Efficacy Of Antifungal Drugs Research Articles

High rate of breakthrough invasive aspergillosis among patients receiving caspofungin for persistent fever and neutropenia

A number of agents are now available for empirical antifungal treatment (EAFT) of patients with persistent fever and neutropenia. We carried out a study of efficacy of antifungal drugs to prevent breakthrough invasive aspergillosis by reviewing the medical records of all consecutive patients who received EAFT from November 2005 to February 2006. Patients' characteristics and the type, dose and duration of antifungal therapy were recorded. Breakthrough invasive fungal infections were documented according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) definition. Fifty-six episodes of persistent fever with neutropenia requiring EAFT were recorded among 49 patients. All patients received high-dose chemotherapy for acute myeloid leukaemia (51%), acute lymphoid leukaemia (12%), lymphoma (14%) or other haematologic conditions (22%). Fourteen (29%) and five (10%) patients were allogeneic and autologous haematopoietic stem cell transplant recipients, respectively. Caspofungin was prescribed initially in 40 episodes (71%), amphotericin B (AmB) desoxycholate and liposomal AmB being prescribed in six (10%) and ten (18%) episodes, respectively. Six patients were switched from liposomal AmB to caspofungin because of adverse events. The median duration of antifungal therapy was 9 days. During follow-up, six patients (12%) were diagnosed with invasive aspergillosis after a median of 8 days (range 3-16 days) of EAFT. Invasive aspergillosis breakthrough occurred in 6/46 (13%) caspofungin recipients and in 0/16 (0%) AmB recipients (OR 3.1, p 0.32). The observed high rate of invasive aspergillosis among caspofungin recipients requires further evaluation.

Antifungal drugs: predicting clinical efficacy with pharmacodynamics

Invasive fungal infections are on the rise, particularly in hospitalized patients and immunocompromised hosts. In recent years, several new antifungals have become available at a rapid pace. Data on pharmacokinetics/pharmacodynamics of the newer/older antifungal drugs are accumulating. As with bacterial infections and antibacterial drugs, predicting the clinical efficacy of antifungal drugs based on pharmacodynamic parameters is becoming feasible. For the echinocandin class, a ratio of 10 or more for peak concentration/minimum inhibitory concentration (MIC) predicts efficacy against most Candida organisms. Increasing the currently used doses does not appear to improve efficacy. For the triazole class, a ratio of 25 or more for the pharmacodynamic parameter, AUC/MIC, predicts efficacy of the antifungal drugs against invasive Candida infections. More data are needed for invasive mold infections. The polyene class of drugs exhibit concentration-dependent activity; doses higher than those used conventionally do not show any clinical advantage. Monitoring serum levels of the newer triazoles, voriconazole and posaconazole, appears to be necessary in clinical practice to ensure efficacy and avoid toxicity. Flucytosine levels help to predict toxicity, but not efficacy. Other classes of drugs do not warrant monitoring serum concentrations. As invasive fungal infections usually occur in the setting of compromised immune defenses, clinical success does not depend upon the activity of the drugs alone; host immune factors need consideration to predict clinical outcome.

Conventional or molecular measurement ofAspergillusload

The quantification of organisms is a standard tool. Measurement of a hyphal organism, like Aspergillus, presents difficulties in that it is problematic to define what constitutes a cell. Growth occurs by hyphal tip extension, in which the hypha elongates and a septum is formed behind the tip to divide it in to a separate compartment. However, communication between compartments and streaming of nuclei makes defining a cell of a hyphal organism difficult and the best method for quantification of the hyphal organism remains controversial. Conventional CFU determination of fungal burden in tissue homogenates is readily done by most laboratories, but CFU recovered temporally tend to show minimal increase, and may indicate that mechanical homogenization does not cause significant fragmentation of the hyphae in the tissues. Does the lack of increase in CFU as infection worsens inadequately reflect fungal load in the tissue? Non-culture based assays including chitin determination, quantitative PCR and enzyme immunoassay (EIA) of cell wall constituents, galactomannan or beta-glucan overcome this difficulty in part. However, qPCR and EIA do not indicate viability, may result in false negatives. qPCR assay may represent a significant over-estimation, because it correlates with number of nuclei present; it also requires specialized equipment and reagents. Temporal studies of infection have demonstrated that qPCR and to some extent EIA reflect an increase in fungal burden not shown by CFU. Although qPCR and CFU may not correlate in those types of studies, comparative studies have shown CFU and qPCR do correlate when determining antifungal drug efficacy, where each method can clearly demonstrate differences in fungal burden; EIA methods have also been shown to reflect this difference. Overall, there remains no optimal, single method for determination of fungal load of Aspergillus and it may be that a combination of methods (e.g., CFU and qPCR) should be used.

Editorial Commentary: AspergillusGalactomannan Index: A Surrogate End Point to Assess Outcome of Therapy?

In this issue of Clinical Infectious Diseases, Miceli et al. [1] have investigated the correlation between circulating Aspergillus galactomannan values and clinical outcomes (survival) using data derived from the published literature on the use of the galactomannan assay. As such, the authors have touched on an important and challenging aspect of clinical trial design: can resolution of galactomannan antigenemia be used as reliable surrogate end point of antifungal drug efficacy in clinical trials? There is little doubt that there are opportunities to improve our current and historical antifungal clinical trial designs, and because future drug development is being threatened by the negative calculated yields of pharmaceutical cost-benefit analyses, clinicians can argue that improved design leading to both more-definitive and more-efficient clinical trials has never been so important. Because the Infectious Diseases Society of America Antimicrobial Availability Task Force recently identified Aspergillus species as 1 of the 6 particularly problematic pathogens for which current outcomes and drug availability necessitate high priority status [2], improving end points in studies of aspergillosis is very timely goal. Clinical trials are performed to provide definitive evidence of the safety and efficacy of drugs or therapeutic approaches (e.g., drug combinations). Without definitive study design, it is arguable whether enrollment of patients and exposure to potentially harmful therapies is even ethical. Defining end points to appropriately measure both efficacy and safety is one of the largest challenges in trial design. To measure the benefit-to-risk ratio, primary clinical end points should be clinical efficacy measures that both are significant (e.g., death, quality of life, and organ function) and adequately reflect the disease process that is impacted by the drug; one working group defined clinical end point as a characteristic or variable that reflects how patient feels or functions, or how long patient survives [3, p. 568]. To facilitate performance of clinical trials, some researchers have advocated for the use of biomarkers or a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic intervention [3, p. 568] . A surrogate end point is biomarker that is used to substitute for clinical end point; it may be reliable when the marker both correlates with clinical outcome and fully captures the treatment effect [4] . A surrogate end point may be most useful when the outcome of chronic disease process can take long time to measure. Studies in oncology and cardiology have relied heavily on the use of surrogate markers [3]. Tumor markers (e.g., prostate-specific antigen and carcinoembryonic antigen) and tumor imaging (e.g., positron emission tomography and CT) are commonly employed in oncology trials. Cardiology trials have used surrogate end points to gain drug approval; for instance, blood pressure monitoring and cholesterol reduction have been accepted because of their correlation with the risks of heart disease and stroke [3]. Although the use of biomarkers may indicate efficacy in 1 biologic process, their use as substitute clinical end points (surrogate end points) has been associated with some controversy. Fleming and DeMets [5, 6] have reminded us that correlates do not necessarily make surrogates, largely because clinical outcomes are affected by multiple causative pathways, some of which are not measured by biomarkers that are even closely correlated with drug activity. This scenario comes into play when considering possible toxicities of drug and in situations in which the natural history of the process is not well defined (or impacted by multiple variables). Therefore, it has been suggested that biomarkers are useful for phase 2 Received 10 December 2007; accepted 11 December 2007; electronically published 20 March 2008. Reprints or correspondence: Dr. Kieren A. Marr, Oregon Health and Science University, 3181 SW Sam Jackson Park Rd., Portland, OR 97239 (marrki@ohsu.edu).

Evaluation of combined deactivators-supplemented agar medium (CDSAM) for recovery of dermatophytes from patients with tinea pedis

Using a newly-developed medium, i.e., combined deactivators-supplemented agar medium (CDSAM), the viability of dermatophytes in skin scales was evaluated. Culture studies were conducted with skin scales collected from 44 patients with tinea pedis who had been treated for two weeks with topical antifungal drugs. Sixty-four percent of the specimens were mycologically positive on CDSAM, while only 23% of these same specimens were positive when cultured on conventional Sabouraud's dextrose agar medium (SDA). Alternatively, 36% of all cases were negative on both media. The experimental data from this clinical study indicate that CDSAM was more useful than SDA in accurately evaluating the efficacy of antifungal drugs since the former minimized the residual effects of drug residues remaining in the skin samples.

Posaconazole Prophylaxis in Experimental Systemic Zygomycosis

Three isolates of zygomycetes belonging to two different genera (Rhizopus oryzae and Absidia corymbifera) were used to produce a systemic infection in neutropenic mice. On days -2 and -1 and at 2 h prior to infection, the mice received either posaconazole (POS) at doses ranging from 20 to 80 mg/kg of body weight/day or amphotericin B (AMB) at 1 mg/kg/day. Antifungal drug efficacy was assessed by determination of the prolongation of survival, determination of the percentage of infected organs (brain, lung, spleen, and kidney), and histological examination for the number of infection foci and their sizes in brain and kidney tissues. AMB significantly prolonged the survival of mice infected with all isolates. POS significantly prolonged the survival of mice infected with zygomycetes. Cultured organs from mice infected with R. oryzae were all positive, while treated mice challenged with A. corymbifera generally showed lower percentages of infected organs compared with the percentages for the controls. Zygomycete isolates established an active infection (the presence of hyphae) in the brains and the kidneys of all controls. In mice challenged with R. oryzae, both antifungal drugs were effective at reducing the number and the size of infection foci in the kidneys. Only AMB reduced the numbers, but not the sizes, of infection foci in the brain. Finally, both drugs significantly reduced the numbers and the sizes of infection foci in both tissues of mice infected with A. corymbifera. Our data suggest that prophylaxis with POS has some potential to prevent zygomycosis.

Animal models testing monotherapy versus combination antifungal therapy: lessons learned and future directions

The continued rise in serious fungal infections and rises in therapy failure dictate that more efficacious therapies be developed. Combination therapy using available drugs is an attractive choice, yet primarily only anecdotal clinical data are available. We review here data from animal models as an indicator of future potential. The primary data are from murine studies and we will briefly review chemotherapeutic combination studies, some showing benefit over monotherapy and some showing no benefit over monotherapy. In addition, we will address the potential of immunotherapy in combination with conventional therapy. The data derived from animal model studies of antifungal drug efficacy have proven to be predictive of clinical utility. Studies on combination therapy will prove useful to the clinician in evaluating courses of treatment, especially where clinical-trial data are not available or probable in the future.

Determination of Antifungal Activities in Serum Samples from Mice Treated with Different Antifungal Drugs Allows Detection of an Active Metabolite of Itraconazole

To establish an in vitro method of predicting in vivo efficacy of antifungal drugs against Candida albicans and Aspergillus fumigatus, the antifungal activities of fluconazole, itraconazole, and amphotericin B were determined in mouse serum. The minimum inhibitory concentration (MIC) of each drug was measured using mouse serum as a diluent. For C. albicans, the assay endpoint of azoles was defined as inhibition of mycelial extension (mMIC) and for A. fumigatus, as no growth (MIC). The MICs of amphotericin B for both pathogens were defined as the MIC at which no mycelial growth occurred. Serum MIC or mMIC determinations were then used to estimate the concentration of the drugs in serum of mice treated with antifungal drugs by multiplying the antifungal titer of the serum samples by the serum (m)MIC. The serum drug concentrations were also determined by HPLC. The serum concentrations estimated microbiologically showed good agreement with those determined by HPLC, except for itraconazole. Analysis of the serum samples from itraconazole-treated mice by a sensitive bioautography revealed the presence of additional spots, not seen in control samples of itraconazole. The bioautography assay demonstrated that the additional material detected in serum from mice treated with itraconazole was an active metabolite of itraconazole. The data showed that the apparent reduction in the itraconazole serum concentration as determined by HPLC was the result of the formation of an active metabolite, and that the use of a microbiological method to measure serum concentrations of drugs can provide a method for prediction of in vivo efficacy of antifungal drugs.

Mice with Disseminated Candidiasis Die of Progressive Sepsis

Candida species are among the most common etiologies of nosocomial bloodstream infections, causing a mortality of >40%. The murine model of hematogenously disseminated candidiasis is the standard for investigating both the activity of antifungal agents and the pathogenesis of this disease. However, despite decades of use, little is known about the physiological characteristics of the host in this model, and the cause of death remains unclear. Using i-STAT technology, we measured blood chemistry and hemodynamic parameters to define host physiological characteristics during murine disseminated candidiasis. Mice with hematogenously disseminated candidiasis died of progressive sepsis, as manifested by worsening hypotension, tachycardia, and hypothermia. The mice developed metabolic acidosis, as well as profound acidemia and hypoglycemia. They also developed renal insufficiency, which became severe only shortly before death. Kidney fungal burden was correlated with severity of renal failure and systemic acidosis. The presence of significant weight loss, hypotension, or hypothermia was predictive of imminent death. These findings indicate that the murine model of hematogenously disseminated candidiasis accurately recapitulates the progressive sepsis seen during severe clinical cases. The results underscore the validity of the model for study of the pathophysiological aspects of this disease, as well as for the evaluation of antifungal drug efficacy.

Fruit flies as a minihost model for studying drug activity and virulence inAspergillus

Invasive aspergillosis (IA) is a significant cause of morbidity and mortality in profoundly immunosuppressed patients. The mediocre efficacy of antifungals for IA in clinical practice and an incomplete understanding of the pathogenesis of IA contribute to its overall poor prognosis. Although logistically difficult for large scale use, conventional animal models of IA provide valuable information regarding both antifungal drug efficacy and Aspergillus mutant virulence. However, in the era of introduction of molecular biology techniques for studying Aspergillus and increasing antifungal options, the existing in vivo models of IA might be well complemented by nonvertebrate minihost models such as the Drosophila melanogaster (fruit fly). Drosophila may offer the distinct advantage of performing fast, inexpensive high-throughput screening of compounds for anti-Aspergillus activity and putative Aspergillus mutants for their role in Aspergillus virulence.

Application of Radioisotopes in Anti-Fungal Research and Fungal Disease Studies

The substantial increase in the incidence of fungal infections during the past two decades has been suggested as the major cause of morbidity and mortality among immunocompromised patients. Aggressive immunosuppression, HIV-1 infection, cancer, and organ transplantation have opened the door to pathogenic fungal organisms. Disseminated candidiasis and pulmonary aspergillosis caused by emerging opportunistic fungi are the most common of the serious mycoses. Reliability of radioisotope-based measurements and specificity of immune reactions, radioimmune tests, like radioimmunoassay (RIA) and indirect radioimmunometric assay (IRMA) make the applications of radiotechniques among the most promising methods in the antifungal drug research and diagnostic tools. The following applications are discussed in this article: mechanisms of fungal physiology and biochemistry, transporter systems, evaluation of mycoses, radiolabeled metabolites, antifungal drug efficacy and discovery, and antifungal drug metabolism and pharmacokinetics in animal models, therapeutic drug monitoring and in-vitro diagnosis. Keywords: antifungal, radiolabeled, fungal biochemistry, mycoses, fungal metabolites, drug discovery, diagnosis, and antifungal therapy

Quantification and assessment of viability of Cryptococcus neoformans by LightCycler amplification of capsule gene mRNA

Cryptococcus neoformans is an opportunistic fungal pathogen. It infects the central nervous system causing meningitis, which is fatal if untreated, especially in AIDS and immunosuppressed patients. In this study a method of quantification and assessment of viability of C. neoformans by LightCycler RT-PCR amplification of the capsule gene mRNA is established. The sequence of primers and probes were derived from C. neoformans capsular CAP10 gene mRNA (GenBank accession number AF144574), and were species specific. Agarose gel electrophoresis analysis of LightCycler RT-PCR product showed a single band of 223 bp in length. In order to develop an internal control a 223 bp exon fragment of capsule mRNA was cloned in the pCR2.1 plasmid vector and RNA was generated by in vitro transcription. To determine the sensitivity of the assay, serial dilutions of in vitro-transcribed RNA with known concentrations and copy numbers, and serially diluted cultures of viable and nonviable C. neoformans were used. Under optimal conditions as little as 0.472 fg of capsule mRNA could be detected, corresponding to 1-10 c.f.u. ml(-1) of the sample. No amplification was observed from up to 10(5) heat/UV radiation-killed yeast cells and RNA of other bacterial and fungal pathogens and human genomic DNA or RNA. The amplification of capsule mRNA represents a sensitive, specific and quantitative means of detection of viable C. neoformans in clinical specimens and can be useful in the evaluation of the therapeutic efficacy of antifungal drugs in the treatment of C. neoformans meningitis.

Antifungals in systemic neonatal candidiasis.

Fungal infections are common in the newborn period, especially among premature neonates, and are responsible for considerable morbidity and mortality. Currently, three classes of antifungals are commonly used in the treatment of systemic fungal infections in neonates: the polyene macrolides (e.g. amphotericin B [deoxycholate and lipid preparations]); the azoles (e.g. fluconazole); and the fluorinated pyrimidines (e.g. flucytosine). The echinocandins (e.g. caspofungin and micafungin) are a newer class of antifungals which shows promise in this population.The available kinetic data on amphotericin B deoxycholate in neonates are derived from very small studies and exhibit considerable variability. There are no kinetic data available for the use of lipid preparations in this population and, again, much has been inferred from adult studies. The information available for flucytosine is also limited but appears similar to what is observed in adults. Fluconazole has the most neonatal pharmacokinetic data, which show slightly less variability than the other antifungals. Genomic factors which affect the metabolism of amphotericin B and fluconazole may explain some of the observed variability. Most of the data for the efficacy of antifungal drugs in neonates are derived from retrospective studies and case reports. The data for amphotericin B deoxycholate and flucytosine are limited. There are more data for the liposomal and lipid complex preparations of amphotericin B and for fluconazole in this population. These support the use of these drugs in neonates, but because of their largely noncomparative nature they can not define the optimal dosage or duration of therapy. Amphotericin B deoxycholate is primarily nephrotoxic. It also induces electrolyte abnormalities and is to a lesser degree cardiotoxic. This toxicity in neonates appears similar to published data in older children and adults. While the lipid preparations of amphotericin B owe their existence to a presumed decrease in toxicity, the observed toxicity in neonates appears to be equal to that seen with the deoxycholate, although it should be noted that the lipid preparations are usually given at much higher dosages. Fluconazole toxicity appears to be milder and less frequent in this population than is seen with amphotericin B. In the final analysis, we do not have sufficient data to define the pharmacokinetic profiles, optimal dose or duration of therapy, or toxicity for any of these compounds in neonates. Further studies are necessary if the optimisation of antifungal therapy in this population is to continue.

In Vitro Analysis of Antifungal Impregnated Polymethylmethacrylate Bone Cement

Fungal infection is a rare but devastating complication of total joint arthroplasty. Many patients require removal of the components and resection arthroplasty for cure; however, revision arthroplasty with medicated polymethylmethacrylate bone cement may be used to salvage the joint. Some studies have documented the efficacy of mixing antibiotics with polymethylmethacrylate, but the efficacy of antifungal drugs when mixed with polymethylmethacrylate is unknown. An in vitro agar diffusion method was used in the current study to investigate this potential, and several clinically important conclusions resulted: (1) after incorporation into bone cement, fluconazole and amphotericin B remained active whereas 5-flucytosine did not, (2) inhibitory activity improved with greater drug concentrations, and (3) more drug eluted from Palacos R than Simplex P cement.

Elitra pharmaceuticals: new paradigms for antimicrobial drug discovery

Antibiotic drug resistance and the limited efficacy of antifungal drugs highlight the urgent need for new antimicrobial drugs. Elitra Pharmaceuticals’ original vision was to identify directly all of the essential genes in key pathogens. We have filed patents on over 4000 such targets, and aim to develop cell-based assays for all our targets. In addition, we have begun to shift the paradigm towards massively parallel screening of all possible drug targets.

Histopathologic and mycologic aspects of experimental infection of guinea pigs with Microsporum canis

Dermatophytosis is a zoonosis in whose etiology the dermatophyte Microsporum canis is frequently involved. The fungus can be transmitted to man by dogs and cats. In the present study, guinea pigs were experimentally inoculated with M. canis and the course of the lesions was evaluated. Inoculation resulted in lesions in 100% of the animals, with a clinical course consisting of an incubation period, an inflammatory and a phase of lesion resolution. The histopathologic evaluation of the skin biopsies revealed the presence of acanthosis, hyperkeratosis, spongiosis and marked dermal edema. M. canis spores and hyphae were detected in histologic sections stained with periodic acid-Schiff. The combination of the fluorescent dyes FD and EB permitted the adequate visualization of viable and dead fungal cells in skin fragments of guinea pigs inoculated with M. canis and the identification of the morphologic characteristics of the cells. This experimental model represents a valuable instrument for the study of the pathogenesis of dermatophytic infection with respect to the evaluation of the efficacy of antifungal drugs, and may also be used for the study of the immunology of dermatophytoses and of dermatophyte morphogenes

Adjunctive immune therapy for fungal infections.

Fungal infections in immunocompromised patients can pose difficult problems in clinical management, because the available antifungal chemotherapy is often unable to eradicate the infection in these people. Hence, the use of immune modulating therapy to augment impaired host immune responses--and thus enhance the efficacy of antifungal drugs--is a reasonable approach to improve the prognosis of fungal infections. Advances in biotechnology have produced a variety of biological response modifiers with the potential to serve as adjunctive immune therapy for the treatment of fungal infections, including cytokines, monoclonal antibodies, and cell growth factors. In recent years, immune-modulating therapies have been studied in an effort to define their potential use for the treatment of fungal infections. Much of the available information on the use of this approach is encouraging and invites further investigation--with the caveats that the information is mostly anecdotal and that immune-modulating therapy occasionally has produced adverse effects.

Host Immune Reactivity and Antifungal Chemotherapy: The Power of Being Together

Fungal infections, particularly those in immunocompromised hosts, have been a major therapeutic challenge over recent decades. The difficulty in diagnosing invasive infections, together with the emergence of unusual opportunistic pathogens and a pathogen shift in the spectrum of the causative organisms, have greatly hampered the effectiveness of antifungal therapy. Knowledge of the immunoregulation of fungal infections may provide new insights for therapeutic interventions. The appreciation of the importance of T helper cells and cytokines in the overall coordination of the effector immune response to fungi has offered novel opportunities to manipulate these processes and to alter the outcome of fungal infections while improving the therapeutic efficacy of antifungal drugs. Cytokine and cytokine antagonists, alone or in combination with antifungals, have the potential to overcome the specific defects of host immune reactivity predisposing to fungal infections. The current challenge lies in translating much of the information obtained in experimental and preclinical studies into therapeutic strategies providing prospects for the ultimate treatment of fungal infections.

Tuftsin-bearing liposomes as drug vehicles in the treatment of experimental aspergillosis

Encapsulation of amphotericin B in tuftsin-bearing liposomes greatly increased its efficacy in treatment of human aspergillosis in mice. Also, the drug efficacy was significantly increased by pretreating the animals with drug-free tuftsin-bearing liposomes. These results demonstrate that macrophage activation can considerably enhance the therapeutic efficacy of antifungal drugs, like amphotericin B.