Specific Opsonic Activity for Staphylococci in Peritoneal Dialysis Effluent During Continuous Ambulatory Peritoneal Dialysis Henrik Nielsen, Frank Espersen, Arsalan Kharazmi, Steen Antonsen, Ellen Ejlersen, Preben Joffe, and Fritz Bangsgaard Pedersen • In a prospective study of intraperitoneal opsonins in 30 patients undergoing continuous ambulatory peritoneal dialysis (CAPO), the IgG concentration, the fibronectin concentration, the specific antistaphylococcal antibody level, and the opsonic activity against Staphylococcus epidermidis were measured in peritoneal dialysis effluent from the initiation of CAPO and monthly for 6 months. Significant correlation was found between the four assays, but the interindividual and intraindividual variations were considerable. No statistically significant correlation was observed between susceptibility of the patients to CAPO-related infectious peritonitis and any of the above-mentioned parameters of humoral defense. We conclude that at the present time it is not feasible to use these assays for the establishment of prognosis with regard to peritonitis in CAPO. © 1992 by the National Kidney Foundation, Inc. INDEX WOROS: Continuous ambulatory peritoneal dialysis; opsonin; peritonitis; staphylococci.
P
ERITONITIS remains one of the major complications for patients treated with continuous ambulatory peritoneal dialysis (CAPD).I The risk of peritonitis probably results from the presence of the peritoneal dialysis catheter and from relatively impaired immune defenses in the peritoneal cavity. Furthermore, the frequent removal of opsonins and phagocytic cells from the peritoneal cavity is likely to predispose the patient to infection.2 Peritoneal phagocytic cells ofCAPD patients in general show normal functions, although incubation in commercial dialysis fluids impairs their function. 3-6 The opsonic capacity of peritoneal dialysis effluent (PDE) has been investigated by several groups and both IgG concentrations and opsonic capacity were reported to predict a later peritonitis episode/- IO although recently other investigators have failed to confirm this correlation. II
The most frequent microbial pathogen isolated from PDE is Staphylococcus epidermidis, and a correlation between immunoglobulin concentration and frequency of S epidermidis peritonitis has been observed in some studies/,8 but not in others. IO This may relate to the fact that the immunoglobulin content ofPDE with S epidermidis specificity varies between patients and suggests that a more relevant prognostic factor could be the level of specific antistaphylococcal immunoglobulin in PDE. Moreover, previous studies have measured immunological parameters at a single random day without examining the fluctuations in these assays during CAPD. We report here a longitudinal study of CAPD patients monitored by monthly evaluation of PDE IgG concentration, fibronectin concentration, opsonic activity, and specific antistaphylococcal antibody titer. MATERIALS AND METHODS
From the Department of Clinical Microbiology, Rigshospitalet, Stat ens Seruminstitut, Copenhagen; the Department of Clinical Chemistry, Odense University Hospital; the Department of Nephrology, Herlev University Hospital; the Department of Nephrology, Hvidovre University Hospital; and the Department of Nephrology, Odense University Hospital, Denmark. Received January 2, 1992; accepted in revised form June 2, 1992. Supported by the Danish Medical Research Council (Grants No. 12-7813 and 12-9510) and Danish Hospital Foundation for Medical Research, Region of Copenhagen, The Faroe Islands and Greenland. Address reprint requests to Henrik Nielsen, MD, PhD, Department of Clinical Microbiology 7806, Rigshospitalet, 20 Tagensvej, DK-2200 Copenhagen N, Denmark. © 1992 by the National Kidney Foundation, Inc. 0272-6386/92/2004-0009$3.00;0 372
Patient Population Thirty patients with end-stage renal failure (eight women and 22 men; age range, 24 to 80 years) were prospectively studied from the start of CAPD and for a minimum of 6 months or until CAPD was stopped. The diagnoses were as follows: glomerulonephritis 11, diabetic nephropathy 5, pyelonephritis 4, polycystic renal disease 3, miscellaneous 7. Twenty-four patients completed treatment for 6 months or more. The reasons for stopping the CAPD treatment before 6 months were dead of other causes in three, recurrent peritonitis in two, and renal transplantation in one. The patients were treated with standard 2-L exchanges four times daily. The dialysis solutions used contained 1.5% or 2.5% dextrose (Dianeal, Baxter-Healthcare). Informed consent was obtained from the patients and the study was approved by the Ethical Committee of Copenhagen County.
American Journal of Kidney Diseases, Vol XX, No 4 (October), 1992: pp 372-375
373
INTRAPERITONEAL OPSONIN IN CAPO
Dialysis Fluid Samples
Measurement of Opsonic Activity of Dialysate
PDE samples were obtained from the first overnight dwell after start of CAPD and monthly thereafter for 6 months. Only samples from uninfected patients were investigated. After centrifugation, cell-free samples were stored frozen in small aliquots. In addition, samples ofPDE from five CAPD subjects unrelated to this study were collected and frozen in small aliquots (pooled normal PDE).
A chemiluminescence assay was used to quantitate opsonic activity by measuring the oxidative burst response of PMNs following stimulation with bacteria. In brief, 105 PMNs were incubated with lOB opsonized bacteria in a final volume of 1.0 mL Krebs-Ringer solution containing luminol 10-4 mol/ L in glass scintillation vials. Control vials with nonopsonized bacteria and without bacteria were run in parallel. Sequential counts were measured in an LKB (Turku, Finland) 12511uminometer every 2 minutes for 45 minutes at 22°e. Results are expressed as the peak cpm of opsonized bacteria relative to the peak response of serum-opsonized bacteria. Responses of PMNs stimulated with nonopsonized bacteria and with medium alone were very low «5% of peak response). Interassay variations due to differences in donor PMN reactivity (coefficient of variation for this assay, -46%) were eliminated, as the results are expressed as relative to the response of serumopsonized bacteria. The intraassay coefficient of variation was always less than 10%.
Quantitation of JgG in Eifluent Dialysate The concentrations of IgG in PDE were determined by turbidimetry with antibodies from Dakopatts, Glostrup, Denmark. The interassay coefficient of variation was 2%.
Measurement of S epidermidis Antibodies IgG against S epidermidis antigens was measured using an enzyme-linked immunoadsorbent assay (ELISA) modified after a previously published method. 12 Briefly, immunoplates (Nunc, Copenhagen, Denmark) were coated with ultrasonic extract of S epidermidis, ATCC 14990, diluted to 50 /lg proteinjmL phosphate-buffered saline (PBS) for I hour at 20°e. After washing and blocking with PBS containing 0.1 % Tween 20, PDEs were diluted I: 100 in the same buffer and applied in triplicates. Incubation was performed for I hour at 20°e. After washing IgG bound to S epidermidis, antigens were detected by addition of peroxidase-conjugated rabbit antibodies to human IgG and subsequent staining reaction. Interassay and intraassay coefficients of variation were 14% and 12%, respectively. 12 The pool of PDEs from the five persons not involved in this study were tested in each plate to standardize the assay. The results were expressed as ELISA ratios by dividing the result of the test sample by the result of the pool. A specific ELISA ratio was calculated by dividing the ELISA ratio by the corresponding IgG concentration.
Preparation of Polymorphonuclear Neutrophils Peripheral venous blood from healthy donors was separated by dextran sedimentation followed by metrizoate-polysucrose density gradient centrifugation (Lymphoprep, Nyegaard, Oslo, Norway) to isolate polymorphonuclear neutrophils (PMNs). The cells were washed twice in Gey's solution, and erythrocytes were removed by hypotonic lysis. Purity ofPMNs was greater than 98% by morphological criteria, and the viability was greater than 95% assessed by a trypan blue exclusion test. The PMNs were used immediately after isolation.
Opsonization of Bacteria A clinical isolate of S epidermidis obtained from dialysate during a CAPD peritonitis was used. The bacteria culture was grown overnight in Mueller-Hinton broth, washed in KrebsRinger solution, and resuspended to a final concentration of 109jmL as determined by a spectrophotometric method. Serum from healthy donors was kept frozen in small aliquots and the same batch was used throughout the study. Bacteria were opsonized for 30 minutes at 37°C with PDE or with pooled human serum, washed in Krebs-Ringer solution, and resuspended to a final concentration of 109jmL.
Fibronectin Measurement The concentration offibronectin in PDE was measured by immune electrophoresis with human purified fibronectin as standard, as previously described. 13 The interassay coefficient of variation was 18%Y
Statistical Analysis For calculation of differences between initial and subsequent measurements in the same patient, Wilcoxon one-sample test was used. For differences between patient subgroups, MannWhitney test was used. Correlation between different assays was evaluated by Spearman rank correlation test. A significance level of 5% was chosen for all calculations.
RESULTS
The concentration ofIgG in PDE after the first overnight dwell was 299 ± 408 mg/mL (mean ± SD) in all the patients investigated, with a range from 37 to 2,100 mg/mL. After 1 month of CAPD, the IgG concentration was 169 ± 115 mg/mL (n = 27). The specific antistaphylococcal antibody ELISA was 0.0190 ± 0.0130 in the first overnight sample (n = 30) and 0.0196 ± 0.0137 after I month on CAPD (n = 27). The opsonic activity of PDE was 26% ± 27% of the response of pooled human serum in the first overnight sample (n = 30), with a range from 0% to 116%, and 24% ± 26% after 1 month ofCAPD (n = 27). The level offibronectin in PDE after 1 month of CAPD was 0.21 ± 0.08 p,mol/L (n = 24). The IgG concentration, the ELISA values, and the opsonic activity did not change significantly during the following 6 months of CAPD (Wilcoxon one-sample test). The correlation between assays of PDE from the first overnight dwell after start of CAPD was
374
statistically significant between ELISA titer and opsonic activity and highly significant between IgG concentration and ELISA titer. However, the correlation between IgG and opsonic activity was not statistically significant. In samples from POE after 1 month of CAPO, the correlations showed an even higher degree of significance, whereas IgG and opsonic activity was without statistical significant correlation (Table 1). For all parameters, a substantial interindividual and intraindividual variation was observed. The range of IgG concentration was 37 to 529 mg/mL in the patient with the most pronounced variation, and the median standard deviation of IgG variation was 63 mg/mL (38% of the median IgG concentration) in 24 patients, who had six or more monthly measurements. For opsonic activity, the intraindividual variation was considerable as well, and the median standard deviation of opsonic activity was 8.5% (49% of the median opsonic activity) in 24 patients, who had six or more monthly measurements. Of 29 patients monitored for more than 1 month, 15 had one or more episodes of peritonitis during 222 months of observation, whereas 14 patients had no infectious complications during an observation period of 150 months. The microbiological etiology of the infectious episodes showed S epidermidis in 13 cases (eight patients), Staphylococcus aureus in three cases (three patients), streptococci in two cases (two patients), gram-negative bacteria in two cases (one patient), and fungal peritonitis in one case. The two groups of patients, with and without peritonitis, had no statistically significant difference in POE IgG, fibronectin concentration, ELISA titer, or opsonic activity in the samples from the first overnight dwell or from samples taken after 1 month of CAPO. Comparing the group of eight patients with S epidermidis peritonitis with the group of 14 patients without any peritonitis episodes also failed to detect statistically significant differences in IgG, fibronectin concentration, ELISA titer, or opsonic activity against S epidermidis. Moreover, we could not identify clinical or paraclinical features distinguishing patients with recurring S epidermidis peritonitis from the other patient groups. It was not possible to identify significant alterations in immunological parameters in POE obtained immediately before or after an episode of peritonitis in these patients. Last, no differences
NIELSEN ET AL Table 1. Correlation Between Humoral Immune Parameters in POEs From Patients Treated With CAPO for 1 Month Anti-8 epidermidis
IgG
p
= 0.82
P < 0.00001
Anti-8 epidermidis Opsonic activity
Opsonic Activity
Fibronectin
= 0.30
NO
p
P = 0.13 p
p
= 0.50
= 0.50
NO
P = 0.018
P = 0.018
p
= 0.41
P = 0.04
NOTE. Analyses of correlations were performed by Spearman rank correlation test (n = 26). Abbreviation: NO, not determined.
in the immunological parameters could be demonstrated between diabetic and nondiabetic patients. DISCUSSION
A reliable prognostic parameter of local peritoneal immunocompetence in CAPO patients would be of potential clinical interest, since patients with a high risk of peritonitis could be offered prophylactic measures. If this should have practical application, the evaluation has to be performed at the initiation (eg, within 1 month) of CAPO. Several investigators have focused on the IgG concentration and the opsonic activity of POE. A significant correlation between these immunological parameters and bacterial peritonitis has been reported,7.9 but other studies have failed to confirm this. IO • 11 A common feature of these previous studies has been the inclusion of patients already undergoing CAPO for a variable time period and the performance of only one measurement in the evaluation of its clinical significance. The present study was designed to give information in a prospective manner on the possible fluctuations in these areas of peritoneal humoral immunity from the very beginning of CAPO and during a longitudinal follow-up. A surprisingly high intraindividual variation during monthly assessments was observed without identifiable association with the clinical course of the CAPO treatment. This fluctuation may have been missed by the previous investigators in the field/-II and the discrepancy between those reporting correlation7-9 and those failing to confirm such
375
INTRAPERITONEAL OPSONIN IN CAPO
a correlation 10, 11 may simply be a matter of random sampling of POE with a high variation of intraindividual measurements. It was found that strong correlations existed between the different assays, that is, opsonic activity against S epidermidis was linked to specific IgG levels toward that microorganism and to fibronectin concentration. Noteworthy, the total IgG concentration of POE was not correlated to opsonic activity in our study, in contrast to previous observations. 7 ,10 However, it has been noted that POE samples with high IgG concentration might have low opsonic activity anyway, 7 suggesting that qualitative factors such as specificity are important. One of the non-IgG humoral factors active in opsonization is fibronectin, since this molecule has a binding site for staphylococci. Fibronectin was present in the POE samples and contributed to the opsonic activity, as a significant correlation was demonstrated. The concentration of fibronectin in POE has been correlated to the infection rate during CAPO, 14 but, like the other humoral factors studied by us, fibronectin did not have
prognostic value concerning peritonitis. However, the comparison on fibronectin is difficult, since the levels of fibronectin reported by Goldstein et al 14 are several-fold lower than those reported by us. A possible explanation could be the well-known instability of fibronectin during the thawing process if not performed at 37°C, or the aggregation in cold. We conclude that for the four different assays of humoral immunity evaluated prospectively no significant correlation with the clinical susceptibility to peritonitis could be established. This relates primarily to a considerable variation during the CAPO treatment, which may also explain the discrepancy with the results of other studies, which evaluated only one sample per patient. Thus, none of the assays of the present study are suitable for predicting the prognosis of bacterial peritonitis in CAPO. ACKNOWLEDGMENT The expert technical assistance of Hanne Tamstorf and Lotte Corneliussen is appreciated. Inge Clemmensen, MD, PhD, is thanked for help with the measurements of fibronectin.
REFERENCES 1. Peterson PK, Matzke G, Keane WF: Current concepts in the management of peritonitis in patients undergoing continuous ambulatory peritoneal dialysis. Rev Infect Dis 9:604612, 1987 2. Verbrugh HA, Keane WF, Hoidal JR, et al: Peritoneal macrophages and opsonins: Antibacterial defense in patients undergoing chronic peritoneal dialysis. J Infect Dis 147: 10181029, 1983 3. Duwe AK, Vas SI, Weatherhead JW: Effects of the composition of peritoneal dialysis fluid on chemiluminescence, phagocytosis and bactericidal activity in vitro. Infect Immun 33:130-135,1981 4. Peterson PK, Gaziano E, Suh HJ, et al: Antimicrobial activities of dialysate-elicited and resident human peritoneal macrophages. Infect Immun 49:212-218, 1985 5. Alobaidi HM, Coles GA, Davies M, et al: Host defense in continuous ambulatory peritoneal dialysis: The effect of the dialysate on phagocyte function. Nephrol Dial Transplant 1:16-21,1986 6. van Bronswijk H, Verbrugh HA, Heezius HCJM, et al: Dialysis fluids and local host resistance in patients on continuous ambulatory peritoneal dialysis. Eur J Clin Microbiol Infect Dis 7:368-373, 1988 7. Keane WF, Comty CM, Verbrugh HA, et al: Opsonic deficiency of peritoneal dialysis effluent in continuous ambulatory peritoneal dialysis. Kidney Int 25:539-543, 1984 8. Lamperi S, Carozzi S: Defective opsonic activity of peri-
toneal effluent during continuous ambulatory peritoneal dialysis (CAPD): Importance and prevention. Perit Dial Bull 6: 87-92,1986 9. Coles GA, Alobaidi HMM, Topley N, et al: Opsonic activity of dialysis effluent predicts those at risk of Staphylococcus epidermidis peritonitis. Nephrol Dial Transplant 2: 359-365, 1987 10. McGregor SJ, Brock JH, Briggs JD, et al: Relationship of IgG, C3 and transferrin with opsonising and bacteristatic activity of peritoneal fluid from CAPD patients and the incidence of peritonitis. Nephrol Dial Transplant 2:551-556, 1987 11. Gordon DL, Rice JL, Avery VM: Surface phagocytosis and host defense in the peritoneal cavity during continuous ambulatory peritoneal dialysis. Eur J C1in Microbiol Infect Dis 9:191-197, 1990 12. Espersen F, Wheat U, Bemis AT, et al: Enzyme-linked immunosorbent assay for detection of Staphylococcus epidermidis antibody in experimental S. epidermidis endocarditis. J Clin Microbiol 23:339-342, 1986 13. Eriksen HO, Clemmensen I, Hansen MS, et al: Plasma fibronectin concentration in normal subjects. Scand J Clin Lab Invest 42:291-295,1982 14. Goldstein CS, Garrick RE, Polin RA, et al: Fibronectin and complement secretion by monocytes and peritoneal macrophages in vitro from patients undergoing continuous ambulatory peritoneal dialysis. J Leukoc Bioi 39:457-464, 1986
P
ERITONITIS remains one of the major complications for patients treated with continuous ambulatory peritoneal dialysis (CAPD).I The risk of peritonitis probably results from the presence of the peritoneal dialysis catheter and from relatively impaired immune defenses in the peritoneal cavity. Furthermore, the frequent removal of opsonins and phagocytic cells from the peritoneal cavity is likely to predispose the patient to infection.2 Peritoneal phagocytic cells ofCAPD patients in general show normal functions, although incubation in commercial dialysis fluids impairs their function. 3-6 The opsonic capacity of peritoneal dialysis effluent (PDE) has been investigated by several groups and both IgG concentrations and opsonic capacity were reported to predict a later peritonitis episode/- IO although recently other investigators have failed to confirm this correlation. II
The most frequent microbial pathogen isolated from PDE is Staphylococcus epidermidis, and a correlation between immunoglobulin concentration and frequency of S epidermidis peritonitis has been observed in some studies/,8 but not in others. IO This may relate to the fact that the immunoglobulin content ofPDE with S epidermidis specificity varies between patients and suggests that a more relevant prognostic factor could be the level of specific antistaphylococcal immunoglobulin in PDE. Moreover, previous studies have measured immunological parameters at a single random day without examining the fluctuations in these assays during CAPD. We report here a longitudinal study of CAPD patients monitored by monthly evaluation of PDE IgG concentration, fibronectin concentration, opsonic activity, and specific antistaphylococcal antibody titer. MATERIALS AND METHODS
From the Department of Clinical Microbiology, Rigshospitalet, Stat ens Seruminstitut, Copenhagen; the Department of Clinical Chemistry, Odense University Hospital; the Department of Nephrology, Herlev University Hospital; the Department of Nephrology, Hvidovre University Hospital; and the Department of Nephrology, Odense University Hospital, Denmark. Received January 2, 1992; accepted in revised form June 2, 1992. Supported by the Danish Medical Research Council (Grants No. 12-7813 and 12-9510) and Danish Hospital Foundation for Medical Research, Region of Copenhagen, The Faroe Islands and Greenland. Address reprint requests to Henrik Nielsen, MD, PhD, Department of Clinical Microbiology 7806, Rigshospitalet, 20 Tagensvej, DK-2200 Copenhagen N, Denmark. © 1992 by the National Kidney Foundation, Inc. 0272-6386/92/2004-0009$3.00;0 372
Patient Population Thirty patients with end-stage renal failure (eight women and 22 men; age range, 24 to 80 years) were prospectively studied from the start of CAPD and for a minimum of 6 months or until CAPD was stopped. The diagnoses were as follows: glomerulonephritis 11, diabetic nephropathy 5, pyelonephritis 4, polycystic renal disease 3, miscellaneous 7. Twenty-four patients completed treatment for 6 months or more. The reasons for stopping the CAPD treatment before 6 months were dead of other causes in three, recurrent peritonitis in two, and renal transplantation in one. The patients were treated with standard 2-L exchanges four times daily. The dialysis solutions used contained 1.5% or 2.5% dextrose (Dianeal, Baxter-Healthcare). Informed consent was obtained from the patients and the study was approved by the Ethical Committee of Copenhagen County.
American Journal of Kidney Diseases, Vol XX, No 4 (October), 1992: pp 372-375
373
INTRAPERITONEAL OPSONIN IN CAPO
Dialysis Fluid Samples
Measurement of Opsonic Activity of Dialysate
PDE samples were obtained from the first overnight dwell after start of CAPD and monthly thereafter for 6 months. Only samples from uninfected patients were investigated. After centrifugation, cell-free samples were stored frozen in small aliquots. In addition, samples ofPDE from five CAPD subjects unrelated to this study were collected and frozen in small aliquots (pooled normal PDE).
A chemiluminescence assay was used to quantitate opsonic activity by measuring the oxidative burst response of PMNs following stimulation with bacteria. In brief, 105 PMNs were incubated with lOB opsonized bacteria in a final volume of 1.0 mL Krebs-Ringer solution containing luminol 10-4 mol/ L in glass scintillation vials. Control vials with nonopsonized bacteria and without bacteria were run in parallel. Sequential counts were measured in an LKB (Turku, Finland) 12511uminometer every 2 minutes for 45 minutes at 22°e. Results are expressed as the peak cpm of opsonized bacteria relative to the peak response of serum-opsonized bacteria. Responses of PMNs stimulated with nonopsonized bacteria and with medium alone were very low «5% of peak response). Interassay variations due to differences in donor PMN reactivity (coefficient of variation for this assay, -46%) were eliminated, as the results are expressed as relative to the response of serumopsonized bacteria. The intraassay coefficient of variation was always less than 10%.
Quantitation of JgG in Eifluent Dialysate The concentrations of IgG in PDE were determined by turbidimetry with antibodies from Dakopatts, Glostrup, Denmark. The interassay coefficient of variation was 2%.
Measurement of S epidermidis Antibodies IgG against S epidermidis antigens was measured using an enzyme-linked immunoadsorbent assay (ELISA) modified after a previously published method. 12 Briefly, immunoplates (Nunc, Copenhagen, Denmark) were coated with ultrasonic extract of S epidermidis, ATCC 14990, diluted to 50 /lg proteinjmL phosphate-buffered saline (PBS) for I hour at 20°e. After washing and blocking with PBS containing 0.1 % Tween 20, PDEs were diluted I: 100 in the same buffer and applied in triplicates. Incubation was performed for I hour at 20°e. After washing IgG bound to S epidermidis, antigens were detected by addition of peroxidase-conjugated rabbit antibodies to human IgG and subsequent staining reaction. Interassay and intraassay coefficients of variation were 14% and 12%, respectively. 12 The pool of PDEs from the five persons not involved in this study were tested in each plate to standardize the assay. The results were expressed as ELISA ratios by dividing the result of the test sample by the result of the pool. A specific ELISA ratio was calculated by dividing the ELISA ratio by the corresponding IgG concentration.
Preparation of Polymorphonuclear Neutrophils Peripheral venous blood from healthy donors was separated by dextran sedimentation followed by metrizoate-polysucrose density gradient centrifugation (Lymphoprep, Nyegaard, Oslo, Norway) to isolate polymorphonuclear neutrophils (PMNs). The cells were washed twice in Gey's solution, and erythrocytes were removed by hypotonic lysis. Purity ofPMNs was greater than 98% by morphological criteria, and the viability was greater than 95% assessed by a trypan blue exclusion test. The PMNs were used immediately after isolation.
Opsonization of Bacteria A clinical isolate of S epidermidis obtained from dialysate during a CAPD peritonitis was used. The bacteria culture was grown overnight in Mueller-Hinton broth, washed in KrebsRinger solution, and resuspended to a final concentration of 109jmL as determined by a spectrophotometric method. Serum from healthy donors was kept frozen in small aliquots and the same batch was used throughout the study. Bacteria were opsonized for 30 minutes at 37°C with PDE or with pooled human serum, washed in Krebs-Ringer solution, and resuspended to a final concentration of 109jmL.
Fibronectin Measurement The concentration offibronectin in PDE was measured by immune electrophoresis with human purified fibronectin as standard, as previously described. 13 The interassay coefficient of variation was 18%Y
Statistical Analysis For calculation of differences between initial and subsequent measurements in the same patient, Wilcoxon one-sample test was used. For differences between patient subgroups, MannWhitney test was used. Correlation between different assays was evaluated by Spearman rank correlation test. A significance level of 5% was chosen for all calculations.
RESULTS
The concentration ofIgG in PDE after the first overnight dwell was 299 ± 408 mg/mL (mean ± SD) in all the patients investigated, with a range from 37 to 2,100 mg/mL. After 1 month of CAPD, the IgG concentration was 169 ± 115 mg/mL (n = 27). The specific antistaphylococcal antibody ELISA was 0.0190 ± 0.0130 in the first overnight sample (n = 30) and 0.0196 ± 0.0137 after I month on CAPD (n = 27). The opsonic activity of PDE was 26% ± 27% of the response of pooled human serum in the first overnight sample (n = 30), with a range from 0% to 116%, and 24% ± 26% after 1 month ofCAPD (n = 27). The level offibronectin in PDE after 1 month of CAPD was 0.21 ± 0.08 p,mol/L (n = 24). The IgG concentration, the ELISA values, and the opsonic activity did not change significantly during the following 6 months of CAPD (Wilcoxon one-sample test). The correlation between assays of PDE from the first overnight dwell after start of CAPD was
374
statistically significant between ELISA titer and opsonic activity and highly significant between IgG concentration and ELISA titer. However, the correlation between IgG and opsonic activity was not statistically significant. In samples from POE after 1 month of CAPO, the correlations showed an even higher degree of significance, whereas IgG and opsonic activity was without statistical significant correlation (Table 1). For all parameters, a substantial interindividual and intraindividual variation was observed. The range of IgG concentration was 37 to 529 mg/mL in the patient with the most pronounced variation, and the median standard deviation of IgG variation was 63 mg/mL (38% of the median IgG concentration) in 24 patients, who had six or more monthly measurements. For opsonic activity, the intraindividual variation was considerable as well, and the median standard deviation of opsonic activity was 8.5% (49% of the median opsonic activity) in 24 patients, who had six or more monthly measurements. Of 29 patients monitored for more than 1 month, 15 had one or more episodes of peritonitis during 222 months of observation, whereas 14 patients had no infectious complications during an observation period of 150 months. The microbiological etiology of the infectious episodes showed S epidermidis in 13 cases (eight patients), Staphylococcus aureus in three cases (three patients), streptococci in two cases (two patients), gram-negative bacteria in two cases (one patient), and fungal peritonitis in one case. The two groups of patients, with and without peritonitis, had no statistically significant difference in POE IgG, fibronectin concentration, ELISA titer, or opsonic activity in the samples from the first overnight dwell or from samples taken after 1 month of CAPO. Comparing the group of eight patients with S epidermidis peritonitis with the group of 14 patients without any peritonitis episodes also failed to detect statistically significant differences in IgG, fibronectin concentration, ELISA titer, or opsonic activity against S epidermidis. Moreover, we could not identify clinical or paraclinical features distinguishing patients with recurring S epidermidis peritonitis from the other patient groups. It was not possible to identify significant alterations in immunological parameters in POE obtained immediately before or after an episode of peritonitis in these patients. Last, no differences
NIELSEN ET AL Table 1. Correlation Between Humoral Immune Parameters in POEs From Patients Treated With CAPO for 1 Month Anti-8 epidermidis
IgG
p
= 0.82
P < 0.00001
Anti-8 epidermidis Opsonic activity
Opsonic Activity
Fibronectin
= 0.30
NO
p
P = 0.13 p
p
= 0.50
= 0.50
NO
P = 0.018
P = 0.018
p
= 0.41
P = 0.04
NOTE. Analyses of correlations were performed by Spearman rank correlation test (n = 26). Abbreviation: NO, not determined.
in the immunological parameters could be demonstrated between diabetic and nondiabetic patients. DISCUSSION
A reliable prognostic parameter of local peritoneal immunocompetence in CAPO patients would be of potential clinical interest, since patients with a high risk of peritonitis could be offered prophylactic measures. If this should have practical application, the evaluation has to be performed at the initiation (eg, within 1 month) of CAPO. Several investigators have focused on the IgG concentration and the opsonic activity of POE. A significant correlation between these immunological parameters and bacterial peritonitis has been reported,7.9 but other studies have failed to confirm this. IO • 11 A common feature of these previous studies has been the inclusion of patients already undergoing CAPO for a variable time period and the performance of only one measurement in the evaluation of its clinical significance. The present study was designed to give information in a prospective manner on the possible fluctuations in these areas of peritoneal humoral immunity from the very beginning of CAPO and during a longitudinal follow-up. A surprisingly high intraindividual variation during monthly assessments was observed without identifiable association with the clinical course of the CAPO treatment. This fluctuation may have been missed by the previous investigators in the field/-II and the discrepancy between those reporting correlation7-9 and those failing to confirm such
375
INTRAPERITONEAL OPSONIN IN CAPO
a correlation 10, 11 may simply be a matter of random sampling of POE with a high variation of intraindividual measurements. It was found that strong correlations existed between the different assays, that is, opsonic activity against S epidermidis was linked to specific IgG levels toward that microorganism and to fibronectin concentration. Noteworthy, the total IgG concentration of POE was not correlated to opsonic activity in our study, in contrast to previous observations. 7 ,10 However, it has been noted that POE samples with high IgG concentration might have low opsonic activity anyway, 7 suggesting that qualitative factors such as specificity are important. One of the non-IgG humoral factors active in opsonization is fibronectin, since this molecule has a binding site for staphylococci. Fibronectin was present in the POE samples and contributed to the opsonic activity, as a significant correlation was demonstrated. The concentration of fibronectin in POE has been correlated to the infection rate during CAPO, 14 but, like the other humoral factors studied by us, fibronectin did not have
prognostic value concerning peritonitis. However, the comparison on fibronectin is difficult, since the levels of fibronectin reported by Goldstein et al 14 are several-fold lower than those reported by us. A possible explanation could be the well-known instability of fibronectin during the thawing process if not performed at 37°C, or the aggregation in cold. We conclude that for the four different assays of humoral immunity evaluated prospectively no significant correlation with the clinical susceptibility to peritonitis could be established. This relates primarily to a considerable variation during the CAPO treatment, which may also explain the discrepancy with the results of other studies, which evaluated only one sample per patient. Thus, none of the assays of the present study are suitable for predicting the prognosis of bacterial peritonitis in CAPO. ACKNOWLEDGMENT The expert technical assistance of Hanne Tamstorf and Lotte Corneliussen is appreciated. Inge Clemmensen, MD, PhD, is thanked for help with the measurements of fibronectin.
REFERENCES 1. Peterson PK, Matzke G, Keane WF: Current concepts in the management of peritonitis in patients undergoing continuous ambulatory peritoneal dialysis. Rev Infect Dis 9:604612, 1987 2. Verbrugh HA, Keane WF, Hoidal JR, et al: Peritoneal macrophages and opsonins: Antibacterial defense in patients undergoing chronic peritoneal dialysis. J Infect Dis 147: 10181029, 1983 3. Duwe AK, Vas SI, Weatherhead JW: Effects of the composition of peritoneal dialysis fluid on chemiluminescence, phagocytosis and bactericidal activity in vitro. Infect Immun 33:130-135,1981 4. Peterson PK, Gaziano E, Suh HJ, et al: Antimicrobial activities of dialysate-elicited and resident human peritoneal macrophages. Infect Immun 49:212-218, 1985 5. Alobaidi HM, Coles GA, Davies M, et al: Host defense in continuous ambulatory peritoneal dialysis: The effect of the dialysate on phagocyte function. Nephrol Dial Transplant 1:16-21,1986 6. van Bronswijk H, Verbrugh HA, Heezius HCJM, et al: Dialysis fluids and local host resistance in patients on continuous ambulatory peritoneal dialysis. Eur J Clin Microbiol Infect Dis 7:368-373, 1988 7. Keane WF, Comty CM, Verbrugh HA, et al: Opsonic deficiency of peritoneal dialysis effluent in continuous ambulatory peritoneal dialysis. Kidney Int 25:539-543, 1984 8. Lamperi S, Carozzi S: Defective opsonic activity of peri-
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