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Preliminary Report on Drug Research Involving

Acanthamoeba and Naegleria

by Tony Chapdelaine 7/14/84 under Robert J. Neff, Ph.D,

Vanderbilt University, Nashville, TN

The following is a preliminary report of work done with the

assistance of Dr. Robert Neff, Department of Molecular Biology,

Vanderbilt University, Nashville, Tennessee. These are broad range

dilution studies intended to show which drugs, and what dilutions, to

investigate further. Most drugs were chosen based on reported anti-

amoebic or anti-protozoal efficacy.

The following information was presented by Tony Chapdelaine,

B.A., now M.D., at The Rheumatoid Disease Foundation’s 1985

Seminar July 18, 1985

A series of drug tests was performed on two species of the

genus Acanthamoeba. Acanthamoeba castellanni (Neff strain) and

the pathogenic Acanthamoeba culberrtsoni, called A5, received from

Ann Stevens who obtained it from Dr. Clyde Culbertson, the man

who isolated it from contaminated tissue cultures in the late fifties.

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

hours.

Solvent dilution tests were first performed with both species. A

final 0.8 molar (6.25%) solution was the highest concentration aad

after twofold dilutions O.OO625 molar (8 one-thousandths of a

percent) the lowest for both DMSO and DMF (dimethylformamide).

The tubes of amoebae were counted each 24 hours for 4 days

and observations made of any morphological changes, such as clump-

ing of cells or excystment. The A5 strain was not affected by a final

concentration as high as 0.78% DMSO and 0.39% DMF whereas

the maximum for the Neff strain was0.39% DMSO and 0.195%

DMF.

A maximum of 0.195% DMSO and DMF was subsequently

used for solubilizing drugs which were not soluble in water.

Counts of anoebae were made in counting chambers on micro-

scope slides with ruled grid lines, each charriber being one-tenth mm

deep and 0.4 microliters (one-tenth cubic mm). Each slide contains

two of these chambers.

A concentration of one hundred thouiand cells per milliter was

chosen since cells needed to be in the log phase of growth. This

number represents a good sample for counting under the microscope

and also avoids the problem of older cultures containing a mixture of

dividing and non-dividing or encysting cells, which would make it

difficult to know whether a drug was stopping growth.

A cell-counter or haemocytometer was not used because it is apt

to count lysed cells and fragments of cells as well as viable whole

cells, and does not allow one to observe important morphological

changes in the amoebae as they occur.

The procedure was as follows:

Fifteen ml of grown; medium containing peptone-protease and

glucose was placed into a 150 ml flask. From a tube containing one

ml of amoebae sufficient cells were withdrawn and then inoculated

into the 150 ml flask to provide approximately 100,000 cells per ml

after a few days growth. Three to four drops of penicillin and strep-

tomycin were added to discourage contamination by bacteria which

tend to overgrow the medium and use up the oxygen. After carefully

rotating 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

0.78% DM F (which gives a final concentration of 0.195% to match

that of the drugs) was added as appropriate. The tubes were gently

shaken to mix the drug and growth medium and then placed into a

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.

Generally a magnification of 200 times was used for counting, al-

though 400 times was used when the viability of the cell was in

question or details and verification of cyst structure was desired.

(Cysts contain cellulose and thus are birefringent. A microscope with

birefringence filters was at times used to verify cells as encysted.)

After inoculating tlne tubes a count was made with a minimum

of 3 separate samples taken from each tube, each sample representing

a 0.4 microliter count. Often 4 or more samples were taken to provide

a good statistical base. Samples were then counted approximately

each 24 hours for 72 to 96 hours.

The concentration of cells per ml was calculated and a standard

deviation made for each count. A graph showing the growth curve

for both species and each drug dilution and control was made with

the standard deviation shown. Separate sheets were prepared with

comments on morphological changes, especially clumping and en-

cystment.

The drugs tested which showed little or no effect on growth or

morphology, and the highest drug concentration (which was some-

times limited by the requirement not to go beyond the initial 0.78%

concentration of DMSO or DMF in preparing the dilution) are as

follows:

Sulfamethoxazole 49.3 micrograms/ ml (David Casemore had

only an inhibitory effect at 100 micrograms/ml when used with

Trimethoprim)

Trimethoprim 113.1 micrograms/ ml.

Copper Aspirinate 41 micrograms/ ml,

Sulfadiazine 680 micrograms/ml. Ve:y slight inhibition of

growth for A5. Casemore had a slight inhibition effect with it at 100

micrograms/ ml and mentions that Dr. Culberston used it with good

protective results in mice experimentally infected with the pathogenic

strain of A. culbertsoni. Ann Stevens showed it has no effect )n

various species of Acanthcmoeba at 100 micrograms/ml.

Chloroquine diphosphate 1290 micrograms/ ml had a very

slight inhibitory effect. 12,900 micrograms/ ml killed them within 24

hours but this represents a 25 rnillimolar concentration and osmotic

effects are the likely reason. Casemore had slight unspecified mor-

phological changes at 100 micrograms/ml.

Metronidazole 428 micrograms/ml. Casemore shawed vari-

able results at 100 micrograms/ml. Prasad showed no effect at 1000

micrograms/ mil. The mechanism with other protozoa involves inter-

ference with electron transfer in the pyruvate phosphoroclastic reac-

tion involving reduced ferredoxin. In effect its nitro group is reduced

and it acts as an electron sink. Since Acanthamoeba have an alternate

pathway available in their electron transport system, metronidazole

will have a very limited effect on this genus.

Allopurinol 250 micrograms/ml. There was an inhibitory ef-

fect on A5 only.

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