Pharmacodynamics of levofloxacin and ciprofloxacin against Streptococcus pneumoniae

Philip D. Lister* and Christine C. Sanders

Center for Research in Anti-Infectives and Biotechnology, Department of Medical Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NB 68178, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An in-vitro pharmacokinetic model was used to compare the pharmacodynamics of levofloxacin and ciprofloxacin against four penicillin-susceptible and four penicillin-resistant Streptococcus pneumoniae.Logarithmic-phase cultures were exposed to the peak concentrations of levofloxacin or ciprofloxacin observed in human serum after 500 mg and 750 mg oral doses, human elimination pharmacokinetics were simulated, and viable bacterial counts were measured at 0, 1, 2, 4, 6, 8, 12, 24 and 36 h. Levofloxacin was rapidly and significantly bactericidal against all eight strains evaluated, with eradication of six strains occurring despite area under the inhibitory curve over 24 h (AUIC24) values of only 32– 64 SIT -1•h (serum inhibitory titre over time). The pharmacodynamics of ciprofloxacin were more variable and the rate of bacterial killing was consistently slower than observed with levofloxacin. Ciprofloxacin eradicated five strains despite having an AUIC24 of only 44 SIT-1•h. These data suggest that the increased potency of levofloxacin and more favourable pharmacokinetics compared with ciprofloxacin provide enhanced pharmacodynamic activity against S. pneumoniae. Furthermore, these data suggest that the minimum AUIC required for clinical efficacy against and eradication of S. pneumoniae with levofloxacin and ciprofloxacin may be well below the 125 SIT -1•h identified by other studies.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The fluoroquinolones are considered a reasonable therapeutic option for the treatment of community-acquired respiratory tract infections because of their potent activity against the most common Gram-negative pathogens in this setting. 1 However, the role of ciprofloxacin and ofloxacin in the treatment of pneumococcal infections has been controversial because of their marginal in-vitro activity. Susceptibility studies have demonstrated that ciprofloxacin MIC90s for S. pneumoniae are two to four times the 1 mg/L susceptible breakpoint, whereas ofloxacin MIC90s are either equal to or twice the 2 mg/L susceptible breakpoint. 2,3,4,5 Furthermore, area under the inhibitory curve (AUIC) values achieved with currently recommended doses against S. pneumoniae are frequently below the 125 SIT-1•h (serum inhibitory titre over time) level which has been proposed by some investigators as the minimum required for the treatment of infections with fluoroquinolones. 6,7 Nevertheless, ofloxacin has proven efficacious in the treatment of pneumococcal infections. 8,9 For ciprofloxacin, clinical experience has been more variable, 1,10 and the development of bacteraemia during treatment of pneumococcal pneumonia with ciprofloxacin 11 and the development of pneumococcal superinfection during therapy of a nosocomial P. aeruginosa pneumonia with ciprofloxacin 12 have been reported.

Levofloxacin, the active L-isomer of racemic ofloxacin, has greater in-vitro activity against S. pneumoniae than ciprofloxacin and its parent compound, ofloxacin. In comparisons with ciprofloxacin and ofloxacin, levofloxacin is generally twice as potent, with MIC90s for S. pneumoniae at or just below the 2 mg/L susceptible breakpoint.2,3,5 In addition to its enhanced anti-pneumococcal potency, levofloxacin has a pharmacokinetic advantage over ciprofloxacin, with higher peak serum levels and a lower rate of elimination. 13,14 The pharmacokinetic advantages of levofloxacin, together with its enhanced activity against pneumococci, suggest that levofloxacin may have a clinical advantage over ciprofloxacin in the treatment of S. pneumoniae respiratory tract infections. This study was designed to compare the in-vitro pharmacodynamics of levofloxacin and ciprofloxacin against S. pneumoniae in an in-vitro pharmacokinetic model of infection when oral doses of levofloxacin 500 mg and ciprofloxacin 750 mg were simulated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and culture conditions

The experimental strains evaluated in this study included eight clinical isolates of S. pneumoniae, four of which were resistant to penicillin (MICs = 2–4 mg/L). Each clinical isolate was obtained from a different patient. Logarithmic-phase cultures were prepared by suspending ten colonies from a 14 h culture on trypticase soy agar supplemented with 5% sheep blood (BBL Microbiology Systems, Cockeysville, MD, USA) into 6 mL of Todd–Hewitt broth (Unipath/Oxoid, Ogdensburg, NY, USA) supplemented with 0.5% yeast extract (THY). 15 Viable bacterial counts after 10 h of incubation at 37°C in 5% CO2 ranged from 1 x 108 cfu/mL to 5 x 108 cfu/mL.

Antibiotic preparations and susceptibility testing

Levofloxacin powder was supplied by R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ, USA. Ciprofloxacin powder was supplied by Bayer Corporation, West Haven, CT, USA. Levofloxacin powder was dissolved in 0.2 mL of 0.1 M NaOH, diluted to final volume with distilled water, and sterilized by passage through an Acrodisc syringe filter membrane (0.20 µm pore size; Gelman Sciences, Ann Arbor, MI, USA). Ciprofloxacin powder was reconstituted with distilled water and filter-sterilized.

Susceptibility tests with levofloxacin and ciprofloxacin were performed by broth microdilution according to the procedure recommended by the National Committee for Clinical Laboratory Standards.16

In-vitro pharmacokinetic model

The basics of the in-vitro pharmacokinetic model used in this study have been described in detail previously, 15,17 and a schematic representation of the model is presented in Figure 1. A hollow-fibre cartridge (Unisyn Fibertech, San Diego, CA, USA) was connected by a continuous loop of silicone tubing to a central reservoir. At the start of each experiment, peak antibiotic concentrations in THY in the central reservoir were pumped through the hollow fibres of the cartridge and back into the central reservoir. As drug-containing THY passed through the hollow fibres, pores in the fibre walls allowed antibiotic and nutrients to diffuse freely from the lumen of the fibres into the space surrounding the hollow fibres within the cartridge (peripheral compartment) and back into the lumen of the hollow fibres. The exclusion size of the pores in the fibre walls (mol. wt cut-off 30,000) prohibited bacteria introduced into the peripheral compartment from entering the lumen of the hollow fibres. Thus, the drug concentration within the peripheral compartment space could be altered without disrupting bacterial growth. The bacterial culture within the peripheral compartment was continuously circulated through a loop of silicone tubing attached to two ports entering and exiting the peripheral compartment, and samples were removed from the peripheral compartment through a three-way stopcock connected within the loop of silicone tubing. The initial volume of culture circulated through the peripheral compartment and silicone tubing was 30– 35 mL.



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Figure 1. Schematic representation of the two-compartment pharmacokinetic model. Each arrow represents a peristaltic pump within the system. Peak concentrations of levofloxacin or ciprofloxacin were dosed into the central reservoir and were pumped through the lumen of hollow fibres in the hollow-fibre cartridge (HFC). Pores (mol. wt cut-off 30,000) in the fibre walls allowed antibiotic to diffuse freely from the lumen of the hollow fibres into the peripheral compartment of the HFC where bacteria were inoculated. Antibiotic was eliminated from the central reservoir by the addition of drug-free broth from a diluent reservoir and elimination of drug-containing broth into the elimination reservoir. As antibiotic concentrations in the central reservoir decreased, antibiotic concentrations within the peripheral compartment also decreased as drug diffused into the lumen of the hollow fibres to maintain equilibrium between the two compartments.

 
A comparison of antibiotic concentrations between the central reservoir and the peripheral compartment every 15 min after dosing demonstrated that equilibrium was established between the compartments at approximately 0.5 h. After peak concentrations were achieved within the peripheral compartment, human elimination pharmacokinetics of ciprofloxacin and levofloxacin were simulated by a process of dilution and elimination of drug in the central reservoir. Drug concentrations in the central reservoir (and peripheral compartment as equilibrium was maintained) were decreased by the addition of drug-free THY from a dilution reservoir. To maintain a constant volume in the central reservoir, drug-containing THY was pumped from the central reservoir into an elimination reservoir. The rate at which drug concentrations in the central reservoir and peripheral compartment were decreased by this method was determined by the flow rate of the peristaltic pumps. This rate was calculated from an equation for clearance by monoexponential decline, based on the serum elimination half-life of ciprofloxacin or levofloxacin and the volume of medium in the central reservoir. Using this methodology, elimination half-lives of 7.5 h were simulated for levofloxacin 13,18 and 4 h for ciprofloxacin. 14,19

Pharmacokinetics of levofloxacin and ciprofloxacin in the in-vitro pharmacokinetic model

Peak levels of levofloxacin and ciprofloxacin achieved in human serum after oral doses of levofloxacin 500 mg 13,18 and ciprofloxacin 750 mg 14,19 were targeted in these studies. To evaluate the pharmacokinetics of levofloxacin and ciprofloxacin, peak concentrations of levofloxacin and ciprofloxacin were dosed into the central reservoir of the in-vitro pharmacokinetic model and samples were removed from the peripheral compartment at 0, 0.5, 1, 2, 4, 8, 12, 12.5, 13, 14, 16, 20 and 24 h. Drug concentrations in each sample were measured by disc diffusion bioassay using a susceptible strain of Escherichia coli. The linear range of the bioassay was 0.1–0.9 mg/L. The area under the concentration curve over 24 h (AUC24) for levofloxacin and ciprofloxacin was calculated using the trapezoidal rule. The AUIC24 for levofloxacin and ciprofloxacin against specific strains was calculated by dividing the AUC24 by the MIC of the antibiotic for the target strain of S. pneumoniae.20

Pharmacodynamic experiments

Logarithmic-phase cultures were diluted into fresh THY (prewarmed to 37°C) for a final inoculum of 1 x 106 to 1 x 107 cfu/mL, introduced into the peripheral compartment of the in-vitro pharmacokinetic model and exposed to levofloxacin or ciprofloxacin as described above. Pharmacodynamic experiments were performed in ambient air at 37°C. At 0, 1, 2, 4, 6, 8, 12, 24 and 36 h, samples were removed from the peripheral compartment and viable bacterial counts were measured by plating serial ten-fold dilutions of each sample into Todd–Hewitt agar (THA; BBL) and incubating plates overnight at 37°C in 5% CO2. The lowest dilution plated was 0.1 mL of undiluted sample from the peripheral compartment. Since 30 colonies is the lower limit of accurate quantification with pour-plate methodology, the lowest number of bacteria that could be accurately counted was 300 cfu/mL. The lowest level of detection, although actual counts were inaccurate, was 10 cfu/mL.

To prevent antibiotic carry-over, 2.5 mM ferric chloride was added to the THA for the least diluted sample.21 Antibiotic carry-over was not a problem with the other dilutions since each antibiotic was diluted to at least 200-fold below the MICs of the drugs against the most susceptible strains. To evaluate the selection of mutants with decreased susceptibility to quinolones, samples removed from the peripheral compartment at 36 h were also plated into THA containing levofloxacin or ciprofloxacin at a concentration of 4 x MIC.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Susceptibility of S. pneumoniae and characterization of in-vitro pharmacokinetic model

Levofloxacin and ciprofloxacin MICs were similar or within one two-fold dilution of each other against all eight strains of S. pneumoniae, ranging from 0.5 to 2 mg/L for levofloxacin and from 1 to 2 mg/L for ciprofloxacin. When a difference in MIC was observed, the MIC of ciprofloxacin was always twice that of levofloxacin.

The pharmacokinetic profiles of levofloxacin and ciprofloxacin within the peripheral compartment of the in-vitro pharmacokinetic model are shown in Figure 2. Peak concentrations (mean ± S.D.) of levofloxacin and ciprofloxacin in the peripheral compartment at 0.5 h were 6.6 ± 0.2 mg/L and 4.6 ± 0.1 mg/L, respectively. The AUC24 for levofloxacin was 64 mg•h/L and that for ciprofloxacin was 44 mg•h/L. The AUIC24 for levofloxacin was 32 SIT– 1•h against one strain, 64 SIT– 1•h against five strains, and 128 SIT– 1•h against two strains. In comparison, the AUIC24 for ciprofloxacin was 44 SIT– 1•h against five strains and 22 SIT 1•h against three strains.



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Figure 2. Pharmacokinetics of levofloxacin ({square}) and ciprofloxacin ({circ}) in the peripheral compartment of the in-vitro pharmacokinetic model after dosing into the central reservoir. Each datum point represents the mean drug level in the peripheral compartment (in mg/L) for three experimental runs. Error bars show standard deviations.

 
Pharmacodynamics against penicillin-susceptible S. pneumoniae

The pharmacodynamics of levofloxacin and ciprofloxacin against representative penicillin-susceptible strains, S. pneumoniae 212 and S. pneumoniae 213, are shown in Figure 3. Levofloxacin was rapidly bactericidal against all four penicillin-susceptible S. pneumoniae, with viable counts falling 5– 6 logs to undetectable levels and remaining below this limit for the remainder of the 36 h experimental period (Figure 3). The time required to achieve a significant 3-log kill of the initial inoculum (time to 99.9% kill) ranged from 2 to 4.5 h. The time required to decrease viable counts below the 10 cfu/mL limit of detection ranged from 6– 8 h in studies with S. pneumoniae 212 (Figure 3a) and S. pneumoniae 257 (data not shown), to 12– 24 h in studies with S. pneumoniae 213 (Figure 3b).



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Figure 3. Time- kill pharmacodynamics of levofloxacin ({square}) and ciprofloxacin ({triangleup}) against (a) penicillin-susceptible S. pneumoniae 212 and (b) S. pneumoniae 213. Each datum point represents the mean cfu/mL of THY from the peripheral compartment for duplicate experiments. {circ}, Control. Error bars show standard deviations.

 
In studies with S. pneumoniae 212 (Figure 3a), the general pharmacodynamics of ciprofloxacin were similar to those of levofloxacin. However, the rate of bacterial killing with ciprofloxacin was lower than with levofloxacin, taking 1.5 h longer to achieve 99.9% kill and taking a substantially longer period of time than levofloxacin to decrease viable counts below the 10 cfu/mL limit of detection. Against S. pneumoniae 257 (data not shown), the rate of killing with ciprofloxacin was also slower, taking 1.5 h longer to achieve 99.9% kill, and failing to decrease viable counts below 10 cfu/mL. In studies with S. pneumoniae strains 213 and 256 the pharmacodynamic differences between levofloxacin and ciprofloxacin were even more apparent. Against S. pneumoniae 213 (Figure 3b), ciprofloxacin failed to decrease viable counts even 2 logs over the first dose interval, and over the second and third dose intervals a gradual net increase in viable counts was observed. However, no mutants with decreased susceptibility to quinolones were detected on drug-selection plates at 36 h. Although a significant 99.9% kill was observed with ciprofloxacin against S. pneumoniae 256, this level of killing was not observed until 7.5 h, as compared with 2.1 h with levofloxacin, and gradual net increases in viable counts were observed over the second and third dose intervals. Similar to studies with S. pneumoniae 213, no mutants with decreased susceptibility to quinolones were detected on drug-selection plates at 36 h.

Pharmacodynamics against penicillin-resistant S. pneumoniae

The pharmacodynamics of levofloxacin and ciprofloxacin against representative penicillin-resistant strains S. pneumoniae 3956 and 3938 are shown in Figure 4. Levofloxacin was rapidly bactericidal against all four strains of penicillin-resistant S. pneumoniae, with viable counts falling 5–6 logs to undetectable levels and remaining below this limit for the remainder of the 36 h experimental period (Figure 4). The time to 99.9% kill ranged from 1.5 to 4 h, and the time required to decrease viable counts below the 10 cfu/mL limit of detection ranged from 4–6 h in studies with S. pneumoniae 3956 (Figure 4a) to 12–24 h in studies with S. pneumoniae 3935 (data not shown).



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Figure 4. Time- kill pharmacodynamics of levofloxacin ({square}) and ciprofloxacin ({triangleup}) against (a) penicillin-resistant S. pneumoniae 3956 and (b) S. pneumoniae 3938. Each datum point represents the mean cfu/mL of THY from the peripheral compartment for duplicate experiments. {circ}, Control. Error bars show standard deviations.

 
The pharmacodynamics of ciprofloxacin against S. pneumoniae3956 (Figure 4a) were similar to those of levofloxacin, taking just 0.5 h longer to achieve 99.9% kill and thereafter decreasing viable counts below 10 cfu/mL within the initial 6– 8 h. Against S. pneumoniae 3935, the pharmacodynamics of ciprofloxacin were almost identical to those of levofloxacin, especially over the first 6 h when the rate of bacterial killing was greatest. After the initial 6 h, the rate of bacterial killing with both drugs decreased substantially such that viable counts did not fall below the 10 cfu/mL limit of detection until 12– 24 h after the first doses. The pharmacodynamics of ciprofloxacin against S. pneumoniae 3938 (Figure 4b) and S. pneumoniae 3933 differed substantially from those of levofloxacin, required 2– 5 h additional time to achieve 99.9% kill and substantially longer to decrease viable counts below 10 cfu/mL.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
An in-vitro pharmacokinetic model was used to simulate the serum pharmacokinetics of levofloxacin and ciprofloxacin and to compare their pharmacodynamics against S. pneumoniae. A comparison of the MICs of levofloxacin and ciprofloxacin against the strains selected for this study demonstrated that the two quinolones were equivalent in their potency against four strains and that levofloxacin was twice as potent as ciprofloxacin against the remaining four strains. A direct comparison of levofloxacin and ciprofloxacin against individual strains of S. pneumoniae in other studies has demonstrated that levofloxacin is generally twice as active as ciprofloxacin against 35– 50% of pneumococcal isolates. 22,23 The S. pneumoniae selected for this study thus provided a realistic representation of the strains that would be encountered clinically.

Levofloxacin was significantly and rapidly bactericidal against all eight strains of S. pneumoniae evaluated in this study. To our knowledge, no other data have been published on the pharmacodynamics of levofloxacin in a pharmacokinetically based model similar to this one. However, using static time– kill methodologies other investigators have also demonstrated significant killing of S. pneumoniae with levofloxacin. 22,23,24 The eradication of six isolates of S. pneumoniae from the in-vitro pharmacokinetic model in this study, despite simulated AUICs of only 32–64 SIT -1•h, suggests that an AUIC of 125 SIT-1•h may not be the minimum required for levofloxacin efficacy against S. pneumoniae. This conclusion is supported by clinical experience with levofloxacin in the treatment of community-acquired pneumonia.25 File and colleagues reported that a once-daily dose of 500 mg of levofloxacin was successful in eradicating all of 30 S. pneumoniae from the lungs and all of nine S. pneumoniae from the bloodstream of patients with pneumococcal pneumonia.25 Furthermore, all 30 patients treated with levofloxacin were either clinically cured (23/30) or clinically improved (7/30) 5–7 days after therapy. These clinical data, combined with the pharmacodynamics observed in the current study simulating the same clinical dose, suggest that a once-daily dose of levofloxacin 500 mg should be effective in treating lower respiratory tract infections and bacteraemias caused by susceptible strains of S. pneumoniae. Although the minimum AUIC required for clinical efficacy appears to be well below 125 SIT –1•h, more systematic studies comparing the pharmacodynamics of levofloxacin over a range of AUICs are required to determine the true minimum which must be achieved.

The pharmacodynamics of ciprofloxacin in this study were much more variable than those of levofloxacin. In contrast to levofloxacin, ciprofloxacin failed to decrease viable counts of three strains below the 10 cfu/mL limit of detection. Although these strains were all from the penicillin-susceptible group, there is no evidence in the literature to suggest that this trend is biologically significant. In studies with two of these strains, gradual net increases in viable counts were observed over the second and third dose intervals. Since no resistant mutants were detected on drug-selection plates at 36 h, the decreased antibacterial activity observed after the first dose of ciprofloxacin probably represents adaptive resistance, or the reversible decrease in susceptibility after first exposure to an antibiotic. 26,27 In direct comparisons between the quinolones against individual strains, the rates of killing observed with ciprofloxacin were consistently lower than those observed with levofloxacin. The decreased rate of killing observed with ciprofloxacin in this study may be related to the lower peak/MIC ratios in the in-vitro pharmacokinetic model since fluoroquinolones have been shown to exhibit dose-response bactericidal activity. 2829Although the pharmacodynamics of ciprofloxacin and levofloxacin may have been more comparable in this study if their pharmacokinetics were similar in the in-vitro pharmacokinetic model (adjusting peak levels to attain similar peak/MIC ratios), the purpose of this investigation was to determine if the enhanced in-vitro potency of levofloxacin and its more favourable pharmacokinetic profile would translate into enhanced pharmacodynamic activity. Data from this investigation do suggest that levofloxacin is pharmacodynamically superior to ciprofloxacin against S. pneumoniae when their highest recommended oral doses are simulated.

Similar to the pharmacodynamics observed with levofloxacin, the ability of ciprofloxacin to eradicate five S. pneumoniae from the in-vitro pharmacokinetic model in this study, despite simulated AUICs of only 44 SIT-1•h, suggests that an AUIC of 125 SIT-1•h may not be the minimum required for clinical efficacy with ciprofloxacin against pneumococci. In fact, a comparison of the pharmacodynamics of ciprofloxacin against the five strains for which the MIC was 1 mg/L (AUIC = 44 SIT -1•h) with the pharmacodynamics observed against the three strains for which the MIC of ciprofloxacin was 2 mg/L (AUIC = 22 SIT-1•h) suggest that the minimum AUIC required for eradication of S. pneumoniae from the in-vitro pharmacokinetic model lies somewhere between 22 and 44 SIT-1•h. Once again, the precise minimum AUIC required for bacterial eradication with ciprofloxacin in this model would require examination of pharmacodynamics over a range of AUICs.

A comparison of the pharmacokinetics of ciprofloxacin in the in-vitro pharmacokinetic model with the MICs of ciprofloxacin against the pneumococcal strains in this study indicates that ciprofloxacin concentrations fell below the 1 mg/L MIC for five strains at 8 h after dosing and below the 2 mg/L MIC for the other three strains at 5 h after dosing. The lack of inoculum regrowth during the first dose interval in these experiments, despite 4–7 h of subinhibitory concentrations of ciprofloxacin, suggests that a substantial post-antibiotic sub-MIC effect (PA-SME) was occurring. The PA-SME is an extension of the post-antibiotic effect period when sub-inhibitory concentrations of antibiotic remain in the environment, in contrast to when antibiotic is virtually eliminated,30 and this pharmacodynamic phenomenon has been reported with both levofloxacin and ciprofloxacin against S. pneumoniae.31 In the current study, the PA-SME intervals were longer than the 1.25–3.25 h reported by Licata and colleagues. 31 However, this is not surprising since antibiotic was removed from the bacterial environment more gradually and by more natural processes in the in-vitro pharmacokinetic model, as opposed to the centrifugation and washing techniques used by Licata and colleagues. In contrast to ciprofloxacin, PA-SME interactions could not be evaluated for levofloxacin since drug levels either remained above the MIC during the entire 24 h dose interval or fell below the MIC only after viable counts fell below detectable levels.

In summary, levofloxacin was pharmacodynamically superior to ciprofloxacin in this study. The increased rates of killing and higher rates of bacterial eradication observed with levofloxacin were probably the result of the increased potency of levofloxacin against many pneumococcal strains in combination with its more favourable pharmacokinetic profile. Data from this study and recent clinical data support the use of a once-daily dose of 500 mg of levofloxacin for the treatment of pneumococcal pneumonia caused by susceptible strains, and suggest that the minimum AUIC required for clinical efficacy with both levofloxacin and ciprofloxacin may be well below the 125 SIT-1•h breakpoint suggested by other studies. Additional studies will be required to address this issue.


    Acknowledgments
 
This work was supported by a grant from R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ, USA. The authors wish to thank Stacey Edward for excellent technical assistance.


    Notes
 
* Tel: +1-402-280-1881; Fax: +1-402-280-1225; E-mail: pdlister{at}creighton.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 8 April 1998; returned 15 June 1998; revised 15 July 1998; accepted 12 August 1998