Randomized Comparison of Safety and Pharmacokinetics of Caspofungin,Liposomal Amphotericin B,and the Combination of Both in Allogeneic Hematopoietic Stem Cell Recipients

The combination of liposomal amphotericin B (LAMB) and caspofungin (CAS) holds promise to improve the outcome of opportunistic invasive mycoses with poor prognosis. Little is known, however, about the safety and pharmacokinetics of the combination in patients at high risk for these infections. The safety and pharmacokinetics of the combination of LAMB and CAS were investigated in a risk-stratified, randomized, multicenter phase II clinical trial in 55 adult allogeneic hematopoietic stem cell recipients (aHSCT) with granulocytopenia and refractory fever. The patients received either CAS (50 mg/day; day 1, 70 mg), LAMB (3 mg/kg of body weight/day), or the combination of both (CASLAMB) until defervescence and granulocyte recovery. Safety, development of invasive fungal infections, and survival were assessed through day 14 after the end of therapy. Pharmacokinetic sampling and analysis were performed on days 1 and 4. All three regimens were well tolerated. Premature study drug discontinuations due to grade III/IV adverse events occurred in 1/18, 2/20, and 0/17 patients randomized to CAS, LAMB, and CASLAMB, respectively. Adverse events not leading to study drug discontinuation were frequent but similar across cohorts, except for a higher frequency of hypokalemia with CASLAMB (P < 0.05). Drug exposures were similar for patients receiving combination therapy and those randomized to monotherapy. There was no apparent difference in the occurrence of proven/probable invasive fungal infections and survival through day 14 after the end of therapy. CASLAMB combination therapy in immunocompromised aHSCT patients was as safe as monotherapy with CAS or LAMB and had similar plasma pharmacokinetics, lending support to further investigations of the combination in the management of patients with invasive opportunistic mycoses.Invasive opportunistic fungal infections are an important cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. Depending on the presence of well-characterized risk factors, rates of infection by opportunistic fungal pathogens are between 10 and 25%. The case fatality rates vary between 35 and 50% for invasive candidiasis and 65 and 90% for invasive aspergillosis and infections by other filamentous fungi (8, 15).The existence of antifungal agents with different molecular targets is providing new opportunities to improve the efficacy of antifungal chemotherapy through combination therapy. Potential target populations include profoundly immunocompromised patients with prolonged granulocytopenia or following allogeneic hematopoietic stem cell transplantation, patients with fulminant or refractory infections, or those with infections in compartments that are difficult to treat and infections by fungal pathogens with decreased microbiological susceptibility (23, 31, 39, 48, 49, 50).Based on its favorable microbiologic and pharmacokinetic properties (16), well-documented efficacy against Candida and Aspergillus infections, and excellent safety (24, 28, 32, 45), the echinocandin lipopeptide caspofungin (CAS) is a suitable candidate for antifungal combination therapies with liposomal amphotericin B (LAMB), a standard agent with broad-spectrum antifungal activity (18) and first-line indications against major opportunistic fungal infections (12, 22, 39). While preclinical studies of the combination of caspofungin and amphotericin B (CASLAMB)) have documented the absence of antagonistic effects in vitro and in animal infection models (3, 4, 5, 10, 19, 20, 30, 38), the clinical experience with combination therapies of caspofungin and lipid formulations of amphotericin B is more difficult to assess (1, 9, 21, 24, 34). Particularly in the setting of allogeneic hematopoietic stem cell transplantation and immunosuppression with cyclosporine, the safety of caspofungin alone and in combination with liposomal amphotericin B and the pharmacokinetic interactions of the combination have not been systematically studied.     

Intrapulmonary Pharmacokinetics and Pharmacodynamics of Micafungin in Adult Lung Transplant Patients

Invasive pulmonary aspergillosis is a life-threatening infection in lung transplant recipients; however, no studies of the pharmacokinetics and pharmacodynamics (PKPD) of echinocandins in transplanted lungs have been reported. We conducted a single-dose prospective study of the intrapulmonary and plasma PKPD of 150 mg of micafungin administered intravenously in 20 adult lung transplant recipients. Epithelial lining fluid (ELF) and alveolar cell (AC) samples were obtained via bronchoalveolar lavage performed 3, 5, 8, 18, or 24 h after initiation of infusion. Micafungin concentrations in plasma, ELF, and ACs were determined using high-pressure liquid chromatography. Noncompartmental methods, population analysis, and multiple-dose simulations were used to calculate PKPD parameters. C_max in plasma, ELF, and ACs was 4.93, 1.38, and 17.41 μg/ml, respectively. The elimination half-life in plasma was 12.1 h. Elevated concentrations in ELF and ACs were sustained during the 24-h sampling period, indicating prolonged compartmental half-lives. The mean micafungin concentration exceeded the MIC₉₀ of Aspergillus fumigatus (0.0156 μg/ml) in plasma (total and free), ELF, and ACs throughout the dosing interval. The area under the time-concentration curve from 0 to 24 h (AUC_0-24)/MIC₉₀ ratios in plasma, ELF, and ACs were 5,077, 923.1, and 13,340, respectively. Multiple-dose simulations demonstrated that ELF and AC concentrations of micafungin would continue to increase during 14 days of administration. We conclude that a single 150-mg intravenous dose of micafungin resulted in plasma, ELF, and AC concentrations that exceeded the MIC₉₀ of A. fumigatus for 24 h and that these concentrations would continue to increase during 14 days of administration, supporting its potential activity for prevention and early treatment of pulmonary aspergillosis.Postoperative invasive pulmonary aspergillosis is a frequent clinical problem among patients who have undergone lung transplantation (13, 23, 38-42). Strategies for management of invasive pulmonary aspergillosis in lung transplant recipients are not well defined. While voriconazole is indicated for the primary treatment of invasive aspergillosis, not all patients are able to tolerate this triazole, and drug interactions may be complicated. The role of echinocandins in treatment and prevention of invasive aspergillosis in lung transplant recipients is unknown. Although most lung transplant centers administer some form of antifungal prophylaxis, these regimens vary widely from center to center, and the optimal strategy for prophylaxis is unknown. Aerosolized amphotericin B, either alone or with systemically administered antifungal agents, may be used for prevention of invasive aspergillosis in lung transplant recipients (13).Micafungin, a member of the echinocandin class of antifungal agents, has in vitro as well as in vivo activities against Candida spp. and Aspergillus spp. in treatment of experimental disseminated candidiasis (3, 5, 6, 33-35) and invasive pulmonary aspergillosis (33). Micafungin is licensed for the treatment of patients with esophageal candidiasis and candidemia (5, 12, 15, 30, 32, 36, 49). Micafungin also is approved for prevention of candidemia in neutropenic hematopoietic stem cell transplant recipients (44). Micafungin has been studied alone or in combination with other antifungal agents for treatment of invasive aspergillosis in hematopoietic stem cell transplant recipients (22). Although studies of micafungin for treatment and prevention of invasive pulmonary aspergillosis in animal models and in patients have demonstrated activity against this serious infection (8, 26, 33, 45), little is known about its intrapulmonary pharmacokinetics in patients at risk for invasive aspergillosis (28). We therefore studied the simultaneous intrapulmonary and plasma pharmacokinetics and pharmacodynamics of micafungin in adult lung transplant recipients.     

Correlation of In Vitro Activity,Serum Levels,and In Vivo Efficacy of Posaconazole against Rhizopus microsporus in a Murine Disseminated Infection

A broth microdilution method was used to evaluate the in vitro activities of seven antifungal agents against 15 clinical strains of Rhizopus microsporus. Amphotericin B (AMB) and posaconazole (POS) were the most active drugs. In a model of disseminated R. microsporus infection in immunosuppressed mice, we studied the efficacy of POS administered once or twice daily against four of the strains previously tested in vitro and compared it with that of liposomal AMB (LAMB). LAMB was the most effective treatment for the two strains with intermediate susceptibility to POS. For the two POS-susceptible strains, LAMB and POS at 20 mg/kg of body weight twice a day orally showed similar efficacies. The in vivo efficacy of POS administered twice a day orally correlated with the in vitro susceptibility data and the serum drug concentrations.Zygomycosis is a frequently lethal invasive infection that occurs predominantly in immunocompromised patients (4), a population with a very poor prognosis and a high mortality rate (8). The clinical manifestations include rhino-orbito-cerebral, cutaneous, pulmonary, gastrointestinal, and disseminated infections (4). In a recent study in which a large number of clinical isolates of zygomycetes from different regions of the United States were molecularly identified, it was demonstrated that Rhizopus oryzae and Rhizopus microsporus were the most common species (3). Traditionally, amphotericin B (AMB) and, more recently, its lipid formulations are the front-line agents for the treatment of zygomycosis (4). Specifically, liposomal amphotericin B (LAMB) is less nephrotoxic than AMB and has better central nervous system penetration than AMB and the other lipid formulations (21). Posaconazole (POS) is a broad-spectrum triazole antifungal with a large volume of distribution into tissues (12). This drug has shown good in vitro activity against zygomycetes (1, 2) and has been used successfully as salvage therapy in some case reports and clinical trials of disseminated zygomycosis (8, 22, 23). However, its effectiveness remains controversial, since in experimental studies it has shown poor activity against R. oryzae, the main species causing zygomycosis (6, 9, 17). Several in vitro studies have shown that POS also exhibits significant activity against R. microsporus, another relevant clinical species (1, 2, 11), and a few clinical (14) and experimental (6) studies seem to demonstrate in vivo efficacy as well.In this study, after confirming the significant in vitro activity of POS and AMB, we evaluated the efficacy of POS against four strains of R. microsporus in a murine model of disseminated infection. Considering that antifungal susceptibility can differ substantially among different strains of a given species, which could explain the variable percentages of success demonstrated by POS and AMB in clinical trials (8, 18, 23), we tested multiple strains exhibiting various in vitro responses to obtain more-robust results.     

In Vitro Antifungal Susceptibilities and Amplified Fragment Length Polymorphism Genotyping of a Worldwide Collection of 350 Clinical,Veterinary, and Environmental Cryptococcus gattii Isolates

The in vitro susceptibilities of a worldwide collection of 350 Cryptococcus gattii isolates to seven antifungal drugs, including the new triazole isavuconazole, were tested. With amplified fragment length polymorphism (AFLP) fingerprinting, human, veterinary, and environmental C. gattii isolates were subdivided into seven AFLP genotypes, including the interspecies hybrids AFLP8 and AFLP9. The majority of clinical isolates (n = 215) comprised genotypes AFLP4 (n = 76) and AFLP6 (n = 103). The clinical AFLP6 isolates had significantly higher geometric mean MICs for flucytosine and fluconazole than the clinical AFLP4 isolates. Of the seven antifungal compounds examined in this study, isavuconazole had the lowest MIC₉₀ (0.125 μg/ml) for all C. gattii isolates, followed by a 1 log₂ dilution step increase (MIC₉₀, 0.25 μg/ml) for itraconazole, voriconazole, and posaconazole. Amphotericin B had an acceptable MIC₉₀ of 0.5 μg/ml, but fluconazole and flucytosine had relatively high MIC₉₀s of 8 μg/ml.The basidiomycetous yeast Cryptococcus gattii is responsible for life-threatening invasive disease in apparently healthy humans and animals (7, 19). A typical C. gattii infection is acquired through the respiratory tract, from which it can further disseminate to the central nervous system, resulting in fatal meningitis (7, 19, 32). Cryptococcosis caused by the primary pathogenic yeast C. gattii was, until a decade ago, a rarely encountered infection outside tropical and subtropical regions (17, 26, 27). However, this changed due to an unprecedented outbreak that emerged in the temperate climate of Vancouver Island (British Columbia, Canada) that subsequently expanded farther into the Pacific Northwest (1, 8, 10, 16). Its sibling species, Cryptococcus neoformans, differs ecologically and epidemiologically from C. gattii since it occurs on a global scale and is linked with disease occurring in immunocompromised individuals, such as HIV-positive patients and transplant patients who receive immune-suppressive medicines (7, 10, 18, 19, 31).Cryptococcus gattii can be discerned from C. neoformans using a wide range of microbiological and molecular techniques (7, 20). A convenient method is the use of canavanine-glycine-bromothymol blue (CGB) medium, which allows C. gattii but not C. neoformans to grow and which changes the pH indicator in the medium from green-yellowish to blue (18). With the increasing use of molecular techniques, such as PCR fingerprinting, restriction fragment length polymorphism (RFLP) analysis of the PLB1 and URA5 loci, and amplified fragment length polymorphism (AFLP) fingerprint analysis, as well as several multilocus sequence typing (MLST) approaches, it became clear that C. gattii could be divided into five distinct genotypes, named AFLP4/VGI, AFLP5/VGIII, AFLP6/VGII, AFLP7/VGIV, and AFLP10 (the last one of which is a recently observed novel genotype) (2, 6, 7, 13, 16, 20, 21, 23). Until recently, a serotype agglutination assay was widely used to distinguish C. neoformans (serotypes A and D) from C. gattii (serotypes B and C) (7, 27). In general, serotype B strains are found in each of the five C. gattii AFLP genotypes, but it seems that C. gattii serotype C strains are restricted to genotypes AFLP5/VGIII and AFLP7/VGIV (2, 6, 16, 21, 27).In addition, it was found that C. gattii and C. neoformans can form interspecies hybrids, named genotype AFLP8 (C. neoformans var. neoformans AFLP2/VNIII serotype D × C. gattii AFLP4/VGI serotype B) and AFLP9 (C. neoformans var. grubii AFLP1/VNI serotype A × C. gattii AFLP4/VGI serotype B). These interspecies hybrids have, until now, been isolated only from clinical samples, and they might have a higher virulence potential than regular C. gattii or C. neoformans isolates (4, 5; F. Hagen, K. Tintelnot, and T. Boekhout, unpublished data).Treatment of cryptococcosis depends on, besides the immune status of the patient, the severity and localization of the infection (11). Severe cases of cryptococcosis in immunocompetent and -compromised patients are treated according to the guidelines of the Infectious Diseases Society of America, according to which treatment consists of an induction therapy for 2 weeks with a combination of amphotericin B and flucytosine, followed by a 10-week consolidation therapy with fluconazole (11, 24).Cryptococcus neoformans has been extensively studied for its in vitro susceptibility to a wide variety of antifungal compounds, including the new triazoles posaconazole, voriconazole, ravuconazole, and isavuconazole (12, 14, 28, 29, 33). Despite the ongoing C. gattii outbreak, only a few studies using relatively small sets of C. gattii isolates have been performed to investigate their in vitro susceptibilities to amphotericin B, flucytosine, fluconazole, and the new triazole antifungals (12, 15, 28-30). A few studies divided the C. gattii isolates into groups according to their serotype or genotype (15, 29).Therefore, we studied the in vitro susceptibilities of each of the C. gattii genotypes from a large worldwide collection, subdivided by AFLP genotyping, to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, and the new experimental broad-spectrum antifungal triazole isavuconazole.     

Biofilm Formation and Effect of Caspofungin on Biofilm Structure of Candida Species Bloodstream Isolates

Candida biofilms are microbial communities, embedded in a polymeric matrix, growing attached to a surface, and are highly recalcitrant to antimicrobial therapy. These biofilms exhibit enhanced resistance against most antifungal agents except echinocandins and lipid formulations of amphotericin B. In this study, biofilm formation by different Candida species, particularly Candida albicans, C. tropicalis, and C. parapsilosis, was evaluated, and the effect of caspofungin (CAS) was assessed using a clinically relevant in vitro model system. CAS displayed in vitro activity against C. albicans and C. tropicalis cells within biofilms. Biofilm formation was evaluated after 48 h of antifungal drug exposure, and the effects of CAS on preformed Candida species biofilms were visualized using scanning electron microscopy (SEM). Several species-specific differences in the cellular morphologies associated with biofilms were observed. Our results confirmed the presence of paradoxical growth (PG) in C. albicans and C. tropicalis biofilms in the presence of high CAS concentrations. These findings were also confirmed by SEM analysis and were associated with the metabolic activity obtained by biofilm susceptibility testing. Importantly, these results suggest that the presence of atypical, enlarged, conical cells could be associated with PG and with tolerant cells in Candida species biofilm populations. The clinical implications of these findings are still unknown.Candida species are opportunistic pathogens that cause superficial and systemic diseases in critically ill patients (8, 22, 44) and are associated with high mortality rates (35%) and costly treatments (8, 19). They rank among the four most common causes of bloodstream infection in U.S. hospitals, surpassing gram-negative rods in incidence (6, 17).Recent studies suggest that the majority of disease produced by this pathogen is associated with a biofilm growth style (7, 16, 28, 48). Biofilms are self-organized communities of microorganisms that grow on an abiotic or biotic surface, are embedded in a self-produced matrix consisting of an extracellular polymeric substance (14, 15, 55), and when associated with implanted medical devices are commonly refractive to antimicrobial therapy.As opportunistic pathogens, Candida species are able to attach to polymeric surfaces and generate a biofilm structure, protecting the organisms from the host defenses and antifungal drugs (11, 16, 45, 48). Candida biofilms are more resistant than their planktonic counterparts to various antifungal agents, including amphotericin B (AMB), fluconazole, itraconazole, and ketoconazole (20, 38, 50). However, the molecular basis for the antifungal resistance of biofilm-related organisms is not completely understood.The complex architecture of Candida biofilms observed both in vitro and in vivo suggests that morphological differentiation to produce hyphae plays an important role in biofilm formation and maturation (7, 32, 33). Baillie and Douglas demonstrated that although mutant cells fixed in either a hyphal or a yeast form can develop into biofilms, the hyphal structure is the essential element for providing the integrity and multilayered architecture of a biofilm (4). It has been reported that Candida parapsilosis, C. glabrata, and C. tropicalis biofilms are not as large as those generated by C. albicans; however, further structural analysis studies are needed to describe biofilm formation by these organisms (30, 31).The mechanisms responsible for the resistance characteristics displayed by Candida biofilms are unclear. Possible mechanisms include a decreased growth rate; nutrient limitation of cells in the biofilm; expression of resistance genes, particularly those encoding efflux pumps; increased cell density; cell aging; or the presence of “persister” cells in the biofilm (1, 3, 5, 29, 34, 36, 38, 43, 46, 48, 50, 51).The echinocandins are a novel class of semisynthetic amphiphilic lipopeptides that display important antifungal activity. The echinocandins that are presently marketed are caspofungin (CAS), micafungin, and anidulafungin. The echinocandins show considerable efficacy in vitro and in vivo in the treatment of candidemia and invasive candidiasis (25, 27, 42). CAS is the first antifungal agent to be licensed that inhibits the synthesis of β-1,3-glucan, the major structural component of Candida cell walls; glucan synthesis might prove to be a particularly effective target for biofilms (29, 31, 38, 48, 50). The paradoxical attenuation of antifungal activity at high echinocandin concentrations is a phenomenon that usually occurs with C. albicans isolates and appears to be specific to CAS among echinocandins. The cells surviving at high concentrations appear to be subject to some drug effect, showing evidence of slowed growth in the presence of CAS (53, 54). Recent studies have described this effect in Candida species biofilms (24, 37, 47); however, we are not aware of studies that have elucidated the effect of CAS on Candida biofilm structure. The present study was designed to (i) characterize the in vitro biofilm growth of Candida species bloodstream isolates and (ii) use scanning electron microscopy (SEM) to obtain visual evidence of the effect of CAS on biofilm morphology changes associated with paradoxical growth (PG).     

Rapamycin Protects Mice from Staphylococcal Enterotoxin B-Induced Toxic Shock and Blocks Cytokine Release In Vitro and In Vivo

Staphylococcal enterotoxins are potent activators for human T cells and cause lethal toxic shock. Rapamycin, an immunosuppressant, was tested for its ability to inhibit staphylococcal enterotoxin B (SEB)-induced activation of human peripheral blood mononuclear cells (PBMC) in vitro and toxin-mediated shock in mice. Stimulation of PMBC by SEB was effectively blocked by rapamycin as evidenced by the inhibition of tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), IL-6, IL-2, gamma interferon (IFN-γ), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and T-cell proliferation. In vivo, rapamycin protected 100% of mice from lethal shock, even when administered 24 h after intranasal SEB challenge. The serum levels of MCP-1 and IL-6, after intranasal exposure to SEB, were significantly reduced in mice given rapamycin versus controls. Additionally, rapamycin diminished the weight loss and temperature fluctuations elicited by SEB.Staphylococcal exotoxins are among the most common etiological agents that cause toxic shock syndrome (28-30, 38, 44). The disease is characterized by fever, hypotension, desquamation of skin, and dysfunction of multiple organ systems (8, 38, 41). These toxins bind directly to the major histocompatibility complex (MHC) class II molecules on antigen-presenting cells and subsequently stimulate T cells expressing specific Vβ elements on T-cell receptors (9, 15, 24, 29, 35, 42). Staphylococcal enterotoxin B (SEB) and the distantly related toxic shock syndrome toxin 1 are also called superantigens because they induce massive proliferation of T cells (29). In vitro and in vivo studies show that these superantigens induce high levels of various proinflammatory cytokines, and these potent mediators cause lethal shock in animal models (1, 6, 22, 27, 37, 39, 45, 51, 55). SEB also causes food poisoning (4, 21, 52) and is a potential bioterrorism threat agent, as humans are extremely sensitive to this superantigen, especially by inhalation (28). There is currently no effective therapeutic treatment for SEB-induced shock except for the use of intravenous immunoglobulins (11). Various in vitro experiments identified inhibitors to counteract the biological effects of SEB, only some of which were successful in ameliorating SEB-induced shock in experimental models (1, 25-27, 51).Rapamycin is a relatively new FDA-approved drug used to prevent graft rejection in renal transplantation, as it shows less nephrotoxicity than do calcineurin inhibitors (14, 40, 43, 48). Recent studies reveal other uses in animal models of cancer (23, 34), diabetic nephropathy (36), bleomycin-induced pulmonary fibrosis (31), liver fibrosis (5), and tuberous sclerosis (32). Rapamycin binds intracellularly to FK506-binding proteins, specifically FKBP12; the rapamycin-FKBP12 complex then binds to a distinct molecular target called mammalian target of rapamycin (mTOR) (reviewed in reference 48). Rapamycin inhibits mTOR activity, prevents cyclin-dependent kinase activation, and affects G₁-to-S-phase transition (16, 48). Other studies identified mTOR as the conserved serine-threonine kinase for sensing cellular stress, and rapamycin promotes anabolic cellular processes in response to stress signals (20, 47, 50, 54). The mTOR pathway regulates myogenesis (13), cell cycle arrest (20), adipocyte differentiation (3), and insulin signaling (47, 50). The immunological effects of rapamycin include regulation of T-cell activation (48); differentiation, expansion, and preservation of regulatory T cells (2, 10, 19, 46); downregulation of dendritic cells (12, 53); and granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced neutrophil migration (17). Rapamycin impairs dendritic cell maturation and function by inhibiting the expression of adhesion molecule ICAM-1 (12, 53). Thus, rapamycin has a broad spectrum of effects and interferes with the activation of multiple cell types of the immune system.Based on the potent immunosuppressive effects of rapamycin, we investigated the therapeutic impact of rapamycin on SEB-mediated toxic shock. The therapeutic efficacy of rapamycin in SEB-induced toxic shock was investigated by using a lethal murine model with intranasal delivery of SEB (22). This “double-hit” murine model relies on two low doses of SEB without the use of sensitizing agents such as lipopolysaccharide (LPS) or galactosamine to induce lethal shock (6, 27, 33, 37, 45). In this “SEB-only” toxic shock model, SEB was administered intranasally (i.n.) and another dose of SEB was strategically given intraperitoneally (i.p.) 2 h later to induce systemic cytokine release and pulmonary inflammation with lethality as an endpoint. We examined the effect of rapamycin on proinflammatory cytokines and chemokines induced by SEB in vitro using human peripheral blood mononuclear cells (PBMC) as a first step to test its immunological effects on SEB activation.     

Borrelia burgdorferi Resistance to a Major Skin Antimicrobial Peptide Is Independent of Outer Surface Lipoprotein Content

We hypothesize a potential role for Borrelia burgdorferi OspC in innate immune evasion at the initial stage of mammalian infection. We demonstrate that B. burgdorferi is resistant to high levels (>200 μg/ml) of cathelicidin and that this antimicrobial peptide exhibits limited binding to the spirochetal outer membrane, irrespective of OspC or other abundant surface lipoproteins. We conclude that the essential role of OspC is unrelated to resistance to this component of innate immunity.Borrelia burgdorferi, a spirochete and the causal organism of Lyme disease, is naturally transmitted to mammals through the bite of infected Ixodes ticks (5, 8). A significant change in B. burgdorferi gene expression accompanies transmission between these diverse environments. This was first described for the inverse relationship between two abundant outer surface proteins of B. burgdorferi, in which synthesis of OspA declines and that of OspC increases during tick feeding (41). We and others have demonstrated the essential nature of OspC for colonization of the murine host (23, 35, 42, 45, 47, 49). These findings suggest a critical role for OspC in evasion of host innate immunity immediately after transmission (47). However, the essential contribution of OspC to early mammalian infection by B. burgdorferi remains undefined.Microorganisms induce a variety of responses from the skin epithelial cells of their hosts, including the production of antimicrobial peptides, which are recognized as integral components of the innate immune system (20, 22). Defensins and cathelicidins comprise two major families of cationic antimicrobial peptides secreted by human and other mammalian skin neutrophils (20). Mouse neutrophils lack α defensins (14, 24), but about 30 cathelicidin members have been identified in various mammalian species, including mice (21, 50). These small, cationic, amphipathic molecules are primarily stored as inactive propeptides in the secretory granules of skin neutrophils. The mature bioactive peptides assume an α-helical structure in solution and preferentially interact with negatively charged cell surface components of a broad spectrum of bacteria and fungi, in which they disrupt cell membrane integrity (6, 9, 12, 20, 34). The importance of the sole murine cathelicidin, known as mCRAMP (mouse cathelin-related antimicrobial peptide) (19, 36), to innate host defense is well established, and mCRAMP has been shown to provide protection against bacterial skin infections in mice (33).     

Pharmacokinetics,Safety, and Tolerability of Voriconazole in Immunocompromised Children

The pharmacokinetics of voriconazole in children receiving 4 mg/kg intravenously (i.v.) demonstrate substantially lower plasma exposures (as defined by area under the concentration-time curve [AUC]) than those in adults receiving the same therapeutic dosage. These differences in pharmacokinetics between children and adults limit accurate prediction of pediatric voriconazole exposure based on adult dosages. We therefore studied the pharmacokinetics and tolerability of higher dosages of an i.v.-to-oral regimen of voriconazole in immunocompromised children aged 2 to <12 years in two dosage cohorts for the prevention of invasive fungal infections. The first cohort received 4 mg/kg i.v. every 12 h (q12h), then 6 mg/kg i.v. q12h, and then 4 mg/kg orally (p.o.) q12h; the second received 6 mg/kg i.v. q12h, then 8 mg/kg i.v. q12h, and then 6 mg/kg p.o. q12h. The mean values for the AUC over the dosing interval (AUCτ) for 4 mg/kg and 6 mg/kg i.v. in cohort 1 were 11,827 and 22,914 ng·h/ml, respectively, whereas the mean AUCτ values for 6 mg/kg and 8 mg/kg i.v. in cohort 2 were 17,249 and 29,776 ng·h/ml, respectively. High interpatient variability was observed. The bioavailability of the oral formulation in children was approximately 65%. The safety profiles were similar in the two cohorts and age groups. The most common treatment-related adverse event was increased gamma glutamyl transpeptidase levels. There was no correlation between adverse events and voriconazole exposure. In summary, voriconazole was tolerated to a similar degree regardless of dosage and age; the mean plasma AUCτ for 8 mg/kg i.v. in children approached that for 4 mg/kg i.v. in adults, thus representing a rationally selected dosage for the pediatric population.Invasive fungal infections cause severe morbidity and mortality in immunocompromised children, particularly those with hematological malignancies and those undergoing hematopoietic stem cell transplantation (HSCT) (3, 8, 11, 19, 21, 27, 28). Voriconazole is a broad-spectrum antifungal triazole with in vitro and in vivo activity against yeasts and filamentous fungi (2, 5). Voriconazole is approved for adults for primary treatment of invasive aspergillosis, esophageal candidiasis, and candidemia in nonneutropenic patients. It is also approved for the treatment of serious, refractory infections caused by Scedosporium and Fusarium species (16, 25). Voriconazole is more effective than deoxycholate amphotericin B in treatment of invasive aspergillosis in adults, most of whom suffered from invasive pulmonary aspergillosis (9). In addition, voriconazole has been used successfully to treat aspergillosis of the central nervous system and bone (14, 20).Several case series and single case reports have described the safety and efficacy of voriconazole in pediatric oncology patients and other immunocompromised children with invasive mycoses (10, 24, 26). Voriconazole also has been used to treat Aspergillus airway disease in pediatric patients with cystic fibrosis (10). However, considerably less is known about the pharmacokinetics of this antifungal agent in children than about those in adults.Current studies indicate that there are major differences between the pharmacokinetics of voriconazole in children and those in adults. The first systematic study of the safety, tolerability, and pharmacokinetics of voriconazole in pediatric patients demonstrated linear plasma pharmacokinetics in patients receiving intravenous (i.v.) voriconazole at dosages of 3 mg/kg every 12 h (q12h) or 4 mg/kg q12h (24). By comparison, voriconazole in adults displays nonlinear Michaelis-Menten plasma pharmacokinetics following similar dosages (17, 18). These differences in pharmacokinetics between children and adults limit accurate prediction of pediatric voriconazole exposure (as defined by area under the concentration-time curve [AUC]) based on adult dosages. Indeed, pharmacokinetic modeling studies demonstrated that the AUC was approximately 3-fold lower in children receiving 4 mg/kg of voriconazole q12h than in adults receiving the same dosage.Therefore, in order to approximate the plasma exposure achieved in adults, we studied the safety, tolerability, and plasma pharmacokinetics of voriconazole in pediatric patients receiving dosages of 4, 6, and 8 mg/kg i.v. q12h. We also examined the plasma pharmacokinetics of the oral suspension of voriconazole at 4 and 6 mg/kg q12h.     

The Antituberculosis Drug Pyrazinamide Affects the Course of Cutaneous Leishmaniasis In Vivo and Increases Activation of Macrophages and Dendritic Cells

Antileishmanial therapy is suboptimal due to toxicity, high cost, and development of resistance to available drugs. Pyrazinamide (PZA) is a constituent of short-course tuberculosis chemotherapy. We investigated the effect of PZA on Leishmania major promastigote and amastigote survival. Promastigotes were more sensitive to the drug than amastigotes, with concentrations at which 50% of parasites were inhibited (MIC₅₀) of 16.1 and 8.2 μM, respectively (48 h posttreatment). Moreover, 90% of amastigotes were eliminated at 120 h posttreatment, indicating that longer treatments will result in parasite elimination. Most strikingly, PZA treatment of infected C57BL/6 mice resulted in protection against disease and in a 100-fold reduction in the parasite burden. PZA treatment of J774 cells and bone marrow-derived dendritic cells and macrophages increased interleukin 12, tumor necrosis factor alpha, and activation marker expression, as well as nitric oxide production, suggesting that PZA enhances effective immune responses against the parasite. PZA treatment also activates dendritic cells deficient in Toll-like receptor 2 and 4 expression to initiate a proinflammatory response, confirming that the immunostimulatory effect of PZA is directly caused by the drug and is independent of Toll-like receptor stimulation. These results not only are strongly indicative of the promise of PZA as an alternative antileishmanial chemotherapy but also suggest that PZA causes collateral immunostimulation, a phenomenon that has never been reported for this drug.The leishmaniases are a group of insect-transmitted parasitic diseases prevalent worldwide, endemic in 88 countries; 350 million people are at risk, and 12 million people are affected. Two million new cases are estimated to occur annually, although only 600,000 are officially reported (10). During the last two decades, it has become increasingly apparent that the leishmaniases are much more prevalent than had been previously suspected. With human migration and vector expansion dramatically affecting the spread of disease, dramatic outbreaks have occurred in locations with previously low levels of infection (e.g., Kabul, Afghanistan, with more than 200,000 infected [22]). Immunologically naive individuals from the developed world traveling to areas of endemicity are particularly prone to infection. Leishmaniasis has been found among American soldiers deployed to the Middle East during both Gulf wars, current conflicts in Afghanistan, and Central America (3, 7, 12, 14, 18, 20, 24, 29). Civilians traveling into these areas are also at risk (2).Currently, there are nearly 25 licensed compounds with antileishmanial effects, but only a few are used in humans. Drawbacks associated with conventional treatment with antimonials and amphotericin B include high toxicity, differences in strain sensitivity, and resistance. Moreover, the expense of these drugs often precludes their use. As recently as 2004, liposomal amphotericin B, miltefosine, and paromomycin were identified by WHO/TDR as the three most promising drugs in the market. These drugs are not new: amphotericin B has been used extensively for decades as a second-line drug for treatment of leishmaniasis (in addition to its antifungal activity), miltefosine was developed long ago as an anticancer agent, and paromomycin is more than 50 years old. To date, these three agents, together with antimonials and nonliposomal amphotericin B, are the reference chemotherapeutic agents for the leishmaniases. Oral miltefosine has been shown to be as efficacious against leishmaniasis as the standard amphotericin B treatment in India; however, important side effects, such as teratogenicity, are associated with this drug (28). With this limitation, the need for safer, inexpensive, and widely available treatments continues to be one of the top research priorities for disease control (5).In contrast to other possible strategies (“orphan drugs,” combinatory chemistry, or rational design), we seek new indications for existing drugs, which can be a very fruitful route for drug discovery and development (6). Pyrazinamide (PZA), an essential constituent of short-course tuberculosis chemotherapy (11), was developed as an analog of nicotinamide and used in the late 20th century in the treatment of Mycobacterium tuberculosis. PZA and related analogs have also demonstrated activity against other Mycobacterium spp. (9, 21, 27). In mycobacteria, we found that PZA inhibits the enzyme fatty acid synthase I (FASI) (33) by competitive inhibition of a NADPH binding site (25). By analogy, it could be proposed that PZA interferes with fatty acid synthesis in trypanosomatids. Although no genes homologous to the FASI gene have been identified in the Leishmania genome, this parasite employs microsomal elongases in an iterative manner to synthesize fatty acids (17). This suggests that inhibition of fatty acid synthesis might be an attractive chemotherapeutic target in Leishmania (16, 23).In this article, we demonstrate that PZA has antileishmanial effect in vitro on both promastigotes and amastigotes. More importantly, PZA dramatically decreases lesion development and the parasite burden in C57BL/6 mice infected with Leishmania major. Finally, we show that PZA increases activation of infected macrophages and dendritic cells by increasing expression of costimulatory molecules and secretion of proinflammatory cytokines and nitric oxide. These results not only show that PZA constitutes a very promising alternative therapy for leishmaniasis but also suggest that the drug causes collateral immunostimulation.     

Identification and Characterization of Small-Molecule Inhibitors of Yop Translocation in Yersinia pseudotuberculosis

Type three secretion systems (TTSSs) are virulence factors found in many pathogenic Gram-negative species, including the family of pathogenic Yersinia spp. Yersinia pseudotuberculosis requires the translocation of a group of effector molecules, called Yops, to subvert the innate immune response and establish infection. Polarized transfer of Yops from bacteria to immune cells depends on several factors, including the presence of a functional TTSS, the successful attachment of Yersinia to the target cell, and translocon insertion into the target cell membrane. Here we employed a high-throughput screen to identify small molecules that block translocation of Yops into mammalian cells. We identified 6 compounds that inhibited translocation of effectors without affecting synthesis of TTSS components and secreted effectors, assembly of the TTSS, or secretion of effectors. One compound, C20, reduced adherence of Y. pseudotuberculosis to target cells. Additionally, the compounds caused leakage of Yops into the supernatant during infection and thus reduced polarized translocation. Furthermore, several molecules, namely, C20, C22, C24, C34, and C38, also inhibited ExoS-mediated cell rounding, suggesting that the compounds target factors that are conserved between Pseudomonas aeruginosa and Y. pseudotuberculosis. In summary, we have identified 6 compounds that specifically inhibit translocation of Yops into mammalian cells but not Yop synthesis or secretion.Many pathogenic Gram-negative bacteria express a type three secretion system (TTSS) that translocates effector proteins into the cytosol of their eukaryotic cell targets. Once introduced into host cells, these proteins subvert normal cell functions, e.g., by disrupting innate immune signaling or modulating the phagosomal environment (7, 51, 67, 73). TTSSs are comprised of a base structure, a needle, and a tip/translocon complex (52). The base structure, which spans the inner and outer membranes, shares high structural homology to conserved bacterial flagellar machinery (11). High-resolution microscopy of the base structures of Shigella and Salmonella revealed that the base consists of several ring structures that surround a hollow cavity (10, 41, 46). The needle is comprised of a small protein that polymerizes to form a hollow tube that starts within the base and protrudes from the bacterial surface (30, 41, 71). Effectors are thought to be translocated through the needle (19, 37, 41, 46), although this has not been demonstrated conclusively for many systems. Many TTSSs secrete effectors into culture supernatants with just the base and needle; however, translocation of effectors into mammalian cells requires three additional components, together called the translocon (28, 31). Two proteins (9, 28, 59) are inserted into the eukaryotic cell membrane to form a pore. The third (53) is critical for proper assembly of the translocon and is localized at the distal end of the needle but is not inserted into the host plasma membrane.There are three species of Yersinia which are pathogenic to humans. Yersinia pseudotuberculosis (32) and Y. enterocolitica both cause gastroenteritis and lymphadenitis and are commonly transmitted via the fecal-oral route (66). Y. pestis is the causative agent of bubonic and pneumonic plague and is commonly transmitted by a flea vector from infected rodents to humans (1, 14). It disseminates through the skin to the lymph nodes, where it causes a bubonic disease. Occasionally, Y. pestis disseminates to the lungs of an infected individual, which can lead to a pneumonic transmission from person to person, resulting in a fatal lung infection (42, 66). The TTSS is an essential virulence factor for all three pathogenic Yersinia spp. (6, 17, 32, 56). Yersinia strains lacking this secretion system can function as live attenuated vaccine strains in mice (6, 61). The critical needle and translocation components of the Yersinia TTSS include the needle protein (YscF) (30), the tip protein (LcrV), and the pore-forming proteins (YopB and YopD) (44, 72). The effector proteins translocated by the Yersinia TTSS, called Yops, are targeted to neutrophils, macrophages, and dendritic cells, where they inactivate the bactericidal effects of these cells during murine infection (21, 39, 45). Inactivation of the TTSS leads to defective colonization of systemic organs and clearance of the bacteria by the host organism (6, 29, 74).The process of translocation in Yersinia requires close contact between the host cell and the bacterium (8). For the enteric Yersinia spp., this contact is mediated by two adhesins, YadA and invasin (8, 36, 82). Both of these molecules bind β1 integrins on the surfaces of target cells (22, 35). In cultured cells, stimulation of β1 integrins by ligands activates Src kinases and RhoA, which in turn enhances translocation of Yops (47). In the absence of Yops, activation of β1 integrins leads to actin rearrangements resulting in bacterial internalization (50). However, in Yersinia strains expressing the TTSS and Yops, this process is antagonized by the effector proteins (8). The result is that virulent Yersinia adheres tightly to mammalian cells while remaining extracellular.Since the TTSS is essential for virulence of Yersinia and other Gram-negative pathogens, this system has been a target for development of novel therapeutics (3, 24, 27, 38, 62, 79). Several screens have been designed to identify inhibitors of TTSS synthesis and/or Yop secretion from the bacteria (3, 24, 62). Such inhibitors should also block translocation of effectors into mammalian target cells and therefore abrogate virulence. These screens have led to the identification of several classes of molecules that inhibit not only the TTSS of Yersinia but also the TTSSs of other pathogens, such as Chlamydia, Salmonella, and Shigella (5, 33, 57, 58, 77). Here we describe a screen to identify small molecules that block translocation of effectors into mammalian cells. The small molecules that were identified were unique in that they still permitted secretion of Yops from bacteria, but they reduced the polarized translocation of Yops into target cells and caused excessive leakage of Yops into culture supernatants. These compounds may represent novel agents that target effector translocation, an essential process for virulence in Yersinia and other TTSS-containing pathogens.     

Echinocandin Susceptibility Testing of Candida Species: Comparison of EUCAST EDef 7.1, CLSI M27-A3, Etest,Disk Diffusion,and Agar Dilution Methods with RPMI and IsoSensitest Media

This study compared nine susceptibility testing methods and 12 endpoints for anidulafungin, caspofungin, and micafungin with the same collection of blinded FKS hot spot mutant (n = 29) and wild-type isolates (n = 94). The susceptibility tests included EUCAST Edef 7.1, agar dilution, Etest, and disk diffusion with RPMI-1640 plus 2% glucose (2G) and IsoSensitest-2G media and CLSI M27A-3. Microdilution plates were read after 24 and 48 h. The following test parameters were evaluated: fks hot spot mutants overlapping the wild-type distribution, distance between the two populations, number of very major errors (VMEs; fks mutants misclassified as susceptible), and major errors (MEs; wild-type isolates classified as resistant) using a wild-type-upper-limit value (WT-UL) (two twofold-dilutions higher than the MIC₅₀) as the susceptibility breakpoint. The methods with the lowest number of errors (given as VMEs/MEs) across the three echinocandins were CLSI (12%/1%), agar dilution with RPMI-2G medium (14%/0%), and Etest with RPMI-2G medium (8%/3%). The fewest errors overall were observed for anidulafungin (4%/1% for EUCAST, 4%/3% for CLSI, and 3%/9% for Etest with RPMI-2G). For micafungin, VME rates of 10 to 71% were observed. For caspofungin, agar dilution with either medium was superior (VMEs/MEs of 0%/1%), while CLSI, EUCAST with IsoSensitest-2G medium, and Etest were less optimal (VMEs of 7%, 10%, and 10%, respectively). Applying the CLSI breakpoint (S ≤ 2 μg/ml) for CLSI results, 89.2% fks hot spot mutants were classified as anidulafungin susceptible, 60.7% as caspofungin susceptible, and 92.9% as micafungin susceptible. In conclusion, no test was perfect, but anidulafungin susceptibility testing using the WT-UL to define susceptibility reliably identified fks hot spot mutants.Three echinocandin class drugs, anidulafungin, caspofungin, and micafungin, are licensed for the treatment of invasive candidiasis. They are among the preferred agents for invasive candidiasis, as a number of recent fungemia surveys have reported a considerable proportion of cases involving species with reduced susceptibility to fluconazole (3, 4, 24, 28, 31, 37, 44). Additionally, anidulafungin has been associated with an improved success rate, even in cases involving fluconazole-susceptible species (39). Following increased use, sporadic cases of failures associated with elevated MICs have been reported. In the majority of cases, these failures have been associated with mutations in two hot spot regions of FKS genes, which encode the target and major subunit of the 1,3-ß-d-glucan synthase complex (5, 7, 22, 25, 26, 33, 34). Consequently, close monitoring and robust susceptibility testing methods have become increasingly important.EUCAST and CLSI have developed standard methods based on broth dilution for the susceptibility testing of yeasts (9, 41). Methodological differences include glucose concentration, inoculum size, shape of microtiter wells (flat or round), and end-point reading (visual or spectrophotometric), but the methods are more alike than different and in general generate similar results (11, 42). Recently, CLSI proposed an S value of ≤2 μg/ml as a tentative susceptibility breakpoint for caspofungin, micafungin, and anidulafungin for Candida spp., taking into account analysis of mechanisms of resistance, an epidemiological MIC population distribution, parameters associated with success in pharmacodynamic models, and results of clinical efficacy studies (9, 38). As no significant differences in clinical response were noted among the various species, results for all species were merged, and a susceptibility breakpoint of 2 μg/ml was found to encompass the vast majority of isolates, while not bisecting the population of Candida parapsilosis. The crucial issue is whether current susceptibility testing methods and breakpoints clearly and reliably identify isolates with resistance mechanisms associated with treatment failures (5, 7, 8, 13, 14, 16, 18, 22, 25, 26, 33, 40). Not only have cases involving isolates classified as susceptible using the reference methods been shown to contain resistance mutations (5, 7, 13, 14, 22, 25), but also recent studies suggest that a breakpoint of an S value of ≤2 μg/ml may be too high for anidulafungin and micafungin, considering the 1,3-ß-d-glucan synthase kinetic inhibition data of wild-type and mutant enzymes from resistant strains (17, 18). Finally, we recently reported a resistant Candida albicans isolate that failed to be identified as resistant when the reference methodologies were used, while Etest, agar dilution, and disk diffusion methods correctly identified it (5).We therefore undertook a comparative study of the two references methods, a modified EUCAST microdilution method using IsoSensitest medium, agar dilution, and disk and Etest diffusion using RPMI-1640 as well as IsoSensitest medium to evaluate their ability to reliably discriminate between a well-characterized panel of wild-type and fks hot spot mutant Candida isolates. The semisynthetic IsoSensitest medium was chosen as an alternative medium due to this medium having previously been shown to be appropriate for amphotericin B MIC testing (10).     

Correlation between Azole Susceptibilities,Genotypes, and ERG11 Mutations in Candida albicans Isolates Associated with Vulvovaginal Candidiasis in China

The relationship between susceptibilities to fluconazole and itraconazole and microsatellite CAI genotypes were examined from a total of 154 Candida albicans isolates (97 isolates causing vulvovaginitis in Chinese women and 6 vaginal isolates and 51 oral cavity isolates from asymptomatic carriers). The two dominant genotypes, CAI 30-45 (45 isolates) and CAI 32-46 (33 isolates), associated with vulvovaginitis showed significantly different azole susceptibility patterns with strong statistical support. CAI 32-46 isolates were usually less susceptible to both fluconazole and itraconazole than CAI 30-45 isolates and than the oral isolates with other diversified CAI genotypes. Remarkably different mutation patterns in the azole target gene ERG11 were correspondingly observed among C. albicans isolates representing different genotypes and sources. Isolates with the same or similar CAI genotypes usually possessed identical or phylogenetically closely related ERG11 sequences. Loss of heterozygosity in ERG11 was observed in all the CAI 32-46 isolates but not in the CAI 30-45 isolates and most of the oral isolates sequenced. Compared with the ERG11 sequence of strain SC5314 ({"type":"entrez-nucleotide","attrs":{"text":"X13296","term_id":"2503","term_text":"X13296"}}X13296), two homozygous missense mutations (G487T and T916C) leading to two amino acid changes (A114S and Y257H) in Erg11p were found in CAI 32-46 isolates. The correlation between azole susceptibility and C. albicans genotype may be of potential therapeutic significance.Vulvovaginal candidiasis (VVC) is a common vaginal infection, affecting up to 75% of women of child-bearing age at least once in their lifetime (7, 21, 22). The most frequent cause of VVC is Candida albicans, which is responsible for 70 to 90% of vulvovaginitis cases. Non-C. albicans species of Candida, predominantly Candida glabrata, are responsible for the remainder of cases (21). On the basis of the severity of symptoms, frequency, and causative agents, VVC is usually classified as either uncomplicated (mild and sporadic) or complicated (recurrent, severe, or caused by non-C. albicans species) (7, 21). Ten to 20% of women suffer complicated VVC in their lifetime (21). When properly diagnosed, uncomplicated VVC may be treated easily and reliably. However, complicated VVC often causes long-term physical and mental discomfort, significant economic burden from treatments, and considerable negative effect on sexual relations (21-23).At present, prolonged suppressive therapy using fluconazole is recommended as the standard management for chronic, recurrent Candida vulvovaginitis (23). Therefore, there is a great concern about the emergence and spread of azole resistance of C. albicans isolates associated with VVC. Indeed, susceptibility testing of VVC-causing isolates has been performed in different countries and regions of the world (1, 2, 4, 5, 6, 13-15, 17, 18, 20, 24). Although relatively high frequencies of fluconazole- and/or itraconazole-resistant C. albicans isolates causing VVC have been observed in a few reports (13, 20, 24), most studies failed to identify any clear correlation between azole susceptibility and VVC association among C. albicans isolates (1, 2, 4, 5, 6, 14, 15, 17, 18).Recently, we compared the genotype distribution patterns among independent C. albicans isolates associated with VVC in Chinese women and those from various extragenital sites by using the polymorphic microsatellite locus CAI (8, 11). The results showed that the CAI genotypes of C. albicans isolates from extragenital sites were highly diversified. In contrast, isolates associated with VVC from unrelated patients were more homogeneous and belonged to only a few genotypes, with two genotypes, CAI 30-45 and CAI 32-46, being the most common. These two dominant genotypes were rarely found among isolates from extragenital sites (11). In addition, the distribution of the dominant genotypes correlated positively with the severity of VVC (8, 11). These results suggested that C. albicans isolates with genotypes CAI 30-45 and CAI 32-46 might be more virulent and/or more resistant to the commonly used azole drugs than those with other genotypes as causative agents of vaginal infection.Antifungal susceptibility testing using the Etest method revealed that the C. albicans isolates causing VVC in Chinese women were generally susceptible to fluconazole, amphotericin B, ketoconazole, and flucytosine; however, 19.1% of the isolates could be interpreted as being resistant to itraconazole in vitro. Interestingly, most of the itraconazole-resistant isolates belonged to a specific genotype (13). Contrary to the report described above, recent susceptibility testing and microsatellite typing of vulvovaginitis-causing Candida isolates from Europe did not find an association between azole resistance and any particular genotype cluster among C. albicans isolates (1). In the present study, fluconazole and itraconazole susceptibilities of the C. albicans isolates with the dominant genotypes CAI 30-45 and CAI 32-46 from VVC patients were compared with those of isolates possessing other minor genotypes and of isolates from the oral cavity by using the standard broth microdilution method. Furthermore, ERG11 (encoding lanosterol-14-α-demethylase, the target of azoles) gene sequences of C. albicans isolates representing different genotypes and sources were determined. The correlation between azole susceptibilities, genotypes, and ERG11 mutations was examined.     

Role for Fks1 in the Intrinsic Echinocandin Resistance of Fusarium solani as Evidenced by Hybrid Expression in Saccharomyces cerevisiae

The opportunistic mold Fusarium solani is intrinsically resistant to cell wall synthesis-inhibiting echinocandins (ECs), including caspofungin and micafungin. Mutations that confer acquired EC resistance in Saccharomyces cerevisiae and other normally susceptible yeast species have been mapped to the Fks1 gene; among these is the mutation of residue 639 from Phe to Tyr (F639Y) within a region designated hot spot 1. Fks1 sequence analysis identified the equivalent of Y639 in F. solani as well as in Scedosporium prolificans, another intrinsically EC-resistant mold. To test its role in intrinsic EC resistance, we constructed Fks1 hybrids in S. cerevisiae that incorporate F. solani hot spot 1 and flanking residues. Hybrid construction was accomplished by a PCR-based method that was validated by studies with Fks1 sequences from EC-susceptible Aspergillus fumigatus and paired EC-susceptible and -resistant Candida glabrata isolates. In support of our hypothesis, hybrid Fks1 incorporating F. solani hot spot 1 conferred significantly reduced EC susceptibility, 4- to 8-fold less than that of wild-type S. cerevisiae and 8- to 32-fold less than that of the same hybrid with an F639 mutation. We propose that Fks1 sequences represent determinants of intrinsic EC resistance in Fusarium and Scedosporium species and, potentially, other fungi.Serious fungal infections have increased in recent years as a consequence of increased immunosuppression associated with human immunodeficiency virus infection, organ and tissue transplants, and aggressive treatments for neoplastic and autoimmune disease. These infections typically are treated with ergosterol biosynthesis-inhibiting azole antifungals such as fluconazole. However, azoles have limitations: their activity is fungistatic, acquired resistance in normally susceptible yeast is not uncommon, and intrinsic low- to high-level resistance is demonstrated by many molds (3, 21). The membrane-disrupting antifungal amphotericin B is generally fungicidal and has broad-spectrum activity, and resistance to it is rare, but its use remains limited due to toxicity. The echinocandins (ECs) caspofungin (CSP), micafungin (MCF), and anidulafungin represent the most recently introduced group of antifungals. Importantly, ECs have fungicidal activity against most Candida species (including azole-resistant strains), fungistatic activity against Aspergillus species, and negligible toxicity (5, 20, 24). ECs act by inhibiting the synthesis of the cell wall polysaccharide β-1,3-glucan (7). This can result in cell lysis or more subtle cell wall changes that enhance susceptibility to innate immunity (25, 41).Acquired EC resistance in susceptible fungi is associated with specific mutations in the integral membrane protein Fks1 (or its paralog Fks2) (7, 35). Fks1 is believed to represent the β-1,3-glucan synthase catalytic subunit, although this has not been formally proven since only crude membrane preparations retain catalytic activity. Most resistance-conferring mutations cluster within so-called hot spot 1, which corresponds, within the model yeast Saccharomyces cerevisiae, to Fks1 residues Phe639 to Pro647 (F639-P647) (1, 2, 6, 7, 13, 18, 19, 26, 33, 34, 40). Fortunately, acquired EC resistance is rare. On the other hand, the intrinsic EC resistance of ascomycetous molds such as Fusarium solani and Scedosporium prolificans, zygomycetous molds such as Rhizopus oryzae, and the basidiomycetous yeasts Cryptococcus neoformans and Trichosporon asahii represents a major limitation to EC clinical use. Many of these fungi have emerged in recent years as important opportunistic pathogens (3, 27, 32, 37), and a contributing factor may be their intrinsic resistance to antifungals, including ECs (10, 22, 30).The basis for intrinsic EC resistance has been investigated in several of these fungi, but it remains unclear (14, 15, 28, 39). Notably, Ha et al. (14) reported that F. solani Fks1, when heterologously expressed in A. fumigatus, conferred a modest but potentially significant fourfold decrease in CSP susceptibility, the basis for which was not explored. However, expression in their system was abnormally low, and A. fumigatus itself exhibits some degree of intrinsic EC resistance (5), which together complicate the interpretation of this result. On the other hand, recent studies examining the basis for the intrinsically low EC susceptibility of Candida parapsilosis strongly implicated its Fks1 sequence; specifically, a hot spot 1 substitution equivalent to P647A (12).FKS1 initially was identified as the S. cerevisiae gene whose null mutation confers susceptibility to the calcineurin inhibitor FK506 (7, 8). This susceptibility results from the requirement for the calcineurin-mediated expression of FKS2; relatedly, single fks1Δ and fks2Δ disruptants are viable, but double disruption is lethal (31). Here, we exploit this FK506 susceptibility to construct Fks1 hybrids, replacing hot spot 1 from S. cerevisiae with that from F. solani (and, for comparison, A. fumigatus and Candida glabrata) to further test the hypothesis that intrinsic EC resistance is mediated by Fks1 sequence.     

Inhibitory and Bactericidal Activities of Daptomycin,Vancomycin, and Teicoplanin against Methicillin-Resistant Staphylococcus aureus Isolates Collected from 1985 to 2007

The inhibitory and bactericidal activities of daptomycin, vancomycin, and teicoplanin against a collection of 479 methicillin-resistant Staphylococcus aureus isolates were assessed. The isolates were collected from U.S. and European hospitals from 1985 to 2007 and were primarily from blood and abscess cultures. The MICs and minimum bactericidal concentrations (MBCs) of the three agents were determined, and the MBC/MIC ratios were calculated to determine the presence or absence of tolerance. Tolerance was defined as an MBC/MIC ratio of ≥32 or an MBC/MIC ratio of ≥16 when the MBC was greater than or equal to the breakpoint for resistance. Tolerance to vancomycin and teicoplanin was observed in 6.1% and 18.8% of the strains, respectively. Tolerance to daptomycin was not observed.Although vancomycin and teicoplanin are the standard therapies for staphylococcal bacteremia, tolerance to vancomycin and teicoplanin has been demonstrated in both coagulase-negative staphylococci and Staphylococcus aureus as well as in various Streptococcus species (2, 3, 7, 10, 13, 15, 20, 21, 23, 25). Daptomycin, a lipopeptide antibiotic, has been demonstrated to have rapid bactericidal activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and tolerance to this drug has not been demonstrated (2, 9, 10, 19, 21, 24, 26, 28).The issue of antibiotic tolerance is a complicated one. Some studies have suggested that infections caused by tolerant strains may be more difficult to treat, especially when they cause complicated infections such as endocarditis, meningitis, or osteomyelitis or cause infections in immunocompromised patients (7, 8, 14, 15, 16, 18, 20, 22, 23, 25). Other investigators'' expert analyses do not agree that there is proof of a correlation between tolerant strains and treatment failures or that bactericidal activity is required for the treatment of serious MRSA infections (17, 25, 26, 27, 28). Controversy concerning the appropriate methods for the determination of tolerance in clinical isolates and in the practicality of testing isolates for tolerance in the clinical laboratory also exists.This study looked at MRSA isolates obtained primarily from blood and abscess cultures collected between 1985 and 2007. The main purpose of the study was to determine the in vitro inhibitory and bactericidal activities and the level of tolerance to the three drugs observed by standardized MIC and minimum bactericidal concentration (MBC) tests (4, 5, 19).(This study was presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 17 to 20 September 2007.)     

Pharmacokinetics and Pharmacodynamics of Amphotericin B Deoxycholate,Liposomal Amphotericin B,and Amphotericin B Lipid Complex in an In Vitro Model of Invasive Pulmonary Aspergillosis

The pharmacodynamic and pharmacokinetic (PK-PD) properties of amphotericin B (AmB) formulations against invasive pulmonary aspergillosis (IPA) are not well understood. We used an in vitro model of IPA to further elucidate the PK-PD of amphotericin B deoxycholate (DAmB), liposomal amphotericin B (LAmB) and amphotericin B lipid complex (ABLC). The pharmacokinetics of these formulations for endovascular fluid, endothelial cells, and alveolar cells were estimated. Pharmacodynamic relationships were defined by measuring concentrations of galactomannan in endovascular and alveolar compartments. Confocal microscopy was used to visualize fungal biomass. A mathematical model was used to calculate the area under the concentration-time curve (AUC) in each compartment and estimate the extent of drug penetration. The interaction of LAmB with host cells and hyphae was visualized using sulforhodamine B-labeled liposomes. The MICs for the pure compound and the three formulations were comparable (0.125 to 0.25 mg/liter). For all formulations, concentrations of AmB progressively declined in the endovascular fluid as the drug distributed into the cellular bilayer. Depending on the formulation, the AUCs for AmB were 10 to 300 times higher within the cells than within endovascular fluid. The concentrations producing a 50% maximal effect (EC₅₀) in the endovascular compartment were 0.12, 1.03, and 4.41 mg/liter for DAmB, LAmB, and ABLC, respectively, whereas, the EC₅₀ in the alveolar compartment were 0.17, 7.76, and 39.34 mg/liter, respectively. Confocal microscopy suggested that liposomes interacted directly with hyphae and host cells. The PK-PD relationships of the three most widely used formulations of AmB differ markedly within an in vitro lung model of IPA.Aspergillus fumigatus is an environmentally ubiquitous mold that is a leading cause of morbidity and mortality in immunocompromised patients (18). Despite the advent of newer diagnostic and therapeutic modalities, the mortality rate remains approximately 50% (22). An improved understanding of the pharmacology of existing agents represents an important strategy to improve the outcomes of patients with this rapidly progressive and frequently lethal infectious syndrome.Amphotericin B (AmB) is a polyene derived from Streptomyces nodosus. This compound was discovered in the mid-1950s and remains a first-line agent for the treatment of invasive aspergillosis and other life-threatening invasive fungal infections (23, 24). Amphotericin B is amphipathic; i.e., it has both hydrophilic and hydrophobic moieties that render it insoluble in water. Aqueous solubility is achieved by formulation with deoxycholate or a variety of lipid carriers. Amphotericin B deoxycholate (DAmB) is a highly potent antifungal formulation, but its clinical utility is limited by a high frequency of adverse effects, such as infusional toxicity and nephrotoxicity (3, 27). Lipid formulations are better tolerated than DAmB and are increasingly used for the treatment of invasive pulmonary aspergillosis (IPA). Three licensed lipid-based formulations have been developed for clinical use: liposomal amphotericin (LAmB), amphotericin B lipid complex (ABLC), and amphotericin B colloidal dispersion (ABCD). These formulations differ significantly in their structures and pharmacological properties (1).Here, we describe the pharmacokinetics and pharmacodynamics (PK-PD) of the frequently used clinical formulations of amphotericin B by the use of an in vitro model of IPA. This model enabled assessment of the extent of drug penetration into a number of tissue subcompartments that are relevant to the pathogenesis of IPA.     

Voriconazole Pharmacokinetics in Liver Transplant Recipients

The objective of this study was to evaluate the pharmacokinetics of voriconazole and the potential correlations between pharmacokinetic parameters and patient variables in liver transplant patients on a fixed-dose prophylactic regimen. Multiple blood samples were collected within one dosing interval from 15 patients who were initiated on a prophylactic regimen of voriconazole at 200 mg enterally (tablets) twice daily starting immediately posttransplant. Voriconazole plasma concentrations were measured using high-pressure liquid chromatography (HPLC). Noncompartmental pharmacokinetic analysis was performed to estimate pharmacokinetic parameters. The mean apparent systemic clearance over bioavailability (CL/F), apparent steady-state volume of distribution over bioavailability (V_ss/F), and half-life (t_1/2) were 5.8 ± 5.5 liters/h, 94.5 ± 54.9 liters, and 15.7 ± 7.0 h, respectively. There was a good correlation between the area under the concentration-time curve from 0 h to infinity (AUC_0-∞) and trough voriconazole plasma concentrations. t_1/2, maximum drug concentration in plasma (C_max), trough level, AUC_0-∞, area under the first moment of the concentration-time curve from 0 h to infinity (AUMC_0-∞), and mean residence time from 0 h to infinity (MRT_0-∞) were significantly correlated with postoperative time. t_1/2, λ, AUC_0-∞, and CL/F were significantly correlated with indices of liver function (aspartate transaminase [AST], total bilirubin, and international normalized ratio [INR]). The C_max, last concentration in plasma at 12 h (C_last), AUMC_0-∞, and MRT_0-∞ were significantly lower in the presence of deficient CYP2C19*2 alleles. Donor characteristics had no significant correlation with any of the pharmacokinetic parameters estimated. A fixed dosing regimen of voriconazole results in a highly variable exposure of voriconazole in liver transplant patients. Given that trough voriconazole concentration is a good measure of drug exposure (AUC), the voriconazole dose can be individualized based on trough concentration measurements in liver transplant patients.Due to chronic immunosuppression, infections are common life-threatening complications in organ transplant patients (7). Invasive aspergillosis is one of the most dreaded complications after organ transplantation (21) due to its high mortality rate, which can range up to 88.1% (18).Voriconazole (V-Fend [Pfizer]; formerly known as UK-109496), (2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoropyrimidin-4-yl)-1-(1,2,4-triazol-1-yl)butan-2-ol, is a novel broad-spectrum triazole systemic antifungal agent and an ideal drug to prevent invasive aspergillosis. Compared with other azole antifungal agents, it has potent activity against a broader spectrum of clinically significant fungal pathogens, including Aspergillus, Candida, Cryptococcus neoformans, and some unusual organisms, such as Fusarium and Pseudallescheria boydii (11, 24, 37, 39, 43).Voriconazole is extensively metabolized hepatically, primarily via the cytochrome P450 (CYP) isoenzymes CYP2C19 and, secondarily, CYP2C9 and CYP3A4 (15, 25, 39) to inactive metabolites. Large inter- and intraindividual variabilities in voriconazole plasma concentrations regardless of the route of administration or the type of patient population have been documented and discussed in the literature (2, 17, 19, 22, 23, 33, 38, 40, 42, 45). Factors associated with interindividual variability of voriconazole exposure include liver dysfunction, alcohol abuse in the past (47), concomitant use of potent CYP450 inducers or inhibitors (10, 12), CYP2C19 genetic polymorphisms (including poor as well as ultrarapid metabolizers) (13, 20, 46), gastrointestinal abnormalities (e.g., mucositis or diarrhea) (38) impairing drug absorption, and intake with or without food (15).Voriconazole is approved at our institution for prophylaxis in all liver transplant patients. The pharmacokinetics of voriconazole in liver transplant patients has not been evaluated, and there is limited information about the pharmacokinetics of voriconazole in other solid organ transplant patient populations (3). We hypothesized that the use of a fixed-dosing regimen of voriconazole would lead to a large degree of variability in voriconazole exposure in liver transplant patients. Given that a low voriconazole plasma level of less than 0.25 μg/ml is associated with a poor outcome in patients with aspergillosis (4, 8, 22, 31, 38, 40) and with ultimately death of the patients, while a high voriconazole plasma concentration of over 5.5 μg/ml is correlated with an increased risk for toxicity, including visual disturbances, elevated transaminase levels, central nervous system (CNS) disorders (e.g., encephalopathy), and electrolyte disturbances (2, 4, 38, 41), it is important to optimize the use of voriconazole in this patient population.The objective of this prospective single-center observational study was to characterize the pharmacokinetics of voriconazole in liver transplant patients on a fixed-dose prophylactic regimen in order to determine the extent of interpatient variability in voriconazole exposure among liver transplant patients and to evaluate the potential correlations between pharmacokinetic parameters and certain patient variables that could potentially explain the large interindividual variability in voriconazole pharmacokinetics in liver transplant patients.     

Antifungal Therapy in a Murine Model of Disseminated Infection by Cryptococcus gattii

We have evaluated the efficacy of posaconazole (PSC), voriconazole (VRC), and amphotericin B (AMB) in a murine model of systemic infection by Cryptococcus gattii using immunocompromised animals and three clinical strains of the fungus. AMB was the most effective drug in prolonging the survival of mice and also in reducing tissue burden in all organs tested. To a lesser degree, VRC at 60 mg/kg of body weight in lung tissue and PSC at 40 mg/kg also in spleen demonstrated good efficacy in reducing the fungal load. The PSC and VRC levels in serum and brain tissue, determined by an agar diffusion bioassay method at 4 h after the last dose of the therapy, were above the corresponding MIC values. However, these drugs were not able to reduce the fungal load in brain tissue. Our results demonstrated that PSC and, to a lesser degree, VRC, have fungistatic activity and potential for the treatment of human pulmonary cryptococcosis.Cryptococcosis is an emerging infection commonly involving the lungs, from which it can disseminate to different tissues, usually the central nervous system (CNS) (20, 23). Cryptococcus neoformans and Cryptococcus gattii are the main agents responsible for this disease, which can affect both immunosuppressed and healthy individuals. Despite antifungal therapies, this infection still has mortality rates near 20% (20).The first choice in the primary therapy of CNS infections remains fungicidal drugs, with amphotericin B (AMB) alone or in combination with flucytosine being the most widely used (20, 23). Fungistatic drugs like itraconazole and fluconazole, with less toxicity, are also used in the maintenance of the therapy and in pulmonary cryptococcosis, but their use in CNS infections has been less than satisfactory. In addition, the extended duration of the therapy with these azoles increases the risk of developing drug resistance (11, 23, 26). It has been suggested that that C. gattii has a higher pathogenicity than C. neoformans (27), which emphasizes the importance of the correct species identification and makes it necessary to improve and search for alternatives to the current therapy.On the basis of the promising results obtained with posaconazole (PSC) and voriconazole (VRC) against C. neoformans in animal models (1, 19, 24) and also in a clinical setting (9, 15, 21, 22), we have evaluated in this study the efficacy of PSC, VRC, and AMB in a murine model of disseminated infection by C. gattii.