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Comparative sensitivity of Naegleria fowleri to amphotericin B and amphotericin B methyl ester
476 TRANSACTIONS OF THEROYALSoc~rn OF TROPICAL
MEDICINE
AND
HYGIENE,
VOL.. 76, lqo.
4, 1982
Comparative
sensitivity of Naegleria fuwleri to amphotericin amphotericin B methyl ester
Dept. of Paediatriq
University
B and
A. FERRANTE of
Adelaide,
The Adelaide Australia
Summary
Data presented showedthat Nuegleria fowlen’ is much more sensitive fo amphorericin B (AmB) than to amphotericin B methylester(AME). In vitro, AME waseffectiveat concentrations of 1.0&ml whereas the parentantibiotic, AmB, was effective at concentrations of 0.0125 to 0.02.5 @ml. In tivo, An& at a concentration of 25 mg/kg/day, produced 100% survival in mice infected with N. foderi, but AME failed to urotect infectedmiceevenat concentrations of 30mgikgldai.The resultssupportpreviousobservations with fungiwhichhaveshownrhat AmB is a morepotent antimicrobial agentthan AME. Introduction
is a free-living amoebawhich has been shown to be responsiblefor a well-defined, acutely fatal, meningoencephalitis in man (CARTER, 1972;DUMA, 1972;THONG,1980;MARTINEZ, 1981). CARTER(1969) demonstratedthat N. fowleri was highly sensitiveto amphotericinB (AmB). While the chktiotherapeutic vaiue of this antibiotic in man remainsto be established(THONG.1980:MARTINEZ. 1981)it is clearly evideni from mousekxperimentai Naegletia meningoencephalitis that AmB hasin vivo efficacy (CARTER,1969; CULBERTSON et al., 1968; THONGet al., 1978a, 1979). In mice, a synergistic effect betweenAmB and tetracycline in treatment of Naegleria meningoencephalitis hasbeendemonstrated (THONGet al., 1978a, 1979). One of the major problemswith AmB is its highly toxic sideeffects, of which nephrotoxicity is the most notorious. Amphotericin B methyl ester (AME), a water-solublederivative of AmB, hasbeenreportedto be a promising substitute for the parent antibiotic becauseof its-much lower toxicity-(FISHER et al., 1976; KEIM et al.. 1976; LAWRENCE & HOEPRICH. 1976; CHENet al:, 1977). In view of the marked sensitivity of N. fowleri to AmB and sincethis antibiotic hasremainedthe only suitable agent for treatment of casesof Nue&ria meningoeicephalitis,the presentstudy wasdesrgned to evaluatethe oossiblesubstitutionof AME for AmB in the treatme& of Naegleria meningoencephalitis. The in vitro and in vivo efficaciesof both AME and AmB were examined and results compared.
Children’s
Hospital,
North Adelaide,
S.A. 5006,
Amoeba Naegkria fozvleri wasthe Northcott strain (THONG et al., 1978a).This wasmaintainedaxcnically by a
newlv develooedsvstcm (Ferrante. Rowan-Kellv & Tho&, sub&ittedj. BrieAv, the thchniclueinvolved continuousculture.of amoebae in flat-bottomedtissue culture flasksutilizing Medium ‘A’ (FULTON, 1970) suoolementedwith 10% foetal calf serum (FCS). St&ulturing wasperformedevery fifth day of &et& culture. Amoebaewerepreparedfor experimentaluse from culturesby agitatingthe flasks,centrifuging the amoebae-containing medium (200g for 5 min) and washingthe parasitesin Medium ‘A’.
Naegleria fauleri
Materials Animals
and Methods
Male BALBic mice,, six to eight week old, were obtainedfrom the Insutute of Medical andVeterinary Science,Adelaide, South Australia. Thesewere kept in an air-conditionedanimalhouseand allowed free accessto food and water.
In vitro amoeba sensitivity assay A total of 1 x lo3 trophozoiteswere seededin flat bottomed wells of microtitre plates (96 wells; Micretest II, Falcon, Oxnard, USA) and a rangeof AmB concentrationswas addedto the amoebae.Cellsand antibiotic were suspendedin a 0.2 ml volume of Medium ‘A’ supplementedwith 10% FCS. Before addition of antibiotic, the amoebaewere left to settle for 30 min and a cell count was made. This was accomplished by usingan inverted microscopeand an eye-piecegrid. The cultureswereincubatedat 37°Cin a humidified incubator and at 24 and 48 hours the cells were counted. AME was tested similarly. Mouse protection
studies
Mice were lightly anaesthetizedwith pentobarbital (0.9 mg given intraperitoneally). Amoebaewere instilled intranasallyaspreviously described(THONGet al., 1978b).Each mousereceived20 ~1of a suspensionof N. fowleri (5 x 10’ amoebae).Chemotherapy was begun one day after infection. AmB and AME were given in 0.2 ml volume intraperitoneally and then daily for 10 days. Control mice received an equivalentvolume of saline.All mice were examined daily for symptomstypical of meningoencephalitis. Brainsof someof the deadmice were examinedfor the presenceof N. fmhri. Antimicrobial
agents
AmphotericinB (AmB) for in vitro usewasobtained from Flow Laboratories.and is a product of E. R. Sauibb & SonsInc., New York. AmB for in vivo use (L”Fungizone”)was’obtainedfrom E. R. Squibb & SonsInc., New York. The amphotericinB methyl ester(AME) wasa gift from R. Turner of the above company. AME wassolubilizedin 0.004N HCI and then neutralizedto pH 7.0 with IM Tris acetate,pH 8.0.
A.
Table I-Comparative in virro sensitivity of N. fozuki to amphotericin B (AmB) and amphotericin B methyl ester WW Concentration of antimicrobial agent (&ml) Control AmB
AME
Number of arnoebaekulturex
0 0.1 0.05 0.025 0.0125 0.00625 5.0 1.0 0.5 0.25 0.125
IO-‘*
24 hours
48 hours
54 5 5 12 22 45 5 1 30
320 6 8 24 69 310 6
37
200 310
27 170
51
477
FERRANTE
*Each culture was inoculated with I.0 x 10’ N. for&K Results represent mean of triplicate cultures. Sodium deoxycholate at similar concentrations as rhat contained in the AmB preparations did not show any effect on the amoeba. Results Both amphotericin B (AmB) and amphotericin B methyl ester (AME) were active against N. fowleri (Table I) but AME was much less active than the parent antibiotic. The amoeba was sensitive to a very low concentration of AmB (0~0125-0~025 lug/ml), but a similar effect on the amoeba was observed only at an approximate AME concentration of 1 ug/ml. The in viva efficacies of AmB and AME were compared. In these experiments mice were inoculated with N. fowleri intranasally and chemotherapy was commenced one day later. The methyl ester and the parent antibiotic were given as a daily injection intraperitoneally for ten days. Mice were observed for a five-month period for development of meningoencephalitis. Infected mice (control group), which were not treated with either AmB or AME, developed symp-
toms typical of Naegleria meningoencephalitis by the fifth to the sixth day. Deaths in this group were evident by day eight, with more than 50% of the group dying by day ten (Table II); N. fowleri was recovered from the dead mice. At doses of 2.5 and 7.5 mg/kg/day alI animals in the group survived challenge (Table II). However., AME at a dose of 30 mglkgiday failed to protect mice infected with the amoeba (Table II); all with the exception of one mouse in the group died of Naegleria meningoencephalitis. Discussion Previous studies with pathogenic or potentially pathogenic fungi showed that AmB was much more potent antimicrobial agent than AME, although some studies demonstrated comparable activity against Candiaiz albicans (HOWARTH et al., 1975; LAWRENCE & HOEPRICH, 1976; BANNATYNE & CHEUNG, 1977; CHEN et al., 1977). The present study shows that AmB and AME exhibit marked amoebicidal activity in vitro, with the potency of AmB being 50-fold greater than AME. Although the anti-amoeba activity of AME was much lower than for AmB, the low toxicity of AME compared to the parent antibiotic suggested that it might be of value in chemotherapy. However, the results from the present study showed that a high dose of 30 mg/kg/day of AME failed to prevent the development of Naegleria meningoencephalitis and subsequent death of infected mice while AmB was highly therapeutic at a concentration of 2.5 mg/kg/ day, a greater than 12-fold difference in the effective therapeutic dose. In BALB/c mice we have found that AME is approximately 8-fold less toxic than AmB, showing that even in consideration of the marked toxicity difference between the two, AmB is the choice for Naegleria chemotherapy. The results confirm and strongly support the previous reports that AmB is very effective in the treatment of Naegtia meningoencephalitis (CARTER, 1969; THONG et al., 1978a:, THONG et al., 1979). N. fmleri was shown to be hrghly sensitive to AME in vitro (1 Kg/ml). Perhaps the lack of in tivo efficacy is
Table II-Protection of mice from Naegleria meningoencephalitis B methyl ester (AME) Injections bwkdday)
Number of mice per group
Control AmB AME
10 2.5 7.5 2.5
8 8 8
7.5
8
15.0
8
30.0
8
by amphotericin
Day of death 8, 8, 9, 9, 10, 10, 10, 10, 12, 17 8, 9, 10, 10, 11, 11, 12, 12 9, 9, 10, 10, 11, 11, 11, 12 9, 10, 10, 10, 11, 12, 12, 17 8, 9, 9, 10, 10, 10, 11
B (AmB) and amphotericin
Number of survivors
Mean survival time (daystS.D.)
0
10.3 + 2.6
8 8
10.4 * 1.3 10.4 2 1.0 11.4 ? 2.4
1
-
478
SENSITIVITY
OF
N.
related to the inability of this agent to cross the blood brain barrier, in which case intrathecal inoculation of AME may provide effective chemotherapy. Acknowledgements I wish to thank Professor G. M. Maxwell and Dr. R. F. Carter for their advice and encouragement, and Leanne K. Seeker for technical assistance. The study was supported by a grant from the Research Trust of the Adelaide Children’s Hospital. References Bannatyne, R. M. & Cheung,, R. (1977). Comparative susceptibility of Candida alblcans fo amphotericin B and amphotericin B methyl ester. Antimicrobial Agents and Chenwtherapy, 12, 449-450. Carter, R. F. (1%9). Sensitivity to amphotericin B of a Nue{fia sp. isol+d from a case ?f primary amoebic rn7ynm7toencephahus. Journal of Chntcal Pathology, 22, Carter, R. k. (1972). Primary amoebic meningoencephalitis: An appraisal of present knowledge. Trunsacrions of the Royal Society of Tropical Medicine and Hygiene, 66, 193-213. Chen, W. C., Sud, I. J., Chou, D-L. & Fiengold, D. S. (1977). Selective toxicity of the polyene antibiotics and their methyl ester derivatives. Btihical and Biophysical Research Connmmicakms, 74, 480-487. Culbertson, C. G., Ensminger, P. W. & Overton, W. M. (1968). Pathogenic Naegleria sp.: A study of a strain isolated from human cerebrospinal fluid. 3oumal of Proram&y, 15, 353-363. Duma, R. J. (1972). Primary VFbic meningoencephaliris. ~6s??ritscal Reviews in Chnual Laboratory Sczence, 3, Fisher, P. B., Goldstein, N: !., Bryson, V. & Schaffner, C. P. (1976). Reduced toxlclty of amphotericin B methyl ester (AME) V.S. amphotericin B and fungizone in tissue culture. In Vitro, 22, 133-140.
foWk?iAm9
AND
AME
F&on, C. (1970). Amoebo-flagellates as research partners. In: The Laboratory biology of Naegleria and Tetramirus: Methods in Cell Physiology, Vol. 4. Prescott, D. M. (Editor). New York: Academic Press, pp. 341-376. Howarth, W. R., Tewari, R. P. & Solotorovsky, M. (1975). Comparative in virro antifungal activity of amphorericin B and amphotericin B methyl ester. Antimicrobial Agems and Chemotherapy, 7, 58-63. Keim, G. R., Jr., Sibley, I’. L., Yoon? Y. H., Kulesza, J. S., Zaidi, I. H. Miller, M. M. & Poutsiaka, J. W. (1976). Comparative toxicological studies of amphotericin B methyl ester and amphotcricin B in mice, rats and dogs. An&ukobial Agents and Chemotherapy, 10, 687-690. Lawrence, R. ,M. & Hoeprich, P. D. (1976). Comparison of amphotericin B and amphotericin B methyl ester: Efficacy in murinc coccidioidomycosis and toxicity. 3ournal of Infectious Diseases, 133, 168-174. Martinez, A. J. (1981). Free-living amebas in human infections. Symposium on Clinical Parasitology. Current Concepts in a Changing Science, Philadelphia, Pennsylvania, USA. Thong, Y: H. (1980). Primary amoebic meningoencepha!i;;;3F;fteen years later. Me&al Journal of Australia, i, Thong, Y. ii., Rowan-Kelly, B., Ferrante, A. & Shepherd, C. (1978a). Synergism between tetracycline and amphotcricin B in experimental amoebic meningoencephalitis. Medical Journal of Australia, i, 663-664. Thong, Y. H., Rowan-Kelly, B. & Fcrrante, A. (1979). Delayed treatment of experimental amoebic meningoencephalitis with amphotericin B and tetracycline. Transactions of rhe Rqal Society of Tropical Medicine and Hygiene, 73, 336-337. Thong, Y. H., Shepherd, C., Ferrante, A. & Rowan-Kelly, B. (1978b). Protective immunity to Naegleriu faulti in experimental amebic meningoencephalitis. American 3oumal of Tropical Medicine and Hygiene, 27, 238-240.
Accepted for publication
27th November,
198I.
MEDICINE
AND
HYGIENE,
VOL.. 76, lqo.
4, 1982
Comparative
sensitivity of Naegleria fuwleri to amphotericin amphotericin B methyl ester
Dept. of Paediatriq
University
B and
A. FERRANTE of
Adelaide,
The Adelaide Australia
Summary
Data presented showedthat Nuegleria fowlen’ is much more sensitive fo amphorericin B (AmB) than to amphotericin B methylester(AME). In vitro, AME waseffectiveat concentrations of 1.0&ml whereas the parentantibiotic, AmB, was effective at concentrations of 0.0125 to 0.02.5 @ml. In tivo, An& at a concentration of 25 mg/kg/day, produced 100% survival in mice infected with N. foderi, but AME failed to urotect infectedmiceevenat concentrations of 30mgikgldai.The resultssupportpreviousobservations with fungiwhichhaveshownrhat AmB is a morepotent antimicrobial agentthan AME. Introduction
is a free-living amoebawhich has been shown to be responsiblefor a well-defined, acutely fatal, meningoencephalitis in man (CARTER, 1972;DUMA, 1972;THONG,1980;MARTINEZ, 1981). CARTER(1969) demonstratedthat N. fowleri was highly sensitiveto amphotericinB (AmB). While the chktiotherapeutic vaiue of this antibiotic in man remainsto be established(THONG.1980:MARTINEZ. 1981)it is clearly evideni from mousekxperimentai Naegletia meningoencephalitis that AmB hasin vivo efficacy (CARTER,1969; CULBERTSON et al., 1968; THONGet al., 1978a, 1979). In mice, a synergistic effect betweenAmB and tetracycline in treatment of Naegleria meningoencephalitis hasbeendemonstrated (THONGet al., 1978a, 1979). One of the major problemswith AmB is its highly toxic sideeffects, of which nephrotoxicity is the most notorious. Amphotericin B methyl ester (AME), a water-solublederivative of AmB, hasbeenreportedto be a promising substitute for the parent antibiotic becauseof its-much lower toxicity-(FISHER et al., 1976; KEIM et al.. 1976; LAWRENCE & HOEPRICH. 1976; CHENet al:, 1977). In view of the marked sensitivity of N. fowleri to AmB and sincethis antibiotic hasremainedthe only suitable agent for treatment of casesof Nue&ria meningoeicephalitis,the presentstudy wasdesrgned to evaluatethe oossiblesubstitutionof AME for AmB in the treatme& of Naegleria meningoencephalitis. The in vitro and in vivo efficaciesof both AME and AmB were examined and results compared.
Children’s
Hospital,
North Adelaide,
S.A. 5006,
Amoeba Naegkria fozvleri wasthe Northcott strain (THONG et al., 1978a).This wasmaintainedaxcnically by a
newlv develooedsvstcm (Ferrante. Rowan-Kellv & Tho&, sub&ittedj. BrieAv, the thchniclueinvolved continuousculture.of amoebae in flat-bottomedtissue culture flasksutilizing Medium ‘A’ (FULTON, 1970) suoolementedwith 10% foetal calf serum (FCS). St&ulturing wasperformedevery fifth day of &et& culture. Amoebaewerepreparedfor experimentaluse from culturesby agitatingthe flasks,centrifuging the amoebae-containing medium (200g for 5 min) and washingthe parasitesin Medium ‘A’.
Naegleria fauleri
Materials Animals
and Methods
Male BALBic mice,, six to eight week old, were obtainedfrom the Insutute of Medical andVeterinary Science,Adelaide, South Australia. Thesewere kept in an air-conditionedanimalhouseand allowed free accessto food and water.
In vitro amoeba sensitivity assay A total of 1 x lo3 trophozoiteswere seededin flat bottomed wells of microtitre plates (96 wells; Micretest II, Falcon, Oxnard, USA) and a rangeof AmB concentrationswas addedto the amoebae.Cellsand antibiotic were suspendedin a 0.2 ml volume of Medium ‘A’ supplementedwith 10% FCS. Before addition of antibiotic, the amoebaewere left to settle for 30 min and a cell count was made. This was accomplished by usingan inverted microscopeand an eye-piecegrid. The cultureswereincubatedat 37°Cin a humidified incubator and at 24 and 48 hours the cells were counted. AME was tested similarly. Mouse protection
studies
Mice were lightly anaesthetizedwith pentobarbital (0.9 mg given intraperitoneally). Amoebaewere instilled intranasallyaspreviously described(THONGet al., 1978b).Each mousereceived20 ~1of a suspensionof N. fowleri (5 x 10’ amoebae).Chemotherapy was begun one day after infection. AmB and AME were given in 0.2 ml volume intraperitoneally and then daily for 10 days. Control mice received an equivalentvolume of saline.All mice were examined daily for symptomstypical of meningoencephalitis. Brainsof someof the deadmice were examinedfor the presenceof N. fmhri. Antimicrobial
agents
AmphotericinB (AmB) for in vitro usewasobtained from Flow Laboratories.and is a product of E. R. Sauibb & SonsInc., New York. AmB for in vivo use (L”Fungizone”)was’obtainedfrom E. R. Squibb & SonsInc., New York. The amphotericinB methyl ester(AME) wasa gift from R. Turner of the above company. AME wassolubilizedin 0.004N HCI and then neutralizedto pH 7.0 with IM Tris acetate,pH 8.0.
A.
Table I-Comparative in virro sensitivity of N. fozuki to amphotericin B (AmB) and amphotericin B methyl ester WW Concentration of antimicrobial agent (&ml) Control AmB
AME
Number of arnoebaekulturex
0 0.1 0.05 0.025 0.0125 0.00625 5.0 1.0 0.5 0.25 0.125
IO-‘*
24 hours
48 hours
54 5 5 12 22 45 5 1 30
320 6 8 24 69 310 6
37
200 310
27 170
51
477
FERRANTE
*Each culture was inoculated with I.0 x 10’ N. for&K Results represent mean of triplicate cultures. Sodium deoxycholate at similar concentrations as rhat contained in the AmB preparations did not show any effect on the amoeba. Results Both amphotericin B (AmB) and amphotericin B methyl ester (AME) were active against N. fowleri (Table I) but AME was much less active than the parent antibiotic. The amoeba was sensitive to a very low concentration of AmB (0~0125-0~025 lug/ml), but a similar effect on the amoeba was observed only at an approximate AME concentration of 1 ug/ml. The in viva efficacies of AmB and AME were compared. In these experiments mice were inoculated with N. fowleri intranasally and chemotherapy was commenced one day later. The methyl ester and the parent antibiotic were given as a daily injection intraperitoneally for ten days. Mice were observed for a five-month period for development of meningoencephalitis. Infected mice (control group), which were not treated with either AmB or AME, developed symp-
toms typical of Naegleria meningoencephalitis by the fifth to the sixth day. Deaths in this group were evident by day eight, with more than 50% of the group dying by day ten (Table II); N. fowleri was recovered from the dead mice. At doses of 2.5 and 7.5 mg/kg/day alI animals in the group survived challenge (Table II). However., AME at a dose of 30 mglkgiday failed to protect mice infected with the amoeba (Table II); all with the exception of one mouse in the group died of Naegleria meningoencephalitis. Discussion Previous studies with pathogenic or potentially pathogenic fungi showed that AmB was much more potent antimicrobial agent than AME, although some studies demonstrated comparable activity against Candiaiz albicans (HOWARTH et al., 1975; LAWRENCE & HOEPRICH, 1976; BANNATYNE & CHEUNG, 1977; CHEN et al., 1977). The present study shows that AmB and AME exhibit marked amoebicidal activity in vitro, with the potency of AmB being 50-fold greater than AME. Although the anti-amoeba activity of AME was much lower than for AmB, the low toxicity of AME compared to the parent antibiotic suggested that it might be of value in chemotherapy. However, the results from the present study showed that a high dose of 30 mg/kg/day of AME failed to prevent the development of Naegleria meningoencephalitis and subsequent death of infected mice while AmB was highly therapeutic at a concentration of 2.5 mg/kg/ day, a greater than 12-fold difference in the effective therapeutic dose. In BALB/c mice we have found that AME is approximately 8-fold less toxic than AmB, showing that even in consideration of the marked toxicity difference between the two, AmB is the choice for Naegleria chemotherapy. The results confirm and strongly support the previous reports that AmB is very effective in the treatment of Naegtia meningoencephalitis (CARTER, 1969; THONG et al., 1978a:, THONG et al., 1979). N. fmleri was shown to be hrghly sensitive to AME in vitro (1 Kg/ml). Perhaps the lack of in tivo efficacy is
Table II-Protection of mice from Naegleria meningoencephalitis B methyl ester (AME) Injections bwkdday)
Number of mice per group
Control AmB AME
10 2.5 7.5 2.5
8 8 8
7.5
8
15.0
8
30.0
8
by amphotericin
Day of death 8, 8, 9, 9, 10, 10, 10, 10, 12, 17 8, 9, 10, 10, 11, 11, 12, 12 9, 9, 10, 10, 11, 11, 11, 12 9, 10, 10, 10, 11, 12, 12, 17 8, 9, 9, 10, 10, 10, 11
B (AmB) and amphotericin
Number of survivors
Mean survival time (daystS.D.)
0
10.3 + 2.6
8 8
10.4 * 1.3 10.4 2 1.0 11.4 ? 2.4
1
-
478
SENSITIVITY
OF
N.
related to the inability of this agent to cross the blood brain barrier, in which case intrathecal inoculation of AME may provide effective chemotherapy. Acknowledgements I wish to thank Professor G. M. Maxwell and Dr. R. F. Carter for their advice and encouragement, and Leanne K. Seeker for technical assistance. The study was supported by a grant from the Research Trust of the Adelaide Children’s Hospital. References Bannatyne, R. M. & Cheung,, R. (1977). Comparative susceptibility of Candida alblcans fo amphotericin B and amphotericin B methyl ester. Antimicrobial Agents and Chenwtherapy, 12, 449-450. Carter, R. F. (1%9). Sensitivity to amphotericin B of a Nue{fia sp. isol+d from a case ?f primary amoebic rn7ynm7toencephahus. Journal of Chntcal Pathology, 22, Carter, R. k. (1972). Primary amoebic meningoencephalitis: An appraisal of present knowledge. Trunsacrions of the Royal Society of Tropical Medicine and Hygiene, 66, 193-213. Chen, W. C., Sud, I. J., Chou, D-L. & Fiengold, D. S. (1977). Selective toxicity of the polyene antibiotics and their methyl ester derivatives. Btihical and Biophysical Research Connmmicakms, 74, 480-487. Culbertson, C. G., Ensminger, P. W. & Overton, W. M. (1968). Pathogenic Naegleria sp.: A study of a strain isolated from human cerebrospinal fluid. 3oumal of Proram&y, 15, 353-363. Duma, R. J. (1972). Primary VFbic meningoencephaliris. ~6s??ritscal Reviews in Chnual Laboratory Sczence, 3, Fisher, P. B., Goldstein, N: !., Bryson, V. & Schaffner, C. P. (1976). Reduced toxlclty of amphotericin B methyl ester (AME) V.S. amphotericin B and fungizone in tissue culture. In Vitro, 22, 133-140.
foWk?iAm9
AND
AME
F&on, C. (1970). Amoebo-flagellates as research partners. In: The Laboratory biology of Naegleria and Tetramirus: Methods in Cell Physiology, Vol. 4. Prescott, D. M. (Editor). New York: Academic Press, pp. 341-376. Howarth, W. R., Tewari, R. P. & Solotorovsky, M. (1975). Comparative in virro antifungal activity of amphorericin B and amphotericin B methyl ester. Antimicrobial Agems and Chemotherapy, 7, 58-63. Keim, G. R., Jr., Sibley, I’. L., Yoon? Y. H., Kulesza, J. S., Zaidi, I. H. Miller, M. M. & Poutsiaka, J. W. (1976). Comparative toxicological studies of amphotericin B methyl ester and amphotcricin B in mice, rats and dogs. An&ukobial Agents and Chemotherapy, 10, 687-690. Lawrence, R. ,M. & Hoeprich, P. D. (1976). Comparison of amphotericin B and amphotericin B methyl ester: Efficacy in murinc coccidioidomycosis and toxicity. 3ournal of Infectious Diseases, 133, 168-174. Martinez, A. J. (1981). Free-living amebas in human infections. Symposium on Clinical Parasitology. Current Concepts in a Changing Science, Philadelphia, Pennsylvania, USA. Thong, Y: H. (1980). Primary amoebic meningoencepha!i;;;3F;fteen years later. Me&al Journal of Australia, i, Thong, Y. ii., Rowan-Kelly, B., Ferrante, A. & Shepherd, C. (1978a). Synergism between tetracycline and amphotcricin B in experimental amoebic meningoencephalitis. Medical Journal of Australia, i, 663-664. Thong, Y. H., Rowan-Kelly, B. & Fcrrante, A. (1979). Delayed treatment of experimental amoebic meningoencephalitis with amphotericin B and tetracycline. Transactions of rhe Rqal Society of Tropical Medicine and Hygiene, 73, 336-337. Thong, Y. H., Shepherd, C., Ferrante, A. & Rowan-Kelly, B. (1978b). Protective immunity to Naegleriu faulti in experimental amebic meningoencephalitis. American 3oumal of Tropical Medicine and Hygiene, 27, 238-240.
Accepted for publication
27th November,
198I.