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glucose was placed into a 150 ml flask. From a tube containing oneml of amoebae sufficient cells were withdrawn and then inoculatedinto the 150 ml flask to provide approximately 100,000 cells per mlafter a few days growth. Three to four drops of penicillin and strep-tomycin were added to discourage contamination by bacteria whichtend to overgrow the medium and use up the oxygen. After carefullyrotating the flask to remove the amoebae which cling to the bottom(by changing the surface tension), three quarter ml samples were pipetted into screw-cap tubes. Then one-quarter ml of the drug dilu-tion, or in the case of controls either water or 0.78% DMSO or Preliminary Report on Drug Research Involving
0.78% DM F (which gives a final concentration of 0.195% to match Acanthamoeba and Naegleria
that of the drugs) was added as appropriate. The tubes were gentlyshaken to mix the drug and growth medium and then placed into a by Tony Chapdelaine 7/14/84 under Robert J. Neff, Ph.D, horizontal position in a rack to provide sufficient oxygen diffusion.
The microscope used for counting is a phase-contrast type.
Slight color changes tell several things about the state of the cell.
The following is a preliminary report of work done with the Generally a magnification of 200 times was used for counting, al- assistance of Dr. Robert Neff, Department of Molecular Biology, though 400 times was used when the viability of the cell was in Vanderbilt University, Nashville, Tennessee. These are broad range question or details and verification of cyst structure was desired.
dilution studies intended to show which drugs, and what dilutions, to (Cysts contain cellulose and thus are birefringent. A microscope with investigate further. Most drugs were chosen based on reported anti- birefringence filters was at times used to verify cells as encysted.) amoebic or anti-protozoal efficacy. After inoculating tlne tubes a count was made with a minimum The following information was presented by Tony Chapdelaine, of 3 separate samples taken from each tube, each sample representing B.A., now M.D., at The Rheumatoid Disease Foundation’s 1985 a 0.4 microliter count. Often 4 or more samples were taken to provide a good statistical base. Samples were then counted approximatelyeach 24 hours for 72 to 96 hours.
A series of drug tests was performed on two species of the The concentration of cells per ml was calculated and a standard genus Acanthamoeba. Acanthamoeba castellanni (Neff strain) and deviation made for each count. A graph showing the growth curve the pathogenic Acanthamoeba culberrtsoni, called A5, received from for both species and each drug dilution and control was made with Ann Stevens who obtained it from Dr. Clyde Culbertson, the man the standard deviation shown. Separate sheets were prepared with who isolated it from contaminated tissue cultures in the late fifties.
comments on morphological changes, especially clumping and en- The doubling time for a growing culture of this strain of A. castellanni is about 14 hours, while that of A. culbertsoni is about 25 The drugs tested which showed little or no effect on growth or morphology, and the highest drug concentration (which was some- Solvent dilution tests were first performed with both species. A times limited by the requirement not to go beyond the initial 0.78% final 0.8 molar (6.25%) solution was the highest concentration aad concentration of DMSO or DMF in preparing the dilution) are as after twofold dilutions O.OO625 molar (8 one-thousandths of a percent) the lowest for both DMSO and DMF (dimethylformamide).
Sulfamethoxazole 49.3 micrograms/ ml (David Casemore had
The tubes of amoebae were counted each 24 hours for 4 days only an inhibitory effect at 100 micrograms/ml when used with and observations made of any morphological changes, such as clump- ing of cells or excystment. The A5 strain was not affected by a final Trimethoprim 113.1 micrograms/ ml.
concentration as high as 0.78% DMSO and 0.39% DMF whereas Copper Aspirinate 41 micrograms/ ml,
the maximum for the Neff strain was0.39% DMSO and 0.195% Sulfadiazine 680 micrograms/ml. Ve:y slight inhibition of
growth for A5. Casemore had a slight inhibition effect with it at 100 A maximum of 0.195% DMSO and DMF was subsequently micrograms/ ml and mentions that Dr. Culberston used it with good used for solubilizing drugs which were not soluble in water.
protective results in mice experimentally infected with the pathogenic Counts of anoebae were made in counting chambers on micro- strain of A. culbertsoni. Ann Stevens showed it has no effect )n scope slides with ruled grid lines, each charriber being one-tenth mm various species of Acanthcmoeba at 100 micrograms/ml.
deep and 0.4 microliters (one-tenth cubic mm). Each slide contains Chloroquine diphosphate 1290 micrograms/ ml had a very
slight inhibitory effect. 12,900 micrograms/ ml killed them within 24 A concentration of one hundred thouiand cells per milliter was hours but this represents a 25 rnillimolar concentration and osmotic chosen since cells needed to be in the log phase of growth. This effects are the likely reason. Casemore had slight unspecified mor- number represents a good sample for counting under the microscope phological changes at 100 micrograms/ml.
and also avoids the problem of older cultures containing a mixture of Metronidazole 428 micrograms/ml. Casemore shawed vari-
dividing and non-dividing or encysting cells, which would make it able results at 100 micrograms/ml. Prasad showed no effect at 1000 difficult to know whether a drug was stopping growth.
micrograms/ mil. The mechanism with other protozoa involves inter- A cell-counter or haemocytometer was not used because it is apt ference with electron transfer in the pyruvate phosphoroclastic reac- to count lysed cells and fragments of cells as well as viable whole tion involving reduced ferredoxin. In effect its nitro group is reduced cells, and does not allow one to observe important morphological and it acts as an electron sink. Since Acanthamoeba have an alternate changes in the amoebae as they occur.
pathway available in their electron transport system, metronidazole The procedure was as follows:
will have a very limited effect on this genus.
Fifteen ml of grown; medium containing peptone-protease and Allopurinol 250 micrograms/ml. There was an inhibitory ef-
Medical data is for informational purposes only. You should always consult your family physician, or one of our referral physicians prior to treatment.
Chenodeoxycholic acid 306.5 micrograms/ ml had a slightly
to 125 micrograms/ml. Some caution that this may not be a desireable Dimetridazole 275 micrograms/rnl had no effect.
Clotrimazole is chemically similar to another synthetic imida-
Niridazole 83.5 micrograms/ml had no effect. Also, it is a po-
zole, miconazole, being antifungal and antibacterial. According to tent, long-lasting suppressor of cell-mediated immunity.
Sawyer et al, like miconazole and Amphotericin B it is preferentially Idoquinol or Diiodohydroxyquin 38.8 micrograms/ml had
bound to the cell membrane where it probably interacts with the no effect. I was unable to obtain a higher concentration and do not phospholipid bilayer altering membrane permeability, resulting in a loss of various precursors, metabolites and ions and thus inhibits Diphenylhydantoin 6,850 micrograms/ml showed an initial slight macromolecular synthesis. Very limited resistance to Clotrimazole inhibition which disappeared after thirty hours. This is a 25 millimo- has been observed. Serum levels after oral adminisrtation reach a mean peak of 1.29 micrograms/ml, with about 0.3 micrograms/ml Tlie drugs which nave a pronounced effect are: mean serum level after continued 100 milligram oral doses.
Copper Sulfate 400 micrograms/ml was amoebicidal for both
Low Toxicity, with gastro-intestinal disturbances are the most species and at 40 micrograms/ml inhibitory for A5 for 48 hours but frequent complaint after oral administration. 67.2 micrograms/ml was amoebicidal after 24 hours for both species. Some shrinkage Ornidazole 1,372 micrograms/mil was inhibitory for both spe-
occured and encystment for the Neff strain was about 33% after 48 cies but caused 17% encystment, for the Neff strain. Cells were hours. At 6.7 and 0.67 micrograms/ml the amoebae were inhibited generally small indicating blocked protein synthesis. The highest with shrinkage and some encystment. For the A5, 6.7 micrograms/ concentration (50 millimolar or 2,745 micrograms/ rnl) was ml showed inhibition, with the 0.67 micrograms/ml showing slight amoebicidal, probably an osmotic effect. At 549 micrograms/ml no inhibition after 24 hours. There is a rounding which indicates pos- sible preencystment at the 67 micrograms/ml dose. Duma showed no Paromomycin sulfate is an antibiotic aminoglycoside of the
effect for Acanthamoeba at 100 micrograms/ml, but Jones showed neomycin group which acts directly on amobae and is also antibacte- inhibition at 12.5 micrograms/ml and an amoebicidal effect at 25 rial to normal and pathogenic microorganisms in the gut. Little of the micrograms/ml, while Stevens showed that 0.5 to 5.0 micrograms/ drug is absorbed into systemic circulation. It is ineffective against E.
ml was amoebicidal for A. castellanni, A. culbertsoni and two other histolytica outside the gut. Hound that at 90 micrograms/ml it was Acanthamoeba species. The cells remaining were cysts which were inhibitory for the first 48 hours and amoebicidal afterwards for both viable on subculture to fresh medium.
species. Concentrations lower than 90 have not been tried yet, but Naefleria according to Duma are inhibited or killed at 0.39 to 39 Casemore showed A. castellanni inhibited at 100 micrograms/ml micrograms/ml depending on the strain, while Jamieson showed and .Jones showed it to be amoebicidal at 12.5 micrograms/ml and Clotrimazole was amoebicidal to N. fowleri at 0.12 to 1.0 micro- inhibitory at 5 micrograms/ml for A. polyphaga. There is probably Discussion
Rifampin or rifamycin is a marcrocyclic antibiotic. After 96
Different researchers unfortunately use differing testing tech- hours at 321 micrograms/ml, slight inhibition was seen with 3 to 5% niques and differing definitions of “inhibition” and “amoebicidal” so encystment and at 80 micrograms/ml no inhibition occured but 19% it is not always practical or wise to use the current literature data, encystment was induced for the Neff strain, inhibition for the A5 other than to get a “ballpark” idea of a drug’s potency. However, it is occured at 321 micrograms/ml. Peak plasma concentrations of 7 clear that Acanthamoebae are much more resistant to the drugs that micrograms/ml and a half-life of 1.5 to 5 hours occur for this drug.
have been tested than Naegleria. Also, in vitro tests with amoebae do Some immunosuppression has been shown in animal models (re- not always project what happens in vivo, Culbertson’s sulfadiazine/ lated to its inhibition of protein synthesis by cells in the immune mouse challenge being a good example. Even the two similar process). When used alone for tuberculosis it induces rapid resis- Acanthamoebae used in this study show widely varying responses tance. There are many minor side effects possible but they are sup- to the same drugs. On the other hand, drugs which have been shown to be effective in vitro against amoebae thus far have been effective 5-flurocytosine or flucytosine at 3 micrograms/ml up to 30
micrograms/ml showed inhibition for the Neff strain. Most cells Clotrimazole shows the best results at low dosages against both were shrunken, but encystment occured at 30 and 300 micrograms/ Acanthamoebae and Naegleria. Amphotericin B shows some prom- ml concentrations, the 300 micrograms/ml being amoebicidal after ise if the dosage can be lowered through synergism with one of the 48 hours. Tnhibition for the A5 required a minimum of 15 micro- protein blockers, otherwise its use is limited to cases of primary grams/ml and 300 micrograms/ml was inhibitory but not amoebicidal.
Clumping showed evidence for pre-encystment for A5. Casemore Further testing needs to be done on these two drugs to see if the showed flucytosine to be inhibitory at 12.5 micrograms/ml and other drugs showing promise (Paromomycin sulfate, rifampin, amoebicidal at 100 micrograms/ml. Stevens showed the Neff strain flucytosine and perhaps some others) will work synergistically, one to be killed at 40 micrograms/ml and the A. culbertsoni at 10 micro- drug altering membrane permeability, allowing the other to kill the amoebae. This would also allow a lower dosage and thus less toxic- Flucytosine is a nucleotide analogue and is antifungal. Peak ity for the host. One major difficulty which must be overcome is plasma levels reach 70 to 80 micrograms/ml with a half-life of 3 to 6 encystment. We hope to find an effective drug or combination of hours. Bone marrow functions may be depressed with anemia, leu- drugs which do not also induce differentiation or encystmcnt.
kopenia and thrombocytopenia, usually in patients with underlying References
hematological disorder or undergoing radiation treatment or drugs Casemore, David. “Sensitivity of Hartmannella that injure bone marrow. Five percent of patients have heptomegaly (Acanthamoeba) to 5-fluorocytosine, hydroxystilbamidine, and other or elevation of hepatic enzymes in the plasma which is reversible substances.” J. Clin. Path. Vol. 23, 1970, 649-652.
when therapy is stopped. All these complications are more frequent Duma, Richard. “In Vitro Susceptibility of Pathogenic Naegleria in patients with azotemia or when plasma levels of the drug reach 100 and Acanthamoeba Species to a Variety of Therapeutic Agents.” Medical data is for informational purposes only. You should always consult your family physician, or one of our referral physicians prior to treatment.
Antimicrobial Agents and Chemotherapy, Vol. 10, No. 2, Aug. 1976,
Goodman, L.S. editor. the Pharmocological Basis of Thera- peutic., N.Y., 1980, Weinstein Publ.
Jones, D., Visvesvara, G, & Robinson, N.
“Acanthamoebapolyphaga Keratitis and Acanthamoeba UveitisAssociated with Fatal Meningoencephalitis.” Transactions of theOpthalmological Society of the United Kingdom, Vol. 93, 197.` ,221-232.
Prasad, B.N. “In vitro Effect of Drugs Against Pathogenic and Non-pathogenic Free-Living Amoebae and on Anaerobic Amoebae.”Indian Journal of Experimental Biology, Vol. 10, Jan. 1972, 43-45.
Sawyer, P. et al “Clotrimazole: A Review of its Antifungal Activity and Therapeutic Efficacy.” Drugs, Vol. 9, 1975, 424-447.
Stevens, A.R. & E. Willaert. “Drug Senisitivity and Resistance of FourAcanthamoeba species.” Trans. Roy. Soc. Trop Med, Hyg.
Vol, 74, No. 6, 806-808, 1980.
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