Susceptibility Breakpoints for Amphotericin B and Aspergillus Species in an In Vitro Pharmacokinetic-Pharmacodynamic Model Simulating Free-Drug Concentrations in Human Serum

Although conventional amphotericin B was for many years the drug of choice and remains an important agent against invasive aspergillosis, reliable susceptibility breakpoints are lacking. Three clinical Aspergillus isolates (Aspergillus fumigatus, Aspergillus flavus, and Aspergillus terreus) were tested in an in vitro pharmacokinetic-pharmacodynamic model simulating the biphasic 24-h time-concentration profile of free amphotericin B concentrations in human serum with free peak concentrations (fC_max) of 0.1, 0.3, 0.6, 1.2, and 2.4 mg/liter administered once daily. Drug concentrations were measured with a bioassay, and fungal growth was monitored for 72 h with galactomannan production. The fC_max/MIC corresponding to half-maximal activity (P₅₀) was determined for each species, and the percentage of target attainment was calculated for different MICs for the standard (1 mg/kg of body weight) and a lower (0.6-mg/kg) dose of amphotericin B with Monte Carlo simulation analysis. The fC_max/MICs (95% confidence intervals) corresponding to P₅₀ were 0.145 (0.133 to 0.158), 0.371 (0.283 to 0.486), and 0.41 (0.292 to 0.522) for A. fumigatus, A. flavus, and A. terreus, respectively. The median percentages of P₅₀ attainment were ≥88%, 47%, and 0% for A. fumigatus isolates with MICs of ≤0.5, 1, and ≥2 mg/liter, respectively, and ≥81%, 24%, and 0% and ≥75%, 15%, and 0% for A. flavus and A. terreus isolates with MICs of ≤0.25, 0.5, and ≥1 mg/liter, respectively. The lower dose of 0.6 mg/kg would retain efficacy for A. fumigatus, A. flavus, and A. terreus isolates with MICs of ≤0.25, ≤0.125, and ≤0.125 mg/liter, respectively. The susceptibility, intermediate susceptibility, and resistance breakpoints of ≤0.5, 1, and ≥2 mg/liter for A. fumigatus and ≤0.25, 0.5, and ≥1 mg/liter for A. flavus and A. terreus were determined for conventional amphotericin B with a pharmacokinetic-pharmacodynamic model simulating free-drug serum concentrations.     

In Vitro Activity of ASP2397 against Aspergillus Isolates with or without Acquired Azole Resistance Mechanisms

ASP2397 is a new compound with a novel and as-yet-unknown target different from that of licensed antifungal agents. It has activity against Aspergillus and Candida glabrata. We compared its in vitro activity against wild-type and azole-resistant A. fumigatus and A. terreus isolates with that of amphotericin B, itraconazole, posaconazole, and voriconazole. Thirty-four isolates, including 4 wild-type A. fumigatus isolates, 24 A. fumigatus isolates with alterations in CYP51A TR/L98H (5 isolates), M220 (9 isolates), G54 (9 isolates), and HapE (1 isolate), and A. terreus isolates (2 wild-type isolates and 1 isolate with an M217I CYP51A alteration), were analyzed. EUCAST E.Def 9.2 and CLSI M38-A2 MIC susceptibility testing was performed. ASP2397 MIC₅₀ values (in milligrams per liter, with MIC ranges in parentheses) determined by EUCAST and CLSI were 0.5 (0.25 to 1) and 0.25 (0.06 to 0.25) against A. fumigatus CYP51A wild-type isolates and were similarly 0.5 (0.125 to >4) and 0.125 (0.06 to >4) against azole-resistant A. fumigatus isolates, respectively. These values were comparable to those for amphotericin B, which were 0.25 (0.125 to 0.5) and 0.25 (0.125 to 0.25) against wild-type isolates and 0.25 (0.125 to 1) and 0.25 (0.125 to 1) against isolates with azole resistance mechanisms, respectively. In contrast, MICs for the azole compounds were elevated and highest for itraconazole: >4 (1 to >4) and 4 (0.5 to >4) against isolates with azole resistance mechanisms compared to 0.125 (0.125 to 0.25) and 0.125 (0.06 to 0.25) against wild-type isolates, respectively. ASP2397 was active against A. terreus CYP51A wild-type isolates (MIC 0.5 to 1), whereas MICs of both azole and ASP2397 were elevated for the mutant isolate. ASP2397 displayed in vitro activity against A. fumigatus and A. terreus isolates which was independent of the presence or absence of azole target gene resistance mutations in A. fumigatus. The findings are promising at a time when azole-resistant A. fumigatus is emerging globally.     

Amphotericin B- and Voriconazole-Echinocandin Combinations against Aspergillus spp.: Effect of Serum on Inhibitory and Fungicidal Interactions

Antifungal combination therapy with voriconazole or amphotericin B and an echinocandin is often employed as primary or salvage therapy for management particularly of refractory aspergillosis. The pharmacodynamic interactions of amphotericin B- and voriconazole-based combinations with the three echinocandins caspofungin, micafungin, and anidulafungin in the presence of serum were tested against 15 Aspergillus fumigatus complex, A. flavus complex, and A. terreus complex isolates to assess both their growth-inhibitory and fungicidal activities. The in vitro activity of each drug alone and in combination at a 1:1 fixed concentration ratio was tested with a broth microdilution colorimetric method, and interactions were assessed by isobolographic analysis. Synergy was found for all amphotericin B- and voriconazole-based combinations, with amphotericin B-based combinations showing strong inhibitory synergistic interactions (interaction indices of 0.20 to 0.52) and with voriconazole-based combinations demonstrating strong fungicidal synergistic interactions (interaction indices of 0.10 to 0.29) (P < 0.001). Drug- and species-specific differences were found, with caspofungin and the A. fumigatus complex exhibiting the weakest synergistic interactions. In the presence of serum, the synergistic interactions were reduced in the order (from largest to smallest decrease) micafungin > anidulafungin > caspofungin, and A. flavus complex > A. fumigatus complex > A. terreus complex, resulting in additive interactions, particularly for inhibitory activities of amphotericin B-echinocandin combinations and fungicidal activities of voriconazole-echinocandin combinations. Drug- and species-specific differences were found in the presence of serum for inhibitory activities of antifungal drugs, with the lowest interaction indices being observed for amphotericin B-caspofungin (median, 0.77) and for the A. terreus complex (median, 0.56). The present in vitro data showed that serum had a major impact on synergistic interactions of amphotericin B-echinocandin and voriconazole-echinocandin combinations, resulting in additive interactions and explaining the indifferent outcomes usually observed in vivo.     

Optimization of Polyene-Azole Combination Therapy against Aspergillosis Using an In Vitro Pharmacokinetic-Pharmacodynamic Model

Although amphotericin B-azole combination therapy has traditionally been questioned due to potential antagonistic interactions, it is often used successfully to treat refractory invasive aspergillosis. So far, pharmacodynamic (PD) interactions have been assessed with conventional in vitro tests, which do not mimic human serum concentrations and animal models using limited doses. We therefore simulated the human serum concentration profiles of amphotericin B and voriconazole in an in vitro dialysis/diffusion closed pharmacokinetic-pharmacodynamic (PK-PD) model and studied the pharmacodynamic interactions against an azole-resistant and an azole-susceptible Aspergillus fumigatus isolate, using Bliss independence and canonical mixture response surface analyses. Amphotericin B dosing regimens with the drug administered every 24 h (q24h) were combined with voriconazole q12h dosing regimens. In vitro PK-PD combination data were then combined with human PK data by using Monte Carlo analysis. The target attainment rate and the serum concentration/MIC ratio were calculated for isolates with different MICs. Synergy (20 to 31%) was observed at low amphotericin B-high voriconazole exposures, whereas antagonism (−6 to −16%) was found at high amphotericin B-low voriconazole exposures for both isolates. Combination therapy resulted in 17 to 48% higher target attainment rates than those of monotherapy regimens for isolates with voriconazole/amphotericin B MICs of 1 to 4 mg/liter. Optimal activity was found for combination regimens with a 1.1 total minimum concentration of drug in serum (tC_min)/MIC ratio for voriconazole and a 0.5 total maximum concentration of drug in serum (tC_max)/MIC ratio for amphotericin B, whereas the equally effective monotherapy regimens required a voriconazole tC_min/MIC ratio of 1.8 and an amphotericin B tC_max/MIC ratio of 2.8. Amphotericin B-voriconazole combination regimens were more effective than monotherapy regimens. Therapeutic drug monitoring can be employed to optimize antifungal combination therapy with low-dose (≤0.6 mg/kg) amphotericin B-based combination regimens against resistant isolates for minimal toxicity.     

Posaconazole Prophylaxis in Experimental Azole-Resistant Invasive Pulmonary Aspergillosis

We investigated the efficacy of posaconazole prophylaxis in preventing invasive aspergillosis due to azole-resistant Aspergillus fumigatus isolates. Using a neutropenic murine model of pulmonary infection, posaconazole prophylaxis was evaluated using three isogenic clinical isolates, with posaconazole MICs of 0.063 mg/liter (wild type), 0.5 mg/liter (F219I mutation), and 16 mg/liter. A fourth isolate harboring TR₃₄/L98H (MIC of 0.5 mg/liter) was also tested. Posaconazole prophylaxis was effective in A. fumigatus with posaconazole MICs of ≤0.5 mg/liter, where 100% survival was reached. However, breakthrough infection was observed in mice infected with the isolate for which the posaconazole MIC was >16 mg/liter.     

Comparative Pharmacodynamics of Posaconazole in Neutropenic Murine Models of Invasive Pulmonary Aspergillosis and Mucormycosis

We used two established neutropenic murine models of pulmonary aspergillosis and mucormycosis to explore the association between the posaconazole area under the concentration-time curve (AUC)-to-MIC ratio (AUC/MIC) and treatment outcome. Posaconazole serum pharmacokinetics were verified in infected mice to ensure that the studied doses reflected human exposures with the oral suspension, delayed-release tablet, and intravenous formulations of posaconazole. Sinopulmonary infections were then induced in groups of neutropenic mice with Aspergillus fumigatus strain 293 (posaconazole MIC, 0.5 mg/liter) or Rhizopus oryzae strain 969 (posaconazole MIC, 2 mg/liter) and treated with escalating daily dosages of oral posaconazole, which was designed to achieve AUCs ranging from 1.10 to 392 mg · h/liter. After 5 days of treatment, lung fungal burden was analyzed by quantitative real-time PCR. The relationships of the total drug AUC/MIC and the treatment response were similar in both models, with 90% effective concentrations (EC₉₀s) corresponding to an AUC/MIC threshold of 76 (95% confidence interval [CI], 46 to 102) for strain 293 versus 87 (95% CI, 66 to 101) for strain 969. Using a provisional AUC/MIC target of >100, these exposures correlated with minimum serum posaconazole concentrations (C_mins) of 1.25 mg/liter for strain 293 and 4.0 mg/liter for strain 969. The addition of deferasirox, but not liposomal amphotericin or caspofungin, improved the activity of a suboptimal posaconazole regimen (AUC/MIC, 33) in animals with pulmonary mucormycosis. However, no combination was as effective as the high-dose posaconazole monotherapy regimen (AUC/MIC, 184). Our analysis suggests that posaconazole pharmacodynamics are similar for A. fumigatus and R. oryzae when indexed to pathogen MICs.     

In Vitro Activity of a New Oral Glucan Synthase Inhibitor (MK-3118) Tested against Aspergillus spp. by CLSI and EUCAST Broth Microdilution Methods

MK-3118, a glucan synthase inhibitor derived from enfumafungin, and comparator agents were tested against 71 Aspergillus spp., including itraconazole-resistant strains (MIC, ≥4 μg/ml), using CLSI and EUCAST reference broth microdilution methods. The CLSI 90% minimum effective concentration (MEC₉₀)/MIC₉₀ values (μg/ml) for MK-3118, amphotericin B, and caspofungin, respectively, were as follows: 0.12, 2, and 0.03 for Aspergillus flavus species complex (SC); 0.25, 2, and 0.06 for Aspergillus fumigatus SC; 0.12, 2, and 0.06 for Aspergillus terreus SC; and 0.06, 1, and 0.03 for Aspergillus niger SC. Essential agreement between the values found by CLSI and EUCAST (±2 log₂ dilution steps) was 94.3%. MK-3118 was determined to be a potent agent regardless of the in vitro method applied, with excellent activity against contemporary wild-type and itraconazole-resistant strains of Aspergillus spp.     

Determination of Isavuconazole Susceptibility of Aspergillus and Candida Species by the EUCAST Method

Isavuconazole is a novel expanded-spectrum triazole, which has recently been approved by the FDA as an orphan drug to treat invasive aspergillosis and is currently being studied in phase III clinical trials for invasive candidiasis. The susceptibility of relatively few clinical isolates has been reported. In this study, the isavuconazole susceptibilities of 1,237 Aspergillus and 2,010 Candida geographically diverse clinical isolates were determined by EUCAST methodology at four European mycology laboratories, producing the largest multicenter data set thus far for this compound. In addition, a blinded collection of 30 cyp51A mutant Aspergillus fumigatus clinical isolates and 10 wild-type isolates was tested. From these two data sets, the following preliminary epidemiological cutoff (ECOFF) values were suggested: 2 mg/liter for Aspergillus fumigatus, Aspergillus terreus, and Aspergillus flavus; 4 mg/liter for Aspergillus niger; 0.25 mg/liter for Aspergillus nidulans; and 0.03 mg/liter for Candida albicans, Candida parapsilosis, and Candida tropicalis. Unfortunately, ECOFFs could not be determined for Candida glabrata or Candida krusei due to an unexplained interlaboratory MIC variation. For the blinded collection of A. fumigatus isolates, all MICs were ≤2 mg/liter for wild-type isolates. Differential isavuconazole MICs were observed for triazole-resistant A. fumigatus isolates with different cyp51A alterations: TR₃₄/L98H mutants had elevated isavuconazole MICs, whereas isolates with G54 and M220 alterations had MICs in the wild-type range, suggesting that the efficacy of isavuconazole may not be affected by these alterations. This study will be an aid in interpreting isavuconazole MICs for clinical care and an important step in the future process of setting official clinical breakpoints.     

Impact of Nonlinear Interactions of Pharmacokinetics and MICs on Sputum Bacillary Kill Rates as a Marker of Sterilizing Effect in Tuberculosis

The relationships between antituberculosis drug exposure and treatment effects on humans receiving multidrug therapy are complex and nonlinear. In patients on treatment, an analysis of the rate of decline in the sputum bacillary burden reveals two slopes. The first is the α-slope, which is thought to reflect bactericidal effect, followed by a β-slope, which is thought to reflect sterilizing activity. We sought to characterize the effects of standard first-line treatment on sterilizing activity. Fifty-four patients receiving combination therapy for pulmonary tuberculosis in a clinical trial had drug concentrations measured and Mycobacterium tuberculosis isolates available for MIC identification. Sputum sample cultures were performed at baseline and weekly for 8 weeks. A time-to-event model based on the days to positivity in the liquid cultures was used to estimate the β-slope. The pharmacokinetic parameters of rifampin, isoniazid, ethambutol, and pyrazinamide were determined for each patient. Multivariate adaptive regression splines analyses, which simultaneously perform linear and nonlinear analyses, were used to identify the relationships between the predictors and the β-slope. The potential predictors examined included HIV status, lung cavitation, 24-h area under the concentration-time curve (AUC), peak drug concentration (C_max), AUC/MIC ratio, C_max/MIC ratio, and the time that that concentration persisted above MIC. A rifampin C_max of >8.2 mg/liter and a pyrazinamide AUC/MIC of >11.3 were key predictors of the β-slope and interacted positively to increase the β-slope. In patients with a rifampin AUC of <35.4 mg · h/liter, an increase in the pyrazinamide AUC/MIC and/or ethambutol C_max/MIC increased the β-slope, while increasing isoniazid C_max decreased it, suggesting isoniazid antagonism. Antibiotic concentrations and MICs interact in a nonlinear fashion as the main drivers of a sterilizing effect. The results suggest that faster speeds of sterilizing effect might be achieved by omitting isoniazid and by increasing rifampin, pyrazinamide, and ethambutol exposures. However, isoniazid and ethambutol exposures may only be of importance when rifampin exposure is low. These findings need confirmation in larger studies. (This study has been registered at controlled-trials.com under registration no. ISRCTN80852505.)     

Pharmacokinetics and Pharmacodynamics of Continuous-Infusion Meropenem in Pediatric Hematopoietic Stem Cell Transplant Patients

This study explored the pharmacokinetics and the pharmacodynamics of continuous-infusion meropenem in a population of pediatric hematopoietic stem cell transplant (HSCT) patients who underwent therapeutic drug monitoring. The relationship between meropenem clearance (CL_M) and estimated creatinine clearance (CL_CR) was assessed by nonlinear regression. A Monte Carlo simulation was performed to investigate the predictive performance of five dosing regimens (15 to 90 mg/kg of body weight/day) for the empirical treatment of severe Gram-negative-related infections in relation to four different categories of renal function. The optimal target was defined as a probability of target attainment (PTA) of ≥90% at steady-state concentration-to-MIC ratios (C_SS/MIC) of ≥1 and ≥4 for MICs of up to 8 mg/liter. A total of 21 patients with 44 meropenem C_SS were included. A good relationship between CL_M and estimated CL_CR was observed (r² = 0.733). Simulations showed that at an MIC of 2 mg/liter, the administration of continuous-infusion meropenem at doses of 15, 30, 45, and 60 mg/kg/day may achieve a PTA of ≥90% at a C_SS/MIC ratio of ≥4 in the CL_CR categories of 40 to <80, 80 to <120, 120 to <200, and 200 to <300 ml/min/1.73 m², respectively. At an MIC of 8 mg/liter, doses of up to 90 mg/kg/day by continuous infusion may achieve optimal PTA only in the CL_CR categories of 40 to <80 and 80 to <120 ml/min/1.73 m². Continuous-infusion meropenem at dosages up to 90 mg/kg/day might be effective for optimal treatment of severe Gram-negative-related infections in pediatric HSCT patients, even when caused by carbapenem-resistant pathogens with an MIC of up to 8 mg/liter.     

Multicenter Study of Isavuconazole MIC Distributions and Epidemiological Cutoff Values for Aspergillus spp. for the CLSI M38-A2 Broth Microdilution Method

Epidemiological cutoff values (ECVs) were established for the new triazole isavuconazole and Aspergillus species wild-type (WT) MIC distributions (organisms in a species-drug combination with no detectable acquired resistance mechanisms) that were defined with 855 Aspergillus fumigatus, 444 A. flavus, 106 A. nidulans, 207 A. niger, 384 A. terreus, and 75 A. versicolor species complex isolates; 22 Aspergillus section Usti isolates were also included. CLSI broth microdilution MIC data gathered in Europe, India, Mexico, and the United States were aggregated to statistically define ECVs. ECVs were 1 μg/ml for the A. fumigatus species complex, 1 μg/ml for the A. flavus species complex, 0.25 μg/ml for the A. nidulans species complex, 4 μg/ml for the A. niger species complex, 1 μg/ml for the A. terreus species complex, and 1 μg/ml for the A. versicolor species complex; due to the small number of isolates, an ECV was not proposed for Aspergillus section Usti. These ECVs may aid in detecting non-WT isolates with reduced susceptibility to isavuconazole due to cyp51A (an A. fumigatus species complex resistance mechanism among the triazoles) or other mutations.     

In Vitro Activity of the Echinocandin Antifungal Agent LY303,366 in Comparison with Itraconazole and Amphotericin B against Aspergillus spp.

LY303,366 (LY) is a novel derivative of the echinocandin class of antifungal agents. The in vitro activities of LY, itraconazole (ITZ), and amphotericin B (AMB) were assessed against 60 Aspergillus isolates, including 35 isolates of A. fumigatus, eight isolates of A. terreus, eight isolates of A. flavus, eight isolates of A. niger and one isolate of A. nidulans. Four A. fumigatus isolates were resistant to ITZ. Susceptibility testing for all drugs was performed with a broth microdilution procedure. LY was tested in two media: antibiotic medium 3 (AM3) and Casitone with 2% glucose (CAS) with an inoculum of 2 × 10³ spores/ml. ITZ and AMB were tested in RPMI 1640 with 2% glucose with an inoculum of 1 × 10⁶ spores/ml. All tests were incubated at 37°C for 48 h. A novel end point was used to determine a minimal effective concentration (MEC) for LY, i.e., almost complete inhibition of growth save a few tiny spherical colonies attached to the microplate. MICs were measured for ITZ and AMB with a no-growth end point. Ranges and geometric mean (GM) MECs were from 0.0018 to >0.5 and 0.0039 mg/liter and from 0.0018 to >0.5 and 0.008 mg/liter for LY in AM3 and LY in CAS, respectively. Differences between species were apparent, with A. flavus being significantly less susceptible to LY than any other species tested with both media (P ≤ 0.05). Ranges and GM MICs were from 0.125 to >16 and 0.7 mg/liter for ITZ and from 0.25 to 16 and 1.78 mg/liter for AMB. Minimal fungicidal concentrations (MFCs) were also determined for all drugs. GM MFCs were 0.018, 0.09, 19.76, and 12.64 mg/liter for LY in AM3, LY in CAS, ITZ, and AMB, respectively. LY in AM3 and LY in CAS were fungicidal for 86.7 and 68% of isolates, respectively (98% killing). In comparison, ITZ and AMB were fungicidal for 35 and 70% of isolates, respectively (99.99% killing). A reproducibility study was performed on 20% of the isolates. For 12 isolates retested, the MEC or MIC was the same or was within 1 dilution of the original value for 11, 11, 10, and 9 isolates for LY in AM3, LY in CAS, ITZ, and AMB, respectively. In conclusion, LY seems to be a promising antifungal agent with excellent in vitro activity against Aspergillus spp.

Invasive aspergillosis is now one of the most common fungal infections found in immunocompromised patients (1) and is also one of the most fatal (2). Treatment of Aspergillus infections is still not ideal, and the two currently used antifungal drugs have a variety of associated problems. Amphotericin B (AMB) can cause serious side effects due to its toxicity and itraconazole (ITZ) is not always absorbed in high enough quantities to be therapeutic, especially in certain patient groups, e.g., AIDS patients.The rise in serious fungal infection over the past decade has prompted the development of new antifungal agents with novel modes of action. LY303,366 (LY) is a semisynthetic derivative of a natural product class of antifungal agents belonging to the new class of drugs known as echinocandins. Echinocandins are noncompetitive inhibitors of (1,3)-β-d-glucan synthase which produces glucan polymers, a major component of the fungal cell wall (3). LY has been reported to have excellent activity against a wide range of fungal pathogens, including Aspergillus species (12) and Candida species (8, 10, 12).In this study we evaluated the in vitro activity of LY against a variety of Aspergillus species and compared it with the activity of currently used antifungal agents, ITZ and AMB.     

Caspofungin at Catheter Lock Concentrations Eradicates Mature Biofilms of Candida lusitaniae and Candida guilliermondii

The antibiofilm activities of caspofungin, anidulafungin, micafungin, and liposomal amphotericin B were studied against Candida lusitaniae, Candida guilliermondii, and a Candida albicans control strain. While anidulafungin and micafungin (0.007 to 2,048 mg/liter) showed reduced activity against biofilms of both test species, caspofungin displayed concentration-dependent antibiofilm activity, reaching complete and persistent eradication at concentrations achievable during lock therapy (512 to 2,048 mg/liter, P < 0.05). Although liposomal amphotericin B strongly inhibited mature biofilms, it possessed lower antibiofilm activity than caspofungin (P < 0.05).     

Pharmacodynamic modeling of aminoglycosides against Pseudomonas aeruginosa and Acinetobacter baumannii: identifying dosing regimens to suppress resistance development

To facilitate optimal dosing regimen design, we previously developed a mathematical model using time-kill study data to predict the responses of Pseudomonas aeruginosa to various pharmacokinetic profiles of meropenem and levofloxacin. In this study, we extended the model to predict the activities of gentamicin and amikacin exposures against P. aeruginosa and Acinetobacter baumannii, respectively. The input data were from a time-kill study with 10⁷ CFU/ml of bacteria at baseline. P. aeruginosa ATCC 27853 was exposed to gentamicin (0 to 16× MIC; MIC = 2 mg/liter), and A. baumannii ATCC BAA 747 was exposed to amikacin (0 to 32× MIC; MIC = 4 mg/liter) for 24 h. Using the estimates of the best-fit model parameters, bacterial responses to various fluctuating aminoglycoside exposures (half-life, 2.5 h) over 72 h were predicted via computer simulation. The computer simulations were subsequently validated using an in vitro hollow-fiber infection model with similar aminoglycoside exposures. A significant initial reduction in the bacterial burden was predicted for all gentamicin exposures examined. However, regrowth over time due to resistance emergence was predicted for regimens with a maximum concentration of the drug (C_max)/MIC (dosing frequency) of 4 (every 8 h [q8h]), 12 (q24h), and 36 (q24h). Sustained suppression of bacterial populations was forecast with a C_max/MIC of 30 (q12h). Similarly, regrowth and suppression of A. baumannii were predicted and experimentally verified with a three-dimensional response surface. The mathematical model was reasonable in predicting extended bacterial responses to various aminoglycoside exposures qualitatively, based on limited input data. Our approach appears promising as a decision support tool for dosing regimen selection for antimicrobial agents.     

Efficacy of LY303366 against Amphotericin B-Susceptible and -Resistant Aspergillus fumigatus in a Murine Model of Invasive Aspergillosis

LY303366 is a novel antifungal echinocandin with excellent in vitro activity against Aspergillus spp. We compared four doses (1, 2.5, 10, and 25 mg/kg of body weight) of LY303366 with amphotericin B (0.5 to 5 mg/kg) in a temporarily neutropenic murine model of invasive aspergillosis against an amphotericin B-susceptible (AF210) and an amphotericin B-resistant (AF65) Aspergillus fumigatus isolate based on in vivo response. Mice were immunosuppressed with cyclophosphamide (200 mg/kg) and infected 3 days later. Treatment started 18 h after infection and lasted for 10 days. LY303366 was given once daily intravenously for 10 days, and amphotericin B (at 0.5, 2, and 5 mg/kg) was given once daily intraperitoneally for 10 days, or only on days 1, 2, 4, and 7 (at 5 mg/kg). Kidneys and lungs from survivors were cultured on day 11. Control mice in both experiments had 90 to 100% mortality. Amphotericin B at 0.5 mg/kg and LY303366 at 1 mg/kg yielded 10 to 20% survival rates for mice infected with either AF210 or AF65. Amphotericin B at 2 and 5 (both regimens) mg/kg yielded a 70 to 100% survival rate for mice infected with AF210 but a 10 to 30% survival rate for mice infected with AF65 (P = 0.01 to 0.04 compared with AF210). Against AF210 and AF65, LY303366 at 2.5, 10, and 25 mg/kg produced a survival rate of 70 to 80%, which was as effective as amphotericin B for AF210, but superior to amphotericin B for AF65 (P < 0.03 to 0.0006). For AF65, LY303366 at 10 and 25 mg/kg/day was superior to amphotericin B at 2 and 5 mg/kg/day in reducing tissue colony counts (P = 0.01 to 0.003), and for AF210, amphotericin B at 5 mg/kg/day and at 5 mg/kg in four doses was more effective than all four regimens of LY303366 in reducing renal culture counts (P = 0.01 to 0.0001). The present study shows, for the first time, that in vivo resistance of A. fumigatus to amphotericin B exists, although this could not be detected by in vitro susceptibility assays. Furthermore, LY303366 appears to be effective against amphotericin B-susceptible and -resistant A. fumigatus infection in this model and should be further evaluated clinically.     

Effects of Iron Chelators on the Formation and Development of Aspergillus fumigatus Biofilm

Iron acquisition is crucial for the growth of Aspergillus fumigatus. A. fumigatus biofilm formation occurs in vitro and in vivo and is associated with physiological changes. In this study, we assessed the effects of Fe chelators on biofilm formation and development. Deferiprone (DFP), deferasirox (DFS), and deferoxamine (DFM) were tested for MIC against a reference isolate via a broth macrodilution method. The metabolic effects (assessed by XTT [2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt]) on biofilm formation by conidia were studied upon exposure to DFP, DFM, DFP plus FeCl₃, or FeCl₃ alone. A preformed biofilm was exposed to DFP with or without FeCl₃. The DFP and DFS MIC₅₀ against planktonic A. fumigatus was 1,250 μM, and XTT gave the same result. DFM showed no planktonic inhibition at concentrations of ≤2,500 μM. By XTT testing, DFM concentrations of <1,250 μM had no effect, whereas 2,500 μM increased biofilms forming in A. fumigatus or preformed biofilms (P < 0.01). DFP at 156 to 2,500 μM inhibited biofilm formation (P < 0.01 to 0.001) in a dose-responsive manner. Biofilm formation with 625 μM DFP plus any concentration of FeCl₃ was lower than that in the controls (P < 0.05 to 0.001). FeCl₃ at ≥625 μM reversed the DFP inhibitory effect (P < 0.05 to 0.01), but the reversal was incomplete compared to the controls (P < 0.05 to 0.01). For preformed biofilms, DFP in the range of ≥625 to 1,250 μM was inhibitory compared to the controls (P < 0.01 to 0.001). FeCl₃ at ≥625 μM overcame inhibition by 625 μM DFP (P < 0.001). FeCl₃ alone at ≥156 μM stimulated biofilm formation (P < 0.05 to 0.001). Preformed A. fumigatus biofilm increased with 2,500 μM FeCl₃ only (P < 0.05). In a strain survey, various susceptibilities of biofilms of A. fumigatus clinical isolates to DFP were noted. In conclusion, iron stimulates biofilm formation and preformed biofilms. Chelators can inhibit or enhance biofilms. Chelation may be a potential therapy for A. fumigatus, but we show here that chelators must be chosen carefully. Individual isolate susceptibility assessments may be needed.     

An Open,Randomized, Single-Center,Crossover Pharmacokinetic Study of Meropenem after Intraperitoneal and Intravenous Administration in Patients Receiving Automated Peritoneal Dialysis

The objective of this study was to determine the pharmacokinetic profile of meropenem in automated peritoneal dialysis (APD) patients. In 6 patients without peritonitis, a single dose of 0.5 g of meropenem was applied intraperitoneally (i.p.) or intravenously (i.v.) and concentrations in serum and dialysate were measured at specified intervals over 24 h with high-performance liquid chromatography-mass spectrometry. The mean maximum concentrations of meropenem in serum (C_max) were 27.2 mg/liter (standard deviation [SD], ±6.9) and 10.1 mg/liter (SD, ±2.5) and in dialysate were 3.6 mg/liter (SD, ±2.3) and 185.8 mg/liter (SD, ±18.7) after i.v. and i.p. administrations, respectively. The mean areas under the curve from 0 to 24 (AUC_0–24) of meropenem in serum were 173.5 mg · h/liter (SD, ±29.7) and 141.4 mg · h/liter (SD, ±37.5) (P = 0.046) and in dialysate were 42.6 mg · h/liter (SD, ±20.0) and 623.4 mg · h/liter (SD, ±84.1) (P = 0.028) after i.v. and i.p. administrations, respectively. The ratios for dialysate exposure over plasma exposure after i.v. and i.p. treatments were 0.2 (SD, ±0.1) and 4.6 (SD, ±0.9), respectively (P = 0.031). A mean target value of 40% T>MIC (time for which the free meropenem concentration exceeds the MIC) for clinically relevant pathogens with EUCAST susceptibility breakpoints of 2 mg/liter was reached in serum after i.p. and i.v. administrations and in dialysate after i.p. but not after i.v. administration. The present data indicate that low i.p. exposure limits the i.v. use of meropenem for PD-associated peritonitis. In contrast, i.p. administration not only results in superior concentrations in dialysate but also might be used to treat systemic infections.     

Changes in MIC Alter Responses of Pseudomonas aeruginosa to Tobramycin Exposure

The pharmacokinetic parameters determining antibiotic efficacy are peak concentrations (C_max), minimum (trough) concentrations (C_min), and area under the concentration-time curve (AUC). There is general agreement about the importance of C_max and AUC for aminoglycosides, but this is not so for maintenance of C_min. With in vitro exposures modelling in vivo administration, Pseudomonas aeruginosa reference strain ATCC 27853 (MIC, 1 mg/liter) and a higher-MIC (relatively resistant) clinical isolate (MIC, 4 mg/liter) were used to explore bacteriostatic and bactericidal outcomes. With P. aeruginosa ATCC 27853, kill followed a complete bolus profile with a 30-min postdistribution peak (C_peak30) of 10 mg/liter. The clinical isolate required a C_peak30 bolus profile of 20 mg/liter for kill, and there was no difference between the efficacies of the bolus and infusion exposures. Bolus profiles that were truncated at 8.5 h and producing sublethal effects were then combined with a wide range of C_mins. With a C_peak30 profile of 8 mg/liter, P. aeruginosa ATCC 27853 showed a graded bacteriostatic response until a C_min of ≥0.8 mg/liter, when complete kill resulted. In contrast, bactericidal effects on the clinical isolate required a C_peak30 profile of 18 mg/liter with a C_min of ≥1.0 mg/liter. Therefore, C_min also contributes to the bactericidal effect of tobramycin, with requirements showing minor variation with change in MIC. Dosing principles for relatively resistant (higher-MIC) organisms are suggested from the data. Relatively higher aminoglycoside doses via infusion regimens are likely to be needed to generate higher peak concentrations and higher AUC values necessary for bactericidal effect in resistant organisms. Maintenance of trough concentrations on the order of 1.0 mg/liter during the interdose interval will tend to guard against the possibility of inadequate peak and AUC exposures for kill.     

Artificial Intelligence and Amikacin Exposures Predictive of Outcomes in Multidrug-Resistant Tuberculosis Patients

Aminoglycosides such as amikacin continue to be part of the backbone of treatment of multidrug-resistant tuberculosis (MDR-TB). We measured amikacin concentrations in 28 MDR-TB patients in Botswana receiving amikacin therapy together with oral levofloxacin, ethionamide, cycloserine, and pyrazinamide and calculated areas under the concentration-time curves from 0 to 24 h (AUC_0–24). The patients were followed monthly for sputum culture conversion based on liquid cultures. The median duration of amikacin therapy was 184 (range, 28 to 866) days, at a median dose of 17.30 (range 11.11 to 19.23) mg/kg. Only 11 (39%) patients had sputum culture conversion during treatment; the rest failed. We utilized classification and regression tree analyses (CART) to examine all potential predictors of failure, including clinical and demographic features, comorbidities, and amikacin peak concentrations (C_max), AUC_0–24, and trough concentrations. The primary node for failure had two competing variables, C_max of <67 mg/liter and AUC_0–24 of <568.30 mg · h/L; weight of >41 kg was a secondary node with a score of 35% relative to the primary node. The area under the receiver operating characteristic curve for the CART model was an R² = 0.90 on posttest. In patients weighing >41 kg, sputum conversion was 3/3 (100%) in those with an amikacin C_max of ≥67 mg/liter versus 3/15 (20%) in those with a C_max of <67 mg/liter (relative risk [RR] = 5.00; 95% confidence interval [CI], 1.82 to 13.76). In all patients who had both amikacin C_max and AUC_0–24 below the threshold, 7/7 (100%) failed, compared to 7/15 (47%) of those who had these parameters above threshold (RR = 2.14; 95% CI, 1.25 to 43.68). These amikacin dose-schedule patterns and exposures are virtually the same as those identified in the hollow-fiber system model.     

Ceftaroline Increases Membrane Binding and Enhances the Activity of Daptomycin against Daptomycin-Nonsusceptible Vancomycin-Intermediate Staphylococcus aureus in a Pharmacokinetic/Pharmacodynamic Model

New antimicrobial agents and novel combination therapies are needed to treat serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to daptomycin and vancomycin. The purpose of this study was to evaluate the combination of ceftaroline plus daptomycin or vancomycin in an in vitro pharmacokinetic/pharmacodynamic model. Simulations of ceftaroline-fosamil at 600 mg per kg of body weight every 8 h (q8h) (maximum free-drug concentration in serum [fC_max], 15.2 mg/liter; half-life [t_1/2], 2.3 h), daptomycin at 10 mg/kg/day (fC_max, 11.3 mg/liter; t_1/2, 8 h), vancomycin at 2 g q12h (fC_max, 30 mg/liter; t_1/2, 6 h), ceftaroline plus daptomycin, and ceftaroline plus vancomycin were evaluated against a clinical, isogenic MRSA strain pair: D592 (daptomycin susceptible and heterogeneous vancomycin intermediate) and D712 (daptomycin nonsusceptible and vancomycin intermediate) in a one-compartment in vitro pharmacokinetic/pharmacodynamic model over 96 h. Therapeutic enhancement of combinations was defined as ≥2 log₁₀ CFU/ml reduction over the most active single agent. The effect of ceftaroline on the membrane charge, cell wall thickness, susceptibility to killing by the human cathelicidin LL37, and daptomycin binding were evaluated. Therapeutic enhancement was observed with daptomycin plus ceftaroline in both strains and vancomycin plus ceftaroline against D592. Ceftaroline exposure enhanced daptomycin-induced depolarization (81.7% versus 72.3%; P = 0.03) and killing by cathelicidin LL37 (P < 0.01) and reduced cell wall thickness (P < 0.001). Fluorescence-labeled daptomycin was bound over 7-fold more in ceftaroline-exposed cells. Whole-genome sequencing and mutation analysis of these strains indicated that change in daptomycin susceptibility is related to an fmtC (mprF) mutation. The combination of daptomycin plus ceftaroline appears to be potent, with rapid and sustained bactericidal activity against both daptomycin-susceptible and -nonsusceptible strains of MRSA.