ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2004, p. 1719–1726 0066-4804/04/$08.00⫹0 DOI: 10.1128/AAC.48.5.1719–1726.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 5
Peritoneal Fluid Titer Test for Peritoneal Dialysis-Related Peritonitis Christine Strijack,1 Godfrey K. M. Harding,2,3 Robert E. Ariano,1,2,4 and Sheryl A. Zelenitsky1,2,4* Faculties of Pharmacy1 and Medicine,2 University of Manitoba, and Department of Pharmacy4 and Microbiology Laboratory,3 St. Boniface General Hospital, Winnipeg, Manitoba, Canada Received 26 November 2003/Accepted 13 January 2004
Standard microbiological tests (i.e., MIC) do not account for the unique factors of peritoneal dialysis (PD)related peritonitis which can significantly influence treatment response. Our goals were to develop a peritoneal fluid titer (PFT) test and to conduct a pilot study of its association with clinical outcome. The methodology was developed by using spent dialysate collected from patients with bacterial PD-related peritonitis prior to the initiation of antibiotics. Dialysate was processed and spiked with antibiotic to simulate two standard intraperitoneal regimens: cefazolin plus tobramycin and cefazolin alone. Thirty-six clinical isolates, including Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa, were tested. In the pilot study, dialysate was collected from 14 patients with bacterial PD-related peritonitis. Titers were determined by using each patient’s dialysate and infecting pathogen. Titers were highly reproducible, with discrepancies in only 1% of cases. Overall, PFTs were notably higher against gram-positive bacteria (P < 0.0001). The addition of tobramycin increased titers significantly from zero to values of 1/16 to 1/64 against E. cloacae and P. aeruginosa (P < 0.0001). In the pilot study, peritoneal fluid inhibitory titers were significantly associated with clinical outcome, with a median value of 1/96 for patients who were cured compared to 1/32 for those who failed treatment (P ⴝ 0.036). In conclusion, this study provides preliminary support for the PFT as a pharmacodynamic index specific to the treatment of PD-related peritonitis. With further characterization and validation in patients, the PFT test may advance the study of antibiotic therapies for PD-related peritonitis. a pilot study of its association with clinical outcome for PDrelated peritonitis.
Peritonitis is a common and potentially serious infection of patients undergoing continuous ambulatory peritoneal dialysis (PD). Significant complications include hospitalization, protracted or recurrent infections, catheter removal, and an attributable mortality approaching 10% (4, 9, 10). Over the past decades, there have been significant reductions in the incidence of PD-related peritonitis (32) but only modest improvements in treatment, which fails in 20 to 30% of cases. Clinical studies are often restricted by relatively small patient numbers and inadequate power to detect significant differences among antibiotic therapies. Furthermore, delays in the identification and susceptibility testing of infecting pathogens lead to most, if not all, patients receiving empirical, broad-spectrum antibiotics before allocation to pathogen-directed treatments (14). Alternative methods to assess and compare antibiotic therapies for PD-related peritonitis are needed. Therapeutic decisions for the management of PD-related peritonitis are often guided by the standard microbiological test of antibiotic susceptibility, MIC. However, MIC interpretations do not account for the unique factors in PD-related peritonitis including (i) much higher intraperitoneal antibiotic concentrations than those achieved in serum, (ii) interactions of antibiotic combinations, and (iii) variable compositions and effects of dialysate on antibacterial activity. As a result, a microbiological test other than MIC which incorporates peritonitis-specific variables into its measure of antibacterial effect may be most predictive of treatment response. Our goals were to develop the peritoneal fluid titer (PFT) test and to conduct
MATERIALS AND METHODS Dialysate. Spent dialysate was collected from 32 patients with bacterial PDrelated peritonitis prior to the initiation of antibiotics. These samples were used to develop the processing procedure. Dialysate bags were shaken to disperse cellular debris. Access ports were swabbed with alcohol, the clamps were released, and 50-ml aliquots were collected. The pH of the fluid was measured. Samples were centrifuged at 2,500 ⫻ g for 10 min, and the supernatant (35 ml) was centrifuged for an additional 10 min. The resultant supernatant (25 ml) was transferred to a sterile tube. To ensure adequate removal of infecting pathogens, 1 ml of processed dialysate (supernatant) was added to 1 ml of Mueller-Hinton broth (MHB) and incubated at 35°C for 48 h. Samples (0.1 and 0.5 ml) were then plated on solid tryptic soy agar (TSA), incubated for 24 h, and examined for bacterial colony growth. The dialysate supernatant was also analyzed for pH, sodium, chloride, potassium, calcium, magnesium, bicarbonate, phosphate, urea, creatinine, total protein, and glucose. Antibiotics. Cefazolin and tobramycin were used to develop the PFTs, whereas vancomycin and ceftazidime were also required in the pilot study. Pharmaceutical-grade cefazolin, vancomycin, and tobramycin obtained from Sigma-Aldrich Inc. (Oakville, Ontario, Canada) were used to prepare 1,000-g/ml stock solutions in sterile distilled water. Aliquots (2 ml) were stored at ⫺70°C for a maximum duration of 2 months. Ceftazidime obtained from Eli Lilly & Company (Greenfield, Ill.) was used to make 1,000-g/ml solutions by dissolving the drug in anhydrous sodium carbonate solution and diluting it with sterile distilled water. Ceftazidime solutions were used within 1 h of preparation. MICs described below were used to verify antibiotic concentrations (24). Staphylococcus aureus (ATCC 29213) was used to test cefazolin and vancomycin, whereas Pseudomonas aeruginosa (ATCC 27853) was used for tobramycin and ceftazidime. Acceptable MIC ranges were 0.25 to 1 g/ml for cefazolin, 0.5 to 2 g/ml for vancomycin, 0.25 to 1 g/ml for tobramycin, and 1 to 4 g/ml for ceftazidime (24). Bacteria. PFTs were developed by using six clinically relevant pathogens including S. aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and P. aeruginosa (32). Six clinical peritoneal isolates of each pathogen were identified by using the Microbiology Laboratory database (Microscan, Dade Diagnostics Corp., Mississauga, Ontario, Canada) of records between 1999 and 2001. Study isolates were retrieved from frozen (i.e.,
* Corresponding author. Mailing address: Division of Clinical Sciences and Practice, Faculty of Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. Phone: (204) 474-8414. Fax: (204) 474-7617. E-mail:
[email protected]. 1719
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⫺70°C) stock. The Microbiology Laboratory used standard techniques for culture and sensitivity testing of peritoneal fluid samples collected from patients with suspected PD-related peritonitis (1). PFTs require bacterial growth in dialysate broth medium; therefore, initial growth studies were conducted. Bacterial suspensions were prepared by transferring 5 to 10 colonies from solid TSA to 5 ml of MHB and incubating them at 35°C for 2 h. The suspension was adjusted to a 0.5 McFarland standard (i.e., 1 ⫻ 108 to 2 ⫻ 108 CFU/ml) and diluted 100-fold in MHB. One milliliter of bacterial suspension was added to 1 ml of dialysate supernatant, yielding 5 ⫻ 105 CFU/ml in a final 2-ml volume. Test tubes were incubated at 35°C for 24 h. Colony counts were performed by serially diluting a sample (0.1 ml) in normal saline at 4°C and plating aliquots (0.01 and 0.1 ml) on solid TSA. Plates were incubated at 35°C for 24 h, and then viable bacterial colonies were counted. The lower limit of detection was 102 CFU/ml. Bacterial growth in dialysate supernatant:MHB (50:50) was then compared to that in MHB. MICs. For all isolates, macrodilution MICs were determined according to methods described by the NCCLS (24). MHB supplemented with 25 mg of Ca⫹⫹/liter and 12.5 mg of Mg⫹⫹/liter was used. In the first test tube, antibiotic stock solution was added to 2 ml of cation-supplemented MHB to achieve twice the desired starting concentration. In seven subsequent tubes, 1 ml of cationsupplemented MHB was added. Serial dilution began by transferring 1 ml from the first tube and ended by discarding 1 ml from the last tube. All test tubes were vortexed prior to the next transfer. Bacterial suspensions were prepared as described above for growth studies, and colony counts were performed to verify inocula. One milliliter of bacterial suspension was added to each test tube, yielding 5 ⫻ 105 CFU/ml in a final 2-ml volume. Test tubes were incubated at 35°C for 24 h. The MIC was determined by visual inspection as the lowest antibiotic concentration to inhibit visible growth. Measurements were conducted in duplicate on separate occasions. Positive and negative controls were run during all experiments. PFTs. PFTs are measures of overall antibacterial activity, based on the assumption that the antibacterial effect is a complex interaction of antibiotic(s), infecting pathogen, and dialysate. The test was adapted from the serum titer, a standardized test of overall antibacterial activity in serum. Given significant interpatient variability in antibiotic dialysate concentrations as determined by dose, dianeal fluid volumes, dwell durations, and peritoneal membrane status, the PFT test was developed by using dialysate supernatant spiked with standard antibiotic concentrations. Antibiotic was added to simulate average levels achieved during the first 6-h dwell of dianeal fluid (2 liters) containing antibiotic. Two intraperitoneal antibiotic regimens were simulated, including cefazolin (500-mg loading dose followed by 125 mg in every bag) plus tobramycin (60 mg in one bag daily) and cefazolin (see above) alone. Based on a dialysate effluent volume of 2.5 liters and a systemic absorption of 85% for both antibiotics (25), processed dialysate was spiked to produce 120 mg of cefazolin/liter and 20 mg of tobramycin/liter. Two milliliters of spiked dialysate supernatant was added to the first test tube, whereas 1 ml of MHB was placed in the seven subsequent tubes. Unsupplemented MHB was used, since the serial dilution of all dialysate components including cations was desired. Serial dilution began by transferring 1 ml from the first tube and ended by discarding 1 ml from the last tube. All test tubes were vortexed prior to the next transfer. Bacterial suspensions were prepared and adjusted to a 0.5 McFarland standard as described above for growth studies, and colony counts were performed to verify inocula. Next, 0.01 ml of bacterial suspension was injected just below the fluid surface, yielding 106 CFU/ml in a final 1-ml volume. Test tubes were incubated at 35°C for 24 h. The peritoneal fluid inhibitory titer (PIT) was determined by visual inspection as the highest dilution to inhibit visible growth. The peritoneal fluid bactericidal titer (PBT) was determined by plating samples (0.01 and 0.1 ml) from each clear tube and identifying the highest dilution to achieve 99.9% bacterial kill. Titers were recorded as 0, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, or ⬎1/256. All titers were determined in duplicate on separate occasions. Again, positive and negative controls were run during all experiments. Pilot study. The pilot study was approved by the University of Manitoba Health Research Ethics Board. Fourteen patients with microbiologically confirmed bacterial PD-related peritonitis from whom spent dialysate was collected prior to the initiation of antibiotics were studied. Each patient’s spent dialysate was processed and spiked with antibiotic(s) to simulate their own treatment regimen. Cefazolin and tobramycin regimens were replicated as described above. Regimens containing vancomycin (2,000 mg in one bag weekly) or ceftazidime (250-mg loading dose followed by 125 mg in every bag) were simulated based on a dialysate effluent volume of 2.5 liters and systemic absorption of 70% for both antibiotics (11, 15, 20, 21). PITs and PBTs were then determined by using each patient’s dialysate and infecting pathogen. Patient and infection-related infor-
ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. MIC data for 36 study isolatesa Bacteria
Isolate no.
MIC (g/ml) Cefazolin
Tobramycin
S. epidermidis
14 18 20 23 25 26
4 0.25 0.5 2 2 1
⬎128 0.5 0.25 64 64 64
S. aureus
4 7 10 11 21 24
1 0.25 1 1 0.5 1
2 4 2 1 2 8
E. coli
12 15 16 28 30 9
2 2 4 2 16 4
4 4 4 4 64 4
K. pneumoniae
2 6 13 19 31 32
4 2 4 4 4 2
2 1 2 2 1 2
E. cloacae
1 3 22 5 29 17
⬎256 128 ⬎256 256 ⬎256 ⬎256
2 2 2 2 2 2
P. aeruginosa
37 30 33 34 35 36
⬎256 ⬎256 ⬎256 ⬎256 ⬎256 ⬎256
4 4 4 2 2 2
a NCCLS MIC breakpoints for cefazolin are ⱕ8 g/ml, susceptible; 16 g/ml, intermediate; and ⱖ32 g/ml, resistant. NCCLS breakpoints for tobramycin are ⱕ4 g/ml, susceptible; 8 g/ml, intermediate; and ⱖ16 g/ml, resistant.
mation, including demographics (age and weight), dialysis history, clinical presentation, treatment (antibiotic, dose, schedule, and duration) and clinical response, were obtained from medical records. Clinical cure was defined as resolution without clinical signs or symptoms of infection for 14 days after the discontinuation of antibiotic therapy. Failure was defined as inadequate clinical response requiring a modification of treatment or the removal of the catheter, infection relapse with the same pathogen within 14 days, or infection-related death. Statistical analysis. Data analysis was performed by using Microsoft Excel and SPSS version 11.1 software. Paired t test or analysis of variance was used to examine differences in dialysate biochemical compositions. Individual PFTs with medians and interquartile ranges were reported. For comparison, titers were plotted on scattergrams, with values two or more dilutions apart considered significantly different. Titers were also compared by using the nonparametric Mann-Whitney test. The relationship between PIT and MIC was characterized by comparing measured and calculated titers, where calculated titers were the antibiotic dialysate concentration divided by MIC. Since titers could not be calculated for combination regimens, the comparison was made only for the cefazolin regimen. In the pilot study, titers and MICs were compared between cases in which patients were cured and those in which patients failed treatment. All statistical tests were conducted by using an alpha of 0.05.
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FIG. 1. PITs for cefazolin plus tobramycin against single isolates of S. epidermidis (isolate number 20), S. aureus (isolate number 11), E. coli (isolate number 9), K. pneumoniae (isolate number 2), E. cloacae (isolate number 3), and P. aeruginosa (isolate number 30) using six dialysates (1 to 6).
RESULTS The processing procedure removed infecting pathogens from 31 of 32 spent dialysates, with bacteria detected in only one sample infected with Stenotrophomonas maltophilia. The biochemical composition of the six dialysates (pH 7.4 ⫾ 0.3) was as follows: sodium (129 ⫾ 4 mmol/liter), chloride (96 ⫾ 3 mmol/liter), potassium (3.6 ⫾ 0.5 mmol/liter), calcium (1.40 ⫾ 0.11 mmol/liter), magnesium (0.57 ⫾ 0.12 mmol/liter), bicarbonate (21.9 ⫾ 4.1 mmol/liter), phosphate (1.2 ⫾ 0.5 mmol/ liter), urea (16.3 ⫾ 1.2 mmol/liter), creatinine (525 ⫾ 111 mol/liter), protein (1.345 ⫾ 0.791 mg/liter), and glucose (23.0 ⫾ 12.6 mmol/liter). Growth studies confirmed sufficient bacterial growth over 24 h for all study isolates. MIC data for the 36 study isolates are provided in Table 1. Four S. epidermidis isolates were methicillin resistant and four were tobramycin resistant. All S. aureus, E. coli, and K. pneumoniae isolates were susceptible to cefazolin and tobramycin, with the exception of one E. coli (isolate number 30), which was intermediately susceptible to cefazolin and resistant to tobramycin. All E. cloacae and P. aeruginosa strains were resistant to cefazolin but susceptible to tobramycin. PFTs were highly reproducible. The results of 90 titers determined by two individuals in separate laboratories were the
same in 69 cases (77%) and within one dilution in 20 cases (22%). Furthermore, PIT and PBT values were comparable and within one dilution in 89% of the cases. Only PITs are reported since they provided similar measures of antibacterial effect and involved significantly less time and labor. PITs for cefazolin plus tobramycin used in six dialysates are shown in Fig. 1. Since titer results with the different dialysates used against the same isolate were similar (i.e., within one dilution of the median), one dialysate (number 3) was selected to test the remaining study isolates. PITs for cefazolin plus tobramycin against all 36 study isolates are shown in Fig. 2. Overall, PITs were significantly higher against gram-positive bacteria than gram-negative bacteria (P ⬍ 0.0001). Medians and interquartile ranges were similar for P. aeruginosa (median, 1/48; interquartile range, 1/32 to 1/64), E. cloacae (median, 1/32; interquartile range, 1/20 to 1/32), K. pneumoniae (median, 1/48; interquartile range, 1/32 to 1/64), and E. coli (median, 1/64; interquartile range, 1/40 to 1/64) and were higher for S. aureus (median, 1/128; interquartile range, 1/128 to 1/128) and S. epidermidis (median, 1/96; interquartile range, 1/64 to 1/224). PITs for cefazolin alone are depicted in Fig. 3. Again, PITs were significantly higher against gram-positive pathogens (P ⬍ 0.0001). There was no measurable activity against E.
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FIG. 2. PITs for cefazolin plus tobramycin against all study isolates.
cloacae or P. aeruginosa. PITs for cefazolin were relatively consistent against K. pneumoniae (median, 1/32; interquartile range, 1/32 to 1/32), E. coli (median, 1/32; interquartile range, 1/32 to 1/32), and S. aureus (median, 1/128; interquartile range, 1/128 to 1/128) and more variable against S. epidermidis (median, 1/48; interquartile range, 1/32 to 1/208). The scattergram in Fig. 4 compares PITs for cefazolin to those of cefazolin plus tobramycin (P ⫽ 0.007). The addition of tobramycin increased titers significantly from zero to values of 1/16 to 1/64 against E. cloacae and P. aeruginosa (P ⬍ 0.0001). There was no difference between measured and calculated PITs for cefazolin alone (Fig. 5). P. aeruginosa and E. cloacae were excluded from this analysis since cefazolin alone was inactive. In the pilot study, the processing procedure removed infecting pathogen from all spent dialysates, including one containing S. maltophilia. Patient demographics, infecting pathogen, treatment, PITs, and clinical outcome are presented in Table 2. The male-to-female ratio was 1:1.8, and the mean age was 65 ⫾ 12 years. Infecting pathogens included the Klebsiella species (2), Staphylococcus warneri (3), E. coli (2), S. aureus (2), S. epidermidis (2), Staphylococcus capitis (1), Aeromonas hydrophilia (1), and S. maltophilia (1). Antibiotic treatments also varied. Ten patients received empirical cefazolin (1,500 mg)
and tobramycin (60 mg) in a 6-h dianeal fluid (2 liters) dwell once daily, and another patient was given this regimen plus ceftazidime (250 mg) in each dwell. Two patients were treated with vancomycin (2,000 mg) once weekly plus tobramycin (60 mg) once daily, and another received cefazolin (1,500 mg) once daily plus ceftazidime (250 mg) in each dwell. Treatment failure was documented for four patients. As shown in Fig. 6, PITs were significantly associated with clinical outcome (P ⫽ 0.036). The median and interquartile ranges were 1:96 (1:64 to 1:224) for patients who were cured and 1:32 (1:24 to 1:40) for those who failed treatment. Cure rates were 90% (9 out of 10) for patients with PITs of ⱖ1/64 and only 25% (1 out of 4) for those with values of ⱕ1/32. There was not a significant association between the MIC of cefazolin and vancomycin (P ⫽ 0.11) or tobramycin (P ⫽ 0.32) and clinical response. DISCUSSION This study provides preliminary support of a pharmacodynamic association between PFT and treatment response for PD-related peritonitis. The significant results in the pilot study were despite a relatively small sample of patients with various infecting pathogens and antibiotic therapies. Traditionally,
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FIG. 3. PITs for cefazolin against all study isolates.
pharmacodynamic studies have related indices of antibiotic concentrations in serum and MIC (e.g., Cmax/MIC and %T ⬎ MIC, where T ⬎ MIC is time of dosing interval during which concentrations are above MIC) to microbiologic or clinical outcome. These data have then been used to suggest optimal antibiotic dosing in the management of surgical site (34), respiratory tract (13, 26), bloodstream (35), and other serious infections (8). Since MIC interpretations do not account for the unique factors in PD-related peritonitis, traditional pharmacodynamic indices may have limited, if any, relevance to this infection. First, MICs do not consider the much higher antibiotic concentrations achieved with intraperitoneal administration. Futhermore, MICs do not apply to antibiotic combinations often used for empirical therapy in PD-related peritonitis. Finally, there is evidence in vitro that antibiotic activity is different in dianeal fluid than in standard broth. Significant impairment in the activities of aminoglycosides, fluoroquinolones, cephalosporins, and vancomycin has been shown in the comparison of MICs and minimum bactericidal concentrations in dianeal fluid to standard broth (12, 28, 30, 31, 33).
The PFT test was adapted from the serum titer which uses patient serum collected during antibiotic treatment, usually at times of peak or trough concentrations (23). Serum titers are used to evaluate in vivo antibiotic pharmacodynamics by administering antibiotics to healthy volunteers, collecting serum samples, and measuring titers against various bacteria (16, 27). The test accounts for interpatient variability with regard to antibiotic concentrations and other serum-related variables such as complement factors. However, the role of serum titers in clinical practice has not been established (17). Initial investigations for infections like endocarditis (5, 18, 19) and osteomyelitis (29) have shown that serum titers provide variable results which are of limited clinical value. It is important to acknowledge, however, that clinical applicability depends on (i) the use of biological samples representative of an infection site, (ii) the provision of information not available through standard MICs or antibiotic concentrations, and (iii) an association with clinical outcome. For example, a study of patients with cystic fibrosis being treated for pulmonary infections found that peak serum bactericidal titers of 1/128 were over 90% predictive of cure, whereas those less than 1/16 were
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FIG. 4. PITs for cefazolin versus cefazolin plus tobramycin against all study isolates. The dashed line is the line of unity and solid lines are ⫾1 dilution. Solid squares indicate S. epidermidis, solid triangles indicate S. aureus, open squares indicate E. coli, open triangles indicate K. pneumoniae, open circles indicate E. cloacae, and closed circles indicate P. aeruginosa.
100% predictive of treatment failure in patients (2). Given the unique factors of PD-related peritonitis, the potential for PFTs is to provide an infection-specific and clinically relevant method of evaluating antibiotic therapies. Since PIT and PBT results were consistently similar, the former method was adopted based on ease and convenience. Although relatively uncommon, some cases such as those involving tolerant bacteria may be more appropriately tested with bactericidal titers. In a recent study, antibiotic activity against P. aeruginosa was measured in standard broth, fresh dianeal fluids (i.e., dextrose and icodextrin), and spent dialysate collected from patients with culture-negative peritonitis (33). Tolerance to ceftazidime and piperacillin was observed with MICs which were unchanged but minimum bactericidal concentrations which were significantly higher in dianeal fluid than in broth. In the present study, there was no evidence of antibiotic tolerance. There were no differences in titers with six different dialysates used against the same isolates (Fig. 1). Since antibiotic concentrations were standardized, differences among dialysate would be due to other dialysate-related factors. Numerous studies have described the effects of variables like pH, CO2, cations, and glucose on antibiotic activity in dianeal fluids (3, 7, 22). There may be direct effects on antibiotics, such as Ca⫹⫹ and Mg⫹⫹ facilitating aminoglycoside uptake, or indirect effects on bacterial growth. PFTs incorporate the dialysate and its components into the measure of overall antibacterial effect. Through serial dilution, however, the dialysate component is rapidly reduced, with 1/32, for example, representing a sample diluted in half five times. Hence, it was not surprising that dialysate-related factors other than antibiotic(s) appeared to have relatively little influence. PFTs for cefazolin alone were consistent with antibiotic concentration in relation to MIC as shown in Fig. 5. In other
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 5. Measured versus calculated PITs for cefazolin against 24 clinical isolates, where the dashed line is the line of unity and solid lines are ⫾1 dilution. Solid squares indicate S. epidermidis, solid triangles indicate S. aureus, open squares indicate E. coli, and open triangles indicate K. pneumoniae.
words, measured titers were very similar to those calculated by dividing the antibiotic dialysate concentration by MIC. However, it is important to consider that using dialysate containing standard antibiotic concentrations would significantly strengthen the correlation between PFT and MIC. It is expected that PFTs using patient dialysate collected during treatment would be more variable because of significant interpatient variability in antibiotic concentrations. Hence, it would also follow that PFTs determined by this method may be more indicative of antibacterial effect and have a stronger association with subsequent clinical outcome. Notably, a significant association with treatment response was observed with PIT but not with MIC. A potential explanation for this finding is the presence of antibiotic combinations which would be considered in the measure of antibacterial activity by PFTs but not by MICs. In other words, it is difficult, if not impossible, to inter-
FIG. 6. PITs determined for 14 patients with PD-related peritonitis by using their dialysate and infecting pathogen.
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TABLE 2. PITs and clinical outcome in pilot study of 14 patients with PD-related peritonitis Patient no.
Age (yr)
Gender
Infecting pathogen
Treatment regimen (MIC [g/ml])
PIT
Outcome
1 2 3 4 5 6 7 8 9 10 11 12 13 14
50 65 52 52 70 71 71 79 60 83 45 72 72 71
Female Male Female Female Male Female Female Female Female Female Male Female Male Male
K. pneumoniae S. maltophilia E. coli E. coli S. warneri A. hydrophila S. capitis S. warneri S. aureus S. epidermidis S. epidermidis K. pneumoniae S. warneri S. aureus
Cefazolin (8) ⫹ tobramycin (16) Cefazolin (⬎256) ⫹ ceftazidime (64) Cefazolin (16) ⫹ tobramycin (8) Cefazolin (8) ⫹ tobramycin (8) Cefazolin (1) ⫹ tobramycin (32) Cefazolin (⬎32) ⫹ tobramycin (16) Cefazolin (64) ⫹ tobramycin (0.25) Cefazolin (2) ⫹ tobramycin (1) Cefazolin (2) ⫹ tobramycin (2) Cefazolin (64) ⫹ tobramycin (1) Cefazolin (64) ⫹ tobramycin (1) Cefazolin (8) ⫹ tobramycin (1) ⫹ ceftazidime (16) Vancomycin (4) ⫹ tobramycin (1) Vancomycin (4) ⫹ tobramycin (8)
1/64 1/2 1/32 1/32 1/64 1/16 1/64 ⬎1/256 1/128 1/128 1/64 1/64 ⬎1/256 ⬎1/256
Failure Failure Failure Failure Cure Cure Cure Cure Cure Cure Cure Cure Cure Cure
pret the pharmacodynamics of antibiotic combinations from multiple MICs. This study exemplifies the potential role of the PFT in evaluating and comparing antibiotic therapies for PD-related peritonitis. PFTs could be used to test empirical antibiotic regimens using similar methods. Spent dialysate could be collected, processed, and spiked with the study regimens. Next, titers against numerous clinical isolates representing the most prevalent peritoneal pathogens could be determined. Alternatively, PFTs could be used to study pathogendirected therapies. For example, various antibiotic regimens (e.g., ceftazidime plus tobramycin and ceftazidime plus ciprofloxacin) could be tested against a representative sample of P. aeruginosa isolated from patients with PD-related peritonitis. The PFT described in this study has important limitations. The test was developed for culture-positive bacterial PD-related peritonitis with single pathogens which grow adequately in spent dialysate and standard broth. For example, alphahemolytic streptococcus, another clinically relevant but fastidious pathogen, was not tested. As a measure of overall antibacterial effect in dialysate, the PFT may not represent antibiotic activity in biofilm, which can form on catheters or abdominal cavity surfaces. Although the prevalence and implications of biofilm in peritonitis are not fully understood, antibiotic activity can be impaired by reductions in drug penetration or alterations in bacterial metabolism (6). Infections which are persistent, relapsing, or associated with exit-site or tunnel infections are considered more likely to have biofilm involvement. In conclusion, this study provides preliminary support for the PFT as a pharmacodynamic index specific to the treatment of PD-related peritonitis. With further characterization and validation in patients, the PFT may advance the study of antibiotic therapies for PD-related peritonitis. ACKNOWLEDGMENTS Funding was provided by the Dr. Paul H. T. Thorlakson Foundation Fund (University of Manitoba). C.S. was supported by graduate student awards from the Kidney Foundation of Canada (Manitoba Branch) and the Leslie F. Buggey Scholarship.
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