An analysis of ten-year trends in infections in adults on continuous ambulatory peritoneal dialysis (CAPD)

Infectious complications are the Achilles heel of CAPD. To determine trends in these events, we analyzed the CAPD related infections of 303 adults on CAPD at a single university center between 1979 and 1989. During this decade the percentage of insulin-dependent diabetics increased from 14% to 39% (p less than 0.005). Peritonitis rates fell from 2.4 episodes/y in 1979 to 0.8 episodes/y in 1989. The proportion of patients with multiple episodes of peritonitis decreased (40% of the patients in 1979-1982 vs 15% in 1983-1989, p = 0.0001) while the proportion of patients with no episodes of peritonitis increased during the same periods (29% vs 49%, p = 0.005). The proportion of peritonitis episodes due to S. aureus rose over the 10-year period (p = 0.005), while those due to S. epidermidis decreased (p less than 0.10). The overall incidence of S. aureus peritonitis remained unchanged. Catheter infection rates initially increased and then fell during the decade; S. aureus remained the predominant cause. The proportion of peritonitis episodes associated with catheter infection rose (13% in 1982 vs 24% in 1989, p = 0.025), and in 1989, 80% of these episodes were caused by S. aureus. Catheter loss was also primarily due to S. aureus infections in 1989 (80%). Infections due to P. aeruginosa were a persistent problem. The proportion of patients transferring to hemodialysis each year paralleled catheter loss rates, which in turn appeared to be more related to catheter infection rates than to peritonitis rates. We conclude that control of S. aureus and P. aeruginosa will be the key to future reductions in the infectious complications of CAPD patients.     

Clonal spread of staphylococci among patients with peritonitis associated with continuous ambulatory peritoneal dialysis

BACKGROUND: Peritonitis is the most important complication of continuous ambulatory peritoneal dialysis (CAPD). Coagulase-negative staphylococci (CNS) are the most common causes of peritonitis, only limited information is available regarding the distribution and epidemiology of different CNS species associated with CAPD peritonitis. METHODS: CNS isolated from dialysis effluent from CAPD patients with peritonitis was identified by species and further analyzed with pulsed-field gel electrophoresis (PFGE). RESULTS: A total of 216 microorganisms (206 bacteria and 10 Candida species) were isolated from 196 consecutive culture-positive CAPD samples obtained from 75 patients. One hundred and twenty-one (56%) isolates represented staphylococci. The four most frequently isolated staphylococcal species were Staphylococcus epidermidis (70 isolates), Staphylococcus aureus (31 isolates), Staphylococcus hemolyticus (10 isolates), and Staphylococcus hominis (4 isolates). PFGE analysis revealed the clonal spread among patients of three different clones of S. epidermidis and one clone of S. aureus among the investigated patients. Indistinguishable isolates of either S. epidermidis, S. hominis, or S. aureus were also isolated in repeated samples from several patients. CONCLUSION: PFGE is a useful method for the epidemiological evaluation of staphylococci-associated CAPD infections and should replace older and less accurate methods, such as antibiotic sensitivity patterns. We recommend that CNS isolates from patients with CAPD-associated peritonitis should be saved for future investigations and typing, which would aid in the management of this patient category.     

Use of pulsed-field gel electrophoresis in the analysis of recurrent Staphylococcus aureus infections in patients on continuous ambulatory peritoneal dialysis

BACKGROUND/AIM: The purpose of this study was to evaluate pulsed-field gel electrophoresis (PFGE) for distinguishing between relapse and reinfection of Staphylococcus aureus infections in patients on continuous ambulatory peritoneal dialysis (CAPD). METHODS: Between July 1993 and May 1997, 4 patients with recurrent CAPD-associated infections caused by S. aureus we enrolled in this study. There were nine episodes of peritonitis, one episode of temporary double lumen catheter infection, and one episode of Hickman catheter infection. A total of eleven S. aureus isolates were collected from peritoneal fluid (n = 9) and blood (n = 2). PFGE typing was applied. RESULTS: In our study, from PFGE typing, the 11 S. aureus isolates were classified into seven patterns. Antibiogram profiling classified only four patterns. Patient A had a reinfection by another strain of S. aureus, and patient B had three episodes of peritonitis caused by the same strain of S. aureus due to exit site infections. Patient C had two episodes of CAPD peritonitis caused by two different strains, respectively. Patient D had four episodes of S. aureus infection (three CAPD peritonitis and one bacteremia); the first two episodes of peritonitis were caused by an identical strain of S. aureus, whereas the subsequent two infections were caused by other organisms. CONCLUSION: PFGE has a high discriminatory power and can be an assistant method to antibiogram profiling for distinguishing relapse from reinfection in CAPD-associated peritonitis.     

Methicillin-resistant staphylococcal infections in an outpatient peritoneal dialysis program

In view of the increasing concern about hospital-acquired methicillin resistance, we examined the sensitivities and outcome of staphylococcal infections related to outpatient peritoneal dialysis over a 5-year period. Data on all episodes of peritonitis (n = 360) and catheter infections (n = 507) were gathered prospectively from January 1984 to December 1988. The numbers of patients on peritoneal dialysis each year ranged from 136 in 1984 to 109 in 1987. Fifteen methicillin-resistant staphylococcal infections (MRSI) related to outpatient peritoneal dialysis occurred. Three were due to methicillin-resistant Staphylococcus aureus found in infected exit sites (2.3% of all S aureus catheter infections). Two of these infections occurred in a continuous ambulatory peritoneal dialysis (CAPD) patient who carried methicillin-resistant S aureus in his nares. The other 12 methicillin-resistant organisms were coagulase-negative staphylococci that caused peritonitis. There was a significant increase in the percentage of episodes of coagulase-negative staphylococci peritonitis caused by methicillin-resistant organisms; from 5% (3/57) in 1984 through 1986 to 28% (9/32) in 1987 through 1988 (P less than 0.005). In view of the high percentage of coagulase-negative staphylococci peritonitis that is methicillin-resistant, vancomycin rather than cephalosporins should be used for initial treatment.     

A prospective evaluation of blood culture versus standard plate techniques for diagnosing peritonitis in continuous ambulatory peritoneal dialysis

Peritonitis remains a major cause of morbidity in patients treated with continuous ambulatory peritoneal dialysis (CAPD). Culture-negative episodes of peritonitis occur at rates of up to 20%, and in part may reflect inadequate culturing techniques of peritoneal effluent. Through a large, prospective study, the improved sensitivity of a blood culture system, when compared with a standard plate technique (P = 0.001), for the detection of bacterial growth in 67 episodes of CAPD peritonitis is demonstrated. Improved recognition of infections caused by gram-positive organisms, primarily Staphylococcus epidermidis, was especially significant using the blood culture system (P = 0.0001). Because of improved sensitivity and a decreased time to organism identification, particularly with infections caused by S epidermidis, the most common cause of bacterial peritonitis in CAPD patients, we suggest that a blood culture system be the standard means of culturing peritoneal fluid in CAPD patients with peritonitis. The lysis-centrifugation system of culturing peritoneal fluid is also discussed in comparison with the blood culture system.     

Peritoneal drainage: an important element in host defence against staphylococcal peritonitis in patients on CAPD.

The growth of Staphylococcus aureus and coagulase-negative staphylococci were studied in fresh and effluent peritoneal dialysate from patients on continuous ambulatory peritoneal dialysis (CAPD). Peritoneal drainage during CAPD removes bacterial contaminants from the peritoneal cavity with an efficiency that depends upon the volume of peritoneal fluid remaining after drainage (residual volume). Combination of our data on the growth of coagulase-negative staphylococci in dialysate with a mathematical model of peritoneal drainage during CAPD shows that a residual volume of less than 800 ml (normal = approximately 400 ml) will prevent survival in the peritoneal fluid. A residual volume of less than 200 ml is required to eliminate S. aureus because of its faster rate of growth in dialysate. Previous work has shown that numbers of macrophages are too few to influence bacterial growth in the peritoneal dialysate. Coagulase-negative staphylococci adhere poorly to mesothelial cells in culture. Survival within the peritoneal cavity during CAPD probably depends on colonization of the PD catheter. Coagulase-negative staphylococcal peritonitis is likely to be localized to areas of the peritoneal membrane in close contact with the PD catheter. S. aureus is able to multiply in the peritoneal dialysate during CAPD and thereby causes generalized peritonitis.     

Staphylococcus aureus skin carriage and development of peritonitis in patients on continuous ambulatory peritoneal dialysis

We conducted a 15-month prospective study to investigate the skin carriage of Staphylococcus aureus and the development of peritonitis in 43 patients undergoing continuous ambulatory peritoneal dialysis (CAPD). Sixteen of 43 patients (37%) were chronic carriers of S. aureus in the anterior nares and/or in the exit-site of the catheter; 12 patients (28%) were intermittent carriers, and 15 (35%) were noncarriers. Fifty episodes of peritonitis occurred during a total of 422 patient-months of observation. S. aureus was responsible for 16 episodes of peritonitis diagnosed in 15 patients. All episodes of S. aureus peritonitis occurred in chronic and intermittent carriers. Phage typing was performed on isolates from 8 patients with S. aureus peritonitis, and they were found to have the same phage type as that previously carried in the skin. We conclude that CAPD patients who are chronic or intermittent carriers of S. aureus are at higher risk of development of peritonitis than noncarriers.     

Analysis of the causative pathogens in uncomplicated CAPD-associated peritonitis: duration of therapy, relapses, and prognosis

We analyzed the frequency with which certain bacteria caused uncomplicated peritonitis in an adult continuous ambulatory peritoneal dialysis (CAPD) program that continued patients on this modality of therapy despite frequent infections. All infections were treated with a commonly employed 10- to 14-day course of narrow spectrum intraperitoneal antibiotics. Although the distribution of bacterial pathogens was similar to previous reports (coagulase-negative staphylococci, 43%; Staphylococcus aureus, 13%), we observed no episodes of fungal peritonitis. Twenty percent of our infections were associated with either "no specimens obtained" or "no growth," a finding similar to the CAPD registry. When the data were available, two thirds of all infections were caused by the same pathogen (genus and species) as in the most immediately preceding infection. Twenty-two of 96 episodes of uncomplicated peritonitis occurred within three weeks of a preceding infection. In all 11 cases where organisms were isolated from both paired episodes, the infecting agent was the same as in the preceding infection and was a staphylococcus. This high rate of apparent relapse and the absence of fungal infections may relate to our treatment protocol and possible explanations are discussed. Lastly, the occurrence of coagulase-negative staphylococcal peritonitis is a harbinger of future episodes of peritonitis caused by a variety of organisms.     

Vancomycin pharmacokinetics in continuous ambulatory peritoneal dialysis patients with peritonitis

Peritonitis has proven to be the major deterrent to the further growth of continuous ambulatory peritoneal dialysis (CAPD) as a treatment strategy for end-stage renal disease. The correct treatment of peritonitis remains unsettled as evidenced by the presence of advocates for oral, intravenous or intraperitoneal antibiotic administration. This study examines the pharmacokinetic parameters of intravenous vancomycin when employed in the therapy of peritonitis. One gram of intravenous vancomycin was administered during 7 episodes of peritonitis in 5 patients. Plasma and end-of-dwell dialysate levels were maintained above the minimum inhibitory concentration for Staphylococcus aureus and S. epidermidis for 7 days following this single dose of vancomycin. These data establish the existence of sustained intraperitoneal entry of intravenous vancomycin during peritonitis and raise for speculation its use as the sole therapy in most episodes of gram-positive peritonitis.     

Vancomycin and tobramycin in the treatment of CAPD peritonitis

Seventy-five episodes of continuous ambulatory peritoneal dialysis (CAPD) peritonitis were studied during a 1 year period at the Queen Elizabeth Hospital, Birmingham. When two simple culture methods were used in parallel, the causative organisms were identified in 97% of cases. Nearly two thirds of episodes of peritonitis were caused by coagulase-negative staphylococci (C-NS), many of which were multiply antibiotic-resistant. On the basis of detailed antibiotic sensitivities, intraperitoneal vancomycin and tobramycin were chosen for the initial treatment of CAPD peritonitis. With this regime, a cure was achieved in 32 of 38 episodes, compared with 15 of 27 episodes when cefuroxime was used. All but 1 of 24 episodes caused by C-NS were cured by vancomycin.     

Increased risk of Staphylococcus epidermidis peritonitis in patients on dialysate containing 1.25 mmol/L calcium.

Peritoneal macrophage function is decreased in vitro in the presence of dialysate with 1.25 mmol/L calcium compared with that containing 1.75 mmol/L calcium. Theoretically, patients using this dialysate may have a higher risk of peritonitis. Nineteen patients on continuous ambulatory peritoneal dialysis (CAPD) or continuous cycling peritoneal dialysis (CCPD) were converted from dialysate with 1.75 mmol/L calcium (mean time, 33 +/- 26 months) to that with 1.25 mmol/L calcium, for some or all exchanges (mean time, 10 +/- 4.7 months). Peritonitis rates were compared with 19 control patients who remained on dialysate with 1.75 mmol/L calcium. The two groups were matched for the proportion of diabetics, sex, age, use of the Y-set, and dialysis modality (CAPD, CCPD). Peritonitis rates were similar in the study patients before conversion to 1.25 mmol/L calcium dialysate and in the control patients (0.49 v 0.58 episodes/patient-year, respectively). After conversion to dialysate with 1.25 mmol/L calcium, the peritonitis rate was 0.82 episodes/patient-year contrasted to 0.58 episodes/patient-year in the control patients (P = 0.09). The peritonitis rate due to Staphylococcus epidermidis was 0.51 episodes/patient-year when 1.25 mmol/L calcium dialysate was used, and 0.19 episodes/patient-year for the comparable period in the control patients on 1.75 mmol/L calcium dialysate (P = 0.005). The proportion of peritonitis episodes due to S epidermidis increased from 20% to 61% after conversion to 1.25 mmol/L calcium (P = 0.01). The increased risk of peritonitis due to S epidermidis in patients using dialysate with 1.25 mmol/L calcium is consistent with a previous study demonstrating that clearance of S epidermidis by peritoneal macrophages is less effective with a decrease in the dialysate calcium content.(ABSTRACT TRUNCATED AT 250 WORDS)     

A five-year study of the microbiologic results of exit site infections and peritonitis in continuous ambulatory peritoneal dialysis

We studied the culture results from 321 continuous ambulatory peritoneal dialysis (CAPD) related infections (exit site, tunnel infections, and peritonitis) in 137 patients over a 5-year period to determine the contribution of exit site and tunnel infections to peritonitis and catheter loss. Seventeen percent of peritonitis episodes were associated temporally and by microbiologic results with exit site or tunnel infections. Twenty-one percent of exit site and tunnel infections and 20% of peritonitis episodes resulted in catheter loss. Peritonitis due to Staphylococcus aureus was more likely to be associated with an exit site or tunnel infection and was more likely to result in loss of the catheter than peritonitis due to Staphylococcus epidermidis. Peritonitis and exit site infections due to Pseudomonas sp also frequently resulted in catheter removal. We found that exit site infections cause significant morbidity in CAPD patients. Further studies in this area are needed.     

Staphylococcus aureus induces caspase-independent cell death in human peritoneal mesothelial cells

Bacterial peritonitis remains a serious complication of peritoneal dialysis. Although Staphylococcus epidermidis is the most common pathogen involved, infections with Staphylococcus aureus lead to severe peritoneal damage and are often associated with a dramatic loss of mesothelial cells. Induction of cell death appears to be involved in peritoneal damage and mesothelial cell loss during bacterial infections. Using cultured human peritoneal mesothelial cells (HMCs), we investigated the ability of different S. epidermidis and S. aureus strains to damage the HMC monolayer and to trigger cell death. We show that only a subgroup of live S. aureus isolates, characterized by an invasive and alpha-hemolysin-producing phenotype, induces cell death. None of the tested S. epidermidis strains, which were not invasive or hemolytic, had a cytotoxic effect. After host cell invasion, S. aureus resided within phagocytic vacuoles, and HMCs were apparently able to degrade staphylococci. However, even after prolonged infection, a high percentage of S. aureus remained alive within HMCs and might be released after host cell death. Cell death induced by S. aureus was accompanied by apoptotic alterations, such as DNA fragmentation, but was independent of endogenous FasL and tumor necrosis factor-alpha death ligand expression. Moreover, caspases were not involved in S. aureus-induced mesothelial cell death. In conclusion, our data indicate that mesothelial cell death might represent a major mechanism of S. aureus-induced damage of the peritoneum during bacterial peritonitis.     

Exit-site and tunnel infections in continuous ambulatory peritoneal dialysis patients

One hundred two exit-site infections (ESI) were diagnosed in 63 of 163 (38.6%) patients, with an incidence of one episode every 23.7 patient-months in patients with a history of ESI, whereas in the overall continuous ambulatory peritoneal dialysis (CAPD) population the incidence was one episode every 48.7 patient-months. In diminishing order of frequency, the bacteria isolated were Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli. The probability of remaining free of ESI was 72% at 1 year and 45% at 5 years. The ESI that led to catheter removal were due to S aureus and gram-negative rods. In 13 (48%) of 27 S aureus ESI unresponsive to antibiotics and local care, deroofing and outer cuff shaving completely resolved the ESI. Despite this treatment, the catheters of the remaining 14 patients had to be removed because of peritonitis associated with the tunnel infection. In conclusion, ESI is a major cause of CAPD failure. In our series, shaving the cuff as a rescue treatment was effective for almost 50% of the patients with antibiotic-resistant S aureus ESI.     

Effects of seasonal changes on peritoneal dialysis associated peritonitis in peritoneal dialysis patients

Objectives To investigate the effects of seasonal changes on peritoneal dialysis associated peritonitis (PDAP) in patients on peritoneal dialysis (PD), and to provide evidence for clinical prevention and treatment of PDAP. Methods All episodes of PD-related peritonitis during clinic follow-up in maintenance PD patients from Jan 1st, 2007 to Dec 31st, 2015 in Peking University People's Hospital were reviewed. The incidence of peritonitis, laboratory indexes, pathogens and clinical outcomes in different seasons were recorded and analyzed. One-way ANOVA and chi square test were employed to compare the incidence of PDAP and related data in different seasons, and Pearson correlation was used to analyze correlations between PDAP rate and monthly mean temperature and mean humidity. Results During nine years, a total of 119 PD patients occurred 190 times of peritonitis during home PD. The PDAP rate in summer was the highest, 0.21 episodes/year, followed by spring (0.16 episodes/year) and autumn (0.16 episodes/risk year), but there was no significant difference among peritonitis rates in four seasons. There were significant positive correlation between monthly mean temperature, monthly mean humidity and the peritonitis rate (mean temperature: r=0.828, P＜0.01; mean humidity r=0.657, P＜0.05). (2) As for bacteria, in Summer the PDAP rate caused by Staphylococcus aureus and Coagulase negative staphylococcus (CoNS), and Gram-negative bacteria was higher than that in other seasons, but there was no statistical difference. There were significant positive correlation between monthly mean temperature, mean humidity and the rate of CoNS peritonitis (mean temperature: r=0.704, P＜0.05; mean humidity: r=0.607, P＜0.05). (3) There were no statistical difference among results of PD related peritonitis in different seasons about general situation, clinical manifestation, causes of peritonitis and laboratory index before peritonitis episodes. PD procedure-related problems were the main cause of peritonitis in summer and autumn. (4) The cure rate of all peritonitis was 90%. The highest cure rate was in autumn and winter, while the lowest cure rate was in summer, but no statistical difference. Among the peritonitis episodes with treatment failure, 52.6% occurred in summer. Conclusions There is some correlation between the rate of PDAP and seasons. Higher temperature and higher humidity were significantly correlated with higher peritonitis rate, especially the rate of CoNS peritonitis. The prognosis of PDAP in summer was relatively poor, with higher proportion of hospitalization and lower cure rate.     

Pseudomonas exit site infections in continuous ambulatory peritoneal dialysis patients.

The purpose of this study is to examine the natural history of Pseudomonas aeruginosa exit site infections in continuous ambulatory peritoneal dialysis (CAPD) patients treated with oral ciprofloxacin and local exit site care. A retrospective view was undertaken of 18 episodes of P. aeruginosa exit site infections developing in 17 patients maintained on CAPD during 1989 and 1990. Standardized therapy for the exit site infection consisted of oral ciprofloxacin (500 mg twice daily) and local exit site care with antiseptic agents. Fifteen (83%) of 18 of the pseudomonas exit site infections resolved with therapy. Three episodes (17%) required catheter removal to successfully eradicate the infection. Four of the 15 patients whose exit site infections resolved developed P. aeruginosa peritonitis 2 to 9 months after the clinical resolution of the exit site infection. The majority of pseudomonas exit site infections in CAPD patients can be successfully treated with oral ciprofloxacin and local care. Approximately 17% of the patients in this study required catheter removal to successfully eradicate the infection and an additional 22% of the patients developed pseudomonas peritonitis several months after the resolution of the exit site infection.     

A review of Staphylococcus aureus exit-site and tunnel infections in peritoneal dialysis patients

Staphylococcus aureus peritoneal exit-site and tunnel infections are a source of considerable morbidity for peritoneal dialysis patients. These infections are difficult to resolve, can lead to peritonitis, and often require removal of the peritoneal catheter. Staphylococcal nasal carriage is the major risk factor for S aureus exit-site infections and peritonitis episodes. In the future, the identification of patients who are S aureus nasal carriers and then treatment of the carriage state with rifampin may prove to be a means of decreasing infection rates. The best treatment for S aureus exit-site and tunnel infections has not been established. Treatment regimens in general use include oral antibiotics or intraperitoneal vancomycin. The optimal length of therapy is also unclear. Since the development of the disconnect peritoneal dialysis system, S aureus, rather than the Staphylococcus epidermidis, is the leading cause of peritonitis. To further decrease peritonitis rates, attention must now be directed at catheter-related peritonitis episodes, with S aureus the most common cause of such episodes. Controlled, prospective studies designed to investigate methods of preventing and treating S aureus exit-site infections in peritoneal dialysis patients are needed.     

Vaccination for prevention of CAPD associated staphylococcal infection: results of a prospective multicentre clinical trial.

124 stable CAPD patients from 8 Australian and 3 New Zealand centers were randomly assigned in a blinded fashion to one of two groups to study the effect of vaccination using commercial preparations consisting of a combined staphylococcus toxoid and whole killed staphylococci (SB) or normal saline solution (SS) on the incidence of peritonitis and exit site infection and S. aureus nasal carriage over a 12-month prospective period. In addition, levels of IgG, IgA, IgM, C3 and C4 were monitored during the trial period in serum and dialysate; serum levels of anti-alpha hemolysin and dialysate levels of fibronectin and specific antistaphylococcal antibodies were also measured. Over the period, treatment with SB or SS did not affect the incidence of peritonitis, catheter-related infection or S. aureus nasal carriage. However, vaccination with SB elicited a significant increase in the level of serum anti-alpha hemolysin throughout the 12 month duration of the study, although the level of increase was unrelated to the subsequent rate of peritonitis. Vaccination with SB but not SS elicited a significant increase in the dialysate level of specific antibodies against S. aureus. Serum levels of IgG, IgA, IgM, complement C3 and C4 were within the normal range in the CAPD patients studied and remained unaffected by vaccination with SB. In addition, dialysate levels of IgG, IgA, IgM, complement C3 and C4 were 50-100 times lower than corresponding serum levels and remained unaffected by vaccination. In summary, immunisation with an anti-staphylococcal agent was not successful in reducing peritonitis or exit site infection in CAPD patients.     

Clinical management of dialysis catheter-related bacteremia with concurrent exit-site infection

Dialysis catheter-related bacteremia (CRB) can frequently be treated with systemic antibiotics, in conjunction with an antibiotic lock, in an attempt to salvage the catheter. It is unknown whether CRB associated with an exit-site infection can be treated with such an approach. We retrospectively queried a prospective, computerized vascular access database, and identified 1436 episodes of CRB, of which 64 cases had a concurrent exit site. The frequency of concurrent exit-site infection was 9.6% with Staphylococcus epidermidis, 6.1% with Staphylococcus aureus, and only 0.7% with Gram negative CRB (p < 0.001 for Staphylococcus vs. Gram negative rods). Five serious complications (four major sepses and one endocarditis) occurred in 24 patients with S. aureus infection, but none in 32 episodes of S. epidermidis infection (p = 0.01). Catheter survival was significantly shorter in patients with S. aureus infections. The median catheter survival (without infection or dysfunction) was 14 days with S. aureus vs. 30 days with S. epidermidis infection (p = 0.035). In conclusion, concurrent exit-site infection is seen most commonly in association with Staphylococcal CRB. When the infecting organism is S. epidermidis, attempted salvage with systemic antibiotics and an antibiotic lock is reasonable. However, prompt catheter removal is indicated when the pathogen is S. aureus.