ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1979, p. 252-254 0066-4804/79/09-0252/03$02.00/0

Vol. 16, No.3

Experimental Endocarditis: Prophylaxis of Candida albicans Infections by 5-Fluorocytosine in Rabbits GEORGES DEMIERRE AND LAWRENCE R. FREEDMAN*

Department of Internal Medicine, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland Received for publication 6 June 1979

The prevention of Candida endocarditis in the rabbit was easily accomplished with a single intramuscular injection of 75 mg of 5-FC (a predominantly fungistatic agent) per kg either 40 min before, or at the same time as, the intravenous challenge with Candida albicans. Renal infarcts were observed more often in rabbits with infected valvular vegetations than in control rabbits with sterile endocarditis. The prophylactic effect of 5-FC is greater in aortic vegetations than in the kidneys. This may be related to differences in the pathophysiology of infection and the pharnacokinetics of 5-FC in the two areas. Candida endocarditis is a frequent postsurgical complication of insertion of prosthetic cardiac valves (1, 6, 8). Once established, the infection is difficult to sterilize. The present studies were undertaken to examine the ability of 5-fluorocytosine (5-FC), an antifungal agent well tolerated in humans (10), to prevent Candida endocarditis in rabbits. MATERLALS AND METHODS Endocarditis. Left-sided sterile endocarditis was produced in white New Zealand rabbits (2 to 4 kg) by inserting polyethylene catheters via the carotid artery to the aortic valve (5). Candida infection of these sterile vegetations and pyelonephritis were produced, 4 days later, by the intravenous injection of ca. 108 colony-forming units of Candida albicans. Candida strains. The C. albicans strain (3) was cultured for 30 h at 37°C in tryptone soya broth (Oxoid, Merz and Dade, Bern/CH) and resuspended in normal saline.

Experimental design. The animals were sacrificed 7 days after infection. The treated groups received 5FC (Ancotil, Roche Diagnostic, Div. Hoffman-La Roche, Inc., 10 mg/ml) intramuscularly in a sole dosage at times varying in relation to the intravenous injection of Candida. The minimal inhibitory concentration of 5-FC for the Candida strain is 0.05 Ig/ml at 2 days and 0.2 ,vg/ml at 7 days. In addition to the production of endocarditis due to Candida, the intravenous injection of this fungus in rabbits regularly produces pyelonephritis (4). Advantage was taken of this fact to observe the effectiveness of chemoprophylaxis of infection in two different organs.

RESULTS Serum levels of 5-FC in rabbits. Serum levels were determined by the method of microbiological agar diffusion after an intramuscular injection of 75 mg of 5-FC per kg. Figure 1 summarizes the results. In 252

all cases, the maximum level was observed in less than 1 h. The serum level of 5-FC was in excess of the minimal inhibitory concentration for the test Candida for at least 8 h after a single injection. Role ofthe quantity of 5-FC in the prophylactic effect. To assure peak serum levels at the time of induction of infection, 5-FC was injected intramuscularly 40 min before the intravenous Candida injection. Cultures of cardiac vegetations and kidneys were performed 1 week later, thus allowing time for the regrowth of Candida still viable after exposure to 5-FC. The effect of different quantities of 5-FC injected 40 min before challenge on the frequency of infection of cardiac vegetations and kidneys is shown in Table 1. A significant prophylactic effect was produced by 75 mg/kg, or more. Role of the chronology in the prophylactic effect. 5-FC (75 mg/kg) was injected intramuscularly 2 h before, 40 min before, at the same time as, 40 min after, or 6 h after the Candida injection (Table 2). A significant difference was observed between the rates of infection of the vegetations and kidneys where the prophylactic regimen was administered 40 min before or at the same time as the injection. At 40 min before the Candida injection, the prophylactic effect was evident in kidneys and vegetations. At zero time, the ability to prevent endocarditis remained unchanged, but virtually no prophylactic effect was seen in the kidneys. In the infected vegetations, the number of Candida colony-forming units is diminished when 5FC is given 2 h or 40 min -before or at the same time as the Candida. In the kidneys, this effect is present even when 5-FC is given 40 min after the injection of Candida. Effect of 5-FC on the appearance of renal emboli. The presence of one or more visible renal infarcts was correlated with the quantity and the freqeuncy of 5-FC administered (Tables 1 and 2). There was a reduction in the number of renal infarcts proportional to the prophylactic effect of the 5-FC. Infection with resistant Candida strain. To test whether the prophylactic effect of 5-FC was related to

5-FLUOROCYTOSINE IN CANDIDA ENDOCARDITIS

VOL. 16, 1979

its antifungal effect, we administered 75 mg of 5-FC per kg to a group of nine rabbits 40 min before intravenous challenge with Candida resistant to the antimicrobial properties of 5-FC in vitro. An additional 10 rabbits served as controls, receiving the same Candida challenge but no 5-FC. No difference was detected in the frequency of infection or in the number of Candida NICG/Mi

70

60

40 30 20

10

0

240 MJUTES

4060

360

480

FIG. 1. Serum levels of 5 FC in four rabbits (2.7 to 3.0 kg) after intramuscular injection of 75 mg/kg.

253

colony-forming units recovered from infected vegetations or kidneys in treated and untreated animals. DISCUSSION

In these experiments, 5-FC, a predominantly fungistatic agent, was effective in preventing Candida endocarditis when given in quantities recommended for humans (75 mg/kg) in a single injection either 40 min before or at the same time of C. albicans injection. Lesser quantities of 5-FC were ineffective, and the effect depended upon the sensitivity of the strain of Candida employed. The prophylactic effect was restricted to a period immediately before or at the same time of inoculation with Candida. It is not easy to understand why this should be since the quantity of 5-FC in the serum, even when the injection was given 2 h before Candida, assured many hours of a serum concentration far in excess of that needed to inhibit the growth of Candida in vitro. A delay of even 40 min in the administration of 5-FC diminished the percentage of sterile vegetations from 80 to 30%, and a

TABLE 1. Effects of 5 FC prophylaxis 40 min before challenge with Candida Kidney lesions

Endocardial lesions

Doses of 5FC (mg/k) No. of hearts % Sterile . (mg/kg) examined

aNoionear

No fkdesMean log of Mean log of % of kidneys % Sterile S Kidneys . eno%f enlgf ineys of vegeta- No.examined Candida/kidney Candida/g with ifarcts

tion (+SD)a

0 24 12 5.58 ± 0.99 0 0 0 6.73 ± 0.71 16 8 0 25 3 17 32 24 5.19 ± 2.02 50 14 4.0 ± 0460 28 79b 75 75d 12 6 125 100 a standard deviation. Positive culture organs only. SD, b0.01 > p > 0.001. c 0.02 > p > 0.01.

(±SD)a

4.37 3.29 2.74 1.60 1.73

± 0.71 ± 0.95 ± 0.54 ± 0.83d

100 81 78

54b 58b

dP < 0.001. TABLE 2. Effects of 5 FC (75 mg/kg) prophylaxis at various time related to challenge with Candida Endocardial lesions Kidney lesions Time of 5 FO injection , Mean log of Can- No. of kidKidneys mg/kg) ofhearts%St hearts % Sterile dida/g of (75 mg/kg) No. of vegetation neys exam- % Sterile Mean log of Can- with indida/kidney (±SD) arwithsn ined 22 9 3.29 ± 1.38 100 18 0 2.94 ± 0.54b 2 h before 46d 1.60 ± 0.83b 54d 14 79b 4.0 ± Oc 28 40 min before ± 20 5 2.79 044b 65d 3.53 ± 0.63d 80b 10 At the same time 14 0 2.85 ± 0.51b 93 6.37 ± 1.21 7 29 40 min after 81 6 3.06 ± 0.62 12 5.68 ± 1.06 15 8 6 h after 100 24 0 4.37 ± 0.71 12 0 5.58 ± 0.99 Control 19 100 100 10 68d Sterile endocarditis a Positive culture organs only. SD, Standard deviation.

(7eof5 Nnjectio.

examined

b P < 0.001

*

0.02 > P > 0.01 0.01 > P> 0.001

~(±SDYa

254

DEMIERRE AND FREEDMAN

delay of 6 h nullified the prophylactic activity completely. It is noteworthy that the prophylactic effect was less marked for the kidney than for the cardiac vegetations. The factors responsible for the initiation and the natural evolution of infection and perhaps the pharmacokinetics of 5-FC in the two sites might account for these differences. The mechanism by which antimicrobial agents prevent infection of cardiac vegetations in this experimental model is unknown. The prevention ofbacterial infection has been proven to require large quantities of bactericidal antibiotics (2, 9), whereas in the present study, prevention was accomplished by usual quantities of a predominantly bacteriostatic agent. Studies of the susceptibility of 5-FC of Candida recovered from animals which resisted the prophylactic effect of 5-FC might also be helpful in elucidating the mechanisms of the prophylactic effect (7). Experiments directed toward the study of the early events after microbial challenge in pretreated animals might be important in explaining the mechanism of prophylaxis, but have proved difficult to interpret because of the presence of antimicrobial activity in serum and its inevitable influence on microbial growth in vitro. ACKNOW LEDGMEENTS This study was supported by grant 831.272.74 from the Swiss National Fund for Scientific Research and by F. Hoffmann-La Roche, Inc., Basel. We are indebted to A. Polak (Hoffmann-La Roche, Basel)

ANTIMICROB. AGENTS CHEMOTHER. for determining the 5-FC serum levels in the rabbits used in the experiments. LITERATURE CITED 1. Dismukes, W. E., A. W. Karchmer, M. J. Buckley, W. G. Austen, and M. N. Swartz. 1973. Prosthetic valve endocarditis: analysis of 38 cases. Circulation 48:365377. 2. Durak, D. T., and R. G. Petersdorf. 1973. Chemotherapy of experimental streptococcal endocarditis. I. Comparison of commonly recommended prophylactic regimens. J. Clin. Invest. 52:592-298. 3. Freedman, L. R., and M. L. Johnson. 1972. Experimental endocarditis. IV. Tricuspid and aortic valve infection with Candida albicans in rabbits. Yale J. Biol. Med. 45: 163. 4. Freedman, L. R., and R. Prokesh. 1974. Defense mechanisms in the kidney, p. 117-124. In I. Greifer (ed.), Bacteriuria and urinary tract infections. National Kidney Foundation, New York. 5. Garrison, P. K., and L. R. Freedman. 1970. Experimental endocarditis. I. Staphylococcal endocarditis in rabbits resulting from placement of a polyethylene catheter in the right side of the heart. Yale J. Biol. Med. 42:394410. 6. Harford, C. G. 1974. Postoperative fungal endocarditis. Arch. Intern. Med. 134:116-120. 7. Schonebeck, J., and S. Normack. 1972. Studies on Candida infection of the urinary tract and the antimycotic drug 5-Fluorocytosine. Scand. J. Urol. Nephrol. Suppl. 11:1-48. 8. Slaughter, L., J. E. Morris, and A. Starr. 1973. Prosthetic valvular endocarditis. A 12-year review. Circulation 47:1319-1326. 9. Southwick, F. S., and D. T. Durack. 1974. Chemotherapy of experimental streptococcal endocarditis. Part III. Failure of a bacteriostatic agent (tetracycline) in prophylaxis. J. Clin. Pathol. 27:261-264. 10. Eilard, T., K. Alestig, and P. Wahlen. 1974. Treatment of disseminated candidiasis with 5-fluorocytosine. J. Infect. Dis. 130:155-159.

Experimental endocarditis: prophylaxis of Candida albicans infections by 5-fluorocytosine in rabbits.

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1979, p. 252-254 0066-4804/79/09-0252/03$02.00/0 Vol. 16, No.3 Experimental Endocarditis: Prophylaxis o...
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