Search

The in vivo efficacy of posaconazole against 4 clinical Aspergillus fumigatus isolates with posaconazole MICs ranging from 0.03 to 16 mg/liter, as determined by CLSI method M38A, was assessed in a nonneutropenic murine model of disseminated aspergillosis. The underlying resistance mechanisms of the isolates included substitutions in the cyp51A gene at codon 220 (M220I), codon 54 (G54W), and codon 98 (L98H). The latter was combined with a 34-bp tandem repeat in the gene promoter region (TR L98H). The control isolate exhibited a wild-type phenotype without any known resistance mechanism. Oral posaconazole therapy was started 24 h after infection and was given once daily for 14 consecutive days. Mice were treated with four different doses (1 to 64 mg/kg of body weight), and survival was used as the end point. Survival was dependent both on the dose and on the MIC. The Hill equation with a variable slope fitted the relationship between the dose/MIC ratio and 14-day survival well ( R 2 , 0.92), with a 50% effective dose (ED 50 ) of 29.0 mg/kg (95% confidence interval [CI], 15.6 to 53.6 mg/kg). This also applied to the relationship between the area under the plasma concentration-time curve (AUC)/MIC ratio and 14-day survival (50% effective pharmacodynamic index [EI 50 ], 321.3 [95% CI, 222.7 to 463.4]). Near-maximum survival was reached at an AUC/MIC ratio of nearly 1,000. These results indicate that treatment of infections with A. fumigatus strains for which MICs are 0.5 mg/liter requires doses exceeding the present licensed doses. Increasing the standard dosing regimen may have some effect and may be clinically useful if no alternatives are available.

New lipid-associated formulations of amphotericin B (AmB) have been developed in order to reduce toxicity and enhance the efficacy of AmB by allowing administration of higher doses of the drug. We determined the in vivo dose-response relationships of 1 day and 7 day treatment of AmB, Ambisome (AmBi) and Abelcet (ABLC) in a non-neutropenic murine model of invasive aspergillosis by using survival as an endpoint. Female CD-1 mice were infected intravenously 48 h prior to start therapy with Aspergillus fumigatus (1 x 10(7) conidia/mouse). Groups of 10 mice were treated iv for 1 day or 7 days with increasing 2-fold doses of AmB, ABLC and AmBi up to a maximum of 20 mg/kg/day. Mortality was determined twice daily until day 15. Results were analyzed using product-moment survival analysis and by determining the dose response relationships on day 15. Survival at day 15 of mice with 7 day AmBi or ABLC treatment was significantly better than that of controls or AmB. The ED50s of AmBi and ABLC were 0.06 (95% CI: 0.03-0.127) mg/kg and 0.21 (0.06-0.66) mg/kg respectively. In addition, the maximum effect was higher for AmBi than ABLC, 90% survival versus 68%, respectively. Most of the effects of treatment with AmBi were reached after 1 day of treatment, indicating that the first dose given is most important in predicting survival. This study shows that AmBi and ABLC were significantly more efficacious than AmB in a non-neutropenic murine model of invasive aspergillosis, and that the effect observed was primarily dependent on the first dose administered.

Since the origin of an "'International Collaborative Study on Antibiotic Sensitivity Testing'" in 1971, considerable advancement has been made to standardize clinical susceptibility testing procedures of antimicrobial agents. However, a consensus on the methods to be used and interpretive criteria was not reached, so the results of susceptibility testing were discrepant. Recently, the European Committee on Antimicrobial Susceptibility Testing achieved a harmonization of existing methods for susceptibility testing and now co-ordinates the process for setting breakpoints. Previously, breakpoints were set by adjusting the mean pharmacokinetic parameters derived from healthy volunteers to the susceptibilities of a population of potential pathogens expressed as the mean minimum inhibitory concentration (MIC) or MIC90%. Breakpoints derived by the deterministic approach tend to be too high, since this procedure does not take the variabilities of drug exposure and the susceptibility patterns into account. Therefore, first-step mutants or borderline susceptible bacteria may be considered as fully susceptible. As the drug exposure of such sub-populations is inadequate, resistance development will increase and eradication rates will decrease, resulting in clinical failure. The science of pharmacokinetics/pharmacodynamics integrates all possible drug exposures for standard dosage regimens and all MIC values likely to be found for the clinical isolates into the breakpoint definitions. Ideally, the data sets used originate from patients suffering from the disease to be treated. Probability density functions for both the pharmacokinetic and microbiological variables are determined, and a large number of MIC/drug exposure scenarios are calculated. Therefore, this method is defined as the probabilistic approach. The breakpoints thus derived are lower than the ones defined deterministically, as the entire range of probable drug exposures from low to high is modeled. Therefore, the amplification of drug-resistant sub-populations will be reduced. It has been a long journey since the first attempts in 1971 to define breakpoints. Clearly, this implies that none of the various approaches is right or wrong, and that the different approaches reflect different philosophies and mirror the tremendous progress made in the understanding of the pharmacodynamic properties of different classes of antimicrobials.

The pharmacokinetics of amoxicillin were studied in umbilical cord and neonatal sera relative to maternal concentrations in prevention of neonatal group B streptococcus infection. The subjects were 44 pregnant women receiving amoxicillin as 1 or 2 g as an intravenous infusion. To measure the concentrations, blood samples were obtained from the mother, the arterial and venous umbilical cord, and the neonate. The pharmacokinetics were characterized by a five-compartment model by using nonlinear mixed-effects (population) modeling. The population estimates for the clearance, central volume of distribution, and the two peripheral maternal volumes of distribution were 19.7 ± 0.99 liters/h, 6.40 ± 0.61 liters, and 5.88 ± 0.83 liters (mean ± standard error), respectively. The volume of distribution of the venous umbilical cord and the neonatal volume of distribution were 3.40 liters and 11.9 liters, respectively. The pharmacokinetic parameter estimates were used to simulate the concentration-time profiles in maternal, venous umbilical cord, and neonatal sera. The peak concentration in the venous umbilical cord serum was 18% of the maternal peak concentration. It was reached 3.3 min after the maternal peak concentration. The concentration-time profile in neonatal serum was determined by the profile in venous umbilical cord serum, which in turn depended on the profile in maternal serum. Furthermore, the simulated concentrations in maternal, venous umbilical cord, and neonatal sera exceeded the MIC for group B streptococcus for more than 90% of the 4-h dosing interval. In a first approximation, the 2-g infusion to the mother appears to be adequate for the prevention of group B streptococcal disease. However, to investigate the efficacy of the prophylaxis, further studies of the interindividual variability in pharmacokinetics are indicated.

Contains fulltext : 71426.pdf (Publisher’s version ) (Closed access) Ceftazidime and cefotaxime are beta-lactam antibiotics with dose-related affinities for penicillin-binding protein (PBP)-3 and PBP-1. At low concentrations, these antibiotics inhibit PBP-3, leading to filament formation. Filaments are long strands of non-dividing bacteria that contain enhanced quantities of endotoxin molecules. Higher concentrations of ceftazidime or cefotaxime cause inhibition of PBP-1, resulting in rapid bacterial lysis, which is associated with low endotoxin release. In the present study, 37 isolates of Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa and Acinetobacter spp. were studied over a 4-h incubation period in the presence of eight concentrations of ceftazidime or cefotaxime. As resistance of Gram-negative bacteria is an emerging problem in clinical practice, 14 isolates of E. coli and Klebsiella pneumoniae that produced extended-spectrum beta-lactamases (ESBLs) were also investigated. Morphological changes after exposure to the beta-lactam antibiotics revealed recognisable patterns in various bacterial families, genera and isolates. In general, all isolates of Enterobacteriaceae produced filaments within a relatively small concentration range, with similar patterns for E. coli and K. pneumoniae. Pseudomonas and Acinetobacter spp. produced filaments in the presence of clinically-relevant concentrations of both antibiotics as high as 50 mg/L. In all genera, filament-producing capacity was clearly related to the MIC. Ceftazidime induced filament production in more isolates and over wider concentration ranges than did cefotaxime. Interestingly, ESBL-producing isolates were not protected against filament induction. The induction of filament production may lead to additional risks during empirical treatment of severe infections.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT center dot Examples exist that pharmacokinetics of drugs in pregnant women can differ from that in non-pregnant individuals. center dot In pregnant women before the onset of labour, the pharmacokinetics of amoxicillin is similar to that in non-pregnant individuals, but for women during labour this is unknown. WHAT THIS STUDY ADDS center dot Labour influences the pharmacokinetics of amoxicillin. center dot During labour and even more in the immediate postpartum period, the peripheral volume of distribution was decreased compared with pregnant women before the onset of labour. center dot The volume of distribution increases with an increasing amount of oedema. Many physiological changes take place during pregnancy and labour. These might change the pharmacokinetics of amoxicillin, necessitating adjustment of the dose for prevention of neonatal infections. We investigated the influence of labour on the pharmacokinetics of amoxicillin. Pregnant women before and during labour were recruited and treated with amoxicillin intravenously. A postpartum dose was offered. Blood samples were obtained and amoxicillin concentrations were determined using high-pressure liquid chromatography. The pharmacokinetics were characterized by nonlinear mixed-effects modelling using NONMEM. The pharmacokinetics of amoxicillin in 34 patients was best described by a three-compartment model. Moderate interindividual variability was identified in CL, central and peripheral volumes of distribution. The volume of distribution (V) increased with an increasing amount of oedema. Labour influenced the parameter estimate of peripheral volume of distribution (V-2). V-2 was decreased during labour, and even more in the immediate postpartum period. For all patients the population estimates (mean +/- SE) for CL and V were 21.1 +/- 4.1 l h(-1) (CL), 8.7 +/- 6.6 l (V-1), 11.8 +/- 7.7 l (V-2) and 20.5 +/- 15.4 l (V-3) respectively. The peripheral distribution volume of amoxicillin in pregnant women during labour and immediately postpartum is decreased. However, these changes are not clinically relevant and do not warrant deviations from the recommended dosing regimen for amoxicillin during labour in healthy pregnant patients.

Contains fulltext : 53715.pdf (Publisher’s version ) (Closed access) We report on the correlation of the outcomes for two cohorts of patients who had been treated for candidemia (126 episodes) or oropharyngeal candidiasis (110 episodes) with various doses of fluconazole and the MIC of fluconazole obtained by using the EUCAST standard for fermentative yeasts. Of 145 episodes caused by an isolate with a fluconazole MIC < or =2 mg/liter, 93.7% (136 of 145) responded to fluconazole treatment. The response for those infected with a strain with a MIC of 4 mg/liter was 66% but reached 100% when the dose was greater than 100 mg/day, whereas the response for those infected with strains with MICs > or =8 mg/liter was only 12%. Hence, a MIC of 2 mg/liter or 4 mg/liter was able to predict successful treatment. A cure rate of 93.9% (140 of 149) was achieved when the dose/MIC ratio was > or =100 but fell to 14.6% (16 of 109) when the ratio was less. The dose/MIC required to achieve a response rate of 50% (the 50% effective concentration) was 43.7 for the cohort of patients with oropharyngeal candidiasis. Classification and regression analysis indicated that a dose/MIC of 35.5 was the threshold for the prediction of cure or failure. However, an increase in exposure above this threshold further increased the probability of cure, and all patients were cured when the dose/MIC exceeded 100. Monte Carlo simulations showed a probability of target attainment of 99% at MICs < or =2 mg/liter and a pharmacodynamic target of a dose/MIC ratio of 100, which was equivalent to an unbound fraction of the fluconazole area under the curve versus the MIC of 79.

The efficacy of β-lactams is thought to be dependent on the time that the unbound concentrations exceed the MIC ( fT >MIC). However, the pharmacokinetic/pharmacodynamic index (PDI) that correlates best to the selection of resistance is not yet clear. The selection of ceftizoxime (CZX)-resistant Enterobacter cloacae mutant strains during the development of murine mixed-infection abscesses was studied to determine the PDI that is important for the emergence of resistance and the PDI value needed for the prevention of resistance. Studies were carried out 24 h after inoculation with Bacteroides fragilis ATCC 23745 and E. cloacae 22491. Six to 1,536 mg of CZX/kg of body weight/day given every 2 h (q2h), q4h, q6h, or q8h was started 30 min before inoculation and continued for 24 h. Resistant mutants were isolated to determine mutant frequencies (MF). The fT >MIC varied from 9 to 98% for E. cloacae , the peak concentration (unbound fraction) was 0.6 to 578 mg/liter, and the area under the concentration-time curve (unbound fraction) ( f AUC) was 1.9 to 553 mg·h/liter. The f AUC-to-MIC ratio best explained the in vivo efficacy. CZX-resistant B. fragilis and E. cloacae mutants were isolated from untreated controls at an MF of 10 −5 to 10 −7 . The MF of resistant B. fragilis did not increase during therapy. The selection of resistant E. cloacae strains at an MF of 10 −1 to 10 −2 was related to the fT >MIC and the ratio of f AUC to MIC following an inverse U shape. However, the ratio of f AUC to MIC was the stronger driver of resistance. The highest MFs were 0.7 to 0.9 at an f AUC-to-MIC ratio of approximately 250. We conclude that the ratio of f AUC to MIC is the PDI that correlated best to the in vivo efficacy of CZX and probably also to the emergence of resistant E. cloacae mutants. An f AUC-to-MIC ratio of 1,000 was needed to prevent the emergence of this resistance.

Continuous infusion of beta-lactam antibiotics is becoming increasingly popular. The background and current clinical evidence are discussed. Tools to apply continuous infusion are analyzed.One randomized controlled trial in an ICU setting and two nonrandomized controlled trials have shown continuous infusion to be more beneficial than intermittent infusion. One randomized controlled trial in chronic obstructive pulmonary disorder patients, however, showed no difference between the two treatments. The stability of most beta-lactams for use during continuous infusion has been documented.Killing of bacteria by beta-lactam antibiotics is maximal at around four times the minimum inhibitory concentration in vitro. To ensure an optimal effect when treating severe infections, free unbound concentrations at or above four times the minimum inhibitory concentration should be maintained. Although continuous infusion has been demonstrated to be superior in animal studies, randomized clinical trials have failed to confirm this in humans, primarily because of suboptimal design. A better designed randomized clinical trial, set up as a pilot study, recently demonstrated a favorable outcome with continuous infusion. A major issue during continuous infusion is the stability of the antibiotic, which may limit its application. The calculation of the infusion rate necessary to obtain the desired free drug concentration is relatively straightforward.

Aztreonam (AZM) is a monobactam antibiotic with a high level of activity against gram-negative micro-organisms, including Pseudomonas aeruginosa . We evaluated AZM pharmacokinetics and pharmacokinetic-pharmacodynamic relationships in patients with cystic fibrosis (CF) and healthy subjects. Pharmacokinetic data in eight CF patients and healthy subjects that were matched for age, gender, weight, and height were obtained and analyzed by using the nonparametric adaptive grid algorithm. Probabilities of target attainment using percentages of time of unbound concentration above the MIC ( f T>MIC) were obtained by using a Monte Carlo simulation. AZM total body clearance was significantly higher in CF patients (100.1 ± 17.1 versus 76.2 ± 7.4 ml/min in healthy subjects; P < 0.01). The pharmacokinetic parameter estimates for terminal half-life (1.54 ± 0.17 h [mean ± the standard deviation]) and volume of distribution (0.20 ± 0.02 liters/kg in patients with CF patients were not different from those in healthy subjects. Monte Carlo simulations with a target of a f T>MIC of 50 to 60% at a dose of 1,000 mg every 8 h indicated a clinical breakpoint of 4 mg/liter and 1 to 2 mg/liter for healthy subjects and CF patients, respectively. This study using matched controls showed that AZM total body clearance and not the volume of distribution is higher in CF patients as a result of increased renal clearance. Pharmacokinetic parameter estimates in healthy subjects resulted in a clinical susceptibility breakpoint of ≤4 mg/liter for a dose of 1,000 mg every 8 h. Patients suspected of having high clearance rates, such as CF patients, should be monitored closely, with dosing regimens adjusted accordingly.

Objectives: The effect of ceftazidime dosing increments and frequency of dosing on the selection of ceftazidime-resistant Enterobacter cloacae in the intestine was studied in rats, during treatment of a pulmonary infection caused by Klebsiella pneumoniae. Methods: Rats with pulmonary infection (n 5 10 per group) received therapy with doses of ceftazidime at 3.1 to 400 mg/kg per day at a frequency of every 6,12 or 24 h for 18 days, starting 24 h after bacterial inoculation of the lung. Emergence of resistance in intestinal E. cloacae was monitored by culturing fresh stool specimens at days 0, 8, 15, 22, 29, 36 and 43 on agar plates with (6.4 mg/L) and without ceftazidime. Pharmacodynamic indices and time within the mutant selection window (MSW) were assessed in infected rats for each regimen. Ceftazidime-resistant E. cloacae mutants were characterized by determination of the β-lactamase activity under cefoxitin-induced and non-induced conditions. Results: A reduction of intestinal ceftazidime-susceptible E. cloacae was observed and showed a significant correlation with the fAUC/MIC at days 8, 15 and 22 and with the fCmaxon days 8, 15, 22, 29 and 36. More rats treated with 12-25 and 50-100 mg/kg per day every 6 h were found colonized with ceftazidime-resistant E. cloacae mutants than animals treated every 12 h or every 24 h. The proportion of rats colonized with ceftazidime-resistant E. cloacae mutants at days 15, 36 and 43 correlated with the time during which ceftazidime plasma concentrations were within the boundaries of the MSW. Only at day 15 was a correlation demonstrated between the fCmaxand significantly fewer rats colonized with ceftazidime-resistant E. cloacae. Ceftazidime-resistant E. cloacae mutants (MIC ≥ 128 mg/L) were characterized as stable derepressed mutants. Conclusions: Colonization with stable derepressed ceftazidime-resistant E. cloacae mutants particularly occurred when rats were exposed to moderate doses of ceftazidime (12-25 or 50-100 mg/kg per day) administered every 6 h. Emergence of resistance was correlated with time within the MSW.

Volatile Organic Compounds (VOCs) analysis in exhaled breath is proposed as a non-invasive method to detect respiratory infections in cystic fibrosis (CF) patients. Since polymicrobial infections are increasingly common, we assessed whether fingerprints of VOCs emitted by mono-cultures of Pseudomonas aeruginosa (PA) and mono-cultures of Aspergillus fumigatus (AF) are consistent in a polymicrobial environment. Mono-cultures of PA (strain ATCC 27853), mono-cultures of AF (clinical isolate) , and co-cultures of PA and AF were studied. Headspace samples were taken at 16, 24 and 48 hours after inoculation. VOCs in the headspace of the cultures were identified by Gas Chromatography – Mass Spectrometry. Multivariate analysis by Partial Least Squares Discriminant Analysis (PLS-DA) was used to identify unique VOC fingerprints for the presence of PA , AF or both. We identified VOC fingerprints consisting of 104 different compounds in each experimental setting. PLS-DA analysis was able to identify distinct VOC profiles for each of the monocultures and for the co-culture. Within each culture type, there is a large variation in the VOCs detected over time. Forty eight hours after inoculation, methyl thiolacetate is prominent and discriminating in PA cultures, 8-nonen-2-one in AF cultures and 2-nonanone in co-cultures. We can identify specific VOC fingerprints for PA and AF monocultures and co-cultures in vitro . This study shows that VOC fingerprints are time-dependent and that the fingerprints of PA and AF in mono-cultures are different from the VOC fingerprint in co-cultures, complicating the in vivo determination of micro-organisms when only based on VOC patterns.

Avibactam is a new non-β-lactam β-lactamase inhibitor that shows promising restoration of ceftazidime activity against microorganisms producing Ambler class A extended-spectrum β-lactamases (ESBLs) and carbapenemases such as KPCs, class C β-lactamases (AmpC), and some class D enzymes. To determine optimal dosing combinations of ceftazidime-avibactam for treating infections with ceftazidime-resistant Pseudomonas aeruginosa , pharmacodynamic responses were explored in murine neutropenic thigh and lung infection models. Exposure-response relationships for ceftazidime monotherapy were determined first. Subsequently, the efficacy of adding avibactam every 2 h (q2h) or q8h to a fixed q2h dose of ceftazidime was determined in lung infection for two strains. Dosing avibactam q2h was significantly more efficacious, reducing the avibactam daily dose for static effect by factors of 2.7 and 10.1, whereas the mean percentage of the dosing interval that free drug concentrations remain above the threshold concentration of 1 mg/liter (% f T>C T 1 mg/liter) yielding bacteriostasis was similar for both regimens, with mean values of 21.6 (q2h) and 18.5 (q8h). Dose fractionation studies of avibactam in both the thigh and lung models indicated that the effect of avibactam correlated well with % f T>C T 1 mg/liter. This parameter of avibactam was further explored for four P. aeruginosa strains in the lung model and six in the thigh model. Parameter estimates of % f T>C T 1 mg/liter for avibactam ranged from 0 to 21.4% in the lung model and from 14.1 to 62.5% in the thigh model to achieve stasis. In conclusion, addition of avibactam enhanced the effect of ceftazidime, which was more pronounced at frequent dosing and well related with % f T>C T 1 mg/liter. The thigh model appeared more stringent, with higher values, ranging up to 62.5% f T>C T 1 mg/liter, required for a static effect.

The majority of azole resistance mechanisms in Aspergillus fumigatus correspond to mutations in the cyp51A gene. As azoles are less effective against infections caused by multiply azole-resistant A. fumigatus isolates, new therapeutic options are warranted for treating these infections. We therefore investigated the in vitro combination of posaconazole (POSA) and caspofungin (CAS) against 20 wild-type and resistant A. fumigatus isolates with 10 different resistance mechanisms. Fungal growth was assessed with the XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt] method. Pharmacodynamic interactions were assessed with the fractional inhibitory concentration (FIC) index (FICi) on the basis of 10% (FICi-0), 25% (FICi-1), or 53 0% (FICi-2) growth, and FICs were correlated with POSA and CAS concentrations. Synergy and antagonism were concluded when the FICi values were statistically significantly ( t test, P < 0.05) lower than 1 and higher than 1.25, respectively. Significant synergy was found for all isolates with mean FICi-0 values ranging from 0.28 to 0.75 (median, 0.46). Stronger synergistic interactions were found with FICi-1 (median, 0.18; range, 0.07 to 0.47) and FICi-2 (0.31; 0.07 to 0.6). The FICi-2 values of isolates with tandem-repeat-containing mutations or codon M220 were lower than those seen with the other isolates ( P < 0.01). FIC-2 values were inversely correlated with POSA MICs ( r s = −0.52, P = 0.0006) and linearly with the ratio of drug concentrations in combination over the MIC of POSA ( r s = 0.76, P < 0.0001) and CAS ( r s = 0.52, P = 0.0004). The synergistic effect of the combination of POSA and CAS (POSA/CAS) against A. fumigatus isolates depended on the underlying azole resistance mechanism. Moreover, the drug combination synergy was found to be increased against isolates with elevated POSA MICs compared to wild-type isolates.

We present the real-time monitoring of hydrogen cyanide (HCN) production from Pseudomonas aeruginosa (P. aeruginosa) strains in vitro, using laser-based photoacoustic spectroscopy. Simultaneously, the production of ammonia (NH3) was measured, and the influence of different factors (e.g. the medium, temperature and antibiotics treatment) was assessed. Both reference strains and clinical isolates of patients with CF were studied, and compared to other pathogens commonly present in lungs/airways of CF patients. Hydrogen cyanide production starts to rise as soon as P. aeruginosa bacteria reach the stationary phase ((9.0-9.5) × 10(9) colony forming units, CFUs), up to concentrations of 14.5 microliters per hour (µl h(-1)). Different strains of P. aeruginosa produced HCN to varying degrees, and addition of tobramycin strongly reduced HCN production within 2 h from application. Burkholderia cepacia also produced HCN (up to 0.35µl h(-1) in 9.0 × 10(9) CFU) while other pathogens (Aspergillus fumigatus, Stenotrophomonas maltophilia, Mycobacterium abscessus) did not produce detectable levels. Our study reveals for the first time a broad overview of the dynamics of the HCN production in vitro.

Objectives: Nitrofurantoin's use has increased exponentially since recent guidelines repositioned it as first-line therapy for uncomplicated lower urinary tract infection (UTI). We conducted a systematic review and metaanalysis to assess nitrofurantoin's efficacy and toxicity in the treatment of lower UTI. Methods: We performed a systematic review of all human controlled clinical trials published from 1946 to 2014 and assessing short-term (

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 TR 34 /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.

Ceftolozane, formally CXA-101, is a new antipseudomonal cephalosporin that is also active in vitro against Enterobacteriaceae but is vulnerable to extended-spectrum β-lactamases (ESBLs). The addition of tazobactam is intended to broaden coverage to most ESBL-producing Escherichia coli and Klebsiella pneumonia as well as other Enterobacteriaceae . The in vitro activities of ceftolozane-tazobactam combinations against 67 clinically and molecularly characterized ESBL-producing isolates were examined by checkerboard MIC testing to evaluate their potential clinical feasibility and to assess the optimal tazobactam concentrations to be used in MIC determinations of ceftolozane. Isolates included those from E. coli ( n = 32), K. pneumoniae ( n = 19), Enterobacter cloacae ( n = 15), and Citrobacter freundii ( n = 1). Checkerboard experiments were performed to study interactions over the range of 0.008 to 64 mg/liter ceftolozane and 0.063 to 32 mg/liter tazobactam using 2-fold-dilution series. The MIC 50 and MIC 90 of ceftolozane alone for all isolates were 16 and ≥64 mg/liter, respectively. Increasing concentrations of tazobactam resulted in decreasing MICs of ceftolozane. The 50th and 90th percentile concentrations of tazobactam required to reduce the MIC of ceftolozane to 8 mg/liter for all organisms in this ESBL collection were 0.5 and 4 mg/liter, respectively. For E. coli , K. pneumoniae , and E. cloacae , these values were 0.5 and 2, 1 and 16, and 0.5 and 4 mg/liter, respectively. When combined with a fixed amount of 4 mg/liter tazobactam (current CLSI concentration used for susceptibility testing), 90% of the isolates would have an MIC of ≤4 mg/liter. The combination ceftolozane-tazobactam is a promising alternative option for treating infections due to ESBL-harboring isolates.

In the face of increasing antimicrobial resistance and the paucity of new antimicrobial agents it has become clear that new antimicrobial strategies are urgently needed. One of these is to revisit old antibiotics to ensure that they are used correctly and to their full potential, as well as to determine whether one or several of them can help alleviate the pressure on more recent agents. Strategies are urgently needed to ‘re-develop’ these drugs using modern standards, integrating new knowledge into regulatory frameworks and communicating the knowledge from the research bench to the bedside. Without a systematic approach to re-developing these old drugs and rigorously testing them according to today's standards, there is a significant risk of doing harm to patients and further increasing multidrug resistance. This paper describes factors to be considered and outlines steps and actions needed to re-develop old antibiotics so that they can be used effectively for the treatment of infections.

Azole resistance is an emerging problem in Aspergillus fumigatus which translates into treatment failure. Alternative treatments with new azoles may improve therapeutic outcome in invasive aspergillosis (IA) even for strains with decreased susceptibility to current azoles. The in vivo efficacy of 0.25, 1, 4, 16, 64, 128, 256, and 512 mg/kg of body weight/day prodrug isavuconazonium sulfate (BAL8557) (isavuconazole [ISA]-equivalent doses of 0.12, 0.48, 1.92, 7.68, 30.7, 61.4, 122.9, and 245.8 mg/kg/day, respectively) administered by oral gavage was assessed in an immunocompetent murine model of IA against four clinical A. fumigatus isolates: a wild-type isolate (ISA MIC EUCAST , 0.5 mg/liter) and three azole-resistant isolates harboring substitutions in the cyp51A gene: G54W (ISA MIC EUCAST , 0.5 mg/liter), M220I (ISA MIC EUCAST , 4 mg/liter), and TR 34 /L98H (ISA MIC EUCAST , 8 mg/liter). The maximum effect (100% survival) was reached at a prodrug isavuconazonium sulfate dose of 64 mg/kg for the wild-type isolate, 128 mg/kg for the G54W mutant, and 256 mg/kg two times per day (q12) for the M220I mutant. A maximum response was not achieved with the TR 34 /L98H isolates with the highest dose of prodrug isavuconazonium sulfate (256 mg/kg q12). For a survival rate of 50%, the effective AUC 0–24 /MIC EUCAST ratio for ISA total drug was 24.73 (95% confidence interval, 22.50 to 27.18). The efficacy of isavuconazole depended on both the drug exposure and the isavuconazole MIC of the isolates. The quantitative relationship between exposure and effect (AUC 0–24 /MIC) can be used to optimize the treatment of human infections by A. fumigatus , including strains with decreased susceptibility.

Objectives: This study aimed to provide basic pharmacodynamic information for key antibiotics used to treat Mycobacterium avium and Mycobacterium xenopi pulmonary disease. Methods: M. avium subspecies hominissuis IWGMT49 and M. xenopi ATCC 19250 type strains were used; the MICs of clarithromycin, amikacin and moxifloxacin were determined by broth microdilution. Time-kill assays were performed, exposing bacteria to 2-fold concentrations from 0.062xto 32xthe MIC at 37 degrees C for 240 h for M. avium or 42 days for M. xenopi. The sigmoid maximum effect (E-max) model was fitted to the time-kill curve data. Results: Maximum killing of M. avium by amikacin was obtained between 24 and 120 h (0.0180 h(-1)) and was faster and higher than with clarithromycin (0.0109 h(-1)); however, regrowth and amikacin-resistant mutants were observed. Killing rates for M. xenopi were higher, 0.1533 h(-1) for clarithromycin and 0.1385 h(-1) for moxifloxacin, yet required 42 days. There were no significant differences between the Hill's slopes determined for all of the antibiotics tested against M. avium or M. xenopi (P = 0.9663 and P = 0.0844, respectively). Conclusions: The killing effect of amikacin and clarithromycin on M. avium subspecies hominissuis was low, although amikacin activity was higher than that of clarithromycin, supporting its role in a combined therapy. Clarithromycin and moxifloxacin may have similar activity within treatment regimens for M. xenopi disease. Future studies of in vitro and in vivo pharmacokinetic/pharmacodynamic interactions are needed to improve the current regimens to treat these two important slowly growing mycobacteria in pulmonary disease.

In the face of diminishing therapeutic options for the treatment of infections caused by multidrug-resistant, Gram-negative bacteria, clinicians are increasingly using colistin and polymyxin B. These antibiotics became available clinically in the 1950s, when understanding of antimicrobial pharmacology and regulatory requirements for approval of drugs was substantially less than today. At the 1st International Conference on Polymyxins in Prato, Italy, 2013, participants discussed a set of key objectives that were developed to explore the factors affecting the safe and effective use of polymyxins, identify the gaps in knowledge, and set priorities for future research. Participants identified several factors that affect the optimum use of polymyxins, including: confusion caused by several different conventions used to describe doses of colistin; an absence of appropriate pharmacopoeial standards for polymyxins; outdated and diverse product information; and uncertainties about susceptibility testing and breakpoints. High-priority areas for research included: better definition of the effectiveness of polymyxin-based combination therapy compared with monotherapy via well designed, randomised controlled trials; examination of the relative merits of colistin versus polymyxin B for various types of infection; investigation of pharmacokinetics in special patient populations; and definition of the role of nebulised polymyxins alone or in combination with intravenous polymyxins for the treatment of pneumonia. The key areas identified provide a roadmap for action regarding the continued use of polymyxins, and are intended to help with the effective and safe use of these important, last-line antibiotics.

Item does not contain fulltext BACKGROUND: Toll-like receptors (TLRs) play a central role in the innate immune response to complicated skin and skin structure infections (cSSSIs), with TLR10 being the first family member known to have an inhibitory function. This study assessed the role of TLR10 in recognition of cSSSI-related pathogens and whether genetic variation in TLR10 influences susceptibility to cSSSIs. METHODS: Human peripheral blood mononuclear cells (PBMCs) preincubated with anti-TLR10 antibody and HEK-293 cells overexpressing TLRs were exposed to cSSSI pathogens, and cytokine secretion was determined by enzyme-linked immunosorbent assay. A total of 318 patients with cSSSI and 328 healthy controls were genotyped for 4 nonsynonymous single-nucleotide polymorphisms in TLR10, and functional consequences of the TLR10 SNPs were assessed via in vitro stimulation assays. RESULTS: PBMC stimulation with cSSSI pathogens in the presence of TLR10 neutralizing antibody significantly increased interleukin 6 secretion. Overexpression of TLR10 completely abrogated TLR2-induced interleukin 8 secretion by HEK-293 cells in response to cSSSI pathogens. Three polymorphisms in TLR10, I775L, I369L, and N241H, were associated with reduced susceptibility to cSSSIs. The presence of the TLR10 alleles 775L, 369L, or 241H increased interleukin 6 secretion by PBMCs in response to cSSSI pathogens. CONCLUSIONS: TLR10 is a modulatory receptor of innate immune responses to cSSSI-related pathogens, and genetic variants in TLR10 are associated with protection against cSSSIs.

To evaluate the in vitro effects of the combination of ceftazidime and avibactam on the MICs of both compounds, checkerboard assays were performed for 81 clinical strains, including 55 Enterobacteriaceae strains (32 Klebsiella pneumoniae , 19 Escherichia coli , 1 Citrobacter freundii , and 3 Enterobacter cloacae ) and 26 strains of Pseudomonas aeruginosa , all with known resistance mechanisms such as extended-spectrum β-lactamases (ESBLs) and carbapenemases, phenotypically or molecularly determined. Phenotypically ceftazidime-resistant strains ( n = 69) were analyzed in more detail. For the Enterobacteriaceae strains, a concentration-dependent effect of avibactam was found for most strains with a maximum effect of avibactam at a concentration of 4 mg/liter, which decreased all ceftazidime MICs to ≤4 mg/liter. Avibactam alone also showed antibacterial activity (the MIC 50 and MIC 90 being 8 and 16 mg/liter, respectively). For the ceftazidime-resistant P. aeruginosa strains, considerable inhibition of β-lactamases by avibactam was acquired at a concentration of 4 mg/liter, which decreased all ceftazidime MICs except one to ≤8 mg/liter (the CLSI and EUCAST susceptible breakpoint). Increasing the concentration of avibactam further decreased the MICs, resulting in a maximum effect for most strains at 8 to 16 mg/liter. In summary, for most strains, the tested addition of avibactam of 4 mg/liter restored the antibacterial activity of ceftazidime to a level comparable to that of wild-type strains, indicating full inhibition, and strains became susceptible according to the EUCAST and CLSI criteria. Based on these in vitro data, avibactam is a promising inhibitor of different β-lactamases, including ESBLs and carbapenemases.

Item does not contain fulltext We present the real-time monitoring of hydrogen cyanide (HCN) production from Pseudomonas aeruginosa (P. aeruginosa) strains in vitro, using laser-based photoacoustic spectroscopy. Simultaneously, the production of ammonia (NH3) was measured, and the influence of different factors (e.g. the medium, temperature and antibiotics treatment) was assessed. Both reference strains and clinical isolates of patients with CF were studied, and compared to other pathogens commonly present in lungs/airways of CF patients. Hydrogen cyanide production starts to rise as soon as P. aeruginosa bacteria reach the stationary phase ((9.0-9.5) x 10(9) colony forming units, CFUs), up to concentrations of 14.5 microliters per hour (microl h(-1)). Different strains of P. aeruginosa produced HCN to varying degrees, and addition of tobramycin strongly reduced HCN production within 2 h from application. Burkholderia cepacia also produced HCN (up to 0.35microl h(-1) in 9.0 x 10(9) CFU) while other pathogens (Aspergillus fumigatus, Stenotrophomonas maltophilia, Mycobacterium abscessus) did not produce detectable levels. Our study reveals for the first time a broad overview of the dynamics of the HCN production in vitro.

This study aimed to explore any mutation in the CYP51 gene conferring azole resistance in Aspergillus flavus . Two voriconazole-resistant and 45 voriconazole-susceptible isolates were included in the study. Sequence analysis demonstrated a T1025C nucleotide change in CYP51C , resulting in the Y319H amino acid substitution in one resistant isolate. However, the earlier described T788G mutation in CYP51C conferring voriconazole resistance in A. flavus isolates was present in all isolates, irrespective of their susceptibility status.

On March 11, 2015, we all lost a mentor, colleague, and friend, Professor William Alexander Craig. Much has been said about his achievements and how he wanted to be a doctor while also graduating in mathematics (which would help him greatly later in his career). Having practiced medicine in the U.S

Objectives In the heterogeneous group of preterm and term neonates, gentamicin and tobramycin are mainly dosed according to empirical guidelines, after which therapeutic drug monitoring and subsequent dose adaptation are applied. In view of the variety of neonatal guidelines available, the purpose of this study was to evaluate target concentration attainment of these guidelines, and to propose a new model-based dosing guideline for these drugs in neonates. Methods Demographic characteristics of 1854 neonates (birth weight 390–5200 g, post-natal age 0–27 days) were extracted from earlier studies and sampled to obtain a test dataset of 5000 virtual patients. Monte Carlo simulations on the basis of validated models were undertaken to evaluate the attainment of target peak (5–12 mg/L) and trough ( Results Across the entire neonatal age and weight range, the Dutch National Formulary for Children, the British National Formulary for Children, Neofax and the Red Book resulted in adequate peak but elevated trough concentrations (63%–90% above target). The proposed dosing guideline (4.5 mg/kg gentamicin or 5.5 mg/kg tobramycin) with a dosing interval based on birth weight and post-natal age leads to adequate peak concentrations with only 33%–38% of the trough concentrations above target, and a constant AUC across weight and post-natal age. Conclusions The proposed neonatal dosing guideline for gentamicin and tobramycin results in improved attainment of target concentrations and should be prospectively evaluated in clinical studies to evaluate the efficacy and safety of this treatment.

The prodrug isavuconazonium sulfate (BAL8557) is an extended-spectrum water-soluble triazole, developed for the treatment of severe invasive and life-threatening fungal diseases. Its active moiety, BAL4815, is a potent inhibitor of ergosterol biosynthesis, resulting in the disruption of fungal membrane structure and function. The active compound shows broad-spectrum of activity and potency against all major opportunistic fungi, such as Aspergillus spp., Candida spp., Cryptococcus spp., Mucorales, Black yeasts and their filamentous relatives and the true pathogenic fungi, including Histoplasma capsulatum and Blastomyces dermatitidis. It is currently in Phase III clinical development for treatment of aspergillosis, candidiasis and mucormycosis, as well as other rare fungi infections. We reviewed the pharmacokinetic and pharmacodynamic characteristics of isavuconazole, and its microbiological and clinical investigation progress in advanced stages of development.

Contains fulltext : 51087.pdf (Publisher’s version ) (Closed access) This study compared the in-vitro properties and in-vivo effects of Escherichia coli filaments, spheroplasts and normal cells in a murine thigh infection model. E. coli was exposed to ceftazidime, meropenem or saline to obtain filaments, spheroplasts or normal bacilli, which were then injected into neutropenic mice. After 24 h, morphology, CFUs, local and circulating endotoxin levels, cytokine levels and mortality were recorded, and correlations between bacterial and host parameters of infection were investigated. Filaments and spheroplasts contained more endotoxin/CFU than controls. Histological studies showed that morphologically altered bacteria changed into rod-shaped cells in the absence of antibiotics. Bacterial spread to the liver was significantly higher in mice challenged with rod-shaped cells, compared with antibiotic-exposed bacteria (p 0.007). Muscle endotoxin levels correlated significantly with circulating interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha, and both pro-inflammatory cytokines were correlated significantly (p 0.011). Despite a tendency toward higher local and systemic concentrations of endotoxin in the filament group, inflammatory responses and survival did not differ between groups. It was concluded that morphologically altered bacteria contain more endotoxin and can regain a rod shape after withdrawal of antibiotics, while non-antibiotic-exposed bacteria show greater spread to the liver. There was a clear intra-individual relationship between local endotoxin, systemic endotoxin, TNF-alpha and IL-6 production, but these parameters did not differ among groups.

The pharmacokinetic/pharmacodynamic (PK/PD) indices that define the therapeutic effect of the beta-lactam ceftazidime in a rat model of Klebsiella pneumoniae lung infection were investigated in relation to treatment duration and treatment endpoint. Treatment was started 24 h after infection with dosing regimens of 3.1 up to 1,600 mg/kg of body weight/day and dosing every 6, 12, or 24 h. When animals were treated for a relatively short period of 48 h, the duration of time that unbound plasma ceftazidime levels exceeded the MIC of the antibiotic for the infecting strain was the index that best correlated with therapeutic efficacy in terms of significant bacterial killing in the infected lung (microbiological effect). The maximum effect was reached when plasma ceftazidime levels were above the MIC for 60 to 70% of the dosing interval. However, when the treatment duration was extended to a relatively long period of 18 days instead of 48 h and animal survival rate instead of microbiological efficacy was taken as the endpoint, the f AUC/MIC ratio (where AUC is the area under the concentration-time curve) was the PK/PD index that best correlated with therapeutic efficacy. The PK/PD indices that effect 50% survival of rats for the f AUC/MIC ratios were 18.0 (95% confidence interval [95% CI], 16.3 to 19.9), 20.2 (95% CI, 13.8 to 29.4), and 27.9 (95% CI, 21.3 to 36.5) for the schedules of administration of every 6, 12, and 24 h, respectively. The f AUC/MIC needed for 100% survival was >100. We conclude that the PK/PD index that best correlates with outcome is dependent on the duration of treatment and/or the parameter of outcome. The effect of long-term treatment should be studied more extensively in other models of infection.

Contains fulltext : 50982.pdf (Publisher’s version ) (Closed access) Originally, itraconazole for parenteral administration was licensed in a 40% hydroxypropyl-beta-cyclodextrin (HPBCD) solution for intravenous administration. A novel formulation, the NanoCrystal formulation (NCF), was prepared. NCF consists of drug particles of approximately 200 to 300 nm. The pharmacokinetics of itraconazole and its hydroxy metabolite in healthy subjects were evaluated after single and multiple doses of itraconazole as NCF. In the single-ascending-dose (SAD) study, itraconazole doses were planned to range from 50 to 500 mg, while in the multiple-ascending-dose (MAD) study, itraconazole doses of 100, 200, and 300 mg as NCF were studied, as was one dose level (200 mg) as an HBPCD solution. Samples were collected in heparinized tubes at various time points and were analyzed by high-performance liquid chromatography to allow full pharmacokinetic analysis both after the first dose and on day 7. The results of both the SAD and the MAD studies indicated that there was a dose dependency in the half-life of itraconazole from the novel formulation, increasing from 44 h (100 mg) to more than 150 h (300 mg) once steady state was achieved. Similar dose-dependent effects were observed for the hydroxy metabolite. The areas under the concentration-time curves for itraconazole and hydroxyitraconazole were also dose dependent. The pharmacokinetic profiles after 200-mg doses of itraconazole as NCF and HPBCD formulations were comparable with respect to the terminal half-life, both after a single dose and at steady state. NCF may provide an alternative to the HPBCD solution for the further optimization of antifungal treatment with itraconazole.

The therapeutic efficacy of flucytosine (5FC) monotherapy and the pharmacodynamic index predictive of efficacy were evaluated in a nonneutropenic mouse model of acute invasive aspergillosis. Mice were infected intravenously with an Aspergillus fumigatus isolate (the median MICs of 5FC were 128 μg/ml under the standard condition, 0.5 μg/ml at pH 6.0, and 0.031 μg/ml at pH 5.0) 2 h prior to the start of therapy and were treated for 7 days with different 5FC dosing regimens. The total doses ranged from 50 to 800 mg/kg of body weight/day and were administered at 6-, 12-, and 24-h intervals. The efficacy was assessed by means of survival. The survival rates of the treatment groups ranged from 40 to 90%, while the survival rate of the control group was 20%. The efficacy found depended primarily on the total daily dose. However, the power of our sample size may have been too low to exclude an effect of dose fractionation. The pharmacodynamic index that most strongly correlated with the efficacy was the area under the serum concentration-time curve and MIC ratio ( R 2 = 0.86). We conclude that 5FC monotherapy is efficacious in a murine Aspergillus fumigatus infection model.

In this study, we investigated the pH dependency of the in vitro activities of amphotericin B (AMB) and flucytosine (5FC) against Candida spp., Cryptococcus neoformans , Aspergillus fumigatus , Rhizopus spp., and Scedosporium prolificans in RPMI 1640 buffered with citrate buffer (pH 4.0, 5.0, 5.4, and 6.0), citrate-phosphate buffer (pH 5.4, 6.0, 6.4, and 7.0), and 3-[ N -morpholino]propanesulfonic acid (MOPS) (pH 6.4, 7.0, 7.4, and 7.9). For 5FC, no significant differences were found between MICs obtained with the different buffers, while for AMB, significant differences were found. The MICs obtained with citrate-phosphate buffer were approximately 1 twofold-dilution step higher than the MICs obtained with MOPS. We demonstrated that the in vitro activities of AMB and 5FC against yeast and mold isolates were pH dependent. The in vitro activity of AMB decreased when the pH was lowered, while the in vitro activity of 5FC increased. The effect of the pH on the in vitro activities was dependent not only on the antifungal agent tested but also on the microorganism. For AMB, there was a nonlinear relationship (median r 2 , 0.864) for Candida spp., C. neoformans , A. fumigatus , and Rhizopus spp. over the pH range tested. The mean MICs ranged from 0.5 to 2.52 μg/ml at pH 7.0 and from 20.16 to 32 μg/ml at pH 5.0. For S. prolificans , there was no relationship. For 5FC, there was a linear relationship for Candida spp. (median r 2 , 0.767) and a nonlinear relationship for C. neoformans and A. fumigatus (median r 2 , 0.882) over the pH range tested. The mean MIC values ranged from 0.125 to 1,024 μg/ml at pH 7.0 and from 0.02 to 4 μg/ml at pH 5.0. For Rhizopus spp. and S. prolificans , the relationship could not be determined, since the MIC was >1,024 μg/ml over a pH range of 4.0 to 7.9.

Background: Over the past decades, the relationship between the pharmacokinetic (PK) properties of antibiotics, MICs, and clinical effects has been increasingly well understood. Interpatient variability in the PK profile, however, has only recently been recognized as a major factor in predicting the outcome in individual patients and establishing breakpoints for clinical susceptibility. Most predictions to date have used data from healthy volunteers. Objective: The purpose of this study was to perform Monte Carlo simulations of the PK/pharmacodynamic relationships of ceftazidime to assess whether the probability of target attainment (PTA) differed significantly between 3 distinct populations. To that end, population PK models of ceftazidime were developed for the 3 populations. Methods: Serum concentration-time data from earlier studies in healthy volunteers (n = 8), patients with cystic fibrosis (CF) (n = 17), and patients in the intensive care unit (ICU) (n = 6) were used to obtain population PK parameter estimates and covariance matrices using the nonparametric adaptive grid program. The PTA for each group was obtained using 10,000 patient simulations for dosing regimens of 1000 and 2000 mg q8h over a range of MICs and percentages of time that concentrations of unbound drug remained above the MIC (%T>MIC). Results: The relationship between the MIC and the population mean %T>MIC, as well as the PTA profiles, differed markedly between the 3 groups as a result of both differences and variations in V d and Cl. Breakpoints based on a 100% PTA for a %T>MIC of 60% were ≤4, 0.5, and 0.5 mg/L in healthy volunteers, patients with CF, and patients in the ICU, respectively. However, when PTA values between 90% and 100% were reevaluated and differences in clinical dosing regimens were accounted for, the resulting breakpoints were identical in the 3 groups. Conclusions: PK parameter estimates for ceftazidime based on data from a small group of healthy volunteers resulted in a clinical susceptibility breakpoint comparable to those for patients with CF and patients in the ICU. Based on the study findings, this breakpoint would be ≤4 mg/L. Patients suspected of having unusually high rates of clearance should be monitored closely.

Item does not contain fulltext In total, 881 presumptive clinical isolates of Streptococcus pneumoniae collected from throughout The Netherlands were analysed to determine their mechanisms of macrolide resistance. Isolates were identified initially by participating laboratories using their own standard identification technique, followed by determination of MICs with Etests. Only 797 isolates were confirmed as pneumococci following bile-solubility tests, lytA PCR and 16S rRNA sequencing. Of these confirmed pneumococci, 59 (7.4%) isolates were macrolide-resistant. Analysis by PCR indicated that 34 (57.6%) isolates harboured only the erm(B) gene and 16 (27.1%) only the mef gene. Three (5.1%) isolates carried both erm(B) and mef, while six (10.2%) isolates were negative for both mechanisms. Of the six negative isolates, three had a mutation in the 23S rRNA gene, and three were negative for all mechanisms tested. No isolates with the erm(A) subclass erm(TR) gene were detected. Among the 19 mef-positive isolates, 14 (73.7%) carried the mef(A) gene, and only five (26.3%) carried the mef(E) gene. No linezolid cross-resistance or multiresistance (resistance to more than two classes of antibiotics) was observed.

The minimum inhibitory concentration (MIC) is the in vitro reference value to describe the activity of an antibacterial against micro-organisms. It does not represent the dynamic effect of the antimicrobial at any point in time, but rather the total antimicrobial effect over the incubation period at a fixed concentration. To explore the concentration-effect relationship of antimicrobial concentrations against micro-organisms in relation to the MIC. Time-kill curves were generated for ceftazidime, meropenem and tobramycin against Pseudomonas aeruginosa. The Hill equation with variable slope was fit to the time-kill data, and mathematical models of growth and kill were explored with reference to the MIC. With declining concentrations, bacterial killing will decrease until a specific threshold concentration is reached. This concentration, at which bacteria are neither killed nor able to grow, is named the stationary concentration (SC) and is not equal to the MIC. Pharmacokinetic/pharmacodynamic simulations over a range of kill rates, growth rates and slope factors showed that for β-lactam antibacterials, the SC is close to the MIC value, which may explain why concentrations in vivo need to be above the MIC, while regrowth of bacteria occurs when concentrations decline below the MIC. For concentration-dependent antibacterials, such as aminoglycosides and quinolones, the SC is shown to be markedly different from the MIC and, in general, is much lower. The MIC is not a good pharmacodynamic parameter to characterise the concentration effect relationship of a given antimicrobial. For ‘concentration independent’ antimicrobials the SC is likely to be close to the MIC, but may be much lower for ‘concentration dependent’ antimicrobials, and may explain sub-MIC effects.

Dosing of antibiotics in neonates requires finding a delicate balance between maximal efficacy and minimal toxicity. There is a lack of data on efficacy of currently used antibiotics in neonates, and rational dosing therefore needs to be based on gestational- and postnatal-age-dependent pharmacokinetics in combination with surrogate markers. These surrogate markers are: (i) the area-under-the serum concentration time curve to minimum inhibitory concentration ratio (AUC/MIC); (ii) peak concentration to MIC ratio (Cmax/MIC); and (iii) the time the concentration remains above the MIC (T>MIC). Whereas the efficacy of beta-lactam antibiotics (including carbapenems) depends on T>MIC, the efficacy of most other antimicrobials (including aminoglycosides and fluoroquinolones) is related to AUC/MIC and Cmax/MIC. Most modern dosing regimens are adequate when these concentration effect relationships are taken into account. Dosing adjustments in neonates are suggested, based on these relationships. Several antimicrobial combinations for treatment of meningitis and necrotizing enterocolitis exist. Empiric treatment should be based on efficacy, concerns about resistance as well as information from institutional microbiological surveillance.

To the Editor: Aspergillus fumigatus causes a variety of diseases in humans. The drugs recommended for treatment of Aspergillus diseases are the mold-active azole antifungal drugs (1). However, a wide range of mutations in A. fumigatus confer azole resistance, which commonly involves modifications in the cyp51A gene (2), the target for azole antifungal drugs. Azole resistance is thought to be selected for as a result of patient therapy or exposure to azole compounds in the environment; resistance in clinical A. fumigatus isolates has been increasingly reported in several European countries, Asia, and the United States (3–7). The most frequently reported resistance mechanism is a 34-bp tandem repeat (TR34) in combination with a substitution at codon 98 (TR34/L98H) (4); this mechanism is believed to have been selected for through environmental exposure to azole fungicides. Because routine in vitro susceptibility testing of clinical Aspergillus isolates is not common in many centers worldwide, the prevalence of azole resistance might be underestimated. We investigated the prevalence of azole resistance in clinical A. fumigatus isolates stored for 6 years (2003–2009) at Tehran University Mycology Reference Centre and Islamic Azad University, Ardabil Branch, Iran. We investigated 124 clinical A. fumigatus isolates obtained from patients with Aspergillus diseases (Technical Appendix Table 1). We conducted strain identification, in vitro antifungal susceptibility testing, and sequence-based analysis of the Cyp51A gene, as described (4). We performed microsatellite genotyping of all A. fumigatus isolates for which the MIC of itraconazole was ≥16 mg/L (8) by using a short tandem repeat A. fumigatus assay, and we compared the results with those reported for the Netherlands (20 isolates) and other European countries (24 isolates) (Technical Appendix Figure). The distribution of azole-resistant and wild-type A. fumigatus isolates examined in this study, according to year of isolation, is shown in online Technical Appendix Table 1. Of 124 A. fumigatus isolates, 4 grew on the wells containing itraconazole and voriconazole, indicating a multidrug-resistant phenotype. Of these resistant isolates, 3 were from patients with chronic pulmonary aspergillosis and 1 was from a patient with allergic bronchopulmonary aspergillosis (Table). Table Characteristics of 4 azole-resistant clinical Aspergillus fumigatus isolates, Iran* Sequence analysis of the CYP51A gene indicated the presence of TR34/L98H in 3 isolates and no mutations in the other isolate (Table). The first TR34/L98H isolate had been recovered in 2005, which is relatively early compared with reported isolations in other countries (Technical Appendix Table 2). Microsatellite typing of 6 short tandem repeat loci demonstrated identical patterns for 2 of the 3 azole-resistant isolates from Iran, but the TR34/L98H isolates from Iran did not cluster with those from the Netherlands and other European countries, indicating no close genetic relatedness (Technical Appendix Figure). The TR34/L98H azole resistance mechanism was first described in 1998 in the Netherlands; since then, its presence in clinical and environmental A. fumigatus isolates in multiple European countries and recently in Asia has been increasingly reported (Technical Appendix Table 2) (3–7). In the study reported here, prevalence of azole resistance in clinical A. fumigatus isolates obtained from patients in Iran was 3.2%; most isolates exhibited the TR34/L98H resistance mechanism. The fact that the first TR34/L98H isolate was found relatively early, in 2005, underscores the possibility that prevalence of azole resistance might be underestimated in many countries because in vitro susceptibility testing of A. fumigatus is not routinely performed. Microsatellite genotypic analysis of A. fumigatus isolates from the Netherlands and various European countries showed that the genetic diversity of TR34/L98H isolates is lower than that of wild-type controls (8). It has been suggested that TR34/L98H isolates might have a common ancestor that developed locally and subsequently migrated across Europe. In contrast, genotyping of TR34/L98H originating from India suggested a different dynamic; all environmental and clinical TR34/L98H isolates from India shared the same multilocus microsatellite genotype not found in any other analyzed samples, from within India or from the Netherlands, France, Germany, or the People’s Republic of China (9). The molecular epidemiology of the TR34/L98H isolates from Iran suggests that they cluster apart from the European isolates, indicating that migration from Europe to Iran, or vice versa, is unlikely. Genotyping of more TR34/L98H isolates from the Middle East and comparison with those from India would enhance understanding of the origin and geographic spread of TR34/L98H. Our study indicates that TR34/L98H was in Iran in 2005; this finding adds to the growing list of regions where acquired resistance in A. fumigatus of environmental origin is documented. From a global perspective, fungicide use is second highest in the Asia–Pacific regions (24%), preceded only by western Europe (37%) (10). For a bettering understand of the scale of this emerging public health problem and for insight into the dynamics of geographic migration, surveys of fungal culture collections for TR34/L98H and molecular typing studies are warranted. These data would be useful not only for clinical management of Aspergillus diseases but also for enabling policy makers to develop strategies that prevent resistance selection by the environmental route. Technical Appendix: Distribution of azole-resistant and azole-susceptible Aspergillus fumigatus isolates, Iran, 2003–2009; first reports of multiple-triazole-resistant A. fumigatus isolate(s) carrying the TR34/L98H mutations in the CYP51A gene, by country; and minimum spanning tree comparing genotypic relatedness of clinical azole-resistant A. fumigatus isolates carrying TR34/L98H alteration in the CYP51A gene from Iran with those reported from European countries. TR, tandem repeat. Click here to view.(160K, pdf)

Combination therapy of flucytosine (5FC) with other antifungal agents could be of use for the treatment of invasive aspergillosis. However, interpretation of the results of in vitro interactions is problematic. The fractional inhibitory concentration (FIC) index is the most commonly used method, but it has several major drawbacks in characterizing antifungal drug interaction. Alternatively, a response surface approach using the concentration-effect relationship over the whole concentration range instead of just the MIC can be used. We determined the in vitro interactions between amphotericin B (AMB), itraconazole, and 5FC against 21 Aspergillus isolates with a broth microdilution checkerboard method that employs the dye MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H -tetrazolium bromide]. FIC indices based on three different MIC endpoints (MIC-0, MIC-1, and MIC-2) and the interaction coefficient alpha were determined, the latter by estimation from the response surface approach described by Greco et al. (W. R. Greco, G. Bravo, and J. C. Parsons, Pharmacol. Rev. 47:331-385, 1995). The value obtained for the FIC index was found to be dependent on the MIC endpoint used and could be either synergistic, indifferent, or antagonistic. The response surface approach gave more consistent results. Of the three combinations tested, the AMB-5FC combination was the most potent in vitro against Aspergillus spp. We conclude that the use of the response surface approach for the interpretation of in vitro interaction studies of antifungals may be helpful in order to predict the nature and intensity of the drug interaction. However, the correlation of these results with clinical outcome remains difficult and needs to be further investigated.

The effects of ceftizoxime (CZX), piperacillin (PIP), and PIP-tazobactam (PT) concentrations on the antibacterial activity and selection of resistant mutants of Bacteroides fragilis and Enterobacter cloacae were investigated in vitro in a mixed-culture anaerobic time-kill study and in vivo in a mixed-infection abscess model. Mixed cultures were incubated for 24 h with 0.125 to 512 μg of CZX per ml or 0.125 to 2,048 μg of PIP or PT per ml. Mice were treated every 2 h for 24 h with CZX at 6 to 1,536 mg/kg/day or with PIP or PT at 24 to 6,144 mg/kg/day starting 30 min before inoculation with different B. fragilis - E. cloacae combinations. There was a good correlation between the in vitro and in vivo activities of the antibiotics and their MICs obtained with high inocula (10 8 CFU/ml). The respective 50% effective doses (milligrams per kilogram per day) with B. fragilis and E. cloacae 22491 were 771 and 521 for CZX, 416 and 643 for PIP, and 85 and 554 for PT, and with the B. fragilis - E. cloacae 032349 combination, they were 81 and 21 for CZX and 77 and 766 for PT. Resistant mutants of E. cloacae 22491 were preferentially selected in vitro with 2 to 64 μg of CZX per ml and in vivo with CZX at 12 to 384 mg/kg/day. There was no preferential selection of CZX-resistant B. fragilis or E. cloacae 032349. For CZX-resistant E. cloacae 22491, we found a 16- to 512-fold increase in the MIC of CZX and increased MICs of other expanded-spectrum cephalosporins, owing in part to the production of a stably derepressed cephalosporinase. In vitro and in vivo, PT did not select resistant mutants of E. cloacae and B. fragilis . Results demonstrate the adverse microbiological outcome of choosing an expanded-spectrum cephalosporin like CZX for empirical treatment of mixed infections involving a susceptible Enterobacter strain.

BAL9141, a new antimicrobial agent belonging to the class of parenteral pyrrolidinone-3-ylidenemethyl cephalosporins, is active against most gram-positive microorganisms, including methicillin-resistant variants (methicillin-resistant Staphylococcus aureus [MRSA] and methicillin-resistant Staphylococcus epidermidis [MRSE]), as well as against penicillin-resistant pneumococci (PRP) and many gram-negative microorganisms. BAL9141 is administered as the prodrug BAL5788, which is rapidly converted to BAL9141 by plasma esterases. Pharmacokinetic (PK) data obtained in a previous multiple ascending dose study were used to fit a population PK model to using the NPEM2 program, yielding PK parameter estimates and its covariance matrix for BAL9141. These estimates and matrix were used to perform Monte Carlo simulations (MCSs) and obtain unbiased target attainment rates (TARs) for various time periods during which the concentration remains above the MIC ( T >MIC ). Assuming a T >MIC of 40%, TARs of 100% were reached with a dose of 500 mg/liter every 12 h for pathogens with MICs of 2 mg/liter and with a dose of 750 mg/liter every 12 h for pathogens with MICs of 4 mg/liter. Because MICs are ≤2 mg/liter for most strains of MRSA, MRSE, and PRP (with some strains showing an MIC of 4 mg/liter), a dosing regimen of 750 mg every 12 h is proposed for clinical studies. The corresponding provisional breakpoint is S (susceptible) ≤ 4 mg/liter.

During the last decade some studies have shown that the area under the curve (AUC)/MIC ratio is the pharmacodynamic index that best predicts the efficacies of quinolones, while other studies suggest that the predictive value of the peak concentration/MIC (peak / MIC) ratio is superior to the AUC/MIC ratio in explaining clinical and microbiological outcomes. In classical fractionated dose-response studies with animals, it is difficult to differentiate between the AUC/MIC ratio and the peak/MIC ratio because of colinearity. Three different levofloxacin and ciprofloxacin dosing regimens were studied in a neutropenic mouse pneumonia model. The different regimens were used with the aim of increasing the AUC/MIC ratio without changing the peak/MIC ratio and vice versa. The first regimen (RC) consisted of daily doses of 5 up to 160 mg/kg of body weight divided into one, two, or four doses. In the second regimen (R0), mice were given 1.25 mg/kg every hour from 1 to 23 h, while the dose given at 0 h was 2.5, 5, 10, 20, 40, or 80 mg/kg. In the third regimen (R11), mice also received 1.25 mg/kg every hour from 0 to 23 h; but in addition, they also received 2.5, 5, 10, 20, 40, or 80 mg/kg at 11 h. The level of protein binding was also evaluated. The results indicate that the unbound fraction ( f u ) was concentration dependent for both levofloxacin and ciprofloxacin and ranged from approximately 0.67 to 0.88 for both drugs between concentrations of 0.5 and 80 mg/liter. The relationships between the AUC/MIC ratio and the number of CFU were slightly better than those between the peak/MIC ratio and the number of CFU. There was no clear relationship between the amount of time that the concentration remained above the MIC and effect ( R 2 < 0.1). For both drugs, the peak/MIC ratio that resulted in a 50% effective concentration was lower for the R0 and R11 dosing regimens, indicating the importance of the AUC/MIC ratio. The same was true for the static doses. Survival studies showed that for mice treated with the low doses the rate of survival was comparable to that for the controls, but with the higher doses the rate of survival was better for mice receiving the R0 regimen. We conclude that for quinolones the AUC/MIC ratio best correlates with efficacy against pneumococci and that the effect of the peak/MIC ratio found in some studies could be partly explained by concentration-dependent protein binding.

In their report, Ericsson and Sherris stated that "adequately described sensitivity categorization schemes have been based on four main concepts" and went on to describe the advantages and inherent limitations of those four criteria. These limitations are very comparable with what is increasingly viewed as limitations to the current breakpoint systems because those four concepts have always been tried to be brought together in one breakpoint system. In line of this development, the European Committee on Antimicrobial Susceptibility Testing has recently recognized that clinical breakpoints based on PK-PD relationships confer a different meaning to resistance than early detection of microorganisms that do not belong to the natural bacterial population, but somehow have acquired resistance mechanisms and have introduced a wild-type cutoff breakpoint conveying a different meaning than clinical breakpoint. Similarly, there is increasing worry on the setting of clinical breakpoints based on frequency distributions because they do not bear an optimal relationship between dose and bacteriologic or clinical effect. This is especially apparent for older drugs because PK-PD was not used until a few years ago. Because dose-effect relationships have now been reasonably well established for most drugs, these should now be used to reappraise current clinical breakpoints.

The purpose of this study was to describe the nonlinear pharmacokinetics of piperacillin observed during intermittent infusion and continuous infusion by using a nonparametric population modeling approach. Data were 120 serum piperacillin concentration measurements from eight adult cystic fibrosis (CF) patients. Individual pharmacokinetic parameter estimates during intermittent infusion or continuous infusion were calculated by noncompartmental analysis and with a maximum iterative two-stage Bayesian estimator. To simultaneously describe concentration-time data during intermittent infusion and continuous infusion, nonlinear models were parameterized as two-compartment Michaelis-Menten models. Models were fit to the data with the nonparametric expectation maximization algorithm. The calculations were executed on a remote supercomputer. Nonlinear models were evaluated by log-likelihood estimates, residual plots, and R 2 values, and predictive performance was based on bias (mean weighted error [MWE]) and precision (mean weighted square error [MWSE]). A linear pharmacokinetic model could not describe combined intermittent infusion and continuous infusion data well. A good population model fit to the intermittent infusion and continuous infusion data was obtained with the constructed nonlinear models. Maximum a posteriori probability (MAP) Bayesian R 2 values for the nonlinear models were 0.96 to 0.97. Median parameter estimates for the best nonlinear model were as follows: K m , 58 ± 75 mg/liter (mean and standard deviation); V max , 1,904 ± 1,009 mg/h; volume of distribution of the central compartment, 14.1 ± 3.0 liters; k 12 , 0.63 ± 0.41 h −1 ; and k 21 , 0.37 ± 0.19 h −1 . The median bias (MWE) and precision (MWSE) values for MAP Bayesian estimation with the Michaelis-Menten model were 0.05 and 4.6 mg/liters, respectively. The developed nonlinear pharmacokinetic models can be used to optimize piperacillin therapy administered via continuous infusion in patients with CF and have distinct advantages over conventional linear models.

One of the causes of infertility in females is tubal pathology as a result of pelvic inflammatory disease (PID) caused by Chlamydia trachomatis (Ct). Diagnosis and identification of patients is hampered by the lack of rapid, easy, sensitive and specific methods. The introduction of Ct-specific enzyme-linked immunosorbent assay (ELISA) tests, based upon synthetic peptides may subsequently increase the sensitivity and specificity for the detection of tubal factor infertility caused by Ct. In order to determine the value of these tests for serological diagnosis of Ct infections, we evaluated several commercially available assays (C. trachomatis enzyme immunoassay (EIA), Labsystems (CtL); SeroCT, Savyon (CtS); pELISA, Medac (CtMp); and a reference assay rELISA, Medac (CtMr)) in two study populations. The first group consisted of 134 female patients with infertility problems. Tubal factor infertility was observed in 85 of these patients (63%). A higher % positivity was found for Ct-specific IgG for the CtL, CtS and CtMp, 41% vs 10%, 57% vs 18% and 55% vs 25% respectively as compared to patients with infertility due to other problems. A similar trend was observed for Ct-specific IgA. The specificity of Ct-specific IgA and IgG in this patient group varied between 92 to 98% and 76 to 90%, respectively. The second group consisted of 107 consecutive gynaecology patients with fertility problems or suspected PID. In this particular patient group, the specificity of the peptide based tests were around 80% and 90% for Ct-specific IgA and 75% and 85% for Ct-specific IgG, respectively. The negative predictive values exceeded 90%, while the positive predictive values varied from 30% to 47% for Ct-specific IgA and was around 30% for Ct-specific IgG. Testing Ct-specific IgG had no additional value above Ct-specific IgA alone. We conclude that the new synthetic peptide-based EIA tests are able to detect species-specific antibodies, which are correlated to (active) infection, and that in particular IgA may be useful in the serodiagnosis of tubal factor infertility caused by C. trachomatis, and will contribute in simplifying the work-up in patients with infertility.