Study of different off-line sample processing procedures and the measurement of antibiotic and antiviral levels in human serum by high-performance liquid chromatography.

We attempted to devise a preparation method for clinical samples that could be used for all antibiotics and antivirals. We studied thirteen antibiotics, including five penicillins, four cephalosporins, metronidazole, ofloxacin, and sulfamethoxazole and four protease inhibitors including indinavir, retonavir, nelfinavir, and sequinavir. We compared four sample preparation techniques including solvent precipitation, filtration and resin column. We employ HPLC methods based on a minimal number of columns and mobile phases. We were unable to find one sample preparation method that could be used for all antibiotics and antivirals. But, we did develop an algorithm for determining optimal processing procedures for all drugs.

Phenotypic resistance of Staphylococcus aureus, selected Enterobacteriaceae, and Pseudomonas aeruginosa after single and multiple in vitro exposures to ciprofloxacin, levofloxacin, and trovafloxacin.

The phenotypic resistance of selected organisms to ciprofloxacin, levofloxacin, and trovafloxacin was defined as a MIC of > or =4 microg/ml. The dynamics of resistance were studied after single and sequential drug exposures: clinical isolates of methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA), Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens, and Pseudomonas aeruginosa were utilized. After a single 48-h exposure of a large inoculum to four times the initial MIC for the organism, the frequency of selection of resistant mutants of MSSA was greater for trovafloxacin than levofloxacin (P = 0.008); for E. cloacae, the frequency was highest for ciprofloxacin and lowest for levofloxacin and trovafloxacin; for S. marcescens, the frequency was highest for trovafloxacin and lowest for ciprofloxacin (P = 0.003). The results of serial passage experiments were analyzed both by the Kaplan-Meier product-limited method as well as by analysis of variance of mean inhibitory values. By both methods, MSSA and MRSA expressed mutants resistant to ciprofloxacin after fewer passages than were required for either levofloxacin or trovafloxacin. For the aerobic gram-negative bacilli, two general patterns emerged. Mutants resistant to trovafloxacin appeared sooner and reached higher mean MICs than did mutants resistant to levofloxacin or ciprofloxacin. Mutants resistant to ciprofloxacin appeared later and reached mean MICs lower than the MICs of the other two drugs studied. Even though individual strain variation occurred, the mean MICs were reproduced when the serial passage experiment was repeated using an identical panel of E. coli isolates. In summary, the dynamic selection of fluoroquinolone-resistant bacteria can be demonstrated in experiments that employ serial passage of bacteria in vitro.

A randomized comparison of the safety and efficacy of once-daily gentamicin or thrice-daily gentamicin in combination with ticarcillin-clavulanate.

PURPOSE: The primary purpose of the clinical trial was to assess the safety and efficacy of once-a-day compared with three-times-a-day gentamicin in patients with serious infections who had protocol-determined peak serum aminoglycoside concentrations. PATIENTS AND METHODS: A total of 249 hospitalized patients with suspected or proven serious infections were randomized in a 2:2:1 ratio to gentamicin given three times a day with ticarcillin-clavulanate (TC), gentamicin once a day with TC, or ticarcillin-clavulanate (TC) alone. The gentamicin once-a-day dosage for patients with estimated creatinine clearance values of > or =80 mL/min was 5.1 mg/kg. With lower creatinine clearance estimates, the mg/kg dosage of gentamicin was decreased, and the dosage intervals (once daily or three times a day) were maintained. Evaluability required documentation of achievement of protocol-defined peak serum gentamicin levels. RESULTS: Of the total 175 evaluable patients, there were no significant differences found between treatment regimens with respect to clinical or microbiologic efficacy. Bedside audiometry proved impractical due to the frequency of altered mental state in ill patients. Based on the traditional increase in serum creatinine values from baseline values, no differences in renal toxicity between the treatment groups was identified. When changes in renal function were reanalyzed based on maintaining, as opposed to worsening, of renal function, preservation of renal function was better in the gentamicin once-a-day patients as opposed to the gentamicin three-times-a-day patients, P <0.01. CONCLUSIONS: Gentamicin once a day plus TC, gentamicin three times a day plus TC, and TC alone had similar effects in seriously ill hospitalized patients. The incidence of nephrotoxicity was similar in the three treatment groups. Using a nonvalidated post-hoc analysis, renal function was better preserved in gentamicin once-a-day + TC and TC-only patients as opposed to gentamicin three-times-a-day + TC.

Influence of assay methodology on the measurement of free serum ceftriaxone concentrations.

The influence of assay methodology on the measurement of the active free fraction of ceftriaxone in plasma was determined. The free fraction was measured by three methods: agar diffusion bioassay, precipitation of plasma protein with methanol followed by high-performance liquid chromatography (HPLC) of the supernatant, and ultrafiltration of plasma followed by HPLC of the filtrate. In human serum, the free ceftriaxone levels were significantly lower (P = 0.03) when measured on ultrafiltrates compared to the other two methods. This difference disappeared when dolphin serum was studied. After ultrafiltration, human serum was shown, by Scatchard plot analysis, to have two ceftriaxone binding sites. Species differences were also demonstrated. Hence, in humans, determination of free plasma ceftriaxone varies with the assay method employed.

Mycobacterium gordonae in fiberoptic bronchoscopes.

BACKGROUND: Failure of high-level disinfection of bronchoscopes has caused several outbreaks of nosocomial infection or pseudoinfection involving mycobacteria. METHODS: Inocula (10(5) colony-forming units/ml and 10(8) colony-forming units/ml) of a clinical Mycobacterium gordonae isolate were used to contaminate bronchoscopes. Glutaraldehyde, iodophor, and peracetic acid disinfectants were evaluated in manual and automated disinfection procedures after 10- to 20-minute exposures at 20 degrees and > or = 25 degrees C. RESULTS: Four of five manual disinfectant procedures failed to eliminate experimental M. gordonae infection after 10-minute exposure at 20 degrees C. All five manual procedures tested at 20 degrees C were effective after 20-minute exposure to the five disinfectants (two 2% alkaline glutaraldehyde preparations, 3.2% alkaline glutaraldehyde, 75 ppm iodophor, and 0.5% glutaraldehyde-0.03% phenolic). Three of four manual (one 2% glutaraldehyde, 3.2% glutaraldehyde, and 0.5% glutaraldehyde-0.03% phenolic) and three automated (one 2% glutaraldehyde, 0.5% glutaraldehyde-0.03% phenolic, and 0.2% peracetic acid) disinfectant procedures eliminated contamination after a 10- to 12-minute exposure at > or = 25 degrees C. Effective total cycle times for the three automated procedures ranged from 20 to 45 minutes. CONCLUSIONS: Previously Environmental Protection Agency-approved tuberculocidal agents may be ineffective against M. gordonae when used according to label claims under normal clinical conditions. A minimum 20-minute exposure time at 20 degrees C is necessary for manual disinfection methods. Higher temperatures may improve disinfectant efficacy. Newer automatic disinfection machines may be as effective as traditional manual methods and also may reduce hazards to employees.

Staphylococcus aureus septic arthritis in patients on hemodialysis treatment.

We retrospectively reviewed hospital discharge diagnoses of septic arthritis over an 11-year period (1982 through 1992) at 3 medical centers; 11 episodes of septic arthritis were identified in patients on hemodialysis treatment. Of the 11 episodes, 9 were caused by Staphylococcus aureus; in 8 of 9, the blood cultures were positive for the organism and the infection was monoarticular. Concurrent infection of the dialysis access site occurred in 4 cases. Two patients died (22%). We postulate that repeated skin trauma and contact with health care personnel and facilities result in a high rate of nasal carriage of S aureus and, hence, an increased risk of bacteremia with its attendant complications such as septic arthritis. The use of mupirocin nasal ointment is reported to eradicate or suppress carriage in a high percentage of patients; some studies report that long-term suppressive therapy reduces the frequency of S aureus bacteremia.

Pig kidney (LLC-PK1) cell membrane fluidity during exposure to gentamicin or tobramycin.

The surface membrane properties of LLC-PK1 cells grown with and without various amounts of gentamicin or tobramycin for various lengths of time were determined by measuring the diffusion coefficient of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L- alpha-phosphatidylethanolamine (NBD-PE) and the percentage of NBD-PE free to diffuse after photobleaching. One hour of exposure to tobramycin decreased the percentage that was free to diffuse. After 1 day or longer of exposure to either aminoglycoside the percentage that was free to diffuse returned to preexposure levels and the diffusion coefficient decreased.

Aminoglycoside-induced increase of intracellular calcium in LLC-PK1 cells due to an artifact caused by trypsin and EDTA.

Dietary calcium supplements attenuate experimental aminoglycoside nephrotoxicity. In cultured renal tubular cells, intracellular calcium levels have been reported to rise with aminoglycoside addition to the culture medium. In experiments designed to verify the in vitro influence of calcium on cultured kidney cells, we detected an unexpected artifact. When we resuspended cultured LLC-PK1 cells with trypsin and EDTA to measure intracellular calcium levels, our results correlated well with previously reported values. However, we saw no increase in intracellular calcium levels when we measured them by digital imaging video microscopy unless trypsin-EDTA exposure preceded aminoglycoside exposure. This apparent artifact should be considered in any study of the effects of various agents on intracellular calcium levels.

Pharmacologic limits of the protective effect of polyaspartic acid on experimental gentamicin nephrotoxicity.

Polyaspartic acid (PAA) ameliorates experimental gentamicin nephrotoxicity despite marked accumulation of gentamicin in the renal cortex. The experiments described here probe the extent of PAA's nephroprotective action when increasing doses of gentamicin, in excess of an established nephrotoxic dose (40 mg/kg of body weight per day), are administered. After 10 days, virtually complete nephroprotection was conferred by PAA coadministered to animals receiving three times the nephrotoxic dose (120 mg/kg/day) of gentamicin.

Determinants of the in vitro interaction of polyaspartic acid and aminoglycoside antibiotics.

The in vitro interaction of polyaspartic acid (PAA) with aminoglycosides was evaluated using double diffusion in agar, dialysis chambers and changes in the optical density of test solutions. The results document a reversible, presumably electrostatic interaction that is optimized at a pH of approximately 5.0, by the absence of proteins over 800 Da, and at a 20:1 or 10:1 molar ratio of aminoglycoside to PAA. The PAA-aminoglycoside complex lost antibacterial activity and the ability to inhibit pronase E enzymatic activity. These results allow generation of a hypothesis as to the mechanism whereby PAA prevents aminoglycoside experimental nephrotoxicity.

Duration of the protective effect of polyaspartic acid on experimental gentamicin nephrotoxicity.

It is known that daily polyaspartic acid (PAA) protects the kidney from gentamicin nephrotoxicity in a standardized rat model despite marked cortical accumulation of the aminoglycoside. The present experiments address the duration of PAA protection. When administered every other day, PAA provided functional and histologic protection against gentamicin-induced toxicity. A stepwise reduction in nephroprotection occurred as the dosage interval was prolonged.

General guidelines for the clinical evaluation of anti-infective drug products. Infectious Diseases Society of America and the Food and Drug Administration.

This document provides new general guidelines for the design and execution of studies evaluating anti-infective drugs for the prevention or treatment of infectious diseases. The first step in evaluation is the determination of in vitro microbial susceptibility. Next, studies are conducted in animals. Several animal models provide information useful in the prediction of appropriate dosing and activity in humans. If the results of these studies are favorable, staged clinical trials are then conducted. These guidelines reflect changes in the practice of medicine, dealing with topics such as the switch from parenteral to oral drug administration during a course of therapy, treatment in settings other than acute-care hospitals, and the use of alternative comparison drugs for the study of indications or dosing schedules not covered by the product label. Because further changes in practice are anticipated, the present guidelines will need to be updated and revised periodically.

Evaluation of new anti-infective drugs for the treatment of infective endocarditis. Infectious Diseases Society of America and the Food and Drug Administration.

These guidelines describe the design and implementation of clinical trials to assess the safety and efficacy of anti-infective drugs for the treatment of infective endocarditis. Identification and enrollment of patients in clinical trials is based on a modification of traditional criteria. To accrue a sufficient number of patients, only those with streptococcal or staphylococcal endocarditis should be included in studies. Results of treatment with approved drugs allow for projection of expected bacteriologic cure rates and survival rates. Prospective randomized, double-blind studies are recommended. These guidelines are based on the premise that future protocols may include shorter courses of therapy, combinations of drugs, or progression from parenteral to oral therapy. Clinical response is judged as cure, failure, or indeterminate; there is no "improved" category. Microbiologic response is categorized as eradication, persistence, or relapse. When a patient has shown no clinical evidence of active disease for a protracted period, there may be no need to perform a posttreatment blood culture; for such patients, the microbiologic response is termed presumptive eradication. Several months of follow-up may be necessary to detect late relapses.

Zidovudine pharmacokinetics in five HIV seronegative patients undergoing continuous ambulatory peritoneal dialysis.

A few reports in the literature describe zidovudine (AZT) pharmacokinetics in patients undergoing hemodialysis; however, the effect of continuous ambulatory peritoneal dialysis (CAPD) on the drug's disposition has not been studied. The pharmacokinetics of AZT were evaluated in five patients, age 37-62 years, who were seronegative for the human immunodeficiency virus and were undergoing CAPD. Serial plasma, urine, and dialysate samples were collected after oral administration of AZT 200 mg. Samples were assayed using radioimmunoassay (RIA). Model-independent analysis was used to determine total plasma clearance, apparent volume of distribution, mean residence time, and half-life. Net peritoneal dialysis clearance was calculated to measure the effect of CAPD on AZT disposition. We found wide interpatient variability in AZT pharmacokinetics. Peak serum concentrations ranged from 0.3-47.8 microns and area under the curve from 0.5-26.1 mg x hour/L. These differences resulted in corresponding differences in clearance (range 66-3176 ml/min/1.73 m2) and volume of distribution (range 16-825 L). Interpatient variability in glucuronidation may partially account for this variability. Net peritoneal dialysis clearance of AZT was 5 ml/minute. Although the effect of peritoneal dialysis on AZT disposition was negligible, clinicians should be aware of the large differences in the way in which individual patients with renal dysfunction handle this drug.

Long-term protection of polyaspartic acid in experimental gentamicin nephrotoxicity.

Polyaspartic Acid (PAA) protects the kidney from experimental gentamicin nephrotoxicity despite large increases in renal cortical gentamicin content. In these experiments, prominent cytoplasmic vacuoles were noted in all animals that received PAA with or without gentamicin. The present study showed that there were no renal structural or functional consequences of PAA given alone or with gentamicin for up to 14 days, followed by a 16-week washout period. Creatinine clearance was similar to that of controls in animals that received gentamicin and in those that received PAA alone. Thus, complete functional protection was conferred by PAA and gentamicin, confirming previous reports from our laboratory. There was no protection by PAA from the nephrotoxic effects of mercuric chloride and cis-platinum.