Probiotics & Antimicro. Prot.
(2015) 7:38–44
DOI 10.1007/s12602-014-9176-0
Local Probiotic Therapy for Vaginal Candida albicans Infections
Stefan Miladinov Kovachev •
Rossitza Stefanova Vatcheva-Dobrevska
Published online: 2 November 2014
 Springer Science+Business Media New York 2014
Abstract The high rate of vaginal Candida albicans
recurrence is attributed to azole resistance rates as high as
15 %. The aim of this study was to determine the clinical
and microbiological efficacy of standard azole therapy for
treatment of vaginal C. albicans infection alone and in
combination with local probiotic as well as the effects on
vaginal microbiota. This study included 436 women with
vaginal candidiasis randomly assigned to two treatment
groups. The first group, with 207 patients (12 dropouts),
was administered 150 mg fluconazole and a single vaginal
globule of fenticonazole (600 mg) on the same day. The
second group of 209 patients (8 dropouts) followed the
same treatment schedule; however, ten applications of a
vaginal probiotic containing Lactobacillus acidophilus, L.
rhamnosus, Streptococcus thermophilus, and L. delbrueckii
subsp. bulgaricus were also administered beginning the
fifth day after azole treatment. Microbiological analysis of
the therapy efficacy in the first treatment group showed C.
albicans resistance in over 30 % of patients. Clinical
complaints persisted after treatment administration in
79.7 % (n = 165) of women in this group. Clinical com-
plaints in the second group decreased to 31.1 % (n = 65)
and microbiological efficacy also improved among
S. M. Kovachev
Department of Gynecology, Military Medical Academy,
G. Sofijsky Str. 3, 1600 Sofia, Bulgaria
S. M. Kovachev (&)
‘‘P.U.Todorov’’ bul. bl.§ 5, entr.B, fl.§ 25, 1404 Sofia,
Bulgaria
e-mail: stkovachev@abv.bg
R. S. Vatcheva-Dobrevska
Department of Microbiology and Virology, University Hospital
Queen Joanna- ISUL, Bialo More Str. 8, 1527 Sofia, Bulgaria
123
investigated parameters, from 93.7 % (n = 193) to 95.2 %
(n = 198). The local application of probiotics after
administration of combined azoles for treatment of vaginal
C. albicans infections increases therapy efficacy and could
prevent relapse.
Keywords Lactobacilli
Vaginal Candida albicans

Azoles
Probiotic
Introduction
Candida albicans is part of the normal gastrointestinal
tract, oral cavity, and urogenital tract microbiota [1].
Depending on age, geographic location, and socioeco-
nomic status, up to 41 % of women may have one or more
Candida species as a normal constituent of their vaginal
microbiota [2]. Over 400 Candida species have been iso-
lated so far [2, 3]. Although many species have been
identified in the vaginal ecosystem (e.g., C. tropicalis, C.
pseudotropicalis, C. stellatoidea, C. krusei, and C. guil-
liermondii), C. albicans is most commonly isolated [2]. C.
albicans is attributed to more than two-thirds of mycotic
vulvovaginitis cases [4].
Azole antifungal drugs are most commonly administered
for vaginal C. albicans infections [5, 6]. These synthetic
derivatives are divided into two groups, imidazoles and
triazoles [6]. Triazoles have three nitrogen atoms in their
azole ring, while imidazoles have only two [6]. Their main
mechanism of action is related to inhibition of lanosterol
14-alpha-demethylase, an enzyme required for synthesis of
ergosterol, the major component of the fungal cell mem-
brane [5, 6]. Imidazoles include miconazole, ketoconazole,
and clotrimazole. Triazole agents most commonly used to
treat fungal infections include fluconazole, itraconazole,
Probiotics & Antimicro. Prot. (2015) 7:38–44
econazole, terconazole, butoconazole, and tioconazole [6].
New triazoles such as voriconazole, posaconazole, and
ravuconazole are used to treat Candida species infections
resistant to more common azoles [5]. Short-course topical
formulations (i.e., single dose and 1- to 3-day regimens)
effectively treat uncomplicated vulvovaginal candidiasis
(VVC) [12]. Topically applied azole drugs are more effec-
tive than nystatin [12]. Azole treatment results in symptom
relief and negative cultures in 80–90 % of patients who
complete therapy [12].
Individual studies C. albicans have reported up to 15 %
resistance to azole therapies, which contribute to high
recurrence incidence rates [7, 8]. The pathogenesis of
recurrent VVC (RVVC) is poorly understood, and most
women with RVVC have no apparent predisposing or
underlying conditions [12]. Individual RVVC episodes
caused by C. albicans respond well to short-duration oral
or topical azole therapy, but the relapse rate is high, with
approximately 60 % of women relapsing within
1–2 months of discontinuing therapy [12, 14]. To maintain
clinical and mycological control, some specialists recom-
mend longer initial therapy duration or different therapy
type, as well as different indications, schedules, or dosages
of the agents used to treat VVC [8, 9, 12]. Several multi-
center studies have reported primary and secondary resis-
tance to standard treatment regimens [8, 9]. Due to
therapeutic and microbiological failures of standard ther-
apy, probiotics were first introduced to gynecological
practices as new agents for treatment of vaginal C. albicans
infections in 2001. Probiotics containing live lactobacilli
species stabilize vaginal microbial balance, support VCC
therapy, and prevent infection recurrence [10, 11].
This study determined the clinical and microbiological
efficacy of standard azole therapy for vaginal C. albicans
infections alone and in combination with local probiotic
treatment as well as the effects of these treatments on
vaginal microbiota.
Materials and Methods
This single-center, randomized and open-label study was
conducted at the Outpatient Group Practice for Specialized
Care in Obstetrics and Gynecology ‘‘GynArt’’ (OGPSC-Ob
& Gyn, Sofia, Bulgaria) from 2008 to 2013. With approval
from the Local Ethics Committee Information, information
about the study purposes and entry requirements was pro-
vided, and informed consent was obtained from each
patient. A total of 436 women between 17 and 50 years of
age with clinically or microbiologically identified pre-
dominantly C. albicans vaginal infections were enrolled in
the study. Patients with Neisseria gonorrhoeae, herpes
simplex virus (HSV), human papillomavirus (HPV),
39
Chlamydia trachomatis, or human immunodeficiency virus
(HIV) infections were excluded. Pregnant women and
those who took corticosteroids, antibiotics, azoles, or pro-
biotics or who had used vaginal agents within the last
month were not enrolled. Immunocompromised patients
and women with autoimmune or endocrine diseases or
diabetes were not enrolled. Patients with identified malig-
nancies were also excluded.
Clinical Examination
Upon enrollment, a medical history was recorded, and
gynecological examinations and microbiological tests were
performed. During vaginal inspection, the amount, con-
sistency, color, and smell of vaginal discharge (often
defined by the patients as ‘‘flow’’) were assessed and
recorded on an outpatient card. The amount of vaginal
discharge (fluorine) was measured using four grades: 0, ?,
??, and ???. The consistency was assessed and recorded
as normal homogeneous, curdled, foamy and bubbled, or
scarcely homogeneous. Color was categorized as trans-
parent, white, yellow-greenish, or gray-yellowish. The
smell of vaginal content was measured using grades from 0
to (???). During inspection, vaginal tissue changes were
assessed, including erythema, edema (swelling), swollen
papillae, petechiae, and ulcerations. Several vaginal tissue
changes could be described in a single patient. The severity
of each was recorded on an outpatient card using increasing
grades of 0, (?), (??), and (???). Clinical complaints
reported by patients included pruritus vulvae (itching),
vulva and vagina erythema, and dyspareunia and dysuria
resulting from the passage of urine over irritated areas.
Enormous vaginal discharge was a variable finding, and
premenstrual exacerbation was characteristic.
Microbiological Tests
Measurement of Vaginal Acidity (pH)
pH of vaginal discharge was measured using pH test strips
(Merck, Darmstadt, Germany) that measured a pH range of
4–7. pH values above 4.5 were considered pathological. pH
values were recorded on the outpatient card for each patient
upon enrollment and at each subsequent examination.
Native Microscope Slides
Vaginal samples were placed on individual slides. Several
drops of saline were mixed each sample and a number of
fields of this suspension examined microscopically at low
and high magnification (910 and 9100). Lactobacilli were
observable as large and long rods and C. albicans, as
hyphae and blastospores. The presence or absence of
123
40
leukocytes was also recorded. Microscopic findings were
documented on the outpatient card as the presence or
absence of lactobacilli, C. albicans, and leukocytes were
recorded as 1 or 0, respectively.
Gram-Stained Microscope Slides
Vaginal samples were placed on individual slides that were
fixed, Gram-stained (BD Gram Stain Kits and Reagents,
BD, USA), and used to detect lactobacilli, pseudomyce-
lium (hyphae), and blastospores.
Culture
Vaginal discharge samples from all women enrolled in this
study were applied to selective culture media for micro-
biological testing to detect pathogenic microbial species.
Samples were collected using aseptic technique before
other vaginal tests were performed: A sterile speculum was
inserted and discharge collected from the upper side walls
of the vaginal vault using a cotton swab placed in Amies
transport medium (BD BBLTM Culture SwabTM Plus
Amies Gel, Single Swab, Becton–Dickinson, USA).
Sabouraud agar (BD, BBLTM Sabouraud-Dextrose-
Agar, BD, USA) was used for primary isolation of Candida
spp. Cultures were grown at 35–37 C for 40–48 h. A
chromogenic medium (BBLTM CHROMagarTM Candida-
BD, BD, USA) was also used for primary isolation, a solid
culture medium that also allowed tentative genus-level
identification of Candida fungi. Only patients positive for
C. albicans infections were included in our study.
Random Group Assignment
After obtaining informed consent, 436 patients with iden-
tified C. albicans infections were assigned to treatment
groups by stratified random sampling using Research
Randomizer software (version 3.0). Patients were randomly
assigned to two treatment groups in a 1:1 ratio. The first
and second treatment groups contained 219 and 217
women, respectively.
The first treatment group, containing 207 patients (12
dropouts) with vaginal C. albicans infection, followed the
first treatment schedule.
Similarly, the second group of 209 patients (8 dropouts)
with vaginal C. albicans infection followed the second
treatment schedule.
Treatment Schedules
• First treatment schedule: a single oral dose of fluco-
nazole (150 mg Mycomax or Mycosyst) administered
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Probiotics & Antimicro. Prot. (2015) 7:38–44
on the same day as a single vaginal globule of fenti-
conazole (600 mg Lomexin).
• Second treatment schedule: a single oral dose of
fluconazole (150 mg Mycomax or Mycosyst) adminis-
tered the same day as a single vaginal globule of
fenticonazole (600 mg Lomexin). Beginning the fifth
day after the single-day azole topical treatment, ten
doses of a vaginal probiotic agent containing live
lactobacilli species Lactobacillus acidophilus, L.
rhamnosus, Streptococcus thermophilus, L. delbrueckii
subsp. bulgaricus (Lactagyn-vag.capsules—Ecopharm,
Sofia, Bulgaria) were also administered.
Sexual abstinence was advised during treatment until the
first follow-up examination. Additional clinical and
microbiological testing was performed during a follow-up
examination 35–40 days after beginning treatment. This
time period was selected to track the vaginal microbiota of
patients included in this study after one completed men-
strual cycle and because approximately 60 % of women
relapse within 1–2 months of discontinuing therapy. Oral
fluconazole therapy of a single 150-mg dose repeated after
7 days was also prescribed to sexual partners. The United
States Centers for Disease Control and Prevention (CDC)
approved the azole therapy used to treat vaginal C. albi-
cans infections [12]. Resistance of C. albicans to azoles is
rare in vaginal isolates, and susceptibility testing is usually
not warranted for individual treatment guidance [12]. Pri-
mary resistance of C. albicans to fluconazole was not seen
in previous studies [14]. In vitro cross-resistance was
reported between fluconazole and other azoles (ketocona-
zole and itraconazole), but to a lesser extent [14].
Statistical Analysis
Clinical and microbiological data obtained at the follow-up
examination were analyzed using Chi-square test. Statisti-
cal significance was assumed for p \ 0.05. The proportion
(percentage) of patients within each treatment group with
improvements was recorded. The number of patients with
decreases in specific complaints (positive/negative labora-
tory or microbiological tests) due to treatment were
compared.
A two-factor dispersion analysis with repeated obser-
vations (two-way ANOVA with repeated measures) was
used for characteristics observed before and after therapy,
with therapy type a second factor. The proportion of
patients with improvement after therapy compared with
percentages of patients with complaints before therapy was
used as a parameter of improvement. Improvement was
calculated for each complaint type or positive/negative
laboratory or microbiological test results. We calculated a
Probiotics & Antimicro. Prot. (2015) 7:38–44 41

Table 1 Clinical parameters of women in the first treatment group Table 3 Clinical parameters of women in the second treatment group
before and after therapy before and after therapy
Clinical parameters Before Th After Th Improvement p Clinical parameters Before Th After Th Improvement p
N—% n % n % % N—% n % n % %

Clinical complaints 195 94.2 165 79.7 15.38 0.001 Clinical complaints 144 68.9 65 31.1 29.06 0.005
Vaginal fluorine 126 60.9 7 3.4 94.44 0.031 Vaginal fluorine 138 66 2 1 98.55 0.308
(??, ???) (??, ???)
Vaginal tissue 205 99 56 27 72.66 0.387 Vaginal tissue 204 97.6 11 5.3 94.61 0.594
changes changes
pH (alkaline) 88 42.5 2 1 97.73 0.098 pH (alkaline) 88 42.1 3 1.4 98.66 0.757
Th therapy, p p values (Chi-square test for distribution) Th therapy, P p values (Chi-square test for distribution)

Table 2 Microbiological parameters of women in the first treatment Table 4 Microbiological parameters of women in the second treat-
group before and after therapy ment group before and after therapy
Microbiological Before Th After Th Improvement p Microbiological Before Th After Th Improvement p
parameters parameters
N—% n % n % % N—% n % n % %
Native microscope slide
Native microscope slide
Spores, filaments 206 99.5 67 32.4 67.96 0.17 Spores, filaments 206 98.6 12 5.7 94.17 0.67
Lactobacilli 77 37.2 195 94.2 91.54 0.03 Lactobacilli 50 23.9 206 98.6 99.37 0.00
Gram-stained microscope slide Gram-stained microscope slide
Hyphae, spores 207 100 78 37.7 62.32 n.s. Hyphae, spores 206 98.6 13 6.2 93.69 0.63
Lactobacilli 73 35.3 196 94.7 91.79 0.02 Lactobacilli 50 23.9 206 98.6 98.74 0.70
Culture testing Culture testing
C. albicans 207 100 76 36.7 63.29 n.s. C. albicans 208 99.5 10 4.8 95.19 0.82

Th therapy, p p values (Chi-square test for distribution), ns change Th therapy, P p values (Chi-square test for distribution)
was not statistically significant

summary parameter for each group of complaints as the
presence of any complaint from the treatment group.
Results
measured in vaginal C. albicans infections varied widely.
Clinical complaints improved 15.38 %, while 72.6 % of
patients had improved vaginal tissue changes identified
during clinical examination and 94.44 % had vaginal
fluorine improvements (??, ???).

Of 436 patients enrolled in the study, 20 women did not
return for follow-up examination and were excluded from
analysis. Data from the remaining 416 patients who satis-
fied the study requirements were analyzed. A total of 207
and 209 women remained in the first and second treatment
groups, respectively.
Clinical and microbiological therapy efficacy among
women in the first treatment group.
Clinical Therapy Efficacy Measured by Clinical
Examination
Clinical efficacy was measured as the change in subjective
clinical and clinical examination findings at study enroll-
ment and 35–40 days later (Tables 1, 2).
After administration of therapy to the first treatment
group, improvements in clinical parameters typically
Microbiological Testing to Measure Therapy Efficacy
Aggregation of microbiological tests from all 207 women
in the first treatment group at study entry and at follow-up
after 35–40 days made it possible to measure the therapy’s
microbiological efficacy.
Microscopic examination of native and Gram-stained
slides of vaginal discharge showed improvements in
spores/filaments and hyphae/spores of 67.96 and 63.3 %,
respectively. However, slides with lactobacillus-dominated
vaginal microbiota increased from 35.3 to 91.79 %. The
microbiological efficacy of combined azole therapy was
measured based on culture and microscopic examination
results. The efficacy of this treatment schedule ranged from
63.29 to 67.96 %.
Clinical and microbiological efficacy among women in
the second treatment group.

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42 Probiotics & Antimicro. Prot. (2015) 7:38–44

Table 5 Clinical indicator improvements (%) among women in the Table 6 shows microbiological parameter differences
first and second treatment groups before and after therapy before and after therapy in the first and second group as
Clinical parameters Group I n = 207 Group II well as the relative microbiological efficacies (percentages)
n = 209 of combined azole as well as topical probiotic with con-
Improvement Improvement ventional azole therapies (first group) for treatment of
after Th after Th predominantly C. albicans-induced vaginal dysbacteriosis.
n % n %

Clinical complaints 30 15.38 79 29.06 Discussion

Vaginal fluorine (??, ???) 119 94.44 137 98.55
Vaginal tissue changes 149 72.66 193 94.61 Fungal resistance to the most common azole agents and
pH (alkaline) 87 97.73 85 98.66 frequent recurrence of infections determine the failure of
Th therapy C. albicans treatments [9, 13, 14].
Modern azole antifungal therapy failed in 10–20 % of
patients in Bulgaria due to resistant fungal strains [13].
Table 6 Microbiological parameter differences (%) among women Similar resistance prevalence has been reported in other
in the first and second treatment groups before and after treatment
countries [9, 14]. C. albicans resistance to azoles, the
Microbiological parameters Group I n = 207 Group II n = 209 commonly used agents and most accessible in everyday
Improvement Improvement life, has been characterized at a genetic level. Research has
after Th after Th shown resistance to be due to expression of specific genes
n % n % that control transport and accumulation of azole antifungal
agents [2, 8, 15]. Recent studies have shown that the pre-
Native microscope slide dominant C. albicans species genotype and mutations in
Spores, filaments 139 67.96 194 94.17 the ERG11 gene determines azole susceptibility [8]. Most
Lactobacilli 118 91.54 156 99.37 reports have studied the in vitro susceptibility of C. albi-
Gram-stained microscope slide cans to various therapeutic agents [14–17]. Although
Hyphae, spores 129 62.32 193 93.69 Candida strain susceptibility in vitro does not always mean
Lactobacilli 123 91.79 156 98.74 successful treatment, in vitro resistance almost always
Culture testing predicts therapy failure [6, 9]. Therefore, this study eval-
C. albicans 131 63.29 198 95.19 uated in vivo response to antifungal agents used in VVC
Th therapy treatment. Some researchers have hypothesized that
immune response to a recurrent pathogen (C. albicans) is
Therapy Efficacy Measured by Clinical Examination the basis of recurrent C. albicans infections [18, 19]. The
inability of antifungal drugs to influence immunity in such
Table 3 shows the clinical efficacy based on all measured cases results contributes to their inefficacy [20]. Wagner
clinical parameters, showing a higher efficacy in second et al. [21] reported in 2012 that C. albicans infection
treatment group compared with the first, with combined induces a pro-inflammatory immune response in vaginal
azole monotherapy. epithelial cells. Vaginal lactobacilli in the same study
inhibited NF-jB-associated inflammatory genes and also
Therapy Efficacy Measured by Microbiological Testing induced IL-1a and IL-1b expression through an alternative
signal transduction pathway [21]. Lactobacilli activation of
Table 4 shows the treatment microbiological efficacy for an alternative signaling mechanism modulates vaginal
all measured microbiological parameters, with values epithelial cell cytokine production [21]. This is the most
higher in this second treatment group compared with the likely mechanism for probiotic modulation of C. albicans
first. infections [21]. Lactobacilli can modulate the immune
Comparison of clinical and microbiological parameters response and protect the vagina from predominantly Can-
between the first and second treatment groups. dida chronic dysbacteriosis, an important feature for con-
Table 5 shows that the treatment schedule followed by trol of chronic and recurrent fungal infections [19, 20, 22].
patients in the second group had higher clinical efficacies Moreover, lactic acid bacteria competitively block adhe-
compared with the first group based on clinical parameters sion of Candida strains to vaginal epithelial cells and
typical to vaginal dysbacteriosis in C. albicans-predomi- produce antimicrobial substances that inhibit C. albicans
nant infections. Measured differences in improvement of growth and development [23–25]. These lactobacilli fea-
clinical parameters after treatment was low in both groups. tures recommend their use as antifungal agents either alone

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or in combination with other therapeutic agents [16, 17].
Clinical studies have evaluated the abilities of oral or in-
travaginal lactobacilli administration to inhibit fungal
vaginal colonization and reduce the incidence of recurrent
dysbacteriosis. Intravaginal probiotic agents have been
developed and put into practice in recent years; however,
clinical in vivo studies of their effects are limited and
report inconsistent findings on the efficacy of local probi-
otics against C. albicans [26, 27]. Some authors describe
high clinical and microbiological efficacy of probiotic
lactobacilli strains to treat and prevent fungal infections
and recommend their use in modern VVC therapy [26, 28].
Others do not support vaginal or oral administration of
lactobacilli for prevention of predominantly C. albicans
vaginal dysbacteriosis [27]. These studies found that pro-
biotics do not heal or protect patients with this condition
[27].
Fluconazole is a broad-spectrum antifungal drug with an
important role in VVC treatment [12, 29]. It is most often
recommended and administered for primary treatment of
acute vulvovaginal infections [6, 12, 29]. These infections
generally respond well clinically and microbiologically to a
single 150-mg dose of oral fluconazole [6, 12]. Previous
studies have reported 80–90 % fluconazole treatment effi-
cacy and 10–20 % resistance [9, 13, 14]. The presence of
even minimal fluconazole resistance raises doubts about
the full efficacy of a single 150-mg dose for treatment of
acute VVC and rapid resolution of clinical symptoms [6].
We therefore added a single topical dose of 600 mg fen-
ticonazole to a single oral dose of fluconazole for increased
and more rapid antimycotic and clinical efficacies [6].
Fenticonazole is an imidazole derivative with broad-spec-
trum antifungal activity [6]. It is applied topically to the
vagina as suppositories or to the skin as a cream.
Our microbiological efficacy results based on native and
Gram-stained microscope slide and culture analyses
showed C. albicans resistance in over 30 % of patients in
the first treatment group who had received combined azole
therapy. Clinical symptoms persisted in 79.7 % of women
in this group after treatment. A probiotic was administered
locally in addition to short-term combination azole therapy
to enhance the clinical and microbiological efficacy of
antifungal therapy. The results showed good clinical
effects. At follow-up examination, 29.06 % of patients in
the second treatment group had decreased clinical com-
plaints, 94.6 % had vaginal tissue changes, and 98.5 % had
improved vaginal fluor. The therapy was well tolerated by
patients, with no serious side effects reported. The micro-
biological efficacy of the treatment schedule measured by
native and Gram-stained slide examination and culture
testing showed improvement from 93.7 to 95.2 %. To our
knowledge, this is the first study to perform this compar-
ative testing of combination treatment schedules. However,
43
Martinez et al. [10] reported a 2009 study of 55 women
with VVC divided into two groups. The patients in the first
group were treated with a single 150-mg dose of fluco-
nazole and a topical probiotic every morning for 28 days,
while the second group treatment received a single 150-mg
dose of fluconazole and topical placebo for 28 days [10].
Microbiological testing at the end of the study showed
89.7 % efficacy in the probiotic group compared with
61.5 % in the placebo group [10]. When added to the
standard 150 mg oral fluconazole azole treatment, admin-
istration of a vaginal probiotic increased clinical and
microbiological efficacies [10]. The clinical and microbi-
ological results reported by Martinez et al. and this study
demonstrate the importance of lactobacilli in modern VVC
treatment. Lactobacilli inhibition of C. albicans has also
been reported in vitro studies, suggesting their potential as
probiotic agents in modern antifungal therapy [16, 17].
Local application of probiotics after conventional azole
treatment for vaginal C. albicans infections increased the
clinical and microbiological efficacy of the therapy. The
microbial balance in the vaginal ecosystem was restored in
the majority of patients in our study, which is a prerequisite
to minimize vaginal candidiasis relapse.
Conflict of interest Stefan Miladinov Kovachev—‘‘Local Probiotic
Therapy for Vaginal Candida albicans Infections’’. I declare that
there is no conflict of interest.
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