In Vitro And In Vivo Activity of 5-Fluorocytosine on Acanthamoeba

Transcription

1 ANTiMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1974, p Copyright i 1974 American Society for Microbiology Vol. 6, No. 3 Printed in U.S.A. In Vitro And In Vivo Activity of 5-Fluorocytosine on Acanthamoeba A. R. STEVENS AND W. D. O'DELLI Research Service, Veterans Administration Hospital and Department of Biochemistry, University of Florida, Gainesville, Florida Received for publication 7 May 1974 The results of our studies indicated that the avirulent Neff strain of Acanthamoeba was more susceptible to the activity of the anti-metabolite 5-fluorocytosine (5-FC) than was the virulent A-1 strain or a mouse brain reisolate of this strain, designated A-3. Results of competition experiments in which cultures were exposed simultaneously to 5-FC and either uracil, thymidine, or both uracil and thymidine demonstrated that the drug was directed against both deoxyribonucleic acid and ribonucleic acid in the avirulent strain, whereas ribonucleic acid was mainly affected in the virulent amebas. Concentrations > 10,ug of 5-FC per ml were amebicidal to the avirulent strain; lower concentrations of the drug, which only affected growth slightly, significantly impaired the capacity of the cells to spontaneously encyst in stationaryphase cultures. On the other hand, the virulent strains were capable of growing in the presence of 5-FC (40 gg/ml) after an initial period of susceptibility. After a few transfers in growth medium lacking the drug, 5-FC-treated virulent amebas exhibited growth parameters typical of untreated cells. However, after successive subcultures in drug-free medium, 5-FC-treated cells lost their resistance and were again susceptible to the drug. This result suggested that the capacity of the cells to develop resistance resulted from a drug-induced mechanism. Spontaneous encystment, which was normally minimal in stationary-phase A-1 or A-3 cultures, was enhanced in A-3 but not A-1 cultures treated with 5-FC (>30.ug/ml). Results obtained from experiments to determine the effectiveness of 5-FC in protecting mice experimentally infected with either A-1 or A-3 amebas indicated that the clinical usefulness of 5-FC may be limited by the capacity of the amebas to develop resistance. Human primary amebic meningoencephalitis is a fulminating disease process generally associated with the exposure of susceptible individuals to aquatic environments that harbor virulent trophozoites of the genus Naegleria (5, 8). Acanthamoeba, on the other hand, appears to produce cerebral lesions in individuals with predisposing pathological conditions (14, 15, 19) Ȧlthough a variety of drugs has been used to combat infections caused by either Naegleria or Acanthamoeba, treatment has been unsuccessful in all but one case actually verified by isolation. Anderson and Jamieson (1) reported that intensive treatment with the fungicide amphotericin B resulted in the survival of a patient infected with N. fowleri. The effectiveness of this drug on inhibiting in vitro growth of 'Present address: Department of Biology, University of Nebraska at Omaha, Omaha, Neb Naegleria and Acanthamoeba had been noted previously (6, 7, 10). Nevertheless, the severe complications that may be encountered with amphotericin B emphasize the need for continued screening efforts. The antimetabolite 5-fluorocytosine (5-FC), which has both a relatively low toxicity in humans and effective penetration of the bloodbrain barrier (25), has been used successfully in the treatment of fungal infections (2). This and the reported in vitro susceptibility of certain strains of Acanthamoeba to 5-FC (7) prompted an examination of the drug's activity against virulent Acanthamoeba. The present report deals specifically with the effect of 5-FC on: (i) the in vitro growth and encystment of avirulent and virulent strains of Acanthamoeba and (ii) the in vivo activity of 5-FC in mice experimentally infected with virulent Acanthamoeba. Future studies are planned to investigate the in

2 VOL. 6, 1974 vitro and in vivo activity of 5-FC on the genus Naegleria. MATERIALS AND METHODS Strains. Acanthamoeba sp. Neff strain was obtained from R. J. Neff. The virulent A-1 strain of A. culbertsoni was supplied by C. G. Culbertson. A subsequent mouse brain reisolate, designated A-3, which represented a second isolate away from the original A-1 strain, was obtained by animal passage. A description of the reisolation technique will appear elsewhere (A. R. Stevens and W. D. O'Dell, manuscript in preparation). Axenic cultivation. All strains were cultivated in a cell culture area maintained at 29 to 31 C. Cells were counted with a Neubauer-Bright Line hemocytometer. Virulent strains were fixed in 0.1% formalin prior to the counting. Details of the aerated suspension culture system and media were described previously (23). Although use of the medium containing cytosine may lead to fallacious results in the in vitro testing of 5-FC (21), apparently the amount of cytosine originating from the yeast extract used in our growth media is not sufficient to interfere with the activity of 5-FC against Acanthamoeba (see below). In vitro studies. Amebas were inoculated routinely into fresh medium 2 days prior to use and grown to an exponential density of 8 x 104 to 1.8 x 105 cells per ml before addition of the drug. 5-FC, supplied in powder form by W. E. Scott of Hoffman-LaRoche, was dissolved in distilled water at a concentration of 2 mg/ml, and the specified volume was added to the culture through a membrane filter (Swinnex, type HA, 0.45 unm). Cultures treated with growth-inhibitory concentrations of 5-FC were subcultured into fresh media to determine viability of the trophozoites and/or cysts. A second subculture was made of viable cultures to ensure that normal growth had resumed. In some instances, initial subculture from treated cultures was made into fresh medium containing 5-FC or, alternatively, treated cells passed successively in fresh medium without 5-FC were reexposed to the drug at later subcultures to determine retention or loss of 5-FC resistance. To investigate the mechanism of action of 5-FC against Acanthamoeba, we treated exponentially growing cultures simultaneously with 5-FC and either uracil (Ur), thymidine (TdR), or TdR and Ur. An appropriate amount of a concentrated solution of the base and/or nucleoside was membrane-filtered (Swinnex, type HA, 0.45 Mm) into the cultures to give 10 times the final concentration of the 5-FC. Animal protection studies. All animal studies were carried out in an isolator hood (Germfree Laboratories, Miami, Fla.). Amebas from exponentially growing axenic cultures were washed and resuspended in sufficient 0.14 M NaCl to give a final concentration of 2.5 x 105 cells per ml. Lightly etherized ICR female mice (12 to 16 g) were inoculated intranasally by instilling 2,500 trophozoites in a single drop (0.01 ml) according to the method of Culbertson et al. (9). Control mice received an equivalent volume of saline. ACTIVITY OF 5-FLUOROCYTOSINE ON ACANTHAMOEBA 283 Mice were observed daily for. neurological disease symptoms and death. Solutions of 5-FC, prepared daily in 0.14 M NaCl (12 mg/ml), were membrane filter-sterilized (Swinnex, type HA, 0.45 Mm) before being administered subcutaneously. Control mice received similar injections of sterile saline. (See Results for the drug regimen.) Amebic infection was-verified in dead or moribund mice either by direct observation of brain suspensions and cultivation of amebas or by histological examination. Brains obtained aseptically as soon as possible after death were suspended in BBL Trypticase soy broth for 2 to 3 h with occasional shaking. A portion of the Trypticase soy broth suspension was fixed in formalin and observed with phase optics for trophic ameba or cysts. A few drops of the remaining suspension were placed in the center of a plate of non-nutrient agar (1.5% Difco agar in distilled water) spread with a 24- to 48-h culture of Escherichia coli grown in brain heart infusion (Difco). The plates were incubated at 37 C. Growth could be observed through the covered and inverted plates within 24 h. Histological examination was carried out after decalcifying whole skulls in 4% formalin saturated with ethylenediaminetetraacetic acid, embedding in celloidion, sectioning at 25 Mm, and staining with hematoxylin and eosin. RESULTS Strain growth parameters. A detailed description of the growth parameters of the A-1 and Neff strains in aerated suspension culture was given previously (23). The mouse brain reisolate, A-3, readily adapted to axenic cultivation in the aerated suspension system and exhibited growth characteristics nearly identical to those of the already adapted A-1 strain by the third subculture. In vitro studies. The concentrations employed for determining the amebistatic or amebicidal activity of 5-FC in vitro were dictated by reported levels of 5-FC in serum and cerebrospinal fluid of human subjects receiving daily doses of the drug (2). Figure 1 shows the dose response of three strains to 5-FC as determined from the percentage of normal growth at 30 h post-drug treatment. Although growth of the Neff strain occurred in the presence of 5 jig of 5-FC per ml, there was an abrupt decrease in the strain's ability to proliferate in concentrations of 10,ug/ml. The virulent A-1 and A-3 strains, treated with 10 ug of the drug per ml, attained normal levels of growth. Maximal inhibition of growth of strains A-1 and A-3 at 30 h post-drug addition occurred at a dose of 30 jig of 5-FC per ml. Figure 2 illustrates the growth profiles for control and 5-FC-treated (40,ug/ml) Neff cul-

3 284 STEVENS AND O'DELL ANTIMICROB. AG. CHEMOTHER. S 0 0 I r -i in -J 5-FC (ug/ml) FIG. 1. Dose response of 5-FC on avirulent Neff (O) and virulent A-1 (0) and A-3 (0) strains of Acanthamoeba. The percent theoretical growth was calculated at 30 h postaddition of the specified concentration of 5-FC. tures. The growth curve of the drug-treated cells was representative of cultures which received 15 to 50,ug of the drug per ml. The growth rate was unaffected in the first generation after addition of the drug. After 1.5 to 1.7 generations postdrug addition, the growth rate rapidly decreased until cessation of growth at a subnormal stationary phase. The cells began dying without encysting after 25 to 30 h in stationary phase (i.e., 40 h post-drug treatment). The growth response of Neff amebas exposed to 10 MAg of 5-FC per ml was essentially the same as cells treated with 40,g/ml, except that a longer time was required to attain stationaryphase densities at slightly less than two doublings after drug addition. Although limited encystment occurred in the 10 ulg/ml-treated cultures, the majority of cells underwent growth decline. On. the other hand, cultures treated with 5 tig/ml exhibited variable responses ranging from unimpaired growth to slightly lower stationary-phase densities. Neff amebas treated with 10 Mg or less of 5-FC per ml and subcultured early in stationary phase resumed normal growth by the second transfer into fresh medium without drug. Cells treated with higher concentrations showed no recovery. Figure 3 shows the percentage of spontaneous encystment of the Neff strain as a function of the 5-FC concentration in the medium. At drug CULTURE HOURS FIG. 2. Growth profile of the avirulent Neff strain exposed in exponential growth (arrow) to 40 Mg of 5-FC per ml. Symbols: 0, control cultures; *, 5-FC-treated cultures. W 60 Z 50 ae 30 / FC (ug/ml) FIG. 3. Effect of 5-FC on spontaneous encystment of the avirulent Neff (0) and virulent A-3 (0) strains in stationary-phase cultures. The percent encystment represents mature cysts calculated as the percentage of total cells at 100 h after drug addition.

4 VOL. 6, 1974 concentrations greater than 5 ug/ml, encystment was severely curtailed. For example, at 100 h post-drug addition, only 30% of the cells had differentiated into mature cysts in cultures treated with 10 jug of the drug per ml, whereas untreated cultures routinely reached 100% encystment in stationary phase by 35 to 40 h after termination of exponential growth (24). Growth profiles for control and 5-FC-treated (40,g/ml) A-1 cultures are shown in Fig. 4. At 1.8 to 2.1 generations post-drug addition, the treated cells entered a stationary growth phase (SPJ). After approximately 38 to 40 h, however, growth resumed in the cultures at a submaximal rate; addition of a second dose of drug during SP, did not prevent this recovery growth. A-1 amebas treated with 5-FC at concentrations greater than 25 ug/ml grew with a 29- to 30-h generation time during the recovery growth phase and entered a second stationary phase (SP2) at approximately 2 x 106 cells per ml. These recovery growth parameters were altered from those normally exhibited by untreated cell populations that grew with an 8- to 10-h generation time to stationary-phase densities of to 4 x 106 cells per ml (23). However, treated cells (40 tig/ml) subcultured into drug-free medium reassumed the growth characteristics of ACTIVITY OF 5-FLUOROCYTOSINE ON ACANTHAMOEBA 285 J/0 ^ O0 x untreated cells by the second transfer. Cultures of the reisolated A-3 strain, treated with 40,ug of 5-FC per ml (and as high as 80,qg/ml), reacted in a manner similar to that of strain A-1 with three notable differences. A-3 amebas remained in the SP, for less time (24 to 26 h), recovered with a faster growth rate (14 to 16 h), and attained a normal SP2 density (Fig. 5). In addition, the A-3 amebas underwent an increase in spontaneous encystment with increasing concentrations of 5-FC above 30 mg/ml (Fig. 3). The A-1 strain never attained greater than 5% encystment in either untreated or 5-FC-treated cultures. As in the case of the A-1-treated cells, A-3 amebas subcultured into medium without the drug exhibited the same growth characteristics as untreated cells by the second transfer and were insensitive to 5-FC in the first several subcultures. To determine whether the virulent amebas had developed a permanent resistance, however, A-3 amebas that had been exposed to 5-FC (40,g/ml) were treated with the drug after an increased period of growth in the medium lacking the drug. Amebas subcultured from SP2 5-FC-treated cultures and allowed to grow to a cell density of 106 cells per ml prior to each 106 4g0 (n -J -J w CULTURE HOURS FIG. 4. Growth profile of the virulent A-i strain exposed in exponential growth (arrow) to 40 ug of 5- FC per ml. Symbols: 0, control growth cultures; 0, 5-FC-treated cultures CULTURE HOURS FIG. 5. Growth of the virulent A-3 strain exposed in exponential growth (arrow) to 40 ug of 5-FC per ml. Symbols: 0, control growth cultures; 0, 5-FC-treated cultures.

5 286 STEVENS AND O'DELL successive subculture in drug-free medium were still insensitive to 5-FC at the 3rd, 8th, 11th, and 14th subcultures. However, by the 16th subculture, the amebas had lost resistance and were affected by the drug in a manner similar to that of cells originally treated with 5-FC. To acquire information about the biochemical mechanism of 5-FC's effect on Acanthamoeba, we exposed virulent A-3 or avirulent Neff cultures simultaneously to 5-FC and either Ur, TdR, or TdR and Ur. Ur or Ur and TdR appeared to be the most effective in blocking the amebistatic activity of 5-FC on the virulent strain (Fig. 6). In contrast, the activity of 5-FC on the Neff amebas was abolished only by simultaneous addition of both Ur and TdR (Fig. 7). Neff cultures treated with 5-FC and either Ur or TdR, however, were capable of undergoing limited encystment, although the cysts appeared abnormal. As noted previously, Neff cultures treated with 40,ug of 5-FC per ml died with only negligible encystment. Animal protection studies. Results of animal protection studies of 5-FC activity in mice infected intranasally with virulent strains A-1 and A-3 are shown in Table 1. No significant difference could be demonstrated between in- U) -j -J w 106 CULTURE HOURS FIG. 6. Growth profiles of the virulent A-3 strain exposed simultaneously to 40 Ag of 5-FC per ml and either uracil (1.6 x 10' cells per ml) (a), thymidine (9 X 104 cells/ml) (A), or thymidine and uracil (1.3 x 105 cells per ml) (0) at 10 times the drug concentration. -i -J w C ANTIMICROB. AG. CHEMOTHER CULT.JRE HOURS FIG. 7. Growth profiles of the avirulent Neff strain exposed simultaneously to 40 ug of 5-FC per ml and either uracil (1.5 x 10' cells per ml) (0), thymidine (1.7 x 105 cells per ml) (A), or thymidine and uracil (8 x 104 cells per ml) (0) at 10 times the drug concentration. fected control mice receiving daily injections of saline and those receiving no treatment. Mice infected with A-1 amebas and receiving a single 3-mg dose of the drug (200 mg/kilogram of body weight) from the onset of infection (0 h) showed a significant increase in survival over the controls (P = by 2 x 2 contingency tables). When drug treatment was initiated 24 h postinfection or later, no significant difference was obtained. In comparison, mice infected with strain A-3 showed no significant survival when treated with single, daily 3-mg doses from the onset of infection. In addition, increasing the daily dosage to 12 mg administered in equal, divided doses at 12-h intervals and initiated 24 h prior to infection did not result in an increased survival (Table 1). However, the latter treatment appeared to increase the mean day of survival of treated animals above that of the controls. Whether this represents a significant increase will be realized only after additional test to determine the effectiveness of 5-FC in

6 VOL. 6, 1974 TABLE 1. In vivo activity of 5-FC in mice infected with virulent Acanthamoeba Strain Treatmenta Day of death" No. dead/ total A-1 Controlc 6, 7, 7, 7, 7, 9, 9, 10, 11, 12/15 11, 12, 12, 5, S,S A-1 3 mg, 0 h 7, 8, 8, 10,S, S, S, S, S, 4/11 S, S A-1 3 mg, 24 h 7, 9, 10, S, S, S, S 3/7 postinfection A-3 Controlc 4, 4, 4, 4, 5, 6, 6, 6, 7, 8 10/10 A-3 3mg,0h 4,5,5,5,6,6,6,6,7,8 10/10 A-3 6mg twice 6, 6, 6, 6, 7, 7,8,11 9/9 daily, h preinfection a 5-FC treatment was initiated at time indicated and continued daily until death. bs, Survivors which were negative for amebas; brain tissues of dead animals were positive for amebas. S were sacrificed at 21 days postinfection. c Received daily injections of saline. protection of mice challenged with lower doses of the A-3 amebas. DISCUSSION The results of this communication indicated that an avirulent strain of Acanthamoeba showed a greatly enhanced susceptibility to the antimetabolite 5-FC compared to that exhibited by the virulent strains. Concentrations greater than 10 gg/ml were amebicidal to the Neff strain, whereas lower concentrations, which only affected growth slightly, significantly impaired the capacity of the strain to encyst in stationary-phase cultures. In contrast, virulent strains were capable of growing, after an initial period of susceptibility, in the presence of 40 zg of 5-FC per ml. Of the virulent strains tested, A-3 amebas appeared to be somewhat more susceptible initially to the drug but possessed a greater capacity for overcoming the effect of 5-FC growth inhibition. Both of these observations might relate to the faster growth rate of the A-3 strain (6 to 8 h versus 8 to 10 h for the A-1 strain) in the suspension growth system. The rapidity with which growth was resumed and the level of growth attained in the drugtreated virulent cultures make it unlikely that the growth was due to a preexisting, resistant population in the original cultures. Additionally, such a population in the culture would not be expected to exhibit growth characteristics identical to untreated cell populations after ACTIVITY OF 5-FLUOROCYTOSINE ON ACANTHAMOEBA 287 only a few transfers in a drug-free medium. A more plausible explanation for the resumption of growth in the presence of 5-FC was that the cells overcame their susceptibility by a druginduced mechanism. This contention was supported by the observation that A-3 amebas previously exposed to 5-FC did not retain resistance to the drug beyond 15 subcultures in a drug-free growth medium. Examples of druginducible resistance, as opposed to constitutive resistance, have been found for various microorganisms, e.g., bacteria can develop resistance by altering the uptake of a drug (13). Virulent Acanthamoeba may develop resistance to 5-FC by a similar mechanism. The capacity of Acanthamoeba to develop resistance to 5-FC may be characteristic only of highly virulent pathogens such as those used in the present studies. For example, Casemore (7) found that 12.5 to 50 Mg of 5-FC per ml was amebistatic to in vitro growth of various Acanthamoeba isolates that were grown in media essentially identical to those employed in our work. However, these isolates apparently could not adapt to growth with the drug since no outgrowth occurred after 7 days of exposure. The virulence of the isolates, which were obtained from human throat swabs or as contaminants of a mammalian tissue culture, was not tested in mice. The difference in the response of these isolates and the A-1, A-3, and Neff strains, however, indicates that they may have been of low virulence. The results of the competition experiments showed that the drug was mainly affecting ribonucleic acid in virulent amebas, whereas the activity was directed against both ribonucleic acid and deoxyribonucleic acid in the avirulent strain. Endogenous utilization of 5- FC apparently involves an initial deamination of the drug to form 5-fluorouracil, which can be incorporated into ribonucleic acid after conversion to 5-fluoro-uridine-triphosphate and can lead to defective ribonucleic acid species. Alternatively, 5-FC may be metabolized to 5- fluoro-2'-deoxyuridine, which can starve the cell for thymidine by noncompetitive inhibition of the enzyme thymidylate synthetase (11). Since Ur (or Ur and TdR) effectively blocked the amebistatic activity of 5-FC on the virulent A-3 strain, it would appear that the drug was converted largely to 5-fluorouracil and incorporated into ribonucleic acid. However, the A-3 amebas may have converted a small amount of 5-FC to 5-fluoro-2'-deoxyuridine since cells treated with 5-FC and TdR grew to higher levels and resumed growth after a shorter interval in SP, than cells treated only with 5-FC (Fig. 6).

7 288 STEVENS AND O'DELL ANTIMICROB. AG. CHEMOTHER. On the other hand, the avirulent Neff strain the onset of amebic infection, the failure of the must have converted a significantly greater drug to protect animals when treatment was amount of the drug to 5-fluoro-2'-deoxyuridine begun 24 h postinfection indicates that it may since TdR plus Ur, but neither alone, reversed be of limited usefulness in human Acanthamoeba infections. The fact that numerous ame- the effect of 5-FC on this strain (Fig. 7). This enhanced metabolism of 5-FC to 5-fluoro-2'- bas were invariably observed in the brain tissues of either A-1- or A-3-infected dead mice deoxyuridine, which is a potent inhibitor of deoxyribonucleic acid synthesis, undoubtedly that had received intensive treatment with accounted for the much greater susceptibility 5-FC prior to or following infection indicates of the Neff amebas to the drug. that the ineffectiveness of the drug was due to The inhibition of spontaneous encystment by the organisms capacity to develop resistance. 5-FC in a stationary-phase culture of the Neff The use of 5-FC as an antifungal chemotherapeutic has also been limited by the ability strain was readily explained from the results of the competition experiments. In addition to of Candidae and Cryptococci pathogens to develop resistance (3, 12, 22). However, such re- transcription being necessary (18,20), deoxyribonucleic acid synthesis may also be essential sistance may be blocked in these organisms by during encystment of Acanthamoeba (L. W. combined treatment with low concentrations of Roti Roti and A. R Stevens, manuscript in amphotericin B (4, 16, 17). Since Acanthamoeba preparation). Moreover, induction of encystment in trophic amebas appears to require a tion in membrane permeability, it may be possi- may become insusceptible to 5-FC by altera- normal net amount of cellular ribonucleic acid ble to prevent the resistance by simultaneous (Roti Roti and Stevens, manuscript in preparation). In contrast, there is no simple explana- which alone would be ineffective. Whether such exposure to amphotericin B at a concentration tion for the stimulation of spontaneous encystment in SP, cultures of drug-treated A-3 virument of human Acanthamoeba infections, how- combined therapy would be feasible in treatlent cells. Since the cells had become relatively ever, must await future experimental and clinical studies. unsusceptible to the drug, the increased potential for encystment must have been related to the alterations in metabolism that occurred during or subsequent to the development of the 5- FC resistance. It is puzzling, however, that encystment of the A-1 strain was not similarly stimulated in 5-FC-treated.cultures. The increased potential of the A-3 strain to transform into dormant cysts may have partially contributed to the failure of 5-FC to protect mice challenged with A-3 infection and treated according to a drug regimen that resulted in significant survival of A-1-infected mice (Table 1). However, since no cysts were observed in brain tissues of treated animals, the primary reason for the failure of the drug to protect A-3-infected mice, probably related to the greater degree of virulence of the strain as compared to the A-1 amebas. In contrast to the virulence of the A-1 strain, the A-3 amebas consistently produced a higher level of mortalities and a greater degree of neurological damage, and were more numerous in the infected cerebral tissues (A. R. Stevens and W. D. O'Dell, manuscript in preparation). An additional reason for the difference in the results of the A-1- or A-3-infected mice treated from 0 h of infection could be the ability of the A-3 strain to develop resistance more rapidly during therapy. Even though 5-FC promoted survival of A-1- infected mice when treatment was initiated at ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of Barbara O'Dell. This work was supported by designated research funds from the Veterans Administration, project no , and by Public Health Service grants AI , awarded to A. R. Stevens (Larkin), and AI postdoctoral fellowship, awarded to W. D. O'Dell, from the National Institute of Allergy and Infectious Diseases. LITERATURE CMD 1. Anderson, K., and A. 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8 VOL. 6, Culbertson, C. G., P. W. Ensminger, and W. M. Overton Pathogenic Naegleria sp.-study of a strain isolated from human cerebrospinal fluid. J. Protozool. 15: Duma, R., W. I. Rosenblum, R. J. McGehee, M. M. Jones, and E. C. Nelson Primary amoebic meningoencephalitis caused by Naegleria. Two new cases, response to amphotericin B, and a review. Ann. Intem. Med. 74: Heidelberger, C Fluorinated pyrimidines. Progr. Nucl. Acid Res. Mol. Biol. 4: Holt, R. J., and R. L. Newman The antimycotic activity of 5-fluorocytosine. J. Clin. Pathol. 26: Inoue, M., H. Hashimoto, and S. Mitsuhashi Mechanism of tetracycline resistance in strains of Staphylococcus aureus, p In H. Umezawa (ed.), Progress in antimicrobial and anticancer chemotherapy, vol. 1. University Park Press, Baltimore. 14. Jager, B. V., and R. P. Stamm Brain abscesses caused by free-living amoeba probably of the genus Hartmannella in a patient with Hodgkin's disease. Lancet ii: Kenney, M The micro-kolmer complement fixation test in routine screening for soil ameba infection. Health Lab. Sci. 8: Medoff, G., M. Comfort, and G. S. Kobayashi Synergistic action of amphotericin B and 5-fluorocytosine against yeast-like organisms. Proc. Soc. Exp. Biol. Med. 138: Medoff, G., G. S. Kobayashi, C. N. Kwan, D. Schles- ACTIVITY OF 5-FLUOROCYTOSINE ON ACANTHAMOEBA 289 singer, and P. Venkov Potentiation of rifampicin and 5-fluorocytosine as antifungal antibiotics by amphotericin B. Proc. Nat. Acad. Sci. U.S.A. 69: Neff, R. J., and R. H. Neff The biochemistry of amoebic encystment, p In H. W. Woolhouse (ed.), Dormancy and survival. Society for Experimental Biology 23rd Symposium. Academic Press Inc., New York. 19. Robert, V. B., and Rorke, L. B Primary amebic encephalitis, probably from Acanthamoeba. Ann. Intem. Med. 79: Rudick, V. L Relationships between nucleic acid synthetic pattems and encystment in aging unagitated cultures of Acanthamoeba castellanii. J. Cell Biol. 49: Shadomy, S In vitro studies with 5-fluorocytosine. Appl. Microbiol. 17: Shadomy, S Further in vivo studies with 5-FC. Infect. Immunity 2: Stevens, A. R., and W. D. O'Dell The influence of growth medium on axenic cultivation of virulent and avirulent Acanthamoeba. Proc. Soc. Exp. Biol. Med. 143: Stevens, A. R., and P. F. Pachler Synthesis and tumover of RNA during density inhibited growth and encystment of Acanthamoeba castellanii. J. Cell Biol. 57: Utz, J. P Fluorocytosine. N. Engl. J. Med. 286: