A rapid, automatic and accurate assay for quantifying temocillin in human serum and CSF using turbulent flow liquid chromatography coupled to high-resolution mass spectrometry. Clinical application.

Temocillin is a ß-lactamase-resistant penicillin used for the treatment of multiple drug-resistant Gram-negative bacteria. To maximize efficacy and avoid adverse effects, the dose regimen has to be quickly adjusted to the clinical situations. This necessitates the development of a rapid, reliable and accurate analytical method. Temocillin and the stable isotopically labeled internal standard ([13 C6 ]-amoxicillin) were extracted from either serum or cerebrospinal fluid by a turbulent flow liquid chromatographic method and eluted onto an octadecyl-silica phase with polar endcapping. Mass spectrometry was conducted using an exact mass determination method by electrospray positive ionization high-resolution mass spectrometry. The LLOQ and ULOQ of the present method were determined to be 0.4 and 200 µg/ml for serum and cerebrospinal fluid samples, respectively. The total analysis time was <7 min. The recovery ranged from 87.7 to 120.8%. Intra- and inter-day precision and trueness were tested at four concentration levels: 0.4, 8, 40 and 160 µg/ml. Values were 6.33 ± 1.53, 8.8 ± 1.3, 8.8 ± 0.36 and 2.1 ± 0.76%, and 5.0 ± 0.54, 9.9 ± 1.0, 5.8 ± 1.6 and 0.1 ± 1.1%, for inter- and intra-day analysis, respectively. Temocillin was found to be stable under all relevant laboratory conditions. The method was cross-validated with a microbiological assay. This method is suitable for accurate measurement of temocillin concentration in small volumes of serum or cerebrospinal fluid. Thanks to the online extraction procedure, the overall analytical time is compatible with high-throughput analysis for clinical application.

Penicillin and ceftriaxone susceptibility of Streptococcus pneumoniae isolated from cerebrospinal fluid of children with meningitis hospitalized in a tertiary hospital in Israel.

BACKGROUND: Streptococcus pneumoniae is now the predominant pathogen causing meningitis. The resistance of S. pneumoniae to penicillin and third-generation cephalosporins has grown steadily. OBJECTIVES: To assess the antibiotic susceptibility of S. pneumoniae isolated from the cerebrospinal fluid of children with meningitis, and determine the antibiotic regimen appropriate for suspected bacterial meningitis in Israel. METHODS: The study group included 31 children with 35 episodes of meningitis hospitalized from 1998 to 2006. S. pneumoniae isolates from the cerebrospinal fluid were tested for susceptibility to penicillin and ceftriaxone. RESULTS: Of the 35 isolates, 17 (48.6%) showed resistance to penicillin (minimum inhibitory concentration > or = 0.12 microg/ml). Only 3 isolates (8.6%) showed intermediate resistance to ceftriaxone (> or = 0.5 and < (2 microg/ml), and none showed complete resistance (MIC > or = 2 microg/ml). The rates of antibiotic resistance were higher in children who were treated with antibiotics prior to admission (penicillin 88.9% vs. 34.6%, P = 0.007; ceftriaxone 22.2% vs. 3.8%, P = 0.156). CONCLUSIONS: The rate of penicillin resistance is high in children with S. pneumoniae meningitis in Israel, especially in those treated with oral antibiotics prior to admission. Resistance to ceftriaxone is infrequent though not negligible. On the basis of these findings, current recommendations to empirically treat all children with suspected bacterial meningitis with ceftriaxone in addition to vancomycin until the bacterial susceptibility results become available are justified also in Israel.

Riboflavin transport in the central nervous system. Characterization and effects of drugs.

The relationship of riboflavin transport to the transport of other substances including drugs in rabbit choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, and brain cells were studied in vivo and in vitro. In vitro, the ability of rabbit choroid plexus to transport riboflavin from the medium (cerebrospinal fluid surface) through the choroid plexus epithelial cells into the extracellular and vascular spaces of the choroid plexus was documented using fluorescence microscopy. These studies provided further evidence that riboflavin is transported from cerebrospinal fluid to blood via the choroid plexus. The transport of [14C]riboflavin by the isolated choroid plexus was inhibited by thiol agents, ouabain, theophylline, various flavins (lumiflavin and lumichrome > sugar containing flavins), and cyclic organic acids including penicillin and fluorescein. Riboflavin inhibited [14C]penicillin transport competitively and the inhibition constant (K1) for riboflavin equaled the concentration of riboflavin at which the saturable transport system for riboflavin is 50% saturated (KT). These and other data suggest that riboflavin, penicillin, and possibly fluorescein are transported by the same transport system in choroid plexus. In vivo, the intra-ventricular injection or riboflavin and [14C]penicillin inhibited [14C]penicillin transport from cerebrospinal fluid. In vitro, various flavins (riboflavin > other sugar-containing flavins > lumiflavin > lumichrome) inhibited [14C]riboflavin accumulation by brain slices. These studies support the notions that: (a) riboflavin accumulation by choroid plexus (active transport) is quite different from that in brain cells (facilitated diffusion and intracellular trapping), and (b) therapeutically important cyclic organic acids (e.g., penicillin) are transported fom cerebrospinal fluid by the riboflavin transport system in choroid plexus.

Inhibition of penicillin transport from the cerebrospinal fluid after intracisternal inoculation of bacteria.

The effect of intracisternal inoculation of bacteria on the choroid plexus system, which transports penicillin from cerebrospinal fluid (CSF) to blood, was studied in vitro and in vivo. Meningeal and choroid plexus inflammations as well as CSF pleocytosis were induced in rabbits with intracisternal inoculations of Hemophilus influenzae or Staphylococcus aureus. At various times after bacterial inoculation, the choroid plexuses of the inoculated rabbits were removed and incubated in artificial CSF containing [(14)C]penicillin. The ability of the choroid plexuses to accumulate pencillin in vitro was measured and was found to be depressed as compared with controls. This depression of choroid plexus uptake reversed with resolution of the inflammatory process. In vivo on the day after intracisternal inoculation of Hemophilus influenzae, a decrease in the disappearance of penicillin relative to inulin in the inoculated rabbits (as compared to the controls) was observed when [(14)C]penicillin and [(3)H]inulin were injected intraventricularly and cisternal CSF was sampled 2 h later. This decrease could not be explained by penicillin binding to the CSF exudate. However, the choroid plexus transport system for penicillin was only partially depressed in those inoculated rabbits with bacterially induced inflammation, since in vitro the choroid plexuses could still accumulate penicillin and in vivo CSF penicillin levels could be further increased with probenecid pretreatment. These results suggest that CSF penicillin levels are increased in this model due to three factors: a depression of active efflux of penicillin from the CSF, an increase in permeability to penicillin of inflamed meninges, and, less significantly, by CSF binding of penicillin.

Ampicillin and an ester in experimental Hemophilus influenzae meningitis.

A new methoxymethyl ester of hetacillin is highly lipid-soluble, a property which was expected to enhance its penetration into the central nervous system (CNS). We compared the penetration of the ester and ampicillin into the cerebrospinal fluid (CSF) of normal rabbits and those with experimental Hemophilus influenzae meningitis. In normal rabbits treated by constant intravenous infusion, mean per cent penetration (see article) of the ester was four times as great as that of ampicillin (6.6 +/- 3.7% against 1.6 +/- 1.9%), and the difference in CSF drug levels attained was accentuated when bolus IV infusion was used. Drug concentration in brain tissue was three times as great in a rabbit treated with the ester (0.79 mug/gm against 0.26 mug/gm). In rabbits with meningitis, the ester again achieved higher CSF concentrations. Per cent penetration into CSF in infected rabbits was 23% for the ester and 13% for ampicillin. Bactericidal activity of the drugs was comparable; over 8 hr of treatment both drugs significantly reduced the CSF bacterial titers of infected animals. Our data demonstrated that the ester enters the CSF and CNS in higher concentration than ampicillin, with no loss in bactericidal activity.

A study of the penetration of temocillin in the cerebrospinal fluid.

Temocillin concentrations in serum and cerebrospinal fluid (CSF) samples from 8 patients were assayed by high pressure liquid chromatography. It was possible to determine sequential series of ventricular (CSF) and serum concentrations in 4 adult neurosurgical patients with slight to moderate impairment of blood-CSF barriers, because serial CSF samples were obtained from external ventricular drains. In 4 other patients with meningitis, temocillin was given in addition to the regular treatment schedule and 6 'spot' concentrations of temocillin in lumbar CSF and serum samples were determined. Temocillin CSF concentrations in these subjects seemed to be inadequate for the treatment of Gram-positive bacterial meningitis and only partially valuable for the treatment of Gram-negative bacillary meningitis.

Gram-negative bacillary meningitis.

The incidence of gram-negative bacillary meningitis has increased significantly in the past two decades. Approximately two thirds of all reported cases have occurred after neurosurgical procedures. With the development of the newer cephalosporins, the overall mortality rate has decreased from 40 to 80 per cent to 10 to 20 per cent.

Intraventricular cerebrospinal fluid antibiotic concentrations in patients with intraventricular infections.

The antibiotic concentration of the fluid from either lateral ventricle was determined 104 times in 37 patients through direct ventricular puncture, external ventricular drainage (EVD), or cerebrospinal fluid shunt sampling. The patients were 1 month to 12 years old. When the patients were receiving maximal intravenous antibiotic therapy alone, the concentrations for the most part were below 5 microgram/ml, whereas patients receiving an antibiotic through direct ventricular puncture, EVD, or a shunt reservoir usually had concentrations over 5 microgram/ml. However, wide variations from patient to patient were found with all forms of treatment despite similar dosages. Clustering of the concentration tended to occur in each individual patient. The authors conclude that, to obtain a high concentration of an antibiotic in the ventricular fluid, one should administer it directly into the ventricle.

Sequential bacteriological findings in the cerebrospinal fluid of Nigerian patients with pneumococcal meningitis.

Sequential bacteriological observations were made on the cerebrospinal fluid (CSF) of 28 patients with pneumococcal meningitis treated with high doses of penicillin for 2 weeks. The organism was isolated from the CSF of four patients 48 h or more after the start of treatment and from a further patient 48 h after treatment was stopped. Positive cultures were obtained in spite of the demonstration in the CSF of penicillin at a concentration well above the minimum inhibitory concentration for the organism isolated. Persistence of bacteria and their products in the CSF of patients with pneumococcal meningitis contrasts with the rapid clearance of bacteria from the CSF of patients with meningococcal meningitis and may contribute to the difference in the prognosis of these forms of meningitis.

Pharmacokinetics of ampicillin and methoxymethyl ester of hetacillin in dogs.

Concentrations of ampicillin and the methoxymethyl ester of hetacillin were measured in plasma, prostatic fluid, and spinal fluid of dogs receiving ampicillin or the hetacillin ester by continuous intravenous infusion. The ratios of ampicillin in prostatic fluid relative to plasma levels were higher after dosing with the hetacillin ester. Pharmacokinetic parameter values were consistent with urinary excretion characteristics. Ready diffusion of the ester across biological membranes may facilitate eradication of pathogenic organisms in prostatic and spinal fluids.

[Neurosyphilis resistant to high doses of penicillin: report of a case]. / Neurossífilis resistente a altas doses de penicilina: registro de um caso.

A case of neurosyphilis that got worse despite several therapeutical trials with high doses of penicillin is described. The clinical condition was stabilized and cerebrospinal fluid data normalized following treatment with chloramphenicol.

[A patient with Streptococcus intermedius brain abscess treated with high dose penicillin G--susceptibility of the isolate to penicillin G and the concentration of penicillin G in cerebrospinal fluid].

We report here a 2-year-old boy with a Streptococcus intermedius brain abscess and bilateral ventriculitis successfully treated with a high dose penicillin G (200,000 U/kg/dose, 6 times a day, 1 hour continuous infusion). Although hydrocephalus residuced, the high dose penicillin G therapy cured his brain abscess and bilateral ventriculitis. The minimal inhibitory concentration of penicillin G to the isolate was 0.008 microgram/ml. The penicillin G concentration in the cerebrospinal fluid after 2 hours from the infusion was about 5 micrograms/ml. S. intermedius must be considered as one of the causative agents for brain abscess. High dose penicillin G therapy is one choice of treatment for brain abscess due to penicillin-susceptible streptococci.

A comparison study of two therapeutic protocols for neurosyphilis.

In Morocco, neurosyphilis is a serious public health problem. In the neurology service at the specialist hospital in Rabat, two drug treatments were used. Treatment A consisted of infusion over a period of 4 h of 20 MUI of penicillin G per day for 3 weeks. Treatment B consisted of infusion over a period of 6 h of 30 MUI of penicillin G per day for 10 days. Each treatment was tested on a group of eight neurosyphilitic patients as first-line treatment. On the first day of treatment, both blood and CSF pharmacokinetics were sampled for each patient. Blood and CSF were taken within 24 h. Penicillin G concentrations were determined by a microbiological method. The results obtained showed that perfusions of either 20 MUI or 30 MUI of penicillin allowed the achievement of high serum concentrations. These increased progressively until reaching their maximum at T4 h for treatment A (from 92.33 to 106.38 micrograms/ml). For treatment B, maximal concentration is obtained at T6 h (from 108 to 141.52 micrograms/ml). Penicillin concentrations decreased immediately after stopping the perfusion. At CSF levels, penicillin G concentrations were identical to serum concentrations. However, one difference was observed: a one-hour difference between the serum and CSF peaks. The CSF peak was achieved at T5 h for treatment A (0.063 to 2.25 micrograms/ml) and at T7 h for treatment B (0.92 to 2.94 micrograms/ml). The concentrations obtained were largely superior to the CMI of Treponema pallidum for both treatment A and treatment B, at 47 times and 82 times higher respectively. The recovery time of the patients was 14 h for treatment A and 24 h for B treatment. These results have shown that therapeutic method B was more efficient than A. Moreover, the evolution of penicillin G's diffusion in the CSF during treatment, of cell counts of protein level, of the VDRL test and of the gamma-globulin rate was studied.

[Transferability of tazobactam/piperacillin (TAZ/PIPC) to cerebrospinal fluid of rabbit with meningitis caused by Staphylococcus aureus].

The transferability of tazobactam/piperacillin (TAZ/PIPC) to cerebrospinal fluid (CSF) was studied employing rabbits with experimental meningitis caused by Staphylococcus aureus. 125 or 250 mg/kg of TAZ/PIPC was intravenously administered to rabbits with experimental meningitis then concentrations of TAZ and PIPC in CSF and serum were measured. In the group to which 125 mg/kg of TAZ/PIPC was administered, mean concentration of TAZ in CSF was 7.3 and 2.4 micrograms/ml at 30 and 60 min after administration, respectively, and concerning PIPC, it was 10.1 and 3.5 micrograms/ml, respectively. CSF/serum ratio of TAZ was 29.4% and 31.4%, respectively, and that of PIPC was 24.3 and 35.6%, respectively. In the group to which 250 mg/kg of TAZ/PIPC was administered, mean concentration of TAZ in CSF was 16.5 and 12.6% micrograms/ml, respectively, and concerning PIPC, it was 25.6 and 18.2 micrograms/ml, respectively. CSF/serum ratio of TAZ was 22.1 and 56.1%, respectively, and that of PIPC was 12.2 and 51.9%, respectively. Addition of TAZ did not make significant change of transferability of PIPC to CSF. Considering the antibacterial effect of TAZ/PIPC against main causative organism of meningitis, this agent was thought to be effective for the treatment of purulent meningitis.

[20 years (1960-1980) of bacterial meningitis in childhood. I. Epidemiological and clinical data]. / 20 anni (1960-1980) di meningite batterica nell'infanzia. I. Dati epidemiologici e clinici.

One hundred forty six children having suffered bacterial meningitis at any age have been collected and retrospectively studied over a period of a twenty year survey (1960 to 1980). In our series we can confirm the prominent epidemiologic and clinical features emerging from the literature. It is notewborty to outline the great percentage of cases lacking any bacterial identification due to previous antimicrobial treatment for parameningeal foci. This raises some major problems about early diagnosis, validity and signification of lumbar punctures, and usefulness of starting treatment with large spectrum antibiotics.