Pharmacodynamics of Posaconazole in Experimental Invasive Pulmonary Aspergillosis: Utility of Serum Galactomannan as a Dynamic Endpoint of Antifungal Efficacy

Background. Aspergillus galactomannan antigenemia is an accepted tool for the diagnosis of invasive pulmonary aspergillosis (IPA) in neutropenic patients. Little is known, however, about the utility of this biomarker to assess the efficacy of antifungal therapies. / Methods. The pharmacokinetics and pharmacodynamics (PK/PD) of posaconazole in treatment and prophylaxis were investigated in the persistently neutropenic rabbit model of Aspergillus fumigatus IPA at doses between 2 and 20 mg/kg and day. Sparse plasma sampling was used to obtain PK data at steady state, and the serum galactomannan index (GMI), as a dynamic endpoint of antifungal response, was obtained every other day in addition to conventional outcome parameters including survival and fungal tissue burden. Nonparametric PK/PD model building was performed using the Pmetrics Package in R. / Results. A one-compartment model with linear elimination best described the PK of posaconazole. The PD effect of posaconazole exposure in plasma on the GMI in serum was best described by a dynamic Hill-functions reflecting growth and kill of the fungus. Through calculations of the AUC0-24h at steady state, the exposure-response relationship between posaconazole and the GMI for treatment followed a sigmoidal function with an asymptote forming above an AUC0-24h of 30 mg*h/L. All prophylactic doses were able to control the fungal burden. / Conclusions. A nonparametric population PK/PD model adequately described the effect of posaconazole in prophylaxis and treatment of experimental IPA. An AUC0-24h greater than 30 mg*h/L was associated with adequate resolution of the GMI, which is well in support of previously suggested exposure-response relationships in humans

Role of NADPH Oxidase versus Neutrophil Proteases in Antimicrobial Host Defense

NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47phox−/−) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)−/−×cathepsin G (CG)−/− mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47phox−/− mice, whereas NE−/−×CG−/− mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens

Endpoint Assessment in Rabbit Models of Invasive Pulmonary Aspergillosis and Mucormycosis.

Multiple animal models have been developed to study the pathogenesis of invasive pulmonary aspergillosis, as well as to evaluate the efficacy, pharmacokinetics, and pharmacodynamics of various antifungal agents and vaccines. Each model is beneficial depending on the questions that are asked. In this chapter, we will discuss the endpoints assessment of the persistently neutropenic rabbit models of invasive pulmonary aspergillosis and invasive pulmonary mucormycosis

Characterization and Comparison of Galactomannan Enzyme Immunoassay and Quantitative Real-Time PCR Assay for Detection of Aspergillus fumigatus in Bronchoalveolar Lavage Fluid from Experimental Invasive Pulmonary Aspergillosis

Bronchoalveolar lavage (BAL) is widely used for evaluation of patients with suspected invasive pulmonary aspergillosis (IPA). However, the diagnostic yield of BAL for detection of IPA by culture and direct examination is limited. Earlier diagnosis may be facilitated by assays that can detect Aspergillus galactomannan antigen or DNA in BAL fluid. We therefore characterized and compared the diagnostic yields of a galactomannan enzyme immunoassay (GM EIA), quantitative real-time PCR (qPCR), and quantitative cultures in experiments using BAL fluid from neutropenic rabbits with experimentally induced IPA defined as microbiologically and histologically evident invasion. The qPCR assay targeted the rRNA gene complex of Aspergillus fumigatus. The GM EIA and qPCR assay were characterized by receiver operator curve analysis. With an optimal cutoff of 0.75, the GM EIA had a sensitivity and specificity of 100% in untreated controls. A decline in sensitivity (92%) was observed when antifungal therapy (AFT) was administered. The optimal cutoff for qPCR was a crossover of 36 cycles, with sensitivity and specificity of 80% and 100%, respectively. The sensitivity of qPCR also decreased with AFT to 50%. Quantitative culture of BAL had a sensitivity of 46% and a specificity of 100%. The sensitivity of quantitative culture decreased with AFT to 16%. The GM EIA and qPCR assay had greater sensitivity than culture in detection of A. fumigatus in BAL fluid in experimentally induced IPA (P ± 0.04). Use of the GM EIA and qPCR assay in conjunction with culture-based diagnostic methods applied to BAL fluid could facilitate accurate diagnosis and more-timely initiation of specific therapy

In vitro combination of isavuconazole with micafungin or amphotericin B deoxycholate against medically important molds

Whether isavuconazole, an extended-spectrum triazole, possesses synergistic activity in combination therapy with echinocandins or amphotericin B for the treatment of invasive molds infections has not been studied. Our in vitro combination studies showed that isavuconazole and micafungin are synergistically active against Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, and Cunninghamella bertholletiae. These results suggest that isavuconazole, in combination with micafungin, may have a role in the treatment of invasive aspergillosis and warrants further investigation. Copyright © 2014, American Society for Microbiology. All Rights Reserved

Increased virulence of Cunninghamella bertholletiae in experimental pulmonary mucormycosis: Correlation with circulating molecular biomarkers, sporangiospore germination and hyphal metabolism

Members of the order Mucorales are emerging invasive molds that cause infections in immunocompromised patients. However, little is known about the relation between different species of Mucorales and their virulence in invasive pulmonary mucormycosis. Based upon our earlier epidemiological studies, we hypothesized that Cunninghamella bertholletiae would demonstrate increased virulence. Therefore, we studied the relative virulence of C. bertholletiae (CB), Rhizopus oryzae (RO), R. microsporus (RM), and Mucor circinelloides (MC) in experimental invasive pulmonary mucormycosis in persistently neutropenic rabbits in relation to the fungi in vitro sporangiospore germination rate and hyphal metabolic activity. Rabbits infected with CB demonstrated (1) higher lung weights in comparison to RM (P ≤ 0.05), RO and MC (P ≤ 0.001), (2) pulmonary infarcts in comparison to RO and MC (P ≤ 0.001), (3) tissue fungal burden (CFU/g) vs. MC (P ≤ 0.001), and (4) the lowest survival of 0% (0/18), in comparison to 16% (3/18, P ≤ 0.01) of RM, 81% (21/26) of RO, and 83% (15/18) of MC-infected rabbits (P ≤ 0.001). Serum PCR concentration-time- curve showed the greatest amplitude for CB. Virulence correlated directly with sporangiospore germination rate at 4 h among species, i.e., CB (67 - 85%) > RM (14 - 56%) > RO (4 - 30%) > MC (0%), and hyphal metabolic activity, i.e., CB (1.22 - 1.51) > MC (0.54 - 0.64) = RM (0.38 - 0.41) = RO (0.37 - 0.59). C. bertholletiae was significantly more virulent in experimental invasive pulmonary mucormycosis than R. microsporus, R. oryzae, and M. circinelloides. In vivo virulence correlated with species-dependent differences of in vitro germination rate and hyphal metabolic activity. © 2013 ISHAM