Amy M. Kelaher

Combination therapy in treatment of experimental pulmonary aspergillosis: Synergistic interaction between an antifungal triazole and an echinocandin

Invasive pulmonary aspergillosis is an important cause of morbidity and mortality in immunocompromised patients. Simultaneous inhibition of fungal cell-wall and cell-membrane biosynthesis may result in synergistic interaction against Aspergillus fumigatus. We studied the antifungal activity of micafungin, a new echinocandin, in combination with ravuconazole, a second-generation triazole, against experimental invasive pulmonary aspergillosis in persistently neutropenic rabbits. This combination led to significant reductions in mortality (P</=.001), residual fungal burden (P</=.05), and serum galactomannan antigenemia (P</=.01), compared with either agent alone. Combination therapy also resulted in reduction (P</=.05) of organism-mediated pulmonary injury and of pulmonary infiltrates detected by thoracic computed tomography (P</=.001). No toxicity was observed with the echinocandin-triazole combination. An MTT hyphal damage assay demonstrated significant in vitro synergistic interaction between the antifungal triazole and the echinocandin. The combination of an antifungal triazole and echinocandin may represent a new strategy for treatment of invasive pulmonary aspergillosis.

Detection of Galactomannan Antigenemia in Patients Receiving Piperacillin-Tazobactam and Correlations between In Vitro, In Vivo, and Clinical Properties of the Drug-Antigen Interaction

ABSTRACT Recent case reports describe patients receiving piperacillin-tazobactam who were found to have circulating galactomannan detected by the double sandwich enzyme-linked immunosorbent assay (ELISA) system, leading to the false presumption of invasive aspergillosis. Since this property of piperacillin-tazobactam and galactomannan ELISA is not well understood, we investigated the in vitro, in vivo, and clinical properties of this interaction. Among the 12 reconstituted antibiotics representing four classes of antibacterial compounds that are commonly used in immunocompromised patients, piperacillin-tazobactam expressed a distinctively high level of galactomannan antigen in vitro (P = 0.001). After intravenous infusion of piperacillin-tazobactam into rabbits, the serum galactomannan index (GMI) in vivo changed significantly (P = 0.0007) from a preinfusion mean baseline value of 0.27 to a mean GMI of 0.83 by 30 min to slowly decline to a mean GMI of 0.44 24 h later. Repeated administration of piperacillin-tazobactam over 7 days resulted in accumulation of circulating galactomannan to a mean peak GMI of 1.31 and a nadir of 0.53. Further studies revealed that the antigen reached a steady state by the third day of administration of piperacillin-tazobactam. Twenty-six hospitalized patients with no evidence of invasive aspergillosis who were receiving antibiotics and ten healthy blood bank donors were studied for expression of circulating galactomannan. Patients (n = 13) receiving piperacillin-tazobactam had significantly greater mean serum GMI values (0.74 ± 0.14) compared to patients (n = 13) receiving other antibiotics (0.14 ± 0.08) and compared to healthy blood bank donors (0.14 ± 0.06) (P < 0.001). Five (38.5%) of thirteen patients receiving piperacillin-tazobactam had serum GMI values > 0.5 compared to none of thirteen subjects receiving other antibiotics (P = 0.039) and to none of ten healthy blood bank donors (P = 0.046). These data demonstrate that among antibiotics that are commonly used in immunocompromised patients, only piperacillin-tazobactam contains significant amounts of galactomannan antigen in vitro, that in animals receiving piperacillin-tazobactam circulating galactomannan antigen accumulates in vivo to significantly increased and sustained levels, and that some but not all patients receiving this antibiotic will demonstrate circulating galactomannan above the threshold considered positive for invasive aspergillosis by the recently licensed double sandwich ELISA.

The pharmacokinetics and pharmacodynamics of micafungin in experimental hematogenous Candida meningoencephalitis: Implications for echinocandin therapy in neonates

BACKGROUND Hematogenous Candida meningoencephalitis (HCME) is a relatively frequent manifestation of disseminated candidiasis in neonates and is associated with significant mortality and neurodevelopmental abnormalities. The outcome after antifungal therapy is often suboptimal, with few therapeutic options. Limited clinical data suggest that echinocandins may have role to play in the treatment of HCME. METHODS We studied the pharmacokinetics and pharmacodynamics of micafungin in a rabbit model of neonatal HCME and bridged the results to neonates by use of population pharmacokinetics and Monte Carlo simulation. RESULTS Micafungin exhibited linear plasma pharmacokinetics in the range of 0.25-16 mg/kg. Micafungin penetrated most compartments of the central nervous system (CNS), but only with doses >2 mg/kg. Micafungin was not reliably found in cerebrospinal fluid. With few exceptions, drug penetration into the various CNS subcompartments was not statistically different between infected and noninfected rabbits. A dose-microbiological response relationship was apparent in the brain, and near-maximal effect was apparent with doses of 8 mg/kg. Monte Carlo simulations revealed that near-maximal antifungal effect was attained at human neonatal doses of 12-15 mg/kg. CONCLUSIONS These results provide a foundation for clinical trials of micafungin in neonates with HCME and a model for antimicrobial bridging studies from bench to bedside in pediatric patients.

Pathogenesis of Aspergillus fumigatus and the Kinetics of Galactomannan in an In Vitro Model of Early Invasive Pulmonary Aspergillosis: Implications for Antifungal Therapy

BACKGROUND Little is known about the pathogenesis of invasive pulmonary aspergillosis and the relationship between the kinetics of diagnostic markers and the outcome of antifungal therapy. METHODS An in vitro model of the human alveolus, consisting of a bilayer of human alveolar epithelial and endothelial cells, was developed. An Aspergillus fumigatus strain expressing green fluorescent protein was used. Invasion of the cell bilayer was studied using confocal and electron microscopy. The kinetics of culture, polymerase chain reaction, and galactomannan were determined. Galactomannan was used to measure the antifungal effect of macrophages and amphotericin B. A mathematical model was developed, and results were bridged to humans. RESULTS A. fumigatus penetrated the cellular bilayer 14-16 h after inoculation. Galactomannan levels were inextricably tied to fungal invasion and were a robust measure of the antifungal effect of macrophages and amphotericin B. Neither amphotericin nor macrophages alone was able to suppress the growth of A. fumigatus; rather, the combination was required. Monte Carlo simulations showed that human dosages of amphotericin B of at least 0.6 mg/kg were required to achieve adequate drug exposure. CONCLUSIONS This model provides a strategy by which relationships among pathogenesis, immunological effectors, and antifungal drug therapy for invasive pulmonary aspergillosis may be further understood.

Triazole-Polyene Antagonism in Experimental Invasive Pulmonary Aspergillosis: In Vitro and In Vivo Correlation

Combination antifungal therapy is increasingly used in the treatment of invasive aspergillosis. Whether the interaction between amphotericin B and triazoles is antagonistic against invasive aspergillosis is a controversial issue that is not likely to be resolved through a randomized clinical trial. Here, we found both in vitro and in vivo antagonism between liposomal amphotericin B and ravuconazole in simultaneous treatment of experimental invasive pulmonary aspergillosis in persistently neutropenic rabbits. Bliss independence-based drug-interaction modeling showed significant antagonism in vitro and in vivo, with the observed drug effects being 20%-69% lower than would be expected if the drugs were acting independently. These in vitro and in vivo findings of antagonism were consistent with the findings from Loewe additivity-based drug-interaction modeling. No pharmacokinetic interaction was found. The combination of a triazole and polyene may be antagonistic in the treatment of invasive pulmonary aspergillosis.

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

ABSTRACT 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.

Development and Validation of a Quantitative Real-Time PCR Assay Using Fluorescence Resonance Energy Transfer Technology for Detection of Aspergillus fumigatus in Experimental Invasive Pulmonary Aspergillosis

ABSTRACT Invasive pulmonary aspergillosis (IPA) is a frequently fatal infection in immunocompromised patients that is difficult to diagnose. Present methods for detection of Aspergillus spp. in bronchoalveolar lavage (BAL) fluid and in tissue vary in sensitivity and specificity. We therefore developed an A. fumigatus-specific quantitative real-time PCR-based assay utilizing fluorescent resonance energy transfer (FRET) technology. We compared the assay to quantitative culture of BAL fluid and lung tissue in a rabbit model of experimental IPA. Using an enzymatic and high-speed mechanical cell wall disruption protocol, DNA was extracted from samples of BAL fluid and lung tissues from noninfected and A. fumigatus-infected rabbits. A unique primer set amplified internal transcribed spacer regions (ITS) 1 and 2 of the rRNA operon. Amplicon was detected using FRET probes targeting a unique region of ITS1. Quantitation of A. fumigatus DNA was achieved by use of external standards. The presence of PCR inhibitors was determined by use of a unique control plasmid. The analytical sensitivity of the assay was ≤10 copies of target DNA. No cross-reactivity occurred with other medically important filamentous fungi. The assay results correlated with pulmonary fungal burden as determined by quantitative culture (r = 0.72, Spearman rank correlation; P ≤ 0.0001). The mean number of genome equivalents detected in untreated animals was 3.86 log10 (range, 0.86 to 6.39 log10) in tissue. There was a 3.53-log10 mean reduction of A. fumigatus genome equivalents in animals treated with amphotericin B (AMB) (95% confidence interval, 3.38 to 3.69 log10; P ≤ 0.0001), which correlated with the reduction of residual fungal burden in lung tissue measured in terms of log10 CFU/gram. The enhanced quantitative sensitivity of the real-time PCR assay was evidenced by detection of A. fumigatus genome in infarcted culture-negative lobes, by a greater number of mean genome equivalents compared to the number of CFU per gram in tissue and BAL fluid, and by superior detection of therapeutic response to AMB in BAL fluid compared to culture. This real-time PCR assay using FRET technology is highly sensitive and specific in detecting A. fumigatus DNA from BAL fluid and lung tissue in experimental IPA.

Use of Quantitative Real-Time PCR To Study the Kinetics of Extracellular DNA Released from Candida albicans, with Implications for Diagnosis of Invasive Candidiasis

ABSTRACT Quantitative real-time PCR (qPCR) is considered one of the most sensitive methods to detect low levels of DNA from pathogens in clinical samples. To improve the design of qPCR for the detection of deeply invasive candidiasis, we sought to develop a more comprehensive understanding of the kinetics of DNA released from Candida albicans in vitro and in vivo. We developed a C. albicans-specific assay targeting the rRNA gene complex and studied the kinetics of DNA released from C. albicans alone, in the presence of human blood monocytes (H-MNCs), and in the bloodstream of rabbits with experimental disseminated candidiasis. The analytical qPCR assay was highly specific and sensitive (10 fg). Cells of C. albicans incubated in Hanks balanced salt solution (±10% bovine serum albumin [BSA]) or RPMI (±10% BSA) showed a significant release of DNA at T equal to 24 h compared to T equal to 0 h (P ≤ 0.01). C. albicans incubated with H-MNCs exhibited a greater release of DNA than C. albicans cells alone over 24 h (P = 0.0001). Rabbits with disseminated candidiasis showed a steady increase of detectable DNA levels in plasma as disease progressed. Plasma cultures showed minimal growth of C. albicans, demonstrating that DNA extracted from plasma reflected fungal cell-free DNA. In summary, these studies of the kinetics of DNA release by C. albicans collectively demonstrate that cell-free fungal DNA is released into the bloodstream of hosts with disseminated candidiasis, that phagocytic cells may play an active role in increasing this release over time, and that plasma is a suitable blood fraction for the detection of C. albicans DNA.

Cerebrospinal Fluid and Plasma (1→3)-β-d-Glucan as Surrogate Markers for Detection and Monitoring of Therapeutic Response in Experimental Hematogenous Candida Meningoencephalitis

ABSTRACT The treatment, diagnosis and therapeutic monitoring of hematogenous Candida meningoencephalitis (HCME) are not well understood. We therefore studied the expression of (1→3)-β-d-glucan (β-glucan) in cerebrospinal fluid (CSF) and plasma in a nonneutropenic rabbit model of experimental HCME treated with micafungin and amphotericin B. Groups studied consisted of micafungin (0.5 to 32 mg/kg) and amphotericin B (1 mg/kg) treatment groups and the untreated controls (UC). Despite well-established infection in the cerebrum, cerebellum, choroid, vitreous humor (102 to 103 CFU/ml), spinal cord, and meninges (10 to 102 CFU/g), only 8.1% of UC CSF cultures were positive. By comparison, all 25 UC CSF samples tested for β-glucan were positive (755 to 7,750 pg/ml) (P < 0.001). The therapeutic response in CNS tissue was site dependent, with significant decreases of the fungal burden in the cerebrum and cerebellum starting at 8 mg/kg, in the meninges at 2 mg/kg, and in the vitreous humor at 4 mg/kg. A dosage of 24 mg/kg was required to achieve a significant effect in the spinal cord and choroid. Clearance of Candida albicans from blood cultures was not predictive of eradication of organisms from the CNS; conversely, β-glucan levels in CSF were predictive of the therapeutic response. A significant decrease of β-glucan concentrations in CSF, in comparison to that for UC, started at 0.5 mg/kg (P < 0.001). Levels of plasma β-glucan were lower than levels in simultaneously obtained CSF (P < 0.05). CSF β-glucan levels correlated in a dose-dependent pattern with therapeutic responses and with Candida infection in cerebral tissue (r = 0.842). Micafungin demonstrated dose-dependent and site-dependent activity against HCME. CSF β-glucan may be a useful biomarker for detection and monitoring of therapeutic response in HCME.

Efficacy, Plasma Pharmacokinetics, and Safety of Icofungipen, an Inhibitor of Candida Isoleucyl-tRNA Synthetase, in Treatment of Experimental Disseminated Candidiasis in Persistently Neutropenic Rabbits

ABSTRACT Icofungipen (formerly PLD-118) is a synthetic derivative of the naturally occurring β-amino acid cispentacin that blocks isoleucyl-tRNA synthetase, resulting in the inhibition of protein synthesis and growth of fungal cells. We investigated the efficacy, plasma pharmacokinetics, and safety of icofungipen in escalating dosages for the treatment of experimental subacute disseminated candidiasis in persistently neutropenic rabbits. Icofungipen was administered for 10 days starting 24 h after the intravenous inoculation of 103Candida albicans blastoconidia. Study groups consisted of rabbits treated with icofungipen at 4 (ICO-4), 10 (ICO-10), and 25 (ICO-25) mg/kg of body weight/day in two divided dosages, rabbits treated with fluconazole at 10 mg/kg/day, rabbits treated with amphotericin B at 1 mg/kg/day, and untreated controls. Levels of icofungipen in plasma were derivatized by phthaldialdehyde and quantified by high-performance liquid chromatography with fluorescence detection. Rabbits treated with ICO-10 (P < 0.01) and ICO-25 (P < 0.001) showed significant dosage-dependent tissue clearance of C. albicans from the liver, spleen, kidney, brain, vitreous, vena cava, and lung in comparison to untreated controls. ICO-25 cleared C. albicans from all tissues and had activity comparable to that of amphotericin B versus untreated controls (P < 0.001). Pharmacokinetics of icofungipen in plasma approximated a dose-dependent relationship of the maximum concentration of drug in serum and the area under the concentration-time curve. There was no significant elevation of the levels of hepatic transaminases, alkaline phosphatase, bilirubin, urea nitrogen, or creatinine in icofungipen-treated rabbits. Icofungipen followed dose-dependent pharmacokinetics and was effective in the treatment of experimental disseminated candidiasis, including central nervous system infection, in persistently neutropenic rabbits.