcomes, it was observed that caspofungin had efficacy and safety comparable with those in candidaemic patients without malignancy.

Caspofungin as primary antifungal prophylaxis in stem cell transplant recipients.

STUDY OBJECTIVES: To assess the effectiveness and tolerability of caspofungin as primary prophylaxis against invasive fungal infections in stem cell transplant recipients who are poor candidates for triazole or lipid amphotericin B prophylaxis due to renal or hepatic dysfunction, and to determine whether any patient characteristics are independently associated with an increased risk of breakthrough invasive fungal infection during caspofungin prophylaxis. DESIGN: Retrospective medical record review. SETTING: Tertiary care comprehensive cancer center. PATIENTS: One hundred twenty-three adult stem cell transplant recipients who received caspofungin 35-50 mg/day for up to 100 days after transplantation as primary antifungal prophylaxis between January 1, 2002, and June 30, 2005. MEASUREMENTS AND MAIN RESULTS: Data were collected on host and transplant characteristics such as transplant type, neutropenia, graft-versus-host disease (GVHD), and corticosteroid use, as well as evidence of breakthrough invasive fungal infections. Of the 123 patients, 117 (95.1%) were allogeneic recipients, and the median time to engraftment was 12 days (range 6-26 days). Fifty (40.7%) of the patients developed GVHD of grade 2 or greater and received corticosteroids for more than 21 days. Median duration of caspofungin prophylaxis was 73 days (range 10-100 days). Nine patients (7.3%) developed breakthrough invasive fungal infections (two cases of mixed Aspergillus species and one each of Aspergillus terreus, Rhizopus, Exserohilum, an unspecified mold, Cryptococcus, Candida glabrata, and Candida tropicalis). Median time to invasive fungal infection development was 65 days (range 12-88 days). Only one case occurred during the neutropenic period before engraftment. Multivariate analysis showed that Pseudomonas coinfection (p=0.04) and infliximab therapy (p=0.02) were associated with breakthrough invasive fungal infections in patients receiving caspofungin. By day 100, there were five (4.1%) deaths, two of which were directly attributable to invasive fungal infections. No caspofungin-related adverse events were reported. CONCLUSION: Caspofungin seems to be an effective and well-tolerated option for primary antifungal prophylaxis in the highly immunosuppressed stem cell transplant patient population.

Pharmacodynamic activity of amphotericin B deoxycholate is associated with peak plasma concentrations in a neutropenic murine model of invasive pulmonary aspergillosis.

We conducted a dose fractionation study of neutropenic, corticosteroid-immunosuppressed mice to characterize the pharmacodynamic/pharmacokinetic (PK/PD) parameter most closely associated with amphotericin B (AMB) efficacy in the treatment of invasive pulmonary aspergillosis. Pharmacokinetic parameter estimates were determined by a nonparametric population pharmacokinetic analysis of plasma drug concentrations following single intraperitoneal doses (0.25, 1.0, and 3.0 mg/kg of body weight) of amphotericin B deoxycholate. Three dosage groups (0.5, 0.75, and 1.0 mg/kg) fractionated into three dosing intervals (every 8 h [q8h], q24h, or q72h) were tested to discriminate between the PK/PD parameters (the ratio of maximum concentration of drug in serum [Cmax]/MIC, the ratio of area under the concentration-time curve/MIC, and percentage of time above MIC) most closely associated with AMB efficacy over a range of clinically achievable exposures in humans. The efficacy of each regimen was determined by quantitative PCR and survival. Reductions in pulmonary fungal burden and improvements in survival were maximized at the highest peak plasma concentrations in each of the dosage groups. Reductions in pulmonary fungal burden and increased survival were most closely associated with Cmax/MIC, with maximal activity occurring as the Cmax/MIC approached 2.4. In our model, Cmax/MIC is the PK/PD parameter most closely associated with efficacy in the treatment of invasive pulmonary aspergillosis. These data predict that less frequently administered, higher dosages of AMB would optimize efficacy.

Pentamidine is active in a neutropenic murine model of acute invasive pulmonary fusariosis.

We studied the efficacy of pentamidine (PNT) as prophylaxis or early treatment in acute pulmonary fusariosis in neutropenic mice. PNT-preexposed mice had significantly improved survival and reduced fungal burden compared to amphotericin B-preexposed and untreated mice. PNT-treated mice had increased survival but no difference in fungal burden versus untreated mice.

Aspergillus susceptibility testing in patients with cancer and invasive aspergillosis: difficulties in establishing correlation between in vitro susceptibility data and the outcome of initial amphotericin B therapy.

STUDY OBJECTIVES: As the failure of amphotericin B therapy in patients with invasive aspergillosis often exceeds 50%, we sought to determine the relationship between in vitro amphotericin B resistance and clinical failure of amphotericin B against invasive aspergillosis. DESIGN: Retrospective study. SETTING: University cancer center. PATIENTS: One hundred sixteen patients with cancer and invasive aspergillosis. MEASUREMENTS AND MAIN RESULTS: The correlation between in vitro amphotericin B susceptibility, defined by both the National Committee for Clinical Laboratory Standards and Etest methods, and other prognostic factors with failure of initial amphotericin B therapy was retrospectively evaluated. Data for correlation could be clearly assessed in only 18 (16%) of the 116 patients. Admission to the intensive care unit was the only prognostic factor associated with failure of initial amphotericin B treatment in these 18 patients (p = 0.01). CONCLUSIONS: Several important obstacles underlie the correlation of in vitro susceptibility testing with in vivo efficacy of individual antifungal agents in the treatment of invasive aspergillosis. Such correlations could be determined in only a small subset of patients.

Increased frequency of non-fumigatus Aspergillus species in amphotericin B- or triazole-pre-exposed cancer patients with positive cultures for aspergilli.

Invasive aspergillosis (IA) can occur despite prior prophylactic or empiric use of triazoles or amphotericin B (AMB). Although profound immunosuppression may account for breakthrough IA, resistance of Aspergillus to antifungals may also play a role. To examine this question, we measured the minimal inhibitory concentration of 105 Aspergillus isolates recovered from 105 cancer patients (64 with IA, 41 with Aspergillus colonization) to AMB, itraconazole (ITC), and voriconazole (VRC) using the National Committee for Clinical Laboratory Standards (NCCLS) M38-A microdilution and E-test methods. We also determined the minimal fungicidal concentration (MFC) of these agents and the minimal effective concentration (MEC) of caspofungin (CAS) using standardized methods. We then collected information regarding pre-exposure to AMB or triazoles (fluconazole, ITC, VRC) within 3 months before Aspergillus isolation. Pre-exposure of cancer patients to AMB or triazoles was associated with increased frequency of non-fumigatus Aspergillus species. Aspergillus isolates recovered from patients who previously received AMB exhibited higher E-test AMB MICs compared with isolates from patients without prior AMB exposure (P = 0.01). In addition, the AMB MICs by E-test were higher in triazole-pre-exposed patients compared with those not exposed to triazoles (P = 0.001). The ITC and VRC MICs by E-test were not affected by prior AMB or triazole exposure. Finally, neither the AMB, ITC, and VRC MICs and MFCs by NCCLS method nor CAS MECs showed such changes. In conclusion, cancer patients with positive Aspergillus cultures who are pre-exposed to AMB or triazoles have high frequency of non-fumigatus Aspergillus species. These Aspergillus isolates were found to be AMB-resistant by the more sensitive E-test method.

Toll-deficient Drosophila flies as a fast, high-throughput model for the study of antifungal drug efficacy against invasive aspergillosis and Aspergillus virulence.

Invasive aspergillosis (IA) is the most important opportunistic mycosis in immunosuppressed patients. The lack of a sufficient number of effective antifungals and our incomplete understanding of the pathogenesis of IA contribute to its overall unfavorable prognosis. Studies of drug efficacy against IA and Aspergillus virulence rely on conventional animal models that are laborious and use limited numbers of animals; alternative, less cumbersome in vivo models are desirable. Using different inoculation models of IA, we found that Toll-deficient Drosophila flies exposed to voriconazole (VRC), the preferred drug for the treatment of IA in humans, had significantly better survival rates and lower tissue fungal burdens than did those not exposed to VRC. Furthermore, Toll-deficient Drosophila flies infected with an alb1-deleted hypovirulent Aspergillus mutant had significantly better survival rates than did those infected with a wild-type Aspergillus strain. Therefore, the Drosophila fly is a fast, high-throughput in vivo model for the study of drug efficacy against IA and Aspergillus virulence.

Genome-wide expression profiling reveals genes associated with amphotericin B and fluconazole resistance in experimentally induced antifungal resistant isolates of Candida albicans.

OBJECTIVES: The aim of this study was to identify changes in the gene expression profile of Candida albicans associated with the acquisition of experimentally induced resistance to amphotericin B and fluconazole. METHODS: C. albicans SC5314 was passed in increasing concentrations of amphotericin B to generate isolate SC5314-AR. Susceptibility testing by Etest revealed SC5314-AR to be highly resistant to both amphotericin B and fluconazole. The gene expression profile of SC5314-AR was compared with that of SC5314 using DNA microarray analysis. Sterol composition was determined for both strains. RESULTS: Upon examination of MICs of antifungal compounds, it was found that SC5314-AR was resistant to both amphotericin B and fluconazole. By microarray analysis a total of 134 genes were found to be differentially expressed, that is up-regulated or down-regulated by at least 50%, in SC5314-AR. In addition to the cell stress genes DDR48 and RTA2, the ergosterol biosynthesis genes ERG5, ERG6 and ERG25 were up-regulated. Several histone genes, protein synthesis genes and energy generation genes were down-regulated. Sterol analysis revealed the prevalence of sterol intermediates eburicol and lanosterol in SC5314-AR, whereas ergosterol was the predominant sterol in SC5314. CONCLUSION: Along with changes in expression of these ergosterol biosynthesis genes was the accumulation of sterol intermediates in the resistant strain, which would account for the decreased affinity of amphotericin B for membrane sterols and a decreased requirement for lanosterol demethylase activity in membrane sterol production. Furthermore, other genes are implicated as having a potential role in the polyene and azole antifungal resistant phenotype.

Combination chemotherapy for invasive fungal infections: what laboratory and clinical studies tell us so far.

Despite potential benefits, few objective clinical data (with the exception of cryptococcocal meningitis) are available supporting the routine use of combination antifungal regimens in patients with invasive mycoses, importantly aspergillosis or candidiasis. There is considerable debate on what constitutes synergy or antagonism in vitro and whether these laboratory findings are translated to beneficial interactions in patients. Given the lack of rigorous clinical data, a better understanding of the important concepts for the justification of the clinical and pharmacoeconomic threshold of antifungal therapy is needed. Such concepts include standardized methods for screening antifungal combinations in culture or in animals and collaborative efforts to collect clinical data on the efficacy and safety of combination regimens.

Candidemia in a tertiary care cancer center: in vitro susceptibility and its association with outcome of initial antifungal therapy.

Since the 1990s, changing trends have been documented in species distribution and susceptibility to bloodstream infections caused by Candida species in cancer patients. However, few data are available regarding the association between in vitro antifungal susceptibility and outcome of candidemia in this patient population. We therefore evaluated the association of in vitro antifungal susceptibility and other risk factors with failure of initial antifungal therapy in cancer patients with candidemia. Candidemia cases in cancer patients from 1998 to 2001 (n = 144) were analyzed retrospectively along with their in vitro susceptibility to amphotericin B, fluconazole, and itraconazole (National Committee for Clinical and Laboratory Standards M27-A method). Patients were evaluable for outcome analysis if they received continuous unchanged therapy with either fluconazole or amphotericin B for >/=5 days. We excluded cases of mixed candidemia. In vitro susceptibility testing data of the first Candida bloodstream isolate were analyzed. Appropriate therapy was defined as that using an active in vitro antifungal for >/=5 days. For fluconazole susceptible-dose dependent Candida species, we defined appropriate therapy as a fluconazole dose of >/=600 mg/day. The Candida species distribution was 30% Candida albicans, 24% Candida glabrata, 23% Candida parapsilosis, 10% Candida krusei, 9% Candida tropicalis, and 3% other. Overall, amphotericin B was the most active agent in vitro, with only 3% of the isolates exhibiting resistance to it (>1 mg/L). Dose-dependent susceptibility to fluconazole and itraconazole was seen in 13% and 21% of the isolates, respectively, while resistance to fluconazole and itraconazole was seen in 13% and 26%, respectively.Eighty patients were evaluable for outcome analysis. In multivariate analysis, the following factors emerged as independent predictors of failure of initial antifungal therapy: leukemia (p = 0.01), bone marrow transplantation (p = 0.006), and intensive care unit stay at onset of infection (p = 0.02). Inappropriate antifungal therapy, as defined by daily dose and in vitro susceptibility, was not shown consistently to be a significant factor (it was significant in multivariate analysis, p = 0.04, but not in univariate analysis), indicating the complexity of the variables that influence the response to antifungal treatment in cancer patients with candidemia.

The echinocandin antifungals: an overview of the pharmacology, spectrum and clinical efficacy.

For over four decades, the principal target of antifungal therapy has been the fungal cell membrane sterol ergosterol. Although this has proven to be a successful and relatively selective antifungal target, collateral toxicity to mammalian cells (amphotericin B) and drug interactions (azoles) have been by-products of agents that target the fungal cell membrane. In the 1970s, the echinocandins were identified during the screening of fungal fermentation products for new antibiotic agents. These agents were subsequently shown to inhibit production of beta(1,3)-glucan, a key structural component of the fungal cell wall. Subsequent chemical modification of these natural products has led to the development of safer, semi-synthetic beta(1,3)-glucan synthase inhibitors with enhanced microbiological and clinical efficacy against infections caused by Candida and Aspergillus species. In this review, the pharmacology, spectrum and clinical efficacy of the three leading beta(1,3)glucan synthase inhibitors (caspofungin, micafungin and anidulafungin), which have completed phase III clinical trials, will be discussed and a perspective for the role of these agents in the management of life-threatening mycoses will be offered.