Medical data is for informational purposes only. You should always consult your family physician, or one of our referral physicians prior to treatment.
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 From a tube containing oneml of amoebae sufficient cells were withdrawn and then inoculatedinto the 150 ml flask to provide approximately 100,000 cells per mlafter a few days growth.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 wouldmake 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
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, 370-376.
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. Sponsored by The Arthritis Trust of America, http:// www.arthritistrust.org.
Ovulation Defects The release of an egg from the ovary is known as ovulation. It is estimated that problems with ovulation occur in 25% of infertile couples. This is an important problem to identify, as most of these patients can be treated successfully. Normal ovulation The female reproductive cycle is controlled by hormones produced by the hypothalamus and pituitary glands at the bas
The Management of Spasticity; The Evaluation and Treatment of An Interdisciplinary Approach Spasticity in Adults with ND/ID • Decrease spasticity • Infection (including urinary-tract) • Improve functional ability and independence • Constipation • Decrease pain associated with spasticity • Reflux • Prevent or decrease incidence of contractures • D