No Consistent Evidence of Decreased Exposure to Varicella-Zoster Virus Among Older Adults in Countries with Universal Varicella Vaccination.

BACKGROUND: Universal varicella vaccination might reduce opportunities for varicella-zoster virus (VZV) exposure and protective immunological boosting, thus increasing herpes zoster incidence in latently infected adults. We assessed humoral and cell-mediated immunity (CMI), as markers of VZV exposure, in adults aged ≥50 years. METHODS: We repurposed data from placebo recipients in a large multinational clinical trial (ZOE-50). Countries were clustered based on their varicella vaccination program characteristics, as having high, moderate, or low VZV circulation. Anti-VZV antibody geometric mean concentrations, median frequencies of VZV-specific CD4 T cells, and percentages of individuals with increases in VZV-specific CD4 T-cell frequencies were compared across countries and clusters. Sensitivity analyses using a variable number of time points and different thresholds were performed for CMI data. RESULTS: VZV-specific humoral immunity from 17 countries (12 high, 2 moderate, 3 low circulation) varied significantly between countries (P < .0001) but not by VZV circulation. No significant differences were identified in VZV-specific CMI between participants from 2 high versus 1 low circulation country. In 3/5 sensitivity analyses, increases in CMI were more frequent in high VZV circulation countries (.03 ≤ P < .05). CONCLUSIONS: We found no consistent evidence of reduced VZV exposure among older adults in countries with universal varicella vaccination. CLINICAL TRIALS REGISTRATION: NCT01165177.

Clinically- versus serologically-identified varicella: A hidden infection burden. A ten-year follow-up from a randomized study in varicella-endemic countries.

Varicella-zoster virus (VZV) infections cause a substantial disease burden, which is underestimated due to incomplete reporting data and lack of serological surveillance. In this post-hoc analysis of a randomized, Phase IIIb clinical trial (NCT00226499) with a ten-year follow-up period, we report anti-VZV antibody levels and persistence in non-vaccinated children, as a varicella infection estimate in ten European countries with endemic varicella. The present analysis specifically focuses on clinical and serological data from the control group, which included 827 healthy participants aged 12-22 months who received two doses of measles-mumps-rubella (MMR) vaccine. The per-protocol cohort included 744 children for whom varicella occurrence was evaluated by clinical definitions, epidemiological links and PCR test outcomes. Anti-VZV antibody levels were assessed by ELISA. The primary objective of this analysis was to correlate varicella occurrence with anti-VZV antibody levels. Varicella was confirmed in 47% of MMR recipients. Among participants without reported varicella, the percentage of anti-VZV seropositive children increased to 75% and average anti-VZV antibody concentrations increased to 250 mIU/mL at year ten after vaccination, suggesting infection or exposure. An eight-fold increase in anti-VZV antibody concentrations between two consecutive visits, which is also observed after confirmed varicella, was detected in 37% of these participants during the follow-up period. About one-third of children not vaccinated against varicella and not diagnosed with varicella developed an anti-VZV immune response, suggesting subclinical varicella occurrence. Longitudinal studies combining serology and disease incidence are necessary to reliably estimate total varicella burden of infection.

Correlation of protection against varicella in a randomized Phase III varicella-containing vaccine efficacy trial in healthy infants.

BACKGROUND: Varicella vaccination confers high and long-lasting protection against chickenpox and induces robust immune responses, but an absolute correlate of protection (CoP) against varicella has not been established. This study models the relationship between varicella humoral response and protection against varicella. METHODS: This was a post-hoc analysis of data from a Phase IIIb, multicenter, randomized trial (NCT00226499) conducted in ten varicella-endemic European countries. Healthy children aged 12-22 months were randomized 3:3:1 to receive one dose of measles-mumps-rubella and one dose of varicella vaccine (one-dose group) or two doses of measles-mumps-rubella-varicella vaccine (two-dose group) or two doses of measles-mumps-rubella vaccine (control group) six weeks apart. The study remained observer-blind until completion, except in countries with obligatory additional immunizations. The objective was to correlate varicella-specific antibody concentrations with protection against varicella and probability of varicella breakthrough, using Cox proportional hazards and Dunning and accelerated failure time statistical models. The analysis was guided by the Prentice framework to explore a CoP against varicella. RESULTS: The trial included 5803 participants, 5289 in the efficacy (2266: one-dose group, 2279: two-dose group and 744: control group) and 5235 (2248, 2245 and 742 in the same groups) in the immunogenicity cohort. The trial ended in 2016 with a median follow-up time of 9.8 years. Six weeks after vaccination with one- or two-dose varicella-containing vaccine, more than 93.0% of vaccinees were seropositive for varicella-specific antibodies. Estimated vaccine efficacy correlated positively with antibody concentrations. The fourth Prentice CoP criterion was not met, due to predicted positive vaccine efficacy in seronegative participants. Further modelling showed decreased probability of moderate to severe varicella breakthrough with increasing varicella-specific antibody concentrations (ten-year probability <0.1 for antibody concentrations ≥2-fold above the seropositivity cut-off). CONCLUSIONS: Varicella-specific antibody concentrations are a good predictor of protection, given their inverse correlation with varicella occurrence. CLINICAL TRIAL: NCT00226499.

Comparison of a glycoprotein E-based ELISA with a varicella-zoster whole-virus ELISA for the quantification of varicella vaccine immune responses in young children.

Antibody response against varicella-zoster virus (VZV) is frequently assessed by whole-virus- (anti-VZV) or glycoprotein-based ELISAs. This study compared antibody concentrations measured by an assay quantifying anti-VZV glycoprotein E (anti-gE) and anti-VZV ELISA in 12-23-month-olds, receiving two varicella vaccine doses in a phase III trial (NCT02570126). Samples (pre- and 42 days post-each vaccination) initially tested with anti-VZV ELISA were re-tested with anti-gE ELISA. Of 1138 samples from 397 children, 757 were positive by anti-VZV (antibody concentration ≥25 mIU/mL) and 758 by anti-gE ELISA (≥97 mIU/mL). There were 375 double-negative and only 11 discrepant samples. The overall agreement was 99.03% (95% confidence interval: 98.28-99.52; McNemar p-value = 1). The ratio between antibody geometric mean concentrations (anti-gE/anti-VZV) for the 752 double-positive samples was 3.78 overall, 4.75 post-first, and 3.01 post-second vaccination. The anti-gE ELISA is a valid alternative for trials assessing antibody response to new varicella vaccines versus established ones, used as control.

Immunogenicity and Safety of a Measles-Mumps-Rubella Vaccine Administered as a First Dose to Children Aged 12 to 15 Months: A Phase III, Randomized, Noninferiority, Lot-to-Lot Consistency Study.

BACKGROUND: MMR II (M-M-R II [Merck & Co, Inc.]) is currently the only measles, mumps, and rubella (MMR) vaccine licensed in the United States. A second MMR vaccine would mitigate the potential risk of vaccine supply shortage or delay. In this study, we assessed the immunogenicity and safety of another MMR vaccine (MMR-RIT [Priorix, GlaxoSmithKline]) compared with those of the MMR II in 12- to 15-month-old children who received it as a first dose. METHODS: In this phase III, observer-blinded, noninferiority, lot-to-lot consistency clinical trial (ClinicalTrials.gov identifier NCT01702428), 5003 healthy children were randomly assigned to receive 1 dose of MMR-RIT (1 of 3 production lots) or MMR II along with other age-recommended routine vaccines. We evaluated the immunogenicity of all vaccines in terms of antibody concentrations (by using an enzyme-linked immunosorbent assay or electrochemiluminescence assay) and/or seroresponse rates 43 days after vaccination. We also assessed the reactogenicity and safety of the vaccines. RESULTS: Immunoresponses after vaccination with MMR-RIT were robust and noninferior to those after vaccination with the MMR II. Immunogenicity of the 3 production lots of MMR-RIT was consistent; more than 97% of the children had a seroresponse to MMR components. The coadministered vaccines elicited similar immunoresponses in the MMR-RIT and MMR II groups. Both MMR vaccines resulted in comparable reactogenicity profiles, and no safety concerns were detected. CONCLUSIONS: If licensed, the MMR-RIT could provide a valid option for the prevention of measles, mumps, and rubella in children in the United States and would reduce potential risks of a vaccine shortage.

Assessment of Mumps Virus-Specific Antibodies: Comparison of Plaque Reduction Neutralization Test and Enzyme-Linked Immunosorbent Assay Estimates.

BACKGROUND: The plaque reduction neutralization test (PRNT), which measures a subset of immunoglobulin antibodies (functional neutralizing antibodies), and the enzyme-linked immunosorbent assay (ELISA), which measures total immunoglobulin (neutralizing and nonneutralizing antibodies), characterize different aspects of the anti-mumps virus antibody response after vaccination. METHODS: Data from a recent phase 3 clinical trial (NCT01681992) of 2 measles-mumps-rubella vaccines were used to compare anti-mumps antibody responses measured using an unenhanced PRNT (GSK; seropositivity cutoff and threshold, 2.5 and 4 times the 50% end-point dilution, respectively) with those estimated using an ELISA (thresholds, 5 and 10 ELISA units/mL, respectively). RESULTS: Of 3990 initially seronegative samples, 3284 (82.3%) were seropositive after vaccination for anti-mumps antibodies in both assays. The Pearson correlation coefficient for double-positive samples was 0.57, indicative of a moderate correlation. Receiver operating characteristic curve analysis showed that an ELISA threshold of 51.7 ELISA units/mL best corresponded to the PRNT seroresponse threshold. There was no obvious vaccine brand effect on the correlation between assays. CONCLUSIONS: The moderate correlation between the anti-mumps antibody measurements obtained with PRNT and ELISA reflects different aspects of the serological response. In the absence of a well-defined protective serological threshold, PRNT provides complementary information on the antibody response, whereas ELISA remains a critically useful measurement of vaccine immunogenicity.

Long-term immunogenicity of measles, mumps and rubella-containing vaccines in healthy young children: A 10-year follow-up.

Measles and mumps outbreaks still occur in countries that have successfully implemented universal routine immunization programs. Measles outbreaks are mostly associated to absent or incomplete vaccination, whereas for mumps outbreaks the combined effects of waning of immunity and circulating new strains are incriminated. It is therefore increasingly useful to characterize the long-lasting immunity induced by measles-, mumps, and rubella (MMR)-containing vaccines. In this 10-year study, 1887 healthy children aged 12-22 months, randomized to receive 1 or 2 doses of MMR-containing vaccines (Priorix or Priorix-Tetra; GSK), were included in an antibody persistence analysis. A total of 364 children in the 1-dose group received a second dose out of study according to their local vaccination schedule between Years 4 and 10 post-dose 1, and were included in a separate post-hoc analysis to evaluate the effect of the second dose when given later. Anti-measles, -mumps and -rubella antibody titers were measured by commercial ELISA kits (Enzygnost, Siemens) after each vaccine dose and at Years 1, 2, 4, 6, 8 and 10 post-vaccination. Antibodies against measles and rubella declined moderately after vaccination but remained well above the seropositivity threshold after 10 years. The anti-measles antibody titers elicited by Priorix-Tetra remained about 2-fold higher throughout the study as compared with Priorix. A second dose of MMR vaccine later in life had a minor and transient effect on anti-measles and anti-rubella waning titers. In contrast, anti-mumps antibody levels remained relatively stable over the 10-year follow-up and a second dose of MMR vaccine, given anytime over the 10-year period, had a boosting effect on anti-mumps antibody titers and seropositivity rates. In conclusion, 1 or 2 doses of MMR-containing vaccines given to children in their second year of life induced antibody responses against measles, mumps and rubella viruses that persisted at least up to 10 years post-vaccination. Clinical trial registration number: NCT00226499.

The impact of childhood varicella vaccination on the incidence of herpes zoster in the general population: modelling the effect of exogenous and endogenous varicella-zoster virus immunity boosting.

BACKGROUND: A controversy exists about the potential effect of childhood varicella vaccination on Herpes Zoster (HZ) incidence. Mathematical models projected temporary HZ incidence increase after vaccine introduction that was not confirmed by real-world evidence. These models assume that absence of contacts with infected children would prevent exogenous boosting of Varicella-Zoster-Virus (VZV) immunity and they do not include an endogenous VZV immunity-boosting mechanism following asymptomatic VZV reactivation. This study aims to explore the effect of various assumptions on exogenous and endogenous VZV immunity-boosting on HZ incidence in the general population after introduction of routine childhood varicella vaccination. METHODS: An age-structured dynamic transmission model was adapted and fitted to the seroprevalence of varicella in France in absence of vaccination using the empirical contact matrix. A two-dose childhood varicella vaccination schedule was introduced at 12 and 18 months. Vaccine efficacy was assumed at 65%/95% (dose 1/dose 2), and coverage at 90%/80% (dose 1/dose 2). Exogenous boosting intensity was based on assumptions regarding HZ-immunity duration, age-dependent boosting effect, and HZ reactivation rates fitted to observed HZ incidence. Endogenous boosting was the same as pre-vaccination exogenous boosting but constant over time, whilst exogenous boosting depended on the force of infection. Five scenarios were tested with different weightings of exogenous (Exo) - endogenous (Endo) boosting: 100%Exo-0%Endo, 75%Exo-25%Endo, 50%Exo-50%Endo, 25%Exo-75%Endo, 0%Exo-100%Endo. RESULTS: HZ incidence before varicella vaccination, all ages combined, was estimated at 3.96 per 1000 person-years; it decreased by 64% by year 80 post vaccine introduction, for all boosting assumptions. The 100%Exo-0%Endo boosting scenario, predicted an increase in HZ incidence for the first 21 years post vaccine introduction with a maximum increase of 3.7% (4.1/1000) at year 9. However, with 0%Exo-100%Endo boosting scenario an immediate HZ decline was projected. The maximum HZ incidence increases at 10, 3, and 2 years post vaccination were 1.8% (75%Exo-25%Endo), 0.8% (50%Exo-50%Endo) and 0.2% (25%Exo-75%Endo), respectively. CONCLUSIONS: Assuming modest levels of endogenous boosting, the increase in HZ incidence following childhood varicella vaccination was smaller and lasted for a shorter period compared with 100%Exo-0%Endo boosting assumption. Endogenous boosting mechanism could partly explain the divergence between previous HZ-incidence projections and real-world evidence.

Temocillin (6 g daily) in critically ill patients: continuous infusion versus three times daily administration.

OBJECTIVES: The growing incidence of infections caused by Enterobacteriaceae producing ESBLs has led to increased use of carbapenems. Temocillin, which resists most ß-lactamases, may be a useful alternative. The aim of this study was to assess the pharmacokinetics and target attainment rates of 6 g of temocillin daily divided into three administrations every 8 h (three times daily) or administered by continuous infusion in critically ill patients. PATIENTS AND METHODS: This was a prospective, two-centre, randomized, controlled study in patients with intra-abdominal or lower respiratory tract infections caused by Enterobacteriaceae. RESULTS: Thirty-two patients were included and analysed for clinical efficacy, and pharmacokinetics were measured in 29 of them. Four patients undergoing continuous veno-venous haemofiltration (CVVH) were analysed separately. Mean, median and range of percentages of the dosing interval during which the free drug concentration remained >16 mg/L were 76.4, 98 and 18.7-98.9 in patients treated three times daily and 98.9, 89.7 and 36.4-99.9 in patients with continuous infusion, respectively. Clinical cure rates were 79% and 93% in each of these groups, respectively (not significant). Patients with CVVH received a daily dose of 750 mg given by continuous infusion and had a mean free drug concentration of only 13.8 ±â€Š1.9 mg/L. No adverse event attributable to temocillin was observed. CONCLUSIONS: Temocillin (6 g daily) given by continuous infusion allows a larger proportion of critically ill patients to have free drug serum concentrations covering infections caused by Enterobacteriaceae with an MIC of 16 mg/L compared with administration three times daily. Clinical efficacy compared with carbapenems in documented severe infections needs to be further studied.

Phospholipases and cationic peptides inhibit Cryptosporidium parvum sporozoite infectivity by parasiticidal and non-parasiticidal mechanisms.

The apicomplexan parasite Cryptosporidium parvum is an important cause of diarrhea in humans and cattle, and it can persistently infect immunocompromised hosts. No consistently effective parasite-specific pharmaceuticals or immunotherapies for control of cryptosporidiosis are presently available. The innate immune system represents the first line of host defense against a range of infectious agents, including parasitic protozoa. Several types of antimicrobial peptides and proteins, collectively referred to herein as biocides, constitute a major effector component of this system. In the present study, we evaluated lactoferrin, lactoferrin hydrolysate, 5 cationic peptides (lactoferricin B, cathelicidin LL37, indolicidin, ß-defensin 1, ß-defensin 2), lysozyme, and 2 phospholipases (phospholipase A2, and phosphatidylinositol-specific phospholipase C) for anti-cryptosporidial activity. The biocides were evaluated either alone or in combination with 3E2, a monoclonal antibody (MAb) against C. parvum that inhibits sporozoite attachment and invasion. Sporozoite viability and infectivity were used as indices of anti-cryptosporidial activity in vitro. All biocides except lactoferrin had a significant effect on sporozoite viability and infectivity. Lactoferrin hydrolysate and each of the 5 cationic peptides were highly parasiticidal and strongly reduced sporozoite infectivity. While each phospholipase also had parasiticidal activity, it was significantly less than that of lactoferrin hydrolysate and each of the cationic peptides. However, each phospholipase reduced sporozoite infectivity comparably to that observed with lactoferrin hydrolysate and the cationic peptides. Moreover, when 3 of the cationic peptides (cathelicidin LL37, ß-defensin 1, and ß-defensin 2) were individually combined with MAb 3E2, a significantly greater reduction of sporozoite infectivity was observed over that by 3E2 alone. In contrast, reduction of sporozoite infectivity by a combination of either phospholipase with MAb 3E2 was no greater than that by 3E2 alone. These collective observations suggest that cationic peptides and phospholipases neutralize C. parvum by mechanisms that are predominantly either parasiticidal or non-parasiticidal, respectively.

Temocillin use in England: clinical and microbiological efficacies in infections caused by extended-spectrum and/or derepressed AmpC ß-lactamase-producing Enterobacteriaceae.

BACKGROUND: Temocillin, a ß-lactam stable against most ß-lactamases [including extended-spectrum ß-lactamases (ESBLs) and derepressed AmpC cephalosporinases (dAmpC)], has been suggested as an alternative to carbapenems when Pseudomonas can be excluded. Aims To assess temocillin clinical and microbiological cure rates (CCR and MCR) in infection caused by ESBL/dAmpC-producing Enterobacteriaceae and the effects of different dosage regimens. METHODS: Data were collected retrospectively from patients treated for at least 3 days with temocillin for urinary tract infection (n = 42), bloodstream infection (n = 42) or hospital-acquired pneumonia (n = 8) in six centres in the UK. RESULTS: Data on 92 infection episodes were collected. Overall CCR and MCR were 86% and 84% respectively; ESBL/dAmpC status had no effect. Significantly higher CCR and MCR occurred in patients treated with temocillin at optimal dosage [2 g twice daily or renally adjusted equivalent (ORAE)] compared with those treated with a suboptimal dosage (<2 g twice daily ORAE) (CCR 91% and MCR 92% versus CCR 73% and MCR 63%). This difference was more pronounced in the ESBL/dAmpC-positive subset (CCR 97% and MCR 97% versus CCR 67% and MCR 50%). CONCLUSIONS: Clinical and microbiological efficacies of temocillin are unaffected by ESBL/dAmpC production, confirming its potential application as a carbapenem-sparing agent. Both CCR and MCR are optimized by a regimen of 2 g twice daily ORAE in ESBL/dAmpC-positive infection.

Penicillin-binding proteins (PBP) and Lmo0441 (a PBP-like protein) play a role in Beta-lactam sensitivity of Listeria monocytogenes.

While seven penicillin-binding proteins (PBPs) or PBP-like proteins have been identified either by radiolabelled penicillin binding studies or genomic analysis, only PBP3 has been considered of interest for Beta-lactams activity against Listeria monocytogenes. Herein we reveal that both PBP4 and Lmo0441 (a PBP-like protein) play a direct role in cephalosporin activity in L. monocytogenes while PBP4 additionally has a protective affect against both penicillin and carbapenem.

Isolation and 2-D-DIGE proteomic analysis of intracellular and extracellular forms of Listeria monocytogenes.

The pathogenicity of Listeria monocytogenes is related to its ability of invading and multiplying in eukaryotic cells. Its main virulence factors are now well characterized, but limited proteomic data is available concerning its adaptation to the intracellular environment. In this study, L. monocytogenes EGD (serotype 1/2a) grown in human THP-1 monocytes (24 h) were successfully separated from host organelles and cytosolic proteins by differential and isopycnic centrifugation. For control, we used cell homogenates spiked with bacteria grown in broth. Proteomes from both forms of bacteria were compared using a 2-D-DIGE approach followed by MALDI-TOF analysis to identify proteins. From 1684 distinct spots, 448 were identified corresponding to 245 distinct proteins with no apparent contamination of host proteins. Amongst them, 61 show underexpression (stress defense; transport systems, carbon metabolism, pyrimidines synthesis, D-Ala-D-Ala ligase) and 22 an overexpression (enzymes involved in the synthesis of cell envelope lipids, glyceraldehyde-3-phosphate, pyruvate and fatty acids). Our proteomic analysis of intracellular L. monocytogenes (i) suggests that bacteria thrive in a more favorable environment than extracellularly, (ii) supports the concept of metabolic adaptation of bacteria to intracellular environment and (iii) may be at the basis of improved anti-Listeria therapy.

Correlation between free and total vancomycin serum concentrations in patients treated for Gram-positive infections.

Routine therapeutic drug monitoring (TDM) reports only total vancomycin (VAN) concentrations, although protein binding varies and it is generally accepted that only free VAN is active. The aims of this study were to examine the correlation between free and total VAN concentrations in order to estimate whether free VAN levels can be predicted based on its total concentration. A high-performance liquid chromatography (HPLC) method was set up and validated (against routine laboratory immunoassays) for measurement of free [ultrafiltration (Centrifree); cut-off 30 kDa] and total [solid-phase extraction (Oasis MCX cartridge)] VAN in serum. Samples (n=65) from patients (n=15) treated by continuous infusion were analysed. There was a wide variation in free to total VAN ratios [range 12-100%; mean 63.6+/-25.8%, with 59 values falling outside the 95% confidence interval (57.3-69.9%); median 70.2%]. The correlation between free and total VAN was poor (R(2)=0.55). Artefacts such as pH variation of sera could be excluded. Both intrapatient and interpatient variabilities were large and no correlation could be made with patients' clinical conditions. Total VAN concentration is not predictive of free VAN concentration, suggesting that actual determination of free VAN might be recommended as an improved method of TDM.

Temocillin in cystic fibrosis: a retrospective pilot study.

BACKGROUND: Temocillin is currently used in the treatment of acute pulmonary exacerbations caused by Burkholderia cepacia complex and multi-resistant Pseudomonas aeruginosa in cystic fibrosis (CF) patients despite little published clinical data. This study assessed if intravenous (IV) antibiotic therapy including temocillin was equivalent to standard combination therapy for an acute exacerbation. METHODS: A retrospective, pilot cross-over study. Adult patients attending two CF centres between 1997 and 2006 who had received a course of IV antibiotics including temocillin (TIV) and a further IV course (within +/-1 year) which did not include temocillin (NTIV) were included. Outcome measures at the start and end of each IV course were recorded (FEV(1)%, FVC%). RESULTS: Twenty six patients had received temocillin. Baseline values of FEV(1)% predicted were comparable for both groups (TIV: 37(18%), NTIV: 39(20%)). FEV(1)% increased by 7.12(11.67)% after TIV (p<0.01) and 6.65(7.62)% after NTIV (p<0.01). There was no significant difference between the IV courses in mean %change in lung function TIV versus NTIV (FEV(1) 0.46% [95%CI: -4.55 to 5.48%]). CONCLUSION: These data suggest equivalence in the lung function outcome of IV antibiotic therapy includingtemocillin versus standard IV antibiotic therapy.

Continuous versus intermittent infusion of temocillin, a directed spectrum penicillin for intensive care patients with nosocomial pneumonia: stability, compatibility, population pharmacokinetic studies and breakpoint selection.

BACKGROUND AND AIMS: Temocillin, a 6alpha-methoxy-penicillin stable towards most beta-lactamases (including extended-spectrum beta-lactamase), is presented as an alternative to carbapenems for susceptible Enterobacteriaceae in microbiological surveys. We aimed at documenting its potential clinical usefulness in intensive care (IC) patients using pharmacokinetic/pharmacodynamic approaches applied to conventional (twice daily) and continuous infusion (CI) modes of administration. METHODS: (i) In vitro evaluation of temocillin stability and compatibility with other drugs under conditions pertinent of CI in IC patients; (ii) pharmacokinetic study in patients treated by CI (4 g/day; n = 6) versus [twice daily (2 g every 12 h); n = 6]; (iii) population pharmacokinetic analysis of twice daily with Monte Carlo simulations to determine 95% probability of target attainment (PTA(95)) versus MIC (based on time above MIC > or = 40% for measured free drug). RESULTS: Temocillin was stable at 37 degrees C in 8.34% solutions for 24 h and compatible with flucloxacillin and aminoglycosides, but not with several other antibiotic and non-antibiotic drugs. With CI, stable total serum concentrations were 73.5 +/- 3.0 mg/L (SEM) and free concentration 29.3 +/- 2.8 mg/L. With twice daily, Cmax (total drug) was 147 +/- 12.3 mg/L (SEM; free drug: 50.3 +/- 15.8 mg/L), lowest trough (total drug) 12.3 mg/L, and PTA(95) (free drug) obtained for MIC < or = 8 mg/L. CONCLUSIONS: Temocillin (4 g/day) by CI yields stable free serum concentrations above the current breakpoint (16 mg/L), although individual variations may suggest lowering the breakpoint to 8 mg/L (as for twice daily) unless the daily dose or the frequency of administration is increased.

Design and evaluation of analogues of the bacterial cell-wall peptidoglycan motif L-Lys-D-Ala-D-Ala for use in a vancomycin biosensor.

Four small molecular receptors of vancomycin have been designed to make part of a novel biosensor device based on the FTIR-ATR detection: N-Boc (2a) or N-Ac (2b)-6-aminocaproyl-D-Ala-D-Ala and N-Boc (3a) or N-Ac (3b)-6-aminocaproyl-D-Ala-d-Ser. Using an original microbiological approach to assess the competition of compounds with the natural target of vancomycin in bacteria, EC(50) values of 6.3-8.0 x 10(-5)M (2a-b) and 7.1-9.3 x 10(-4)M (3a-b) were determined. Vancomycin:2b complex was characterized by MS.

Cellular pharmacokinetics and pharmacodynamics of the glycopeptide antibiotic oritavancin (LY333328) in a model of J774 mouse macrophages.

The intracellular pharmacokinetics and pharmacodynamics of oritavancin (LY333328) were studied in cultured cells. Oritavancin was avidly accumulated by J774 and THP-1 macrophages and rat fibroblasts and to a lesser extent by LLC-PK1 and Caco-2 cells. In J774 macrophages, the level of accumulation reached a plateau (at 370-fold the extracellular concentration) within 24 h and was partly defeated by a rise in serum protein levels. Efflux was incomplete (with a plateau at two-thirds of the original level at 6 h). In short-term kinetic studies, oritavancin uptake was linear for up to 4 h (as was the case for horseradish peroxidase and small latex beads, used as markers of the fluid phase and adsorptive endocytosis, respectively), which was in contrast to azithromycin and chloroquine uptake (which accumulate in cells by diffusion and segregation). The rates of clearance of oritavancin and latex beads were comparable (150 and 120 microl x mg of protein(-1) x h(-1), respectively) and were approximately 200 times higher than that of horseradish peroxidase. Oritavancin accumulation was partially reduced by monensin but was unaffected by acidic pH (these conditions abolished chloroquine accumulation). Cell-associated oritavancin was found in lysosomal fractions after homogenization of J774 macrophages and fractionation by isopycnic centrifugation. Oritavancin was bactericidal against intracellular Staphylococcus aureus (phagolysosomal infection) but was unable to control the intracellular growth of Listeria monocytogenes (cytosolic infection), even though its cellular concentration largely exceeded the MIC (0.02 mg/liter) and minimal bactericidal concentration (2 mg/liter). We conclude that oritavancin enters cells by adsorptive endocytosis (favored by its lipophilic side chain and/or the presence of three protonatable amines), which drives it to lysosomes, where it exerts antibiotic activity.