ERADICATION 0F CARBIDE ALBICANS
FRO!!! THE GASTROIRTESTIRAL TRACT
OF MICE MD WOMEN

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ABSTRACT
ERADICATION OF CANDIDA ALBICANS FROM

THE GASTROINTESTINAL TRACT
OF MICE AND WOMEN

By

Linda Sue Olsen

Candidosis, most commonly caused by Candida albicans,
is an infection of endogenous nature, originating in most
cases from the intestine. A mouse model was developed to
study removal of the intestinal carrier state of C.
albicans. Nystatin and crystal violet were compared for
efficacy in eradication of intestinal C. albicans in the
mouse. Crystal violet was found to be effective, while
nystatin was not. Human studies were also conducted to
determine if gentian violet would remove intestinal car-
riage of C. albicans. Removal of the intestinal reservoir
of C. albicans could result in a long-lasting period with-
out candidosis. All persons taking 6-10 days of the
prescribed therapy became culturally negative for intes-
tinal C. albicans and remained negative at least one month

after completion of therapy.

ERADICATION 0F CANDIDA ALBICANS FROM
THE GASTROINTESTINAL TRACT

OF MICE AND WOMEN

By

Linda Sue Olsen

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

1975

t

(\o‘k

ACKNOWLEDGEMENTS

I am most thankful to Alvin L. Rogers, PhD, for his
kind and patient guidance and encouragement throughout
this project and for the use of his laboratory facilities.
I also wish to thank Mary H. Ryan, MD, for providing ideas
and patients for the human portion of the study, and
Everett S. Beneke for his kind advice and the use of his
laboratory facilities.

I am deeply grateful to Ronald J. Patterson, PhD,
who allowed me the freedom, while in his employ and as his
student, to explore areas of microbiology other than his
special interests, and whose support and encouragement
helped me bring this portion of my graduate education to
completion.

I wish to thank Ralph Gordon, MD, Gordon Carter, PhD,
and Norman McCullough, MD, PhD, for their advice and sug-
gestions. James Stapleton, PhD, provided aid in statistical
analysis of the data and Sue Rose drew the graphs. The
Department of Microbiology and Public Health provided
financial assistance, for which I am appreciative.

My special friend, Doug Flower, provided emotional
support, encouragement, and understanding through the

major portion of my graduate endeavors, for which I am

ii

most grateful. I would also like to thank all my other
friends for their support and tolerance, especially Barb
Shimei and June Hillelson.

I am deeply indebted to my parents, who allowed and
encouraged me to explore whatever interests I had at any
given time without regard to traditional male or female
roles. Jane Lipe and Gary Smith, my past teachers,
encouraged and magnified my interests in the sciences,

especially microbiology.

iii

TABLE OF CONTENTS

INTRODUCTION.
LITERATURE REVIEW .
Candida albicans

Epidemiology, Ecology, Distribution.
Pathogenicity.

Candidal Vaginitis . . . . .
Experimental Murine Infections
Nystatin

Gentian Violet
LITERATURE CITED.
ARTICLE I - Eradication of Candida albicans from
the Gastrointestinal Tract of Mice:
A Comparison of Nystatin and Crystal
Violet. . . . . . . . . . . .

ARTICLE II - Eradication of Candida albicans from
the Gastrointestinal Tract of Women.

APPENDICES.
I - CULTURE MEDIA.
II - CONSENT FORM.

III - PATIENT INSTRUCTIONS

iv

Page

31

50
66
66
67
68

LIST OF TABLES

Table Page
I Results of gentian violet treatment in
patients with Candida albicans in the
gastrointestinal tract and vagina. . . . . . 58

Figure

LIST OF FIGURES

The number of mice with Candida albicans
in the intestinal tract over a 10-week
period

The percentage of mice with Candida
aZbicans in the intestinal tract during
a 4-week period following treatment of
either 10 or 15 days duration with vary-
ing amounts of nystatin and crystal
violet

The percentage of mice with Candida

aZbicans in feces after treatment with
nystatin and crystal violet.

vi

Page

40

42

44

INTRODUCTION

Candida albicans is a commensal yeast in the intes-
tine of 25-50% of healthy persons [19,112]. It is passed
from mother to neonate at birth during passage through
the birth canal [51,102]. The incidence of Candida infec-
tions and the change from commensal to parasite are
increased in debilitated persons as well as in those with
altered physiological states [92,112].

It is thought that the intestine is a reservoir for
infection of other parts of the body by Candida albicans
[17,67,102]. In debilitated persons systemic dissemina-
tion may occur. More commonly the gut may seed the
perianal area causing cutaneous candidosis of both infant
and adult. The intestine is also a reservoir for the
infection of the vagina and reinfection after tOpical
treatment [110].

To eliminate the reservoir, C. aZbicans must be
eradicated from the alimentary tract. Sternberg et a1.
(1954), as well as other investigators, have suppressed
the intestinal Candida population by administration of
nystatin tablets or capsules. However, they failed to

completely eradicate the Candida population of the

2
alimentary tract as evidenced by reappearance of the yeast
soon after treatment ended [78,98].

Current therapy for candidosis is aimed at the site
of infection rather than the intestinal source of the
infection. Even when oral intestinal therapy with nystatin,
the current drug of choice, is combined with tOpical therapy,
the goal and result is merely to return the population of
C. albicans to commensal proportions.

The mouse has been a useful model for the study of
pathogenesis and treatment of various forms of candidosis.
In the present study, a mouse model for intestinal coloni-
zation by C. aZbicans is described. This model was used
‘to compare two antimycotic agents, crystal violet and
nystatin, with regard to efficacy in removal of an intes-
tinal population of C. albicans. The model is potentially
useful in the laboratory study of human intestinal can-
didosis and the treatment and prevention of candidoses
arising from intestinal sources.

The study was extended to humans, in which the pur-
pose was to determine if the alimentary population of
C. aZbicans could be eradicated through use of gentian

violet.

LITERATURE REVIEW

Candida albicans

 

There are 8 species of Candida which have been
repeatedly associated with human disease. Of these,
Candida aZbicans is the most pathogenic and most commonly
encountered in disease [92]. Its synonyms are Oidium
albicans, Robin 1853; Monilia albicans (Robin) Zopf
1890; Syringospora robinii, Quinquad I869; Endomyces
pinoui, Castellani 1922; Candida genitalis, Batista and
Silveria 1962; and Candida intestinalis, Batista and
Silveria 1959. Lodder lists a total of 100 synonyms [61].

Candida albicans is a yeast-like fungus, which
reproduces by budding and forms a white, creamy, smooth,
opaque colony on Sabouraud's dextrose agar after 3 days
at 25 C. At one month it is "creamy, glistening, waxy,
soft, smooth to somewhat reticulated; old stocks wrinkled
and folded with spicules." Microsc0pic cellular mor-
phology after 3 days at 25 C in Sabouraud's dextrose
broth consists of globose, short, ovoid (5—7 pm), some-
times elongate yeasts (4-6 x 6—10 pm) [92]. When incu-
bated in serum for 2 h at 37 C, the blastospores form
germ tubes (Reynold's—Braude phenomenon), a characteristic
of C. albicans not seen in most other yeasts or other

3

4
species of Candida associated with human disease [91,101].
This phenomenon is probably due to the low glucose, high
undigested protein content of serum. Sabouraud's dextrose
broth is 1% peptone (enzyme-digested denatured proteins)
and 4% glucose. The nitrogen in serum occurs as protein
and the glucose content is less than 0.1% [16]. Germ tube
formation is a rapid, reliable diagnostic technique for
identification of C. albicans [101].

Candida aZbicans is the only yeast occurring in man
which usually forms chlamydospores on chlamydospore agar.
Chlamydospores are "thick-walled, non—deciduous, inter-
calary or terminal asexual spores made by the rounding
up of a cell or cells" [2]. The word chlamydospore is
derived from the Greek word chlamys, meaning mantle or
sheath. Chlamydospores of C. aZbicans are large (7-17
pm), thick-walled cells which appear at the tips and
sides of hyphae. The chlamydospore is borne on an
elongated suspensor cell or a long filament. Both of
these cells are produced by budding. Material is
deposited inside giving rise to a thick wall. Proto-
chlamydospore cells which look as though they will become
chlamydospores must bud once more and the budded cell
becomes the chlamydospore. They concentrate glycogen
and other cellular material which migrates into the
chlamydospore [46]. Chlamydospores are generally formed
in the absence of an easily utilizable carbon source. An

increase in glucose produces a decrease in chlamydospore

5
formation [81]. Glucose at a concentration of 5 mg/ml
has been shown to inhibit chlamydospore production [46].
Chlamydospore formation may be enhanced by inoculation of
media free of reducing sugars. Nickerson-Mankowski
chlamydospore agar or cornmeal agar plus Tween-80 is used
for this purpose. These media are inoculated by furrow-
ing an inoculum into the medium, thus effecting a reduced
oxygen tension in the organism's immediate environment.

Electron micrographs of chlamydospores have shown
them to be double-layered, consisting of a thin, electron-
transparent outer layer surrounding a thick electron-
dense inner layer. The thickness of the inner layer
increases with age. It is 0.4 pm in mature cells. The
outer layer is continuous with the cell wall of the
suspensor cell. A membrane seems to separate the inner
layer from the cytOplasm. It has no structurally dis-
tinguishable cytoplasmic components [46].

Tsuchiya has done serologic studies on the different
species of Candida [108]. There are many antigens
associated with the species of this genus. Charts of the
antigenic structures of each species as determined by
absorbed antisera have been compiled by several researchers
[35,108,113]. As a result of these studies, as well as
others, C. albicans can be divided into 2 serologic
groups, A and B, of equal pathogenicity [92].

Carbohydrate fermentation, assimilation, and mycelial

morphology may also be useful in identification of C.

6
albicans. Candida aZbicans ferments glucose and maltose
with acid and gas production, while only acid is produced
as sucrose is fermented. It assimilates glucose, galactose,
maltose, and sucrose [6,92].

There is no known perfect stage of C. albicans [92].

Epidemiology,gEcology, Distribution

Candida albicans is part of the normal flora of the
alimentary tract, including the oral cavity, of 25-50% of
healthy adult humans [19,112]. The prevalence of C.
aZbicans in the normal vagina of healthy, nonpregnant
women has been reported as 5-20% [68,102,112,113], but
is increased to 15-50% in healthy pregnant women [19,42,
112,113]. In the oral cavity of the newborn the occur-
rence has been reported to be about 4% [102]. In the
feces of 2- to 6-week-old infants the prevalence of C.
aZbicans is 25% [23], which is similar to that in the
vagina of pregnant women and the feces of healthy
adults [112].

Before an oral flora of the neonate has been estab-
lished, even a few organisms indicate imminent thrush
[102]. Kozinn et al. [51] established that the organism
may be swallowed at birth as the fetus passes through
the birth canal. This route of infection not only con-
tributes to the incidence of thrush but also allows the
organism to colonize the intestine. Thrush has also been

found to be passed by cross-contamination to other infants

7
and personnel during a nursery epidemic [113]. Intestinal
colonization of the infant is closely associated with
clinical symptoms, including diarrhea, general irrita-
bility, and perianal colonization resulting in diaper
rash [102].

Although other species of Candida may be found on
normal skin, C. albicans is not part of the normal skin
flora. In one study it was only recovered 3 times in 2444
scrapings from the skin of 118 normal people [32]. Some
reseachers, however, report that a tropical environment
or contact with infected patients resulted in increased
recovery of C. albicans from the skin [69].

Candida albicans is a common inhabitant of the ali-
mentary tract of many mammals and birds. Van Uden et
a1. [110,111] have done extensive studies in this area
and have recovered the organism from horses, sheep, goats,
swine, African bush pigs, African pied crows, baboons,
and African wart hogs.

Candida albicans has rarely been isolated from soil.
When found, it probably represents fecal contamination [92].

The population of C. aZbicans in the adult intestine
is affected by other intestinal flora and by diet [92,110].
The lactobacilli and other lactic acid producing bacteria
act to decrease the yeast population. Competition for
nutrients, oxidation-reduction potentials, and secreted
inhibitory factors may also act to control the yeast popu-

lation. Diet influences the pOpulation of C. aZbiaans in

8
that the type of available nutrients affect growth. Poor
oral hygiene or use of small amounts of antibiotics may
increase the number of organisms in the oral cavity, but

does not usually result in clinical symptoms [92].

Pathogenicity

 

Candida aZbicans is part of the normal flora of many
humans. It is not known whether the presence of the organism
is useful to humans [112]. It is an opportunist, taking
advantage of altered host states. Candidosis may be a
primary or secondary infection. Although other members of
the genus may be involved, C. albicans is most commonly the
causative organism. Physiologic changes or certain states
predispose to candidosis. Among these are oral contracep-
tion [47,87], infancy, pregnancy, antibiotic therapy,
diabetes and other endocrine disorders, immunosuppressive
therapy, immune deficiencies, malignant diseases, and
other debilitated states [92,112].. Candidosis may occur
in infants during the process of establishing normal flora
[92,102]. During pregnancy, oral contraception, or dia—
betes, the glycogen content of the vaginal epithelium is
raised, resulting in optimal conditions for growth of Candida.
The estrogen component of oral contraceptives seems to alter
carbohydrate metabolism, causing an increase in blood glucose
levels due to a slower disappearance rate than in non-oral

contraceptive users [97]. Antibiotic therapy, especially

9
if prolonged, alters the microbial flora, thus removing
the system of bacterial checks and balances, allowing
overgrowth of C. albicans [95]. Experiments in mice sug-
gest that antibiotics may also increase the pathogenicity
of C. albicans by allowing direct invasion through the
renal capsule [114]. Corticosteroids and antibiotics
may suppress the host's immune response, allowing pro-
liferation of Candida [95]. Immunosuppressive agents,
as cytotoxins, and other drugs which alter the host's
defense mechanisms may lead to candidosis [112].

Candidosis encompasses a wide spectrum of symptoms,
diseases, and pathology ranging from superficial to
systemic involvement. Allergies to C. albicans also
cause clinical manifestations [17,92).

Normal human serum contains a factor which is not an
antibody but is lethal to Candida[62]. Some patients
with cirrhosis, hepatitis, diabetic and non-diabetic
renal disease, and with mucocutaneous or systemic candi-
dosis show a reduction in the amount of this factor [62].
Iron-unsaturated transferrin [24] and lactoferrin [50]
have been shown to inhibit growth of C. albicans in vitro.
This inhibition is decreased when transferrin and lacto-
ferrin are saturated with iron. Another serum factor
called clumping factor, present in normal serum, promotes
germ tube formation and causes clumping if many Candida
organisms are present. This factor is not lethal to C.

albicans. It is inhibited by IgG antibodies for C.

10
albicans; however, this IgG does not affect the organism's
growth [13]. Although some of these factors may exhibit
anti-candidal effects in vitro, their in viva role has not
been elucidated.

Circulating antibody does not appear to play a major
role in candidal immunity [112]. The major host defense
lies in the cellular immune system supported by serum
Opsonization factor and the candidacidal capacity of poly-
morphonuclear leukocytes [8,49,59]. This is supported by
the low incidence of candidoses in Bruton-type agamma—
globulinemia, in which cellular immunity is intact but
humoral immunity is defective, and the high incidence of
mucocutaneous candidosis in persons with Swiss-type
agammaglobulinemia or DiGeorge syndrome, a thymus abnor-
mality [38,92].

Candida albicans may exist in 2 phases: a yeast (Y)
form or a mycelial, pseudomycelial (M) form, or both.

It was once thought that only the mycelial phase was the
invasive form; however, it has been shown that the yeast
phase may also be invasive [75]. The transition from Y
to M form is due to available nutrients and environmental
factors [l6,71,80,94]. The yeast phase is the form which
initiates the lesion. There is a change from Y to M form
as tissue invasion begins [112]. When cell division, but
not growth, is inhibited, elongation occurs, resulting in
a pseudomycelium [44,103]. The ratio of M form to Y form

increases as the lesion gets older [92]. It has been

11
suggested that the mycelium may have a capsular aggressin
which is cytotoxic to vascular endothelium [33]. Cellular
components may cause inflammatory and toxic responses in
the host [65].

As with other commensal organisms, if C. albicans
should occur out of its usual habitat or in greater numbers
than normal, it may produce disease. Its growth is better
in some organs than in others. 'The kidney is especially
favorable to growth of C. albicans. Other strains of the
organism will grow well in the brain and myocardium. The
skin is not particularly favorable for growth, nor are the

lungs, except when damaged [112].

Candidal Vaginitis

 

Candidal vaginitis is characterized by the presence
of a yellow to white milky or curdy discharge, patches of
gray-white pseudomembrane on the vaginal epithelium,
marked pruritus, inflammation, and a 3.8-5.0 pH range
[10,92]. "The lesions vary from a slight eczematoid
reaction with minimal erythema to a severe disease process
with pustules, excoriations, and ulcers." Extension to
perineum, vulva, and the entire inguinal area may occur
[92].

Oral contraception [47,87], pregnancy, diabetes,
corticosteroid therapy, antibiotic therapy, and immune

deficiencies predispose to vaginal candidosis [112]. It

12
is more common premenstrually due to the increased estro-
gen level [19].

Treatment is usually topical. Nystatin, 0.25-2.0%
gentian violet solution, and candicidin, among other anti-
fungals, have been used for treatment of candidal vaginitis,
but recurrent infections are common. A high percentage
of patients with recurrent vaginal candidosis have C.
albicans in oral smears [4]. Sexual transmission is
usually not a problem unless the male is uncircumcized
[12]. Balanitis, probably of an allergic nature, occurs
rarely in men whose spouses have candidal vaginitis. It

disappears when the spouse's vaginitis is treated [92].

Experimental Murine Infections

 

The mouse has proven to be a useful model for the
study of pathogenesis and treatment of candidoses. Many
investigators have induced experimental candidosis in
mice. The great majority of these studies were conducted
using the intraperitoneal or intravenous route to estab-
lish infection. Immunity to CL aZbiaans [48,76,77] as
well as enhancement of virulence by various agents [54]
has been studied in the mouse. Even vaginal candidosis
has been established in mice [104].

In most mice, C. albicans shows a predilection for
the kidney. It has been suggested that this is due to the
slower clearance of the organism from the kidney than

from other organs. Penetration of the renal tubules is

13
followed by proliferation in intratubule sites. While
other organs have been found to mount a cellular response
which clears the organism, the renal intratubule position
apparently is not accessible by normal cellular defenses.
The result is intratubule proliferation of the organism
leading to death [63].

The effects of cortisone [64,96], various antibiotics
[95,96], and artificial diabetes [3] have been studied in
mice and were found to enhance candidosis. Experimentally
induced mouse thigh lesions have been used as a model for
the study of the efficacy of various antifungal drugs and
for the study of the effects of various bacterial anti-
biotics and cortisone on the lesions [82,83,105].

Establishment of intestinal colonization by oral
administration of C. albicans has been used by few
investigators as a model. Those using this technique

reported no particular difficulties [64,96].

Nystatin

In 1949, Hazen and Brown isolated the first anti-
fungal antibiotic, which they called fungicidin, from a
soil actinomycete, Streptomyces noursei [36]. The drug
was later named nystatin and is produced by E. R. Squibb
6 Sons under the trade name Mycostatin.R It was recovered
from the surface growth of the organism after fermenta-
tion. The intracellular product was shown to be both

fungistatic and fungicidal. Nystatin is highly insoluble

14
in water and only slightly soluble in the lower aliphatic
alcohols, but this solubility is enhanced if water is
added. It is highly soluble in propylene glycol, N,N-
dimethylformamide, or dimethylsulfoxide. The antifungal
activity is broad, while there is no antibacterial
activity [36].

The empirical formula of nystatin is C46H77N019.
There are four C-methyl groups and one amino sugar moiety,
mycosamine, in the molecule. Nystatin is a tetraene,
having four conjugated double bonds [11,39].

In its purest form, nystatin exists as fine needles,
but is commonly a white, amorphous or partly crystalline
powder that is unstable in the presence of light, oxygen,
and extremes of pH. This instability is due to unsaturated
and unstable parts of the nystatin molecule. Nystatin
is also unstable in suspension but will remain active in
water for 2 weeks if refrigerated. In the dry crystalline
form it is stable at room temperature [39].

Nystatin formulated for clinical use has a potency
of 3500 units/mg. This drug has broad spectrum antifungal
properties against both pathogenic and saprophytic fungi
in vitro [30]. The minimal inhibitory concentration (MIC)
for C. aZbicans and Candida spp. is 1.56-6.25 pg/ml [20].
Nystatin also has an inhibitory effect on Trichomonas
vaginalis and will kill Leishmania [39]. The presence

of blood or plasma decreases its activity [30].

15

In vivo resistance has not been demonstrated with
C. albicans; however, gradual repeated exposure in vitro
may result in resistant strains [60,86]. This is especially
true of species of Candida other than C. albicans. Organ-
isms treated in this way also demonstrate resistance to
amphotericin B, another polyene antifungal agent.

Nystatin binds to sterols present in the cell membrane
of a susceptible organism [56,58]. This binding causes
a change in permeability of the cell membrane, resulting
in leakage of cellular components, including essential
ions such as potassium [55,86]. Inhibition of endogenous
respiration and aerobic and anaerobic glucose utilization
also occur [20,57,58]. Impairment of intracellular pro—
tein synthesis results from interference with phosphate
utilization and accelerated degradation of adenosine
triphosphates. Stimulation of oxygen uptake coupled with
inhibition of respiration result in death of cells.
Nystatin is not bound by bacteria or resistant organisms
[56,58].

Nystatin can be administered orally to laboratory
animals in dosages of 800,000 units/kg body weight without
toxicity. The LD50 for the crude substance given to mice
is 20-26 mg/kg injected intraperitoneally. It is less
toxic when administered subcutaneously [36]. Parenteral
administration of 1.2 mg/kg was tolerated for 30 days in
dogs [39]. In man, doses of up to 12 g/day orally for 6

months were administered with no side effects other than

l6

transitory nausea and diarrhea [79]. Nystatin is not
absorbed from the gastrointestinal tract; thus, nearly
all of the drug is excreted in the feces [30]. Intra-
muscular and intravenous administration have consistently
been associated with severe systemic symptoms, and local
inflammation at the site of injection. When given paren-
terally, shaking, chills, fever, and malaise accompanied
the first injection. Sclerosing of veins was also noted.
Severe pain and tenderness at the site of injection
resulted when given intramuscularly [37]. Nystatin has
been used as an aerosol containing 25,000 units and as
an eye drop suspension of 20,000 units/drop without
toxicity. Topical application in a concentration of
100,000 units/g of vehicle has been used without untoward
effects. No sensitivities have been reported [30,39].

Since nystatin produces toxic effects when given
parenterally and is not absorbed through the gastroin-
testinal tract or the epidermis, the primary use is in
the therapy of superficial candidosis and conjunctival
infections caused by sensitive fungi [39]. Nystatin is
available in oral and vaginal tablets, oral suspension,
ointments, creams, and powders. Oral tablets contain
100,000 units each; the oral suspension contains 100,000
units/ml; ointments, creams and powders contain 100,000
units/g of vehicle. For adults, the oral dose is 500,000
units to 1,000,000 units 3 times daily; for children,

100,000 units 3 or 4 times daily. No toxicities or

l7
sensitivities have been reported from oral or topical
administration [30,39]. No irritation of skin or mucous
membranes and no toxic effects on the blood or blood-
forming organs have been reported [30]. Since nystatin
has no effect on bacteria, secondary bacterial infections
are not encouraged by its use [30,39]. Nystatin has been
used to treat all types of cutaneous candidoses [9,18,
43,115], intestinal candidosis in infants and adults
[18,21], oral candidosis [21,3152], e50phagea1 candidosis
[29,34], vaginal candidosis [4,7,45,85,116], paronychia
[99], urinary tract candidosis [18,21,41], pulmonary
candidosis [5,18,72] and aspergillosis [27], ocular
candidosis [74,93], and keratitis from aspergillosis [27].
It is used as prophylaxis against yeast overgrowth in
persons on corticosteroid therapy [30]. A combination
of tetracycline and nystatin is available to prevent
overgrowth of Candida during tetracycline therapy [30,
39,40]. Nystatin is also available as a sterile powder
for use in the laboratory as an antifungal agent in
bacteriological samples and media, especially in tissue

culture techniques [39,70,73,89].

Gentian Violet

 

Gentian violet (crystal violet, methyl violet,
methylrosaniline chloride) is one of the methylrosaniline
dyes. Introduction of methyl groups into the amino radicals

of pararosaniline hydrochloride (fuscin) results in the

l8
formation of methylrosaniline dyes. The methylrosaniline
dyes used in therapy are mixtures of the hexamethyl,
pentamethyl, and tetramethyl derivatives. Crystal violet
is more refined, consisting of the hexamethyl derivative.
Methyl violet is the pentamethyl derivative. Gentian
violet is a dark green powder with a metallic sheen. It
is moderately soluble in water, alcohol, and glycerine
[30,107]. These solutions are stable [84,100]. The old
proprietary name for gentian violet is pyoktanin [109].

Gentian violet is both bacteriostatic and bacteri—
cidal to gram positive bacteria, but gram negative and
acid—fast bacteria are resistant to it, presumably due to
differences in cell wall structure. Its mechanism of
action is unknown [26,30,100].

Gentian violet has been administered intravenously to
laboratory rabbits in doses of 10 mg/kg body weight with no
toxicity. All mucous membranes were instantly stained
purple, but the stain disappeared [15]. Rabbits that
received 20 mg/kg body weight intravenously died in 5 hours
[117]. In humans, gentian violet is nontoxic and nonirritat-
ing when used topically or orally. Transitory nausea, vomit—
ing, and diarrhea may occur with oral use, but no sensitivities
have been noted [30,39,88]. Gentian violet has been admin—
istered to children and adults intravenously in doses of
5 mg/kg body weight or less, which were well tolerated
and without toxicity [117]. An infant received 3 mg/kg

intravenously, which was well tolerated; however, 5 mg/kg

19

as the initial dose caused a depression in the white blood
cell count [15]. Tattooing of the skin was reported when
gentian violet was applied to granulation tissue [100]. Not
much is known about the metabolism of gentian violet except
that little is absorbed when given by mouth.

Gentian violet is available in powder and solution,
which is 1% of the dye in 10% alcohol. A concentration
of 1:500 to 1:100 is used for direct application to
tissues. The concentration is reduced to 1:1000 for
instillations in closed cavities [30,39,88]. Enteric
coated tablets containing 25 mg are available as an over-
the-counter drug for treatment of enterobiasis. The
adult dosage is 50 mg 3 times daily for 10 days. Vaginal
tampons containing 5 mg gentian violet to be inserted
for 3 to 4 hours 1 or 2 times daily for 12 days are
available for treatment of vaginal candidosis. Vaginal
tablets containing 2 mg gentian violet or inserts con-
taining a 0.4% gentian violet solution to be inserted 1
or 2 times daily for 12 and 14 days, respectively, are
also available for treatment of vaginal candidosis [88].

Gentian violet was the first reasonably effective
treatment for strongyloidiasis. It may be used in treat-
ment of enterobiasis. There are newer, more effective
drugs presently available for treatment of these helmin—
thiases, so gentian violet is not the current drug of
choice in either infection [30,39]. Some therapeutic

uses of gentian violet include topical treatment of

20

pyogenic infections, geotrichosis [26], Vincent's infec-
tion [14,100], leg ulcers [53,90,106], chronic and irri—
tative lesions [30], paronychia due to Candida [28], oral
thrush [l7], diaper rash due to C. albicans [25], topical
therapy for candidal vaginitis [4], pruritus ani [100],
and other nondisseminated candidoses [l]. Intravenous
use has been recommended in cases of systemic candidosis
[25,100]. Crystal violet is used in many bacteriologic

media for the inhibition of gram positive organisms.

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11.

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ARTICLE I
Eradication of Candida albicans from
the Gastrointestinal Tract of Mice:

Comparison of Nystatin
and Crystal Violet

By

Olsen, Linda S. and Rogers, Alvin L.

(Article is to be submitted to Journal of Bacteriology)

ARTICLE I

Abstract

A model for experimental intestinal colonization
with Candida albicans was deveIOped in the mouse. This
model was then used to compare nystatin and crystal violet
with regard to efficacy in their ability to eradicate C.
albicans from the alimentary tract. Crystal violet suc-
cessfully eliminated C. albicans from the mouse gastro-
intestinal tract as evidenced by no growth of this yeast
from feces when placed into culture media. Nystatin
failed to eliminate, but did reduce, the intestinal popu-
lation of C. albicans. Intestinal colonization lasted at
least nine weeks in control animals. Systemic dissemina-
tion probably did not occur as no evidence of growth of
yeast resulted when the macerated kidneys were placed into
culture media. The results obtained may have an important

influence on human therapy.

Introduction

 

Candida albicans is a commensal yeast in the intes-
tine of 25-50% of healthy persons [2,27]. It is passed
from mother to neonate at birth during passage through the

birth canal [9,24]. The incidence of Candida infections

32

33
and the change from commensal to pathogen are
increased in debilitated persons as well as in those with
altered physiological states. Examples of conditions
which predispose to candidosis are: oral contraception
[8,17], pregnancy, infancy, diabetes, immunosuppressive
therapy, immune deficiencies, and antibiotic therapy
[19,27].

It is thought that the intestine is a reservoir for
infection of other parts of the body by C. albicans [1,
12,24]. In debilitated persons, systemic dissemination
may occur. More commonly, the gut may seed the perianal
area causing cutaneous candidosis of both infant and
adult. The intestine is also a reservoir for the infec-
tion of the vagina and reinfection after topical vaginal
treatment [26].

To eliminate the reservoir, C. albicans must be
eradicated from the alimentary tract. Since C. albicans
is most frequently acquired at birth [9,24] and is not
normally found in non-animal environments [19], eradica-
tion should be long lasting. Sternberg et al. [22], as
well as other investigators, have suppressed the intestinal
C. albicans population in mice and humans by administra-
tion of nystatin. However, they failed to completely
eradicate the C. albicans population of the alimentary
tract as evidenced by reappearance of the yeast soon after

treatment ended [13,22].

34

Crystal violet is an old drug which is currently on
the market as gentian violet and is used for therapy of
candidosis. It is effective against organisms with gram
positive cell walls. Not much is known about its metabo-
lism, but it is known that little of the drug is absorbed
when given by mouth. Its mechanism of action is unknown
[4,5,18,23].

Nystatin is an antifungal compound derived from the
soil actinomycete Streptomyces noursei. It is a polyene
with broad spectrum antifungal activity but shows no
effect on bacteria [6]. Since this drug is not absorbed
from the gastrointestinal tract or the epidermis [5,7],
the primary therapeutic use is in the treatment of super—
ficial candidosis and conjunctival infections due to
sensitive fungi [7]. Nystatin is bound to sterols of the
cell membranes present in susceptible organisms. This
binding causes a change in permeability of the cell
membrane, resulting in leakage of cellular components,
especially essential ions such as potassium [10,16].
Stimulation of oxygen uptake, coupled with inhibition of
respiration, results in cell death [3].

The mouse has been a useful model for the study of
pathogenesis and treatment of various forms of candidosis.
Most investigators have induced systemic candidosis through
intraperitoneal or intravenous inoculation. The effects
of cortisone [11,21] and various antibacterial compounds

[20,21] have been studied in mice. An experimentally

35

induced mouse thigh lesion has been used as a model for
the study of the effects and interactions of various anti-
mycotic and antibacterial agents and cortisone [14,15,25].
Establishment of intestinal colonization by oral adminis—
tration of C. albicans has been used by only a few investi-
gators, who reported no particular difficulties [21,22].

In the present study, a mouse model for intestinal
colonization by C. albicans is described. This model was
used to compare two antimycotic agents, nystatin and
crystal violet, with regard to efficacy in removal of an
intestinal population of C. albicans. The model is poten-
tially useful for a comparative study of human intestinal
candidosis, its treatment and prevention when candidoses

arise from intestinal sources.

Materials and Methods

 

Mice

 

Swiss white mice (Spartan Research Animal, 5735
North Shoeman Road, Haslett, Michigan) weighing approxi-

mately 25-30 grams were used.

Candida
Strain "B” of C. albicans from the stock cultures
of A. L. Rogers, PhD, Department of Microbiology and Public
Health, Michigan State University, was used. Inocula were
prepared from organisms grown in Sabouraud's glucose broth

(Appendix I) for 40 hours at 37 C in 250 ml Erlenmeyer

36
flasks rotating at 125 rpm on a shaker (New Brunswick
Laboratory Rotary Shaker, Model G2, New Brunswick Scien-
tific Co., New Brunswick, N.J.). Part of the yeast
suspension was mixed with an equal volume of a 0.5%
solution of trypan blue for counting in a haemacytometer
and for checking viability. The suspension was then
diluted with sterile physiologic saline to obtain the

desired cell count.

Inoculation

 

Volumes of 1.0 ml containing the desired number
of organisms (30 x 106 cells for Experiment I and 8 x 106
cells for Experiment 11) were administered through a
stomach tube. The stomach tube apparatus consisted of

a stainless steel l8-gauge hypodermic needle from which
the beveled edge had been removed. The needle was inserted
1/4" into a 1” length of Tygon Microbore tubing I.D.

0.040" x O.D. 0.070" and attached to a 3 cc syringe for

inoculation of mice.

Drug Administration

 

Volumes of 1.0 ml of either an aqueous solution
of crystal violet (Difco Laboratories, Detroit, Michigan)
or nystatin (Mycostatin Oral Suspension, E. R. Squibb &
Sons, Princeton, New Jersey) containing the desired amount
of drug were administered by the stomach tube. Fresh

nystatin solutions were made every 5 days.

37

Fecal Culture

 

Presence of C. albicans was determined by
inoculation of one average-sized fecal pellet freshly
collected from each mouse into 5.0 ml of Raulin's medium
(Appendix I) which was incubated for 7 days at 24 C.
Some broth from all cultures was then transferred to
cornmeal plus Tween—80 agar (Appendix I) and incubated
for 48 hours to 7 days at 24 C to determine if pseudo-

hyphae and chlamydospores developed.

Kidney Culture

 

Kidneys were removed, trimmed of connective
tissue, minced into pieces approximately 2 mm3 and plated
on Sabouraud’s glucose agar (Appendix I). These plates
were incubated at 24 C and observed daily for 7 days for

presence of yeast-like colonies.

Experiment I

 

In the first experiment 5 male and 6 female mice
were given 30 x 106 cells of C. albicans via stomach tube
for 4 consecutive days. Fecal specimens were collected
from all mice prior to the first inoculation to insure
that they were not previously colonized with C. albicans.
Fecal specimens were collected 1 day after the last
inoculum, 3 days after the last inoculum, 1 week after

the last inoculum, and weekly thereafter for 10 weeks.

38

Experiment II

 

In the second experiment 45 mice were given 8 x 106

cells of C. albicans via stomach tube for 4 consecutive
days. Fecal specimens were obtained prior to the first
inoculation to insure that the mice were not already
colonized with C. albicans. Mice were given a 3-day rest
after the last of the 4 inoculations. Fecal specimens
were collected 3 days after the last inoculum to insure
that the alimentary tracts of all mice were colonized by
C. albicans. At this time inoculated mice were divided
into 5 groups. Four groups of 10 mice each were to
receive drug therapy. The fifth group of 5 mice was to
act as a C. albicans colOnization control. Twenty-five
uninoculated mice were divided into groups of 5 each to
serve as either mouse or drug controls.

Each individual in the group of 10 colonized mice
and their respective controls received the specified
amount of one of the following drugs in l—ml volumes per
day for 10 consecutive days:

3.2 mg nystatin/mouse
1.6 mg nystatin/mouse
0.1% crystal violet (1 m1)

One milliliter amounts of a 1.0% crystal violet solu-
tion were administered to the remaining group of 10
colonized mice and controls for 3 consecutive days, but
the drug was terminated at that time due to several deaths

in both test and control groups.

39

Fecal specimens were collected 1 day after the 10th
dose of each drug was administered, except in the 1.0%
crystal violet group, from which specimens were obtained 1
day after the 3rd (final) dose and weekly thereafter. At
this time the mice from each nystatin group and the 0.1%
crystal violet group were divided into 2 subgroups of 5 mice
each. One subgroup received no further drugs, and their
feces were cultured weekly. The other subgroup received an
additional 5 days of treatment with the same drug and dose
as before for a total of 15 consecutive days treatment.
Feces from these mice were cultured 1 day after the last
(15th) dose of drug and weekly thereafter for 9 weeks.

Ten weeks after the last inoculation of C. albicans,
the kidneys from 5 mice of each drug group were placed in
culture medium to determine if there was evidence of

systemic dissemination.

Results

All mice in Experiment I remained colonized at least
5 weeks after the last inoculation of C. albicans as
evidenced by this yeast in their feces. After 6 weeks,
2 mice (1 male, 1 female) hadnegative fecal cultures,
while 9 remained positive. In the 7th week, 6 mice were
negative (3 males, 3 females) and 5 were positive, while
in the 8th week 9 were negative and only 2 remained
positive (1 male, 1 female). After 9 weeks all mice were
negative for C. albicans (Figure 1). All mice appeared

healthy throughout the experiment.

40

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41

In Experiment 11 the groups of mice received drugs
for a 10- or 15-day period. Figure 2 shows the results
of the individual treatment groups. All of the mice
receiving 0.1% crystal violet solution for 15 days became
culturally negative and remained negative for C. albicans
in the feces for at least 7 weeks after treatment. Eighty
percent of the mice receiving 0.1% crystal violet solu-
tion for 10 days became culturally negative and remained
negative at least 4 weeks after treatment. Three days
of treatment with 1.0% crystal violet solution reduced
the percent colonized to 28% 2 weeks after therapy. Not
any were colonized 4 weeks after therapy. Administration
of the 1.0% solution was terminated after 3 doses due to
several deaths. One mouse in this test group (colonized
and treated mice) and 1 mouse in the control group
(receiving drug only) died within 24 hours after receiving
the second dose. Two mice in the test group and 2 mice
in the control group died within 24 hours after receiving
the third dose. At this time the viable mice had scruffy
coats and no further treatments were given. Dissection
of the dead animals revealed perforated ulcerations along
the entire intestine.

All of the mice receiving 3.2 mg nystatin per day
for 15 days remained intestinally colonized for at least
3 weeks after termination of treatment. Eighty percent
were still colonized 4 weeks after treatment. Results

were similar in the other groups receiving nystatin. All

42

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43

mice in the group receiving 3.2 mg per day for 10 days
remained colonized at least 2 weeks after therapy.
Seventy-five percent were colonized 3 weeks after treat-
ment and 50% remained colonized 4 weeks after treatment.
All of the mice receiving 1.6 mg per day nystatin for
15 days remained colonized 2 weeks after therapy, while
30% remained colonized at least 4 weeks after drug admin-
istration. Eighty percent of the mice receiving 1.6 mg
per day nystatin for 10 days were culturally positive 2
weeks after therapy, while 60% remained colonized at
least 4 weeks after therapy.

The C. albicans control mice remained colonized at
least 9 weeks after the last inoculum of C. albicans.

Figure 3 shows the combined results in Experiment
11 of all groups treated with nystatin and all groups
treated with crystal violet. The majority of mice in
the combined nystatin groups remained colonized after
treatment; 95% were culturally positive 2 weeks after
therapy. However, only 17.6% of the combined crystal
violet groups were still colonized 2 weeks after therapy.
While 68.4% of the mice in the nystatin groups remained
colonized 4 weeks after treatment, only 5.9% of the mice

in the crystal violet groups were colonized at that time.

Discussion

 

The mouse model for intestinal colonization by C.

albicans described in this paper is easy to establish and

44

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ucoEumopp-umom mxoo?

 

 

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1 OO

 

 

 

45
the duration of infection is sufficient to be useful in
the study of effects of drugs used in therapy of candidosis
of the alimentary tract. It appears, from the data

6 cells) results

obtained, that the smaller inoculum (8 x 10
in a longer—lasting colonization than does the larger inocu-
lum (30 x 106 cells). Further work is necessary to determine
the size of inoculum and number of inocula which would
result in the longest period of colonization. At present,
this procedure was established for short-term (9 weeks or
less) studies.

In a similar experiment using only nystatin, Sternberg
et al. [22] showed that while nystatin reduced the number
of C. albicans in feces during therapy, colony counts in
both humans and mice rose after cessation of nystatin
therapy. In the present study, nystatin and crystal
violet were used to determine if either of these drugs
could eradicate the intestinal population of C. albicans.
The dosages selected were comparable to those used in
human therapy.

Nystatin was ineffective in removal of the C. albicans
population in the alimentary tract. Raising the dose,
lengthening the treatment time, or both, had no effect on
the number of mice that remained colonized. The groups of
mice receiving crystal violet, however, became and remained
culturally negative with degrees of success varying with

drug concentration and length of treatment. While all

46
groups receiving crystal violet experienced a marked
reduction in percent colonization, the group receiving
a 0.1% solution for 15 consecutive days showed total
eradication of the intestinal population. This is the
concentration that is recommended for use in closed
instillations in humans. These mice remained culturally
negative at least 7 weeks after treatment (9 weeks after
last inoculation of C. albicans).

The mice receiving a 1.0% solution of crystal violet
did not tolerate this high concentration well. This dose
is 10 times more concentrated than that recommended for
analogous use in humans; it is, however, the recommended
concentration for topical use in humans.

The effect on the intestinal yeast population obtained
with nystatin administration is comparable in mice, other
laboratory animals, and humans [13,22]. Presumably the
results of crystal violet treatment would also be compar-
able. If so, it would be possible to eradicate the
carrier state for C. albicans in humans. This has many
ramifications. Since candidosis is a disease of endogenous
nature, probably originating from intestinal foci, eradi-
cation of the intestinal population coupled with topical
therapy to eliminate any extraintestinal infection would
be of utmost benefit to the patient in prevention of

recurrent infections.

47

References

 

I.

10.

11.

12.

13.

Dobias, R. J. 1957. Moniliasis in pediatrics.
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Drake, T. E. and H. I. Maibach. 1973. Candida and
candidiasis. 1. Cultural conditions, epidemiology
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Drouhet, E. 1970. Basic mechanisms of antifungal
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Ferguson, F. C., Jr. 1962. Drug Therapy. Lea and
Febiger, Philadelphia, p. 76.

Goodman, L. S. and A. Gilman. 1970. The Pharmaco-
logical Basis of Therapeutics. The MacMillan Co.,
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Hazen, E. L. and R. Brown. 1951. Fungicidin, an
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Hildick-Smith, G. 1971. In: Drill's Pharmacology
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1650.

Jensen, H. R., P. L. Hansen, and J. Blom. 1970.
Incidence of Candida albicans in women using oral
contraceptives. Acta Obstet. Gynec. Scand., 49:
293-296. ——

Kozinn, P. J., C. L. Taschdjian, et al. 1960.
Transmission of P-32 labeled Candida albicans to
newborn mice at birth. A.M.A.J. Dis. Child., 88:
31-34.

Lampen, J. O. 1966. Interference by polyenic anti-
fungal antibiotics (esp. nystatin and filipin) with
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Studies of Antimicrobial Drugs. B. A. Newton,

M. W. Reynolds (eds.), Cambridge, London, p. 110.

Louria, D. B. and H. G. Browne. 1960. The effects
of cortisone on experimental fungus infections. Ann.
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Manson, I. 1957. Part I: Clinical, clinical
investigation. Squibb Clinical Summary, pp. 6-27.

Newcomer, V. D., E. T. Wright, and T. H. Sternberg.
1955. The effect of nystatin when administered
simultaneously with tetracycline upon the yeast
flora of the gastrointestinal tract of man. Anti-
biot. Ann. l954-l955:686-690.

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O'Grady, F., R. E. Cotton, and R. E. M. Thompson.
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656-665. __

O'Grady, F. and R. E. M. Thompson. 1958. Compara-
tive effects of chlortetracycline and cortisone on
a local monilial lesion in the mouse. Brit. J.
Pharm. Chemother., 18:1-5.

Patel, P. V. and J. R. Johnston. 1971. Kinetics
of action of nystatin on yeast. J. Appl. Bact.,
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Perl, G. 1970. Monilial vulvovaginitis following
"The Pill." Mt. Sinai J. Med., N.Y., 81:699-701.

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Rippon, J. W. 1974. Medical Mycology, The Patho-
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Seligman, E. 1953. Virulence enhancement of Candida
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Sternberg, T. H., J. E. Tarbet, et al. 1954. Anti-
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49

27. Winner, H. I. 1969. The transition from commensalism
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62-68.

ARTICLE II
Eradication of Candida albicans from

the Gastrointestinal Tract of Women

By

Olsen, Linda S., Rogers, Alvin L., Ryan, Mary H.

(to be submitted to the New England Journal of Medicine)

50

ARTICLE II

Abstract

Candidosis, most commonly caused by Candida albicans,
is an infection of endogenous nature, probably originating
from an intestinal focus. Oral gentian violet therapy
successfully eradicated the intestinal population of C.
albicans in women with recurrent vaginitis due to this
organism. Patients treated with the prescribed regimen
who submitted appropriate specimens remained culturally
negative for intestinal and vaginal C. albicans for at
least 3 months. Since C. albicans is rarely found in non-
animal environments, eradication should be long-lasting.
These findings have implications concerning treatment and
management of debilitated patients as well as normal

patients.

Introduction

 

Candida albicans is a commensal yeast in the intestine
of 25-50% of healthy persons [2,18]. It is passed from
mother to neonate at birth during passage through the
birth canal [8,16]. The incidence of Candida and the
change from commensal to pathogen are increased in debili-

tated persons as well as in those with altered physiological

51

52
states. Examples of conditions which predispose to candi-
dosis are: oral contraception [7,11], pregnancy, infancy,
diabetes, immunosuppressive therapy, immune deficiencies,
and antibiotic therapy [13,18].

It is thought that the intestine is a reservoir for
_infection of other parts of the body by C. albicans [1,9,
16]. In debilitated persons, systemic dissemination may
occur. More commonly, the gut may seed the perianal area
causing cutaneous candidosis of both infant and adult.

The intestine is also a reservoir for the infection of
the vagina and reinfection after topical vaginal treat-
ment [17].

To eliminate the reservoir, C. albicans must be
eradicated from the alimentary tract. Since C. albicans
is most frequently acquired at birth [8,16] and is not
normally found in non-animal environments [13], eradica-
tion should be long lasting. Sternberg et al. [14], as
well as other investigators, have suppressed the intestinal
Candida population by administration of nystatin. However,
they failed to completely eradicate the C. albicans popu-
lation of the alimentary tract as evidenced by reappearance
of the yeast within 2-5 days after treatment ended [14].

Candida albicans can be demonstrated in the normal
oral cavity of 25-50% of healthy persons (2,18] and is
frequently found in the estophagus at autopsy [3]. Thus,
to clear C. albicans from the alimentary tract, foci of

colonization both in and above the intestine must be

53
eliminated. It is possible that this could be done by
administration of gentian violet solution in conjunction
with gentian violet enteric-coated tablets which, together,
would reach C. albicans in the oral, pharyngeal, and
esophageal as well as intestinal areas. Candida albicans,
unlike other Candida species, is rarely found outside the
natural animal hosts [13], so eradication of the intestinal
reservoir should prevent most relapses.

Gentian violet is an old drug which is currently
marketed and used for therapy of candidosis in infants
and adults. It is effective against organisms with gram
positive cell walls. The mechanism of action is unknown
[4,5,6,12,15]. Not much is known about its metabolism,
but little is absorbed when given by mouth. In humans it
is nontoxic and nonirritating when used tOpically or
orally. Up to 5 mg/kg body weight has been administered
intravenously without toxicity [16,19]. Oral tablets are
sold as an over-the-counter drug for the treatment of
enterobiasis. These tablets have been used in treatment
of enterobiasis and strongyloidiasis without untoward
reactions. Hypersensitivities to this drug have not been
reported [5,12,15].

The object of this study was to determine if adminis-
tration of gentian violet solution in conjunction with
gentian violet oral tablets would clear the adult alimen-
tary tract of C. albicans. This has implications con-

cerning the management and prophylaxis of candidosis

54
in the debilitated patient as well as in the healthy
patient with pruritus ani, diaper rash, or vaginitis

due to C. albicans.

Materials and Methods

 

Patients

Healthy non-pregnant female patients 18-30
years of age who presented themselves for treatment at
Olin Health Center (Michigan State University, East
Lansing, Michigan) with recurrent vaginitis due to C.
albicans alone were chosen on a volunteer basis. The
cause of vaginitis was determined by physicians at Olin
Health Center, supported by laboratory examinations by
personnel at Olin Health Center Laboratory. In those
patients in which C. albicans was the sole cause of
vaginitis, an oral swab and rectal swab or fecal specimen
were also collected to determine presence of oral and
intestinal colonization. These and further specimens
were processed in E-216 Fee Hall, Michigan State Uni-
versity. Only those patients exhibiting both vaginal
and intestinal colonization were accepted for participa-
tion in this study. Informed consent (Appendix II) was
obtained from each volunteer after the nature of the pro-
cedure had been fully explained. Patients were free to

withdraw from the study at any time without penalty.

55
Cultures
Raulin's medium (Appendix I) was used for
primary isolation of C. albicans. Chlamydospore forma-
tion on cornmeal plus Tween-80 agar (Appendix I) was
used to identify C. albicans. All cultures were incu-
bated at 24 C for 7 days to determine presence of C.
albicans. The swabs used in collection of oral and
vaginal specimens were inoculated directly into 5 ml of
Raulin's medium. Either fecal specimens or rectal swabs
were obtained before treatment. Only fecal specimens
were obtained after medication was taken. Approximately
1 gram of fecal material was inoculated into 5 ml of
Raulin's medium. Oral swabs, fecal specimens or rectal
swabs, and vaginal swabs were placed into culture media
for C. albicans before therapy, 2 days, 1 week, and 1
month after oral therapy was completed. Specimens were
obtained from 5 patients 3 months after completion of

therapy.

Medication

 

All patients admitted to the study received
the following:

(1) Gentian violet oral tablets (Jayne's P-W Vermi-
fuge, Glenbrook Laboratories, Division of Sterling Drug,
Inc., 90 Park Avenue, New York, N.Y. 10016). Each tablet
contains 25 mg gentian violet. Patients were instructed

to follow the recommended adult dosage of 2 tablets 3

56
times daily during meals for 10 days. Patients were
advised to refrain from alcohol consumption during
treatment, as recommended in the Physician's Desk
Reference [12].

(2) Gentian violet (Merck, Sharp and Dohme, Division
of Merck Co., Inc., West Point, Pa. 19486) in a solution
with a final concentration of 0.1%. Patients were
instructed to dilute 30 cc of a 1.6% solution with 450
cc of grape juice or another strongly flavored non—
alcoholic beverage to produce a final volume of 480 cc,
giving a final concentration of 0.1%. Twice daily for
4 consecutive days, 60 cc of this solution was to be
swished around in the mouth, then swallowed. Patients
were instructed to refrain from eating or drinking for
2-3 hours after administration of the solution.

(3) One course of treatment with gentian violet
tampons (Genapax, Lakeside Laboratories, Inc., 1707 E.
North Avenue, Milwaukee, Wis. 53201). Each tampon con-
tains 5 mg gentian violet. Patients were instructed to
insert 1 tampon for 3-4 hours each day for 12 consecutive
days. Patients were advised to have their partner use a
condom during sexual intercourse, as recommended in the

Physicians' Desk Reference [12].

Results
Twenty-two patients were involved in the study.

Four were unwilling to complete the major portion of the

57
treatment due to nausea, vomiting, diarrhea, or a combina—
tion of these. All patients completing 6 days or more of
therapy became culturally negative for oral and intestinal
C. albicans. Table I shows the results of all patients

studied.

Stool Specimens

 

Of the patients who carefully followed the
prescribed course of treatment (i.e., those who took all
the tablets, at least one dose of the liquid solution,
and used all the tampons), 100% (9) had stool cultures
which were negative for C. albicans 1 month after comple-
tion of therapy. Seven specimens were obtained 2 days
and 1 week after therapy, of which 85.7% (6) were negative
each time. Three of these patients also submitted 2- and
3—month post-treatment specimens which were also negative.

Nine patients did not follow the oral treatment
exactly, but did report completion of 6 days or more of
tablet therapy, used all the tampons, and took at least
1 dose of the liquid solution. All of these patients
were culturally negative 1 month after therapy. Of the
6 specimens obtained 2 days after therapy, 16.6% (1) were
negative. Seven specimens were received 1 week after
therapy, of which 57% (4) were negative. Two of these
patients also submitted specimens which were negative for

C. albicans 2 months after therapy.

58

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:H oeeoweme cheeses :qu mucoHumm :H peoepmopp uoHoH> :mHucom Ho mpHsmom .H oHOme

59
Four patients took 5 days or less of the oral
therapy. One of these patients became culturally nega-
tive 2 days after completion of therapy and remained
negative for at least 1 month after therapy. The others

remained culturally positive for intestinal C. albicans.

Oral Specimens

 

Oral specimens from 19 patients were collected
before administration of medication. At this time 36.84%
(7) of these patients had oral cultures positive for
C. albicans. Twenty-five percent (4) of the 16 specimens
obtained 2 days after completion of total oral medication
were culturally positive at this time. Of the 13 specimens
obtained 1 week after therapy, 7.69% (l) were positive.
All 22 patients, even those who took only 1 dose of liquid
medication (2 patients), had negative oral cultures 1

month after therapy.

Vaginal Specimens

 

Of the patients who completed vaginal therapy,
but not necessarily oral therapy (18 patients), 55.5%
(10) had vaginal cultures which were negative for C.
albicans 1 month after therapy.
Of the 17 patients who both became negative for oral
and intestinal C. albicans and submitted vaginal specimens,
64.7% (11) were culturally negative for vaginal C. albicans

1 month after therapy.

60

All patients who had both negative vaginal and
negative stool cultures 1 week after therapy (4 patients)
remained negative at both sites at least 1 month after
therapy. One of these patients submitted specimens 3
months after completion of therapy which were negative
for C. albicans. Of the 8 patients who had negative
stool cultures 1 week after therapy and who submitted
the appropriate specimens, 75% (6) had negative vaginal

cultures both 1 week and 1 month after therapy.

Discussion

 

Current therapy for candidosis is aimed at the site
of infection rather than the intestinal source of the
infection. Even when oral intestinal therapy with
nystatin, the current drug of choice, is combined with
topical therapy to the extra-intestinal site, the goal
and result is merely to return the population of C.
albicans to commensal proportions. Sternberg et al.[14]
showed that, although nystatin reduced the intestinal
yeast population during therapy, within 2-5 days after
cessation of nystatin the C. albicans population had
returned to numbers as high or higher than those observed
before treatment. The purpose of the present study was
to determine if the alimentary population of C. albicans
could be totally eradicated through use of gentian violet.

In mouse experiments comparing crystal violet (a more

refined form of gentian violet) and nystatin with regard

61
to the ability to remove the intestinal population of
C. albicans, it was shown that nystatin was ineffective
but that crystal violet eradicated the yeast. In the
present study gentian violet was found to be effective
in eradication of the carrier state of C. albicans in
humans. The data indicate that at least 1 dose of a
0.1% gentian violet solution coupled with 6 to 10 days
of gentian violet oral tablet therapy results in long-
lasting removal of the intestinal reservoir of C.
albicans.

In some patients, nausea and at times vomiting and/or
diarrhea accompanied the treatment. These discomforts
were most often associated with the administration of
the liquid solution of gentian violet. Most patients
noting these transitory side effects were willing to
endure the discomfort in light of the benefits they hoped
to obtain. Since the number of doses of the liquid did
not seem to alter removal of intestinal carriage as long
as most of the tablets were taken, only 1 dose of liquid
may be sufficient, or the liquid may not be necessary at
all.

The 35-45% failure rate experienced after 1 course
of the vaginal tampons compares with the failure rates
observed with other vaginal antimycotic agents [10].
However, the failure rate was reduced to 25% (2 of 8) in

those patients whose intestines were negative for C.

62
albicans within a few days of completion of vaginal
therapy.

Since the intestinal source was removed and the
vaginal site of infection treated, these patients would
not be expected to develop recurrent vaginal candidosis
in the future. Administration of another course of
vaginal tampons would be expected to remove the vaginal
population of C. albicans in the vaginal failures. This
removal would be long lasting if the intestinal source
were also removed.

It can be concluded, then, that gentian violet may
be effective in eradicating the intestinal reservoir of
C. albicans. Candidosis is usually a disease of endogenous
origin, spread from gastrointestinal foci; thus, elimina-
tion of the intestinal population coupled with topical
therapy to any extra-intestinal infection would be of
utmost benefit to the patient. Since C. albicans (l)
unlike other Candida species, is rarely found in non-
animal environments [13], (2) is most commonly acquired
at birth by swallowing a portion of the vaginal contents
on passage through the vaginal canal with resultant
intestinal colonization [8,16], and (3) infections are
usually of an endogenous nature, originating from gastro-
intestinal carriage [16,17], eradication of the alimentary
population of C. albicans should result in a long-lasting
cure for such diseases as recurrent vaginitis, pruritus

ani, and diaper rash due to C. albicans. This also has

63

implications concerning treatment and management of the
debilitated patient. Candida albicans is able to spread
by persorption from the intestine to internal organs,
where, in the debilitated person, it may cause a serious
infection [3]. Removal of the intestinal source of
infection could prevent a potentially lethal candidosis.

Gentian violet may be tolerated intravenously in
doses of up to 5 mg/kg body weight without toxicity [15,
19]. In cases of systemic dissemination, intravenous
therapy combined with oral therapy could prove to be a
less toxic and longer-lasting treatment than the current
nephrotoxic drug of choice, amphotericin B.

This study has shown that it is possible to remove
the intestinal reservoir of C. albicans by oral adminis-

tration of gentian violet, a nontoxic, inexpensive drug.

64

References

 

1.

10.

11.

12.

13.

Dobias, B. 1957. Moniliasis in pediatrics. A.M.A.J.
Dis. Child., 84:234-251.

Drake, T. E. and H. I. Maibach. 1973. Candida and
candidiasis. 1. Cultural conditions, epidemiology
and pathogenesis. Postgrad. Med., 88:83-87.

Eras, P., M. J. Goldstein, and P. Sherlock. 1972.
Candida infection of the gastrointestinal tract.
Medicine, 81:367-379.

Ferguson, F. C., Jr. 1962. Drug Therapy. Lea and
Febiger, Philadelphia, p. 76.

Goodman, L. S. and A. Gilman. 1970. The Pharmaco—
logical Basis of Therapeutics. The MacMillan Co.,
New York, London, Toronto, pp. 1299-1300.

Hildick-Smith, G. 1971. Chemotherapy of fungus
infections. Chapter 83 in: Drill's Pharmacology
in Medicine. McGraw-Hill Book Co., New York, pp.
1363-1375, 1650.

Jensen, H. K., P. A. Hansen, and J. Blom. 1970.
Incidence of Candida albicans in women using oral

contraceptives. Acta Obstet. Gynec. Scand., 48:
293-296.

Kozinn, P. J., C. L. Taschdjian, et al. 1960. Trans-
mission of P-32 labeled Candida albicans to newborn
mice at birth. A.M.A.J. Dis. Child., 88:31-34.

Manson, I. 1957. Part I: Clinical, clinical
investigation. Squibb Clinical Summary, pp. 6-27.

Mead, P. 1974. Candida albicans. In: Infectious
Diseases in Obstetrics and Gynecology. G. R. G.
Monif (ed.), Harper and Row, Hagerstown, Md., pp.
242-267.

Perl, G. 1970. Monilial vulvovaginitis following
"The Pill." Mt. Sinai J. Med., New York, 81:699-701.

Physicians' Desk Reference. 1974. Medical Economics
Co., Oradell, N.J., pp. 787,795,819,1280.

Rippon, J. W. 1974. Medical Mycology, The Patho—
genic Fungi and the Pathogenic Actinomycetes. W. B.
Saunders Co., Philadelphia, London, Toronto, pp.
175-204.

14.

15.

16.

17.

18.

I9.

65

Sternberg, T. H., J. E. Tarbet, et al. 1954. Anti-
fungal effects of nystatin on the fecal flora of
animals and man. Antibiot. Ann. 1953-1954:199-209.

Sutton, R. L., Jr. 1938. Gentian violet as a thera-

peutic agent, with notes on case of gentian violet
tattoo. J.A.M.A., 110:1733-1738.

Taschdjian, C. L. and P. J. Kozinn. 1957. Labora-
tory and clinical studies on candidiasis in the
newborn infant. J. Pediatr., 88:426-433.

Van Uden, N. 1960. The occurrence of Candida and
other yeasts in the intestinal tracts of animals.
Ann. N.Y. Acad. Sci., 88:59-68.

Winner, H. 1969. The transition from commensalism
to parasitism. Br. J. Derm., 81(Supplement 1):62-68.

Young, H. H. and J. H. Hill. 1924. The treatment
of septicemia and local infections by the intravenous

injection of mercurochrome 220-soluble and of gentian
violet. J.A.M.A., 81:669.

APPENDICES

APPENDIX I

CULTURE MEDIA

Sabouraud's Glucose Broth

Glucose 40 g
Neopeptone 10 g
Distilled water 1000 m1 .
Adjust to pH 5.6 and autoclave at 121 C for 15 min.

Raulin's Medium

Brown sugar 7
Tartaric acid
Ammonium nitrate
Potassium carbonate
Magnesium carbonate
Ammonium phosphate
Zinc sulfate
(crystals)
Ferrous sulfate
Manganese sulfate .
Distilled water 1500 m1
Adjust to pH 2-3 and autoclave at 121 C for 15 min.

CO COCOA-ho

Cornmeal Agar plus Tween-80

Cornmeal 40 g
Water 1000 m1
Agar 20 g
Tween-80 10 g

Simmer cornmeal and water for one hour. Filter or
decant, bring volume up to 1000 ml and add agar; melt
and filter again if necessary. Add Tween-80 and auto-
clave at 121 C for 15 min.

66

APPENDIX II

CONSENT FORM

I understand that my physician has decided that the
use of the antifungal agent gentian violet is indicated
for the treatment of my disease.

In order to better understand the treatment of
candidosis (yeast infections) with gentian violet,
Doctors Mary Ryan, Alvin L. Rogers, and their associates
at Michigan State University wish to run special studies
on oral, fecal, and vaginal specimens. This will consist
of approximately four oral, fecal, and vaginal specimens
obtained before and at various times after treatment. I
understand and agree that any data collected may be used
for research, teaching, and publication.

I further understand that the medical treatment I
receive will be the same whether or not I participate in
the study. I understand that all records will remain
confidential and that I am free to withdraw from this
study at any time without penalty.

 

The purpose of this study has been fully explained to me
and I have read and understood the above statement. My
participation in this study is freely given.

Signed

 

Witness

 

Date

 

67

APPENDIX III

PATIENT INSTRUCTIONS

1) Mix the entire bottle of gentian violet solution
with 15 oz. of grape juice. We recommend a frozen con-
centrate. When mixing the frozen concentrate, use as
little water as possible. The gentian violet is extremely
distasteful, so the stronger the grape juice taste, the
more the gentian violet taste will be disguised. The
gentian violet will stain clothing and possibly the con-
tainer you put the solution in, so be careful.

The total volume of the final solution (gentian
violet plus grape juice) will be 16 oz. (2 cups). This
will last four days. Take 2 oz. two times per day for
4 days. Stir or shake the solution to make sure it is
evenly mixed before taking each dose. However distaste-
ful, swish it around your mouth and swallow all of it
each time. It will turn the inside of your mouth purple.

Do not eat or drink anything for 2-3 hours after
swallowing this liquid. The bad taste will go away after
a few minutes.

You may keep the solution at room temperature or
you may refrigerate it. As long as you keep it covered
it shouldn't spoil.

2) Take the gentian violet tablets (Jayne's P-W
Vermifuge) as directed on the bottle under "adult dosage";
that is, 2 tablets three times per day for 10 days. Take
these during a meal. They may cause purple feces.

3) Do not drink any alcoholic beverages during the
10 days of oral treatment. The alcohol will wash the
gentian violet out of your system so the medication will
not have a chance to work.

4) Insert one Genapax tampon into the vagina and
leave it in for 3-4 hours once each day for 12 days. Try
to do this at about the same time each day. Any vaginal
discharge produced after the tampon is removed may be
purple and may stain your clothing. A condom may be used

68

69
during intercourse to avoid staining your partner purple.
5) Start all medication on the same day.

6) Follow the diet for the 10 days you take the
tablets. In particular, don't eat any fruit for these
10 days. This helps to cut down on the yeast population
of the intestine. After the 10 days you may resume your
regular eating and drinking habits.

7) Bring a stool specimen to me (Linda Olsen) on
April 21, April 28, and May 19. I will be in room 294
in Giltner Hall on the above days from 3-5 p.m. You may
collect the stool specimens in the containers provided
or any other suitable container (e.g., a small, plastic
margarine tub). When you give me the stool specimen 1
will give you a sterile swab so you can collect a vaginal
specimen in the rest room across from my office. Also
I will collect a mouth swab.

It is very important that you bring these stool
specimens to me on the assigned days. Otherwise, we will
not be able to follow your progress as closely as we
would like.

If, for any reason, you cannot deliver a stool
specimen to me on the appropriate day or time (3-5 p.m.),
please call me at home in the evening so we can make other
arrangements. My phone number is 351-3337.

8) A suggested regimen is as follows:

Morning - 2 tablets during meal
2 oz. solution after meal

Afternoon - 2 tablets during meal

Evening - 2 tablets during meal
Insert Genapax tampon for 3-4 hours
2 oz. solution before bedtime

Be sure to eat some sort of food when taking the tablets
and leave at least a 5-hour interval between doses of
tablets.