Model Of Invasive Aspergillosis Research Articles

In vitro and in vivo evaluation of antifungal combinations against azole-resistant Aspergillus fumigatus isolates.

Azole resistance in Aspergillus fumigatus (Af) has become a widespread threat and a major concern for optimal management of patients with invasive aspergillosis (IA). Combination of echinocandins with azoles is an attractive alternative option for the treatment of IA due to azole-resistant Af strains. The aim of this study was to evaluate the in vitro and in vivo combination of caspofungin (CAS) with either voriconazole (VRZ) or posaconazole (PSZ). In vitro interactions were assessed by two methods, and an animal model of IA in Galleria mellonella was used for in vivo evaluation. Assessment of efficacy was based on larvae mortality. Groups of 10 larvae were infected by 3 clinical strains of Af (azole susceptible, AfS; PSZ resistant, AfR1; VRZ and PSZ resistant strain, AfR2). In vitro, combination of CAS and azoles was indifferent against AfS, and AfR2, and a synergy was found for AfR1. When compared to VRZ monotherapy, the combination of VRZ at 4 µg/larva with CAS at 4 µg/larva improved survival of AfR2-infected larvae (p=0.0066). Combination of PSZ at 4µg/larva with CAS at 4 µg/larva improved survival of AfR1-infected larvae compared to CAS (p=0.0002) and PSZ (0.0024) monotherapy. Antagonism was never observed. In conclusion, the combination of caspofungin with azoles is a promising alternative for the treatment of azole resistant strains of Af.

The novel fungal CYP51 inhibitor VT-1598 displays classic dose-dependent antifungal activity in murine models of invasive aspergillosis.

Aspergillus spp. infections remain a global concern, with ∼30% attributable mortality of invasive aspergillosis (IA). VT-1598 is a novel fungal CYP51 inhibitor designed for exquisite selectivity versus human CYP enzymes to achieve a maximal therapeutic index and therefore maximal antifungal efficacy. Previously, its broad-spectrum in vitro antifungal activity was reported. We report here the pharmacokinetics (PK) and pharmacodynamics (PD) of VT-1598 in neutropenic mouse models of IA. The plasma area-under-the-curve (AUC) of VT-1598 increased nearly linearly between 5 and 40 mg/kg after 5 days of QD administration (155 and 1033 μg*h/ml, respectively), with a further increase with 40 mg/kg BID dosing (1354 μg*h/ml). When A. fumigatus isolates with in vitro susceptibilities of 0.25 and 1.0 μg/ml were used in a disseminated IA model, VT-1598 treatment produced no decrease in kidney fungal burden at QD 10 mg/kg, intermediate decreases at QD 20 mg/kg and maximum or near maximum decreases at 40 mg/kg QD and BID. The PK/PD relationships of AUCfree/MIC for 1-log killing for the two strains were 5.1 and 1.6 h, respectively, similar to values reported for approved CYP51 inhibitors. In a survival study where animals were observed for 12 days after the last treatment, survival was 100% at the doses tested (20 and 40 mg/kg QD), and fungal burden remained suppressed even though drug wash-out was complete. Similar dose-dependent reductions in lung fungal burden were observed in a pulmonary model of IA. These data strongly support further exploration of VT-1598 for the treatment of this lethal mold infection.

12/15-lipoxygenase supports inflammatory cytokine production and PMN recruitment during invasive aspergillosis

Abstract Eicosanoids are a family of inflammatory lipid mediators produced via the metabolism of arachidonic acid by a network of biosynthetic enzymes. We and others have recently shown that pathways within this network, specifically the cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) pathways, participate in induction of protective immune responses during invasive aspergillosis (IA). Based on the high degree of cooperation and interconnection within the eicosanoid network, we hypothesized that a third core enzyme, 12/15-lipoxygenase (12/15-LOX), also may support innate antifungal responses. We report here that mice deficient in the gene encoding 12/15-LOX, Alox15, exhibit decreased survival and increased fungal burden in a model of IA. These defects were associated with decreased PMN numbers in the lung and decreased levels of PMN chemokines (CXCL1, CXCL2, CCL3) in Alox15−/− mice, while fungal killing by PMNs in vitro was unaffected. Alox15−/− mice produced lower levels of the inflammatory cytokines IL-17A, IL-22, IL-1α and IL-6 during infection. iNKT cells, which we have reported to be a major cell source of IL-22 during IA, were present in decreased numbers in Alox15−/− mice. This reduction was associated with lower levels of IL-7, a cytokine we have recently shown to be required for optimal numbers of iNKT cells during IA. Decreased IL-22 levels were further associated with decreased levels of IL-23. Together, these data indicate that 12/15-LOX is a critical player in induction of multiple aspects of the innate immune response to A. fumigatus.

1346. The Tetrazole VT-1598 Is Efficacious in a Murine Model of Invasive Aspergillosis with a PK/PD Expected of a Mold-Active CYP51 Inhibitor

BackgroundVT-1598 is a novel fungal CYP51 inhibitor with potent in vitro activity against yeast, mold, and endemic pathogenic fungi (Wiederhold, JAC, 2017). Its tetrazole-based rational drug design imparts much greater selectivity vs. human CYPs (Yates, BMCL, 2017), which could reduce human CYP-related side effects and DDIs. We report here VT-1598’s in vivo activity in an invasive aspergillosis (IA) model.MethodsMIC was determined as outlined in CLSI M38-A2. Plasma PK was measured after 4 days of oral doses in neutropenic ICR mice without fungal inoculation. In vivo antifungal activity was determined in a tail-vein IA model in neutropenic mice inoculated with A. fumigatus (AF) ATCC 204305 (N = 10 per dose). Two separate studies were conducted, with oral VT-1598 treatment starting either 48 hours prior (prophylaxis) or 5 hours postinoculation (delayed), with 4 days of postinoculation dosing, and kidney fungal burden measured 1 day post last dose by both CFU and qPCR. Drug control was 10 mg/kg AmBisome i.v.ResultsThe MIC for VT-1598 against AF 204305 was 0.25 μg/mL. The plasma PK of VT-1598 was linearly proportional between the 5 and 40 mg/kg once-daily doses, with AUCs of 155 and 1,033 μg h/mL for the two doses, respectively. VT-1598 was similarly effective in reducing fungal burden when given in delayed treatment compared with prophylaxis, and both studies demonstrated a full dose–response (i.e., no to full reduction of fungal burden). When comparing fungal burdens of each dose group to the fungal burden at the start of treatment, the dose of VT-1598 to achieve fungal stasis ranged from 20.5 to 25.9 mg/kg and to achieve a 1-log10 fungal kill ranged from 30.9 to 50.5 mg/kg. Using the previously measured mouse plasma binding (>99.9%), the free AUC /MIC values for stasis and 1-log10 kill ranged from 2.1–2.7 and 3.2–5.2, respectively. These values are within the range of 1–11 that have been reported for posaconazole and isavuconazole (Lepak, AAC, 2013).ConclusionVT-1598 had potent antifungal activity in a murine model of IA. The PK/PD relationship was the same as clinically used mold-active CYP51 agents, suggesting that it could have similar clinical efficacy. If correct, the tetrazole-based greater selectivity may significantly differentiate VT-1598 from current IA therapies.Disclosures E. P. Garvey, Viamet Pharmaceuticals, Inc.: Employee, Salary. A. Sharp, Evotec (UK) Ltd.: Employee, Salary. P. Warn, Evotec (UK) Ltd.: Employee, Salary. C. M. Yates, Viamet Pharmaceuticals, Inc.: Employee, Salary. R. J. Schotzinger, Viamet Pharmaceuticals, Inc.: Board Member and Employee, Salary.

Host immune status-specific production of gliotoxin and bis-methyl-gliotoxin during invasive aspergillosis in mice

Delayed diagnosis in invasive aspergillosis (IA) contributes to its high mortality. Gliotoxin (GT) and bis-methyl-gliotoxin (bmGT) are secondary metabolites produced by Aspergillus during invasive, hyphal growth and may prove diagnostically useful. Because IA pathophysiology and GT’s role in virulence vary depending on the underlying host immune status, we hypothesized that GT and bmGT production in vivo may differ in three mouse models of IA that mimic human disease. We defined temporal kinetics of GT and bmGT in serum, bronchoalveolar lavage fluid (BALF) and lungs of A. fumigatus-infected chronic granulomatous disease (CGD), hydrocortisone-treated, and neutropenic mice. We harvested lungs for assessment of fungal burden, histology and GT/bmGT biosynthetic genes’ mRNA induction. GT levels were higher in neutropenic versus CGD or steroid-treated lungs. bmGT was persistently detected only in CGD lungs. GT, but not bmGT, was detected in 71% of sera and 50% of BALF of neutropenic mice; neither was detected in serum/BALF of CGD or steroid-treated mice. Enrichment of GT in Aspergillus-infected neutropenic lung correlated with fungal burden and hyphal length but not induction of GT biosynthetic genes. In summary, GT is detectable in mouse lungs, serum and BALF during neutropenic IA, suggesting that GT may be useful to diagnose IA in neutropenic patients.

Neutrophil Interactions Stimulate Evasive Hyphal Branching by Aspergillus fumigatus.

Invasive aspergillosis (IA), primarily caused by Aspergillus fumigatus, is an opportunistic fungal infection predominantly affecting immunocompromised and neutropenic patients that is difficult to treat and results in high mortality. Investigations of neutrophil-hypha interaction in vitro and in animal models of IA are limited by lack of temporal and spatial control over interactions. This study presents a new approach for studying neutrophil-hypha interaction at single cell resolution over time, which revealed an evasive fungal behavior triggered by interaction with neutrophils: Interacting hyphae performed de novo tip formation to generate new hyphal branches, allowing the fungi to avoid the interaction point and continue invasive growth. Induction of this mechanism was independent of neutrophil NADPH oxidase activity and neutrophil extracellular trap (NET) formation, but could be phenocopied by iron chelation and mechanical or physiological stalling of hyphal tip extension. The consequence of branch induction upon interaction outcome depends on the number and activity of neutrophils available: In the presence of sufficient neutrophils branching makes hyphae more vulnerable to destruction, while in the presence of limited neutrophils the interaction increases the number of hyphal tips, potentially making the infection more aggressive. This has direct implications for infections in neutrophil-deficient patients and opens new avenues for treatments targeting fungal branching.

Pharmacokinetic-Pharmacodynamic Target Attainment Analysis to Support VL-2397 Dose Selection for a Phase 2 Trial in Patients with Invasive Aspergillosis

BackgroundVL-2397 is a novel antifungal agent in clinical development for treatment of invasive aspergillosis (IA). The analysis objectives were to: 1) develop a population PK model using data from a Phase 1 trial in healthy adult volunteers, 2) define the PK-PD driver of efficacy in a mouse model of IA, and 3) conduct PK-PD target attainment analyses to aid in Phase 2 dose selection for patients with IA.MethodsData from two studies were used: a dose fractionation study in a mouse model of IA and a Phase 1 study in healthy adult volunteers receiving single- and multiple-ascending doses of intravenous (IV) VL-2397 ranging from 3 mg to 1200 mg daily. Mouse lung fungal burden (LFB) measured on Day 4 after infection with Aspergillus fumigatus FP1305 (MIC of 1 µg/mL) was used to assess the predictive value of three measures of VL-2397 exposure: total-drug AUC0-24:MIC (tAUC0-24:MIC), total-drug Cmax:MIC, and total-drug %T>MIC; total drug was used because serum protein binding is similar in mice and humans. A population PK model for VL-2397 in humans was developed in NONMEM v7.1.2 and formed the basis of Monte Carlo simulations. The model was also fit to mouse PK data to estimate the VL-2397 exposure in mice.ResultsVL-2397 concentration-time data in humans revealed nonlinear PK behavior consistent with highly-bound compounds. A robust fit to the data was obtained using a two-compartment model with linear elimination and concentration-dependent binding in both central and peripheral compartments. The results of the dose fractionation study suggested that tAUC0-24:MIC was the most predictive (r2 = 0.713) driver of efficacy, with a value of 21.8 on Day 1 associated with a 2-log reduction in LFB on Day 4. Comparisons of the simulated VL-2397 tAUC0-24:MIC on Day 1 in humans receiving daily dosing regimens of 300 mg, 600 mg, and 900 mg of VL-2397 to the 2-log target (figure) suggested that a dose of 600 mg Q24 would provide robust (99.9%) PK-PD target attainment up to an A. fumigatus MIC of 2 µg/mL.ConclusionThe PK-PD driver of efficacy for VL-2397 was tAUC0-24:MIC and a value of 21.8 was associated with a 2-log reduction in LFB in a mouse model of IA. PK-PD target attainment assessments indicate that a VL-2397 dose of 600 mg IV Q24 will provide adequate target attainment up to an MIC of 2 µg/mL.Disclosures C. M. Rubino, Vical Incorporated: Consultant, Consulting fee; L. R. Smith, Vical Incorporated: Employee, Salary and Stock Options; M. P. Mammen, Vical Incorporated: Employee, Salary and Stock Options; A. M. Hopkins, Vical Incorporated: Employee, Salary and Stock Options; E. A. Lakota, Vical Incorporated: Consultant, Consulting fee; S. M. Sullivan, Vical Incorporated: Employee, Salary and Stock Options

Aspergillus fumigatus virulence through the lens of transcription factors.

Invasive aspergillosis (IA), most commonly caused by the filamentous fungus Aspergillus fumigatus, occurs in immune compromised individuals. The ability of A. fumigatus to proliferate in a multitude of environments is hypothesized to contribute to its pathogenicity and virulence. Transcription factors (TF) have long been recognized as critical proteins for fungal pathogenicity, as many are known to play important roles in the transcriptional regulation of pathways implicated in virulence. Such pathways include regulation of conidiation and development, adhesion, nutrient acquisition, adaptation to environmental stress, and interactions with the host immune system among others. In both murine and insect models of IA, TF loss of function in A. fumigatus results in cases of hyper- and hypovirulence as determined through host survival, fungal burden, and immune response analyses. Consequently, the study of specific TFs in A. fumigatus has revealed important insights into mechanisms of pathogenicity and virulence. Although in vitro studies have identified virulence-related functions of specific TFs, the full picture of their in vivo functions remain largely enigmatic and an exciting area of current research. Moreover, the vast majority of TFs remain to be characterized and studied in this important human pathogen. Here in this mini-review we provide an overview of selected TFs in A. fumigatus and their contribution to our understanding of this important human pathogen's pathogenicity and virulence.

Loss of efficacy of the antifungal caspofungin is mediated by increased chitin exposure and concomitant eosinophilia in invasive pulmonary aspergillosis

Abstract Recently significant interest has focused on the composite recognition of fungal molecular patterns that program innate and adaptive immunity to the human fungal pathogen Aspergillus fumigatus. We observed that decreased relative levels of cell wall β-glucan to chitin in A. fumigatus strains correlated with increased airway eosinophil recruitment associated with decreased fungal clearance by type 2 immune responses in invasive pulmonary aspergillosis. Furthermore, an increase in surface chitin exposure driven by growth and germination in the presence of the anti-fungal, β-glucan targeting drug caspofungin resulted in increased airway eosinophilia upon aspiration of A. fumigatus conidia. More importantly, in a model of invasive aspergillosis, we observed a decrease in fungal clearance upon caspofungin treatment that was reversed in eosinophil-deficient mice. Eosinophil recruitment was partially dependent on γδ T cells, and our preliminary results show a similar increase in caspofungin mediated clearance in γδ-T cell-deficient mice. Our results provide a novel mechanism that may explain the widely reported paradoxical loss of efficacy in response to caspofungin, and we are currently working to define the relationship between drug treatment, eosinophil activation, and fungal burden in clinical samples.

Antibodies generated against Streptococci protect in a mouse model of disseminated aspergillosis.

Invasive aspergillosis (IA) resulting from infection by Aspergillus fumigatus is a leading cause of death in immunosuppressed populations. There are limited therapeutic options for this disease and currently no vaccine. There is evidence that some anti-A. fumigatus mAbs can provide protection against IA. However, vaccine development has been impeded by a paucity of immunological targets on this organism demonstrated to provide protective responses. Sialylated oligosaccharide epitopes found on a variety of pathogens, including fungi and group B streptococci (GBS), are thought to be major virulence factors of these organisms facilitating pathogen attachment to host cells and modulating complement activation and phagocytosis. Because some of these oligosaccharide structures are conserved across kingdoms, we screened a panel of mAbs raised against GBS serotypes for reactivity to A. fumigatus. This approach revealed that SMB19, a GBSIb type-specific mAb, reacts with A. fumigatus conidia and hyphae. The presence of this Ab in mice, as a result of passive or active immunization, or by enforced expression of the SMB19 H chain as a transgene, results in significant protection in both i.v. and airway-induced models of IA. This study demonstrates that some Abs generated against bacterial polysaccharides engage fungal pathogens and promote their clearance in vivo and thus provide rationale of alternative strategies for the development of vaccines or therapeutic mAbs against these organisms.

Bayesian Development of a Dose‐Response Model for Aspergillus fumigatus and Invasive Aspergillosis

Invasive aspergillosis (IA) is a major cause of mortality in immunocompromized hosts, most often consecutive to the inhalation of spores of Aspergillus. However, the relationship between Aspergillus concentration in the air and probability of IA is not quantitatively known. In this study, this relationship was examined in a murine model of IA. Immunosuppressed Balb/c mice were exposed for 60 minutes at day 0 to an aerosol of A. fumigatus spores (Af293 strain). At day 10, IA was assessed in mice by quantitative culture of the lungs and galactomannan dosage. Fifteen separate nebulizations with varying spore concentrations were performed. Rates of IA ranged from 0% to 100% according to spore concentrations. The dose-response relationship between probability of infection and spore exposure was approximated using the exponential model and the more flexible beta-Poisson model. Prior distributions of the parameters of the models were proposed then updated with data in a Bayesian framework. Both models yielded close median dose-responses of the posterior distributions for the main parameter of the model, but with different dispersions, either when the exposure dose was the concentration in the nebulized suspension or was the estimated quantity of spores inhaled by a mouse during the experiment. The median quantity of inhaled spores that infected 50% of mice was estimated at 1.8 × 10(4) and 3.2 × 10(4) viable spores in the exponential and beta-Poisson models, respectively. This study provides dose-response parameters for quantitative assessment of the relationship between airborne exposure to the reference A. fumigatus strain and probability of IA in immunocompromized hosts.

Efficacy and pharmacodynamics of voriconazole combined with anidulafungin in azole-resistant invasive aspergillosis

Azole resistance is an emerging problem in the treatment of Aspergillus fumigatus infections. Combination therapy may be an alternative approach to improve therapeutic outcome in azole-resistant invasive aspergillosis (IA). The in vivo efficacy of voriconazole and anidulafungin was investigated in a non-neutropenic murine model of IA using voriconazole-susceptible and voriconazole-resistant A. fumigatus clinical isolates. Treatment groups consisted of voriconazole monotherapy, anidulafungin monotherapy and voriconazole + anidulafungin at 2.5, 5, 10 and 20 mg/kg body weight/day for 7 consecutive days. In vitro and in vivo drug interactions were analysed by non-parametric Bliss independence and non-linear regression analysis. Synergistic interaction between voriconazole and anidulafungin against the voriconazole-susceptible isolate (AZN 8196) was observed in vitro and in vivo. However, among animals infected with the voriconazole-resistant isolate (V 52-35), 100% survival was observed only in groups receiving the highest doses (20 mg/kg voriconazole + 20 mg/kg anidulafungin). For this isolate, additivity, but not synergy, was observed in vivo. Combination of voriconazole and anidulafungin was synergistic in voriconazole-susceptible IA, but additive in voriconazole-resistant IA. There is a clear benefit of combining voriconazole and anidulafungin, but the reduced effect of combination therapy in azole-resistant IA raises some concern.

Pharmacodynamics of Anidulafungin against Clinical Aspergillus fumigatus Isolates in a Nonneutropenic Murine Model of Disseminated Aspergillosis

Azole resistance is an emerging increasing problem in Aspergillus fumigatus that results in treatment failure. Alternative treatments may improve the therapeutic outcome in patients with azole-resistant invasive aspergillosis (IA). Little is known about the in vivo efficacy of the echinocandin anidulafungin (AFG) in IA. The in vivo efficacy of 2.5, 5, 10, and 20 mg/kg of body weight AFG was assessed against two clinical Aspergillus fumigatus isolates with identical AFG minimum effective concentrations (MECs; 0.03 mg/liter) in a murine model of IA: a wild-type voriconazole (VCZ)-susceptible (VCZ(s)) A. fumigatus isolate (AZN 8196) and a VCZ-resistant (VCZ(r)) A. fumigatus isolate (V52-35) harboring the TR(34)/L98H resistance mechanism (substitution at codon L98 in combination with a 34-bp tandem repeat in the promoter region of the CYP51A gene). The pharmacokinetics of AFG were also assessed for each dose. Increasing doses increased survival for both isolates in a manner dependent on the AFG dose level (R(2) = 0.99 and 0.95, respectively) up to a maximum of 72.7% and 45.45% for the VCZ(s) and VCZ(r) isolates, respectively. The area under the concentration-time curve (AUC) correlated significantly with the dose in a linear fashion over the entire dosing range (R(2) = 0.86). The Hill equation with a variable slope fitted the relationship between the 24-h AUC/MEC ratio and 14-day survival well (R(2) = 0.87; P < 0.05). The 50% effective AUC/MEC for total AFG was 126.5 (95% confidence interval, 79.09 to 202.03). AFG treatment improved the survival of mice in a dose-dependent manner; however, a maximal response was not achieved with either isolate even in those treated with the highest AFG dose.

Efficacy of liposomal amphotericin B for prophylaxis of acute or reactivation models of invasive pulmonary aspergillosis

The efficacy of antifungal prophylaxis for prevention of invasive aspergillosis (IA) may depend on whether IA results from recent inhalation of spores or reactivation of latent colonisation. Compare the efficacy of liposomal amphotericin B (LAmB) for prophylaxis in acute and reactivation models of IA. In the acute model, mice immunosuppressed from day 0 were challenged at day 3 with an aerosol of Aspergillus fumigatus. LAmB (15 mg kg(-1) ) was administered at day 0 or at challenge. In the reactivation model, naïve mice exposed to A. fumigatus remained untreated until clearance of spores from the lungs, then immunosuppressed to induce reactivation. A single LAmB dose was administered at start of immunosuppression. In the acute model, a single administration of LAmB at start of immunosuppression was not effective, but an additional administration resulted in a significant decrease in lung fungal burden (P < 0.05 vs. controls). A significant prophylactic efficacy was observed when LAmB was administered once at challenge (P < 0.01). In the reactivation model, a single LAmB administration at start of immunosuppression significantly reduced both reactivation rate and fungal burden vs. controls (P < 0.01). Our results show that the conditions under which IA develop and timing of administration of LAmB were determinant variables for prophylactic efficacy.

Detection of Urinary Excreted Fungal Galactomannan-like Antigens for Diagnosis of Invasive Aspergillosis

Mortality associated with invasive aspergillosis (IA) remains high, partly because of delayed diagnosis. Detection of microbial exoantigens, released in serum and other body fluids during infection, may help timely diagnosis. In course of IA, Aspergillus galactomannan (GM), a well established polysaccharide biomarker, is released in body fluids including urine. Urine is an abundant, safely collected specimen, well-suited for point-of-care (POC) testing, which could play an increasing role in screening for early disease. Our main objective was to demonstrate GM antigenuria as a clinically relevant biological phenomenon in IA and establish proof-of-concept that it could be translated to POC diagnosis. Utilizing a novel IgM monoclonal antibody (MAb476) that recognizes GM-like antigens from Aspergillus and other molds, we demonstrated antigenuria in an experimental animal IA model (guinea pig), as well as in human patients. In addition, we investigated the chemical nature of the urinary excreted antigen in human samples, characterized antigen detection in urine by immunoassays, described a putative assay inhibitor in urine, and indicated means of alleviation of the inhibition. We also designed and used a lateral flow immunochromatographic assay to detect urinary excreted antigen in a limited number of IA patient urine samples. In this study, we establish that POC diagnosis of IA based on urinary GM detection is feasible. Prospective studies will be necessary to establish the performance characteristics of an optimized device and define its optimal clinical use.

Anti-Group B streptococci Ib antibodies protect against invasive aspergillosis. (166.9)

Abstract Aspergillus fumigatus (A.f.) the cause of invasive Aspergillosis (IA) has become a leading cause of death in immunosuppressed populations, due to the lack of effective therapies or vaccines. Studies using anti-A.f. monoclonal antibodies indicate that they can protect against IA. However, the lack of detailed information on the antigenic targets of protective anti-A.f. antibodies has hampered the development of an effective anti-A.f. vaccine. Sialylated epitopes found on a variety of pathogens including fungi and Group B Streptococci (GBS) are thought to be major virulence factors and are involved in attachment to host cells, uptake by phagocytes, and complement activation. Sialylated epitopes on A.f. may therefore provide a potential target for new therapies. Our preliminary data show that a purified, calcium-dependent GBSIb type-specific antibody, SMB19 (IgM,κ) also binds to A.f. and prolongs survival in a murine model of IA. Similarly, immunization of wild type and SMB19 VH bearing immunoglobulin transgenic mice with a GBSIb vaccine also provides protection. These results support our hypothesis that induction of GBSIb specific antibodies, perhaps supplemented with antifungal drugs, will lead to new vaccines or passive antibody prophylaxis to prevent and treat IA.

RT-qPCR detection ofAspergillus fumigatusRNAin vitroand in a murine model of invasive aspergillosis utilizing the PAXgene®and Tempus™ RNA stabilization systems

Molecular diagnosis of invasive aspergillosis (IA) is a potentially life-saving tool in the care of at-risk individuals. To date, the development of PCR-based diagnostic tests has been hampered by the lack of standardization in the methods for such critical activities. In this study, we used both spiked volunteer blood samples and a murine model of IA to test the utility of the PAXgene and Tempus systems for stabilization and isolation of fungal RNA from blood as part of an evaluation of a new diagnostic strategy. In spiking experiments, RNA isolation followed by RT-qPCR that targeted the 18S gene was compared to a standard DNA isolation and qPCR assay that targeted the ITS ribosomal region. We demonstrated that both PAXgene and Tempus RNA stabilization and extraction systems followed by RT-qPCR had similar performance in detecting fungal RNA in blood samples from Aspergillus fumigates-infected mice. In spiked samples, the Tempus system performed better than the PAXgene system as it detected 100% of all samples spiked with 10 or 20 germinated Aspergillus conidia/ml blood sample as compared to the PAXgene system which detected 33% and 56% of the samples spiked with 10 or 20 conidia/ml, respectively. The stabilization of fungal nucleic acids in blood samples and its efficient isolation by a commercial method is an important step in the development of standardized molecular diagnostic tools that are needed to improve the outcomes for individuals with IA.

Antifungal treatment affects the laboratory diagnosis of invasive aspergillosis

AimsThe purpose of this study was to investigate the performance of non-invasive diagnostic tests such as galactomannan enzyme immunoassay and quantitative PCR in the early diagnosis of invasive aspergillosis (IA),...

Assessment of efficacy of antifungals in experimental models of invasive aspergillosis in an era of emerging resistance: the value of real-time quantitative PCR

Experimental models of invasive aspergillosis (IA) have been used to explore pharmacokinetic and pharmacodynamic (PK/PD) properties of antifungal agents. Survival is still considered the golden standard effect measure but has the disadvantage that a large number of animals are needed to determine the dose-response relationships and PK/PD of antifungals. The feasibility of using fungal load by real-time quantitative PCR (qPCR) as an effect measure has been explored recently. The majority of studies reported convincingly demonstrate a larger dynamic range for qPCR compared to conventional assays. However interpretation and translating the results to guidance in clinical decision making need further study. It is expected that the use of qPCR will become the primary outcome measure for assessment of PK/PD relationships of antifungals in experimental models of IA.

Are Serum Cytokines Sensitive and Specific Enough to Prognosticate in Aspergillosis?

Chai et al [1] are to be commended for their admirable effort to find associations between some (IL-6, IL-8, IL-10, INF-8, and C-reactive protein) but not all serum cytokines and the clinical response (at 12 weeks) and outcome (mortality at day 84) in patients with invasive aspergillosis (IA). Because no IA-attributable mortality was analyzed, the similarity in cytokine profiles with nonresponders and nonsurvivors is reassuring; several competing causes of death exist in severely immunosuppressed hosts who develop IA. However, these interesting data do not address the issue of whether serum cytokine level changes are inherently linked to the natural history of IA (progression or not) per se and not to the underlying disease. As up to 74% of patients in the cohort analyzed had hematologic malignancy, and because information about remission rates and neutropenia status (eg, recovery or not in cytopenic patients) in responders or not is not provided, a question arises: are these cytokine changes merely a sophisticated marker to describe the evolution of underlying hematologic disease? For example, Kornblau et al [2] recently described that there are specific serum cytokine signatures (including IL-8) that impact leukemia-related survival, independent of infections. The possible influence of underlying disease and neutrophil count is reflected by the contrasting findings of an earlier, smaller study by Roilides et al [3] in 7 non-neutropenic patients with IA. Furthermore, because up to 10% of patients with invasive mycoses have another infection (bacterial or fungal) at autopsy [4], it might be premature to ascribe all these cytokine changes to IA alone. Only measurement of a wide spectrum of serum cytokines and chemokines in a well-matched control group (same underlying disease but without IA) and careful association of serum cytokine levels with the quality of hematologic remission, cytogenetic abnormalities, coinfections, and grade of mucositis could address specificity issues convincingly. Furthermore, issues about sensitivity remain. Levels of cytokines would be significantly different (lower or higher) depending on whether measurements were done in serum or plasma samples [5]. Perhaps plasma is a better platform to evaluate the association of cytokine kinetics with IA outcome. Finally, it is uncertain whether serum cytokine levels mirror the pathophysiology of a primarily sinopulmonary infection such as IA. Up to 90% of patients in the study by Chai et al [1] had pulmonary IA. It is unclear how many of these cases were limited to a single lung nodule, compared with extensive pulmonary disease, or sequestration and angioinvasive process, both factors that would influence the spill over of cytokines from the site of primary infection (lungs) to systemic (bloodstream) compartment. Beyond the artificiality of animal models of IA, human IA is a true opportunistic infection that occurs in complex clinical scenarios that could influence, in multiple ways, the proinflammatory signatures. This is an important and complicated area of research that requires prospective, carefully controlled, prospective, large sample sets and casting the net widely by measuring cytokines and/or chemokines to gain more insight on the validity of cytokines as surrogate, early prognostic markers in IA.