LABORATORY DIAGNOSIS AND ORAL TREATMENT OF CAPD PERITONITIS

1301

of Raynaud’s phenomenon,9’lO but we believe this to be the published controlled trial to use nifedipine in the treatment of Raynaud’s phenomenon. In another randomised double-blind trial the calcium antagonist verapamil was found to be ineffective in 16 patients with severe Raynaud’s

LABORATORY DIAGNOSIS AND ORAL TREATMENT OF CAPD PERITONITIS

first

This lack of response may reflect the less potent vasodilator effect of verapamil. Skin temperatures have been reported to take longer to return to normal after cold challenge in patients with Raynaud’s phenomenon than in normal controls.8 In this study, skin temperature recovery times did not correlate with other measures of clinical improvement, although they discriminated fairly well between patients with Raynaud’s phenomenon and normal controls. This lack of correlation may reflect the fact that in most patients the end point (i.e., recovery to baseline skin temperature) was not ’achieved within 45 min of recording. Further studies are underway to determine whether modifications of this method or other techniques are better suited to objective assessment of

phenomenon."

Raynaud’s phenomenon. In general, side-effects with nifedipine were mild and disappeared with continued therapy. No serious side-effects were encountered. These findings accord with those of other large studies suggesting that nifedipine is generally well tolerated.’2 Recent studies in animals13 have suggested a potential teratogenic effect, although no similar experience has been reported in man to date. Contraception should be recommended when nifedipine is prescribed for women of child-bearing age. We believe that the slow calcium channel antagonist nifedipine may considerably improve the treatment of patients with moderate to severe Raynaud’s phenomenon. It remains to be seen whether or not effective therapy of Raynaud’s phenomenon might affect the prognosis of diseases such as systemic sclerosis in which vasospasm may be involved in target-organ damage. We thank Dr G. Anderson for statistical assistance, Mrs S. McCulloch for technical assistance, and Mr$D. Claude for secretarial assistance. C.D.S. is supported by fellowship funding from the Canadian Arthritis Society.

Correspondence should be addressed to R. J. R. McK., Rheumatic Disease Unit, Ottawa General Hospital, 501 Chemin Smyth Road, Ottawa, Ontario

K. R. KNIGHT

A. POLAK

Department of Renal Medicine, Southampton University, and Wessex Renal Unit, St Mary’s Hospital, Portsmouth J. CRUMP Public Health

ROSALIND MASKELL

Laboratory, St Mary’s Hospital, Portsmouth

laboratory diagnosis of 50 consecutive episodes of peritonitis in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) was studied. The technique which yielded the highest rate (84%) of positive bacteriological diagnoses was inoculation and subculture of thioglycollate broth. Cloudiness of fluid to the naked eye was an accurate predictor of a raised white cell count. A minimum laboratory protocol for the bacteriological diagnosis of CAPD peritonitis was devised and has been in use satisfactorily since the completion of the study. Antibiotic treatment was given orally in the first instance in 43 episodes Summary

and

was

The

successful in 34. Introduction

PERITONITIS is the most important complication of continuous ambulatory peritoneal dialysis (CAPD). With greater use of this technique in the management of chronic renal failure a considerable diagnostic burden has been placed upon microbiology laboratories. The low isolation rate of organisms from the peritoneal dialysate in earlier studies has been improved upon with expensive and time-consuming laboratory procedures. We have- studied 50 consecutive episodes of CAPD peritonitis with the aim of designing a minimum laboratory protocol for diagnosis and of providing a guide to management. Treatment protocols usually include parenteral (intraperitoneal and sometimes intramuscular or intravenous) administration of antibiotics.2 We have made some preliminary observations on the efficacy of oral treatment.

KIH 8L6, Canada.

Patients and Methods

REFERENCES

Halperin JL, Coffman JD. Pathophysiology of Raynaud’s disease. Arch Intern Med 1979; 139: 89-92. 2. Editorial. Episodic digital vasospasm—the legacy of Maurice Raynaud. Lancet 1977; i: 1.

1039

FA. Syndrome of symptomatic coronary arterial spasm with nearly normal coronary arteriograms. Am J Cardiol 1980; 45: 873-81. 4. Miller D, Waters DD, Warnica W, Sylachcic J, Kreeft J, Théroux P. Is variant angina the coronary manifestation of a generalised vasospastic disorder? N Engl J Med 1981; 304: 763-66. 5. Coffman JD, Cohen RA. Editorial. Vasospasm-ubiquitous? N Engl J Med 1981; 304: 780-82. 6 Antman EM, Stone PH, Muller JE, Braunwald E. Calcium channel blocking agents in the treatment of cardiovascular disorders. Part I: Basic and clinical electrophysiologic effects. Ann Int Med 1980; 93: 875-85. 7 Stone PH, Antman EM, Muller JE, Braunwald E. Calcium channel blocking agents in the treatment of cardiovascular disorders. Part II: Hemodynamic effects andclinical applications. Ann Int Med 1980; 93: 886-904. 8 Porter JM, Snider RL, Bardana EJ, Rosch J, Eidemiller LR. The diagnosis and treatment of Raynaud’s phenomenon. Surgery 1975; 77(1): 11-23. 9 Kahan A, Weber S, Amor B, Saporta L, Hodara M. Nifedipine and Raynaud’s phenomenon. Ann Int Med 1981; 94: 546 10. Vaissairat M, Capron L, Fiessinger JN, Mathieu J-F, Housset E. Calcium channel blockers and Raynaud’s disease. Ann Int Med 1981; 95: 243. 11. Kinney EL, Nicholas GG, Gallo J, Pontoriero C, Zelis R. The treatment of severe Raynaud’s phenomenon with verapamil. J Clin Pharmacol 1982; 22: 74-76. 12 Ebner F, Dunschede HB. Haemodynamics therapeutic mechanism of action and clinical findings of Adalat use based on worldwide clinical trials. In: Jatene AD, Lichtlen PR. eds. The Third International Adalat Symposium. Amsterdam: Excerpta Medica; 1976: 283-300. 13 Product monograph, "Adalat", February 1982, Miles Laboratories Ltd., Rexdale, Ontario 3

Heupler

Patients

were

instructed

to

present

to

the renal unit if their

dialysate became cloudy, if they had abdominal pain, if fever developed, or if there had been any break in their sterile bagchanging procedure.

Specimen

Collection and Examination

On presentation, the surface of the most recently drained bag of dialysate was cleaned with an antiseptic spray (’Dispray’: Stuart Pharmaceuticals Ltd) and allowed to dry. A dry swab of the surface was taken and 20 ml offluid was aspirated with a sterile syringe. 3 ml of fluid was set aside for immediate microscopy; 2-3 ml was inoculated into thioglycollate broth with 5% CO2 under vacuum (Gibco Ltd), and the remainder was placed in a sterile plastic container. One drop of dialysate was examined under the ward microscope for red and white cells; if none were seen, the reserved 3 ml was centrifuged, the supernatant decanted, and the deposit reexamined. During working hours all specimens were delivered to the laboratory immediately. At night and during weekends the swab and plastic container were stored at 4°C and the thioglycollate bottle at room temperature; they were delivered to the laboratory when it reopened. The appearance of the fluid was then noted. Leucocyte and erythrocyte counts were made with a modified Fuchs-Rosenthal haemocytometer, and a Leishman-stained slide for differential leucocyte count was prepared. With a sterile swab,

1302 the fluid was inoculated onto blood agar (BA) for incubation at 37°C for 48 h in an atmosphere containing 7% CO2; onto electrolytedeficient agar (EDA) and MacConkey agar (McC) for incubation at 37°C for 24 h in air; and onto glucose Sabouraud agar (G/Sab) for incubation at 37°C for 24 h in air followed by 7 days at room temperature. 10 ml of the fluid was centrifuged for 10 min at 700 g. The supernatant was decanted and the deposit was resuspended in the remaining fluid, which was inoculated with a sterile swab onto the same media as those used for the fluid, and incubated in the same way. A gram-stained film was also prepared from the deposit. After the top of the thioglycollate bottle had been wiped with an alcohol swab (’Steret’: Schering Ltd) and air-dried, 0 - 5 ml fluid was aspirated by syringe for culture on BA and for incubation at 370C for 5 days in an atmosphere containing 7% and at 37°C on BA and ’Isosensitest’ agar and at 37°C (ISA) containing lysed blood and 16 g/ml tobramycin for 5 days anaerobically. The thioglycollate bottle was incubated at 37°C and subcultured on the same media after 2 days and 7 days.

CO2,

All

were examined daily for bacterial growth. Organisms identified by standard methods (Cowan and Steel,3 API Products Ltd) and sensitivity tests to appropriate antibiotics were performed. Staphylococci sensitive to a 10 g disc of methicillin were assumed to be sensitive to cephradine;4 gram-negative organisms were tested against a disc containing 30 pg cephradine.

plates

were

The swabs taken from the surface of the bag before sampling were cultured on the same media used for culture of the fluid and the deposit. In addition, a BA plate was inoculated for anaerobic incubation.

Diagnosis, Treatment, and Follow-up A clinical diagnosis of peritonitis was made ifleucocytes were seen on ward microscopy of the dialysate, or if the patient complained of abdominal pain with tenderness and guarding, whether or not leucocytes were seen. The patient was admitted to hospital and given oral cephradine 500 mg 6 hourly (cotrimoxazole two tablets 12 hourly or erythromycin 500 mg 6 hourly if the patient was known to be hypersensitive to cephalosporins). The method of dialysis was changed to intermittent peritoneal dialysis (IPD) for the next 48 h. 1-litre exchanges every 30 min with 5 min dwell time were used. After 48 h CAPD was recommenced and the patient sent home, provided all was well and that the fluid was clear; the 7 day course of cephradine was completed at home. If, however, there were residual symptoms or fever at 48 h, or if the fluid remained cloudy, further specimens were collected by the same technique as that used initially, and processed identically. Failure of clinical response to oral treatment, or culture of a cephradine-resistant organism, usually required a change of treatment. If sensitivity tests were available an appropriate agent was chosen; otherwise, cefotaxime 1 g 12 hourly was given a

intramuscularly. The patients were reviewed at an outpatient clinic 2-7 days after completion of treatment (9-14 days after the onset of peritonitis). A swab and sample of dialysate were collected and examined according to the same protocol.

Results

Over the

study period 50 episodes of clinically diagnosed peritonitis patients (10 male and 17 female, age-range 17-65 years) were studied (table I) and 100 specimens of in 27

TABLE I-EPISODES OF PERITONITIS PER PATIENT

dialysate,

collected for

diagnosis and follow-up,

Presenting Features In 49 episodes the patient presented with cloudy dialysate, and 27 of these were associated with abdominal pain. Pain was the only presenting feature in the remaining episode. A fever of37’5°C or higher was recorded in only 13 episodes (26%). Leucocytes were seen on ward microscopy of the fluid in all episodes. In accordance with the protocol all patients were admitted to the renal unit. However, an assessment of the severity of the clinical illness and the necessity for

admission for clinical reasons was also made. In 9 of the episodes the patient was sufficiently ill for admission to be mandatory-severe abdominal pain was the indication in 7, severe malaise associated with intercurrent Herpes zoster infection in 1, and symptoms associated with generalised atherosclerosis in the other. White Cell Counts White cell counts were performed on 67 samples of cloudy 49 were examined at the onset of an episode (in 1 the initial white cell count was not recorded) and 18 were examined because of apparent failure to respond to treatment. 33 samples of clear fluid were examined; all were collected from well patients at follow-up. Table II shows the relation between white cell count and macroscopic appearance. Differential counting was attempted on all cloudy specimens; it was possible in 39 out of 67. Polymorphonuclear leucocytes predominated in all except 4. 3 of these were from the same patient and all showed a lymphocytosis; the 1 remaining had 95% eosinophils. In the other 28 specimens the cells were degenerate and differential counting was not possible.

fluid;

Gram-staining and Culture No swab taken from the surface of the bag before withdrawal of fluid yielded any organisms. Of the bacteriological investigations done at the onset of the 50 episodes, the gram-stained film was positive on only 8 occasions (16%), and the procedure which yielded the highest percentage (84%) of positive results was subculture of the thioglycollate broth. Culture of fluid and deposit yielded 48 and 54% detection rates respectively. 15 organisms (30%) were isolated from the thioglycollate broth only, all from the 48-h subculture. All procedures were negative in 8 episodes (16%). In one of these episodes the patient had started treatment with cephradine before she presented; in another

TABLE II-WHITE CELL COUNT RELATED TO MACROSCOPIC FLUID APPEARANCE

*WCC 15xl0"/t; red cell

count

80xJO’/l.

were

examined.

1303 TABLE III-ORGANISMS ISOLATED

Clinical Recovery and Antibiotic Sensitivity

(73%) of the 44 organisms isolated at the onset of the 50 episodes were sensitive in vitro to cephradine. Clinical recovery after oral cephradine occurred in 6 episodes from which cephradine-resistant organisms were isolated. In 4 of these episodes the organism was Staph. albus ; in 1 episode there was a mixed culture of two gram-negative organisms, 1 of which was sensitive to cephradine; in the remaining episode the patient presented a week later with peritonitis after apparent clinical recovery, and the same cephradine-resistant gram-negative organism was isolated. Organisms resistant to the oral agent used were isolated in 6 of the 9 episodes in which the patient did not respond to oral treatment. Only one of these organisms was Staph. albus. 32

*42

episodes in 21 patients.

patient was still on intramuscular cefotaxime for a previous episode. Table III shows the organisms isolated. 18 follow-up specimens at 2-3 days after onset of the episode were received, sent either because the fluid remained cloudy or the patient had persistent symptoms. All procedures were negative in 6 (33%); in the remainder the highest positive yield was from the thioglycollate broth (67% compared with 33% from culture of the fluid and of the deposit). 33 specimens were collected at 9-14 days follow-up from patients who were symptomless and had clear fluid. All except 1 were sterile by all procedures. 1 yielded a mixed growth of Staphylococcus albus and Enterobacter cloacai;7 the patient presented 24 h later with cloudy fluid. the

of White Cell Count to Positive Culture With the one exception above, all specimens which yielded an organism were recorded in the laboratory as cloudy and had a raised white cell count. 14 specimens (8 initially diagnostic and 6 sent at 2-3 days) were cloudy and had a raised white cell count, but yielded no organism. In 8 episodes the patient was already on antibacterial treatment; in 5 the patients responded to oral cephradine and the fluid became clear. 1 patient died from causes other than peritonitis. Relation

Specimen Storage 40 specimens with raised white cell counts were examined on the day of collection, and 27 after storage. The correlation between white cell count and macroscopic appearance was as good in the stored specimens as in the fresh ones. Differential counting was possible in 23 (58%) of the former and 16 (60%) of the latter. 8 initial diagnostic specimens yielded no organism; 7 were examined on the day of collection and 1 after storage. 42 yielded organisms; 18 were examined on the day of collection and 24 after storage.

Efficacy of Oral Treatment 1 patient received no treatment for 1 episode and died from other causes. 1 patient refused to take tablets and 3 patients (5 episodes) were considered too ill at presentation to be given oral treatment. The remaining 43 episodes (in 26 patients) were treated orally-37 with cephradine, 3 with cotrimoxazole, 2 with erythromycin, and 1 with ’Augmentin’ (amoxycillin and clavulanic acid). Of these 43 episodes, 34 (79%) subsided within 48 h, 30 of them on cephradine. 9 episodes (in 8 patients) failed to-respond to oral treatment; all were treated parenterally and 5 responded.

Discussion The main aim of this study was to select laboratory tests that would be particularly helpful in the diagnosis and management of peritonitis. The macroscopic appearance of the fluid was a reliable guide to the presence or absence of a raised white cell count (with one exception in which the cloudy appearance was due to red cells). This observation agrees with those of Vas who found that visual cloudiness of the fluid was as sensitive an indicator of increased numbers of cells as ’Cytour’ strips. Differential white cell counting was possible in only 58% of the fluids with raised white cell counts; the proportion was the same whether the fluid was examined on the day of collection or after storage. Only one patient had a high cosinophil count and Staph. albus was isolated. She was treated, her symptoms disappeared, and the cell count fell, suggesting that it was related to infection. All specimens at the onset of the 50 episodes were recorded in the laboratory as cloudy, although the clinician had considered 1 to be clear; 42 (84%) yielded organisms. In 2 of the remaining 8 episodes the patient was already on treatment; 5 of the others who received treatment recovered and their cell counts fell, suggesting that infection may have been present although not detected. All specimens from well patients after treatment were clear and had low white cell counts. Only 1 yielded an organism, and the patient presented a day later with cloudy fluid. None of the swabs taken from the surface of the bag before withdrawal of fluid yielded any organisms; this finding, together with the fact that no organisms were isolated from follow-up specimens from well patients, indicates that the bacteria isolated were in the dialysate and not extraneous contaminants. The distribution of organisms was similar to that described in other studies;56 skin organisms predominated, common bowel organisms were conspicuous by their absence, and the patient with Pasteurella multocida infection kept many pets. The diagnostic procedure which yielded the highest percentage of positive cultures was inoculation and subculture of thioglycollate broth. This procedure was included in the protocol to detect anaerobes; none, however, was isolated. Chan et al. 5 also reported that their isolation rate was increased from 3107o to 79% by introduction of thiolbroth-enriched medium from early in 1981. Possibly organisms stressed by the high dextrose content of the dialysate may be revived or the small numbers of organisms in the fluid may require incubation in a nutrient medium in order to reach detectable levels. Even when direct culture of the fluid or deposit yielded an organism the results of

1304

AEROMONAS-ASSOCIATED GASTROENTERITIS

sensitivity tests were never available until 48 h after specimen collection; thus direct culture gave little time advantage over subculture of the thioglycollate broth. Gram-staining of either centrifuged or uncentrifuged dialysate is seldom helpful in predicting the culture result,7 and

findings confirm this.. Storage of specimens did not alter the correlation between our

naked eye appearance and the white cell content of the fluid (table II). The isolation rate was higher in stored specimens than in those processed immediately. The laboratory diagnosis was delayed by storage, but since all patients must undergo empirical treatment initially, it seems unnecessary to call in laboratory staff at night or at weekends, provided that the specimen is collected and stored appropriately. These findings have allowed our laboratory protocol for examination ofCAPD dialysate to be simplified. Only cloudy specimens, or clear specimens collected from patients with clinical evidence of peritonitis, are submitted for diagnosis; no follow-up specimens are sent if the patient is well and the fluid is clear. The only specimen submitted to the laboratory is 2-3 ml obtained as described and inoculated into thioglycollate broth. If the laboratory is closed the broth is stored at room temperature. In the laboratory it is subcultured and incubated as described. If these cultures are negative, the subcultures are repeated after the broth has been incubated for 48 h. Since the end of the study a further 26 episodes of peritonitis have been investigated in this way and organisms have been isolated in 24. This study was intended to evaluate microbiological diagnosis, and the numbers are too small to draw definite conclusions about clinical management. Oral treatmF:lt could reduce the need for hospital admission and we judged that in 41 of the 50 episodes the patients were well enough to have been treated at home. All of these episodes were treated at presentation with oral antibiotics only, and 34 resolved within 48 h. The choice of cephradine as an oral agent was apparently satisfactory; it has a wide gram-negative and gram-positive spectrum and most isolates were sensitive. 5 patients with organisms reported as resistant in vitro to cephradine recovered during oral treatment with this agent. The organism in 4 of these episodes was Staph. albus. It is possible that sensitivity to methicillin may not always predict accurately the response of Staph., albus to cephradine; alternatively the patients might have recovered without treatment. Most patients from whom cephradine-resistant organisms were isolated did not recover, and required other treatment; only one of the organisms in these episodes was Staph. albus. We thank Prof. H. A. Lee and Dr D. J. Warren, the nursing and medical staff of the Wessex Renal Unit, and the M.L.S.O. staff for their cooperation; Mrs T. Diplock for typing the script and Dr 0. A. Okubadejo for advice. The study was supported by a grant from the Wessex Kidney Research Fund.

Correspondence should be addressed to A. P., Wessex Renal Unit, St Mary’s Hospital, Milton Road, Portsmouth P03 6AD. REFERENCES 1. Vas S Microbiological aspects of peritonitis. Peru Dial Bull 1981; 1: S 11-14. 2. Editorial. Ambulatory peritonitis. Lancet 1982; i: 1104-05. 3. Cowan ST, Steel KJ Manual for the identification of medical bacteria. Cambridge: Cambridge University Press, 1976 4. Waterworth PM. Laboratory control. In: Garrod LP, Lambert HP, O’Grady F, eds. Antibiotic and chemotherapy. Edinburgh Churchill Livingston, 1981: 475.

5. Fenton S, Wu G, Catran D, Wadgymar A, Allen AF. Clinical aspects of peritonitis in patients on CAPD. Perit Dial Bull 1981; 1: S 4-7. 6. Chan MK, Baillod RA, Chuah P, et al. Three years’ experience of continuous ambulatory peritoneal dialysis. Lancet 1981; i: 1409-12. 7. Rubin J, Rogers WA, Taylor HM, et al. Peritonitis during continuous ambulatory peritoneal dialysis. Ann Intern Med 1980; 92: 7-13.

MICHAEL GRACEY

VALERIE BURKE

JENNIFER ROBINSON Gastroenterology and Nutrition Research Unit, Princess Margaret Children’s Medical Research Foundation, Perth, Western Australia Aeromonas spp. were isolated from 118 (10·2%) children with diarrhoea and 7 (0·6%) of those without diarrhoea in a prospective, year-long study of 1156 children with gastroenteritis and the same number of age and sex matched controls. In Perth, Western Australia, aeromonas-associated diarrhoea is distinctly seasonal with a sharp summer peak. The disease most commonly presents in children under two years of age as watery diarrhoea of short duration and mild fever which require no specific treatment. In more than onethird of patients diarrhoea lasted for over 2 weeks and in almost one-quarter there was a dysentery-like illness. In some patients with aeromonas-associated diarrhoea the clinical features could be regarded as suggestive of ulcerative colitis.

Summary

Enterotoxigenic

Introduction SEVERAL reports suggest that Aeromonas spp. may

cause

gastroenteritis; 1,2 this seems to be supported by the finding that some strains off Aeromonas spp. are enterotoxigenic.3 Yet these gram-negative vibrios are not widely accepted as enteric pathogens. This may be because they are easily confused with enterobacteria, unless oxidase testing is done routinely,’ so their frequency in faecal specimens is likely to be underestimated. It may also be partly because some earlier studies have not distinguished between the identification of species of Aeromonas and their ability to produce enterotoxins. The isolation of Aeromonas spp. in patients with and without diarrhoea needs to be assessed in relation to enterotoxin production since diarrhoea may be associated only with enterotoxigenic strains. We report the clinical features and results of testing for enterotoxins in relation to isolation of Aeromonas spp. from more than 1000 children with diarrhoea and their age-matched controls. Patients and Methods Patients All faecal samples submitted to the department of microbiology, Princess Margaret Hospital, Perth, during the 12 months beginning September, 1980, were included in the study. Patients were deemed to have diarrhoea if they had 3 or more loose stools per day within 2 days of collection of the faecal sample. Repeated isolation of enteric pathogens from multiple samples of faeces during an episode of diarrhoea was recorded as a single isolation. Patients allotted to the control group had no diarrhoea within 2 weeks of collection of the stool sample. These children were inpatients or outpatients at Princess Margaret Hospital, matched for age and sex to the patients with diarrhoea. Stool samples were collected from controls within one week of collection of the matched diarrhoeal sample.

Microbiological Methods Isolation and identification of Aeromonas spp. - Faecal specimens plated onto blood agar containing 43mg/dl of para nitro phenyl glycerine (PNPG). All oxidase-positive colonies and those typical of Aeromonas spp. were identified by the MB24E multitest system (Disposable Products, Adelaide, South Australia). Enterotoxin assay. - The suckling mouse assay was used to detect Aeromonas enterotoxin.4 This method measures net intestinal fluid were