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 complaints 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: [email protected] R. S. Vatcheva-Dobrevska Department of Microbiology and Virology, University Hospital Queen Joanna- ISUL, Bialo More Str. 8, 1527 Sofia, Bulgaria
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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 socioeconomic 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 isolated 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. guilliermondii), 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 membrane [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 effective 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 recommend 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 multicenter studies have reported primary and secondary resistance to standard treatment regimens [8, 9]. Due to therapeutic and microbiological failures of standard therapy, 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.
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Chlamydia trachomatis, or human immunodeficiency virus (HIV) infections were excluded. Pregnant women and those who took corticosteroids, antibiotics, azoles, or probiotics 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 malignancies 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, consistency, 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 transparent, 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)
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 provided, 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 predominantly C. albicans vaginal infections were enrolled in the study. Patients with Neisseria gonorrhoeae, herpes simplex virus (HSV), human papillomavirus (HPV),
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
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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, pseudomycelium (hyphae), and blastospores. Culture Vaginal discharge samples from all women enrolled in this study were applied to selective culture media for microbiological 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-DextroseAgar, 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 CandidaBD, 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.
•
on the same day as a single vaginal globule of fenticonazole (600 mg Lomexin). Second treatment schedule: a single oral dose of fluconazole (150 mg Mycomax or Mycosyst) administered 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 menstrual 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. albicans 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]. Primary resistance of C. albicans to fluconazole was not seen in previous studies [14]. In vitro cross-resistance was reported between fluconazole and other azoles (ketoconazole and itraconazole), but to a lesser extent [14].
Random Group Assignment Statistical Analysis After obtaining informed consent, 436 patients with identified 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 fluconazole (150 mg Mycomax or Mycosyst) administered
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Clinical and microbiological data obtained at the follow-up examination were analyzed using Chi-square test. Statistical 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 laboratory or microbiological tests) due to treatment were compared. A two-factor dispersion analysis with repeated observations (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
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Table 1 Clinical parameters of women in the first treatment group before and after therapy
Table 3 Clinical parameters of women in the second treatment group before and after therapy
Clinical parameters
Before Th
After Th
Improvement
Clinical parameters
Before Th
After Th
Improvement
N—%
n
%
n
%
N—%
n
%
n
%
65
31.1
29.06
0.005
2
1
98.55
0.308
%
p
Clinical complaints
195
94.2
165
79.7
15.38
0.001
Clinical complaints
144
68.9
Vaginal fluorine (??, ???)
126
60.9
7
3.4
94.44
0.031
Vaginal fluorine (??, ???)
138
66
Vaginal tissue changes
205
99
pH (alkaline)
88
%
p
56
27
72.66
0.387
Vaginal tissue changes
204
97.6
11
5.3
94.61
0.594
2
1
97.73
0.098
pH (alkaline)
88
42.1
3
1.4
98.66
0.757
42.5
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 group before and after therapy
Table 4 Microbiological parameters of women in the second treatment group before and after therapy
Microbiological parameters N—%
Microbiological Before Th parameters N—% n % Native microscope slide
Before Th
After Th
Improvement
n
n
%
%
%
p
After Th
Improvement
p
n
%
%
12 206
5.7 98.6
94.17 99.37
0.67 0.00
Native microscope slide Spores, filaments Lactobacilli
206
99.5
67
32.4
67.96
0.17
77
37.2
195
94.2
91.54
0.03
Lactobacilli Culture testing C. albicans
207 73
100 35.3
206 50
98.6 23.9
Gram-stained microscope slide
Gram-stained microscope slide Hyphae, spores
Spores, filaments Lactobacilli
78 196
37.7 94.7
62.32 91.79
n.s.
Hyphae, spores
0.02
Lactobacilli
206
98.6
13
6.2
93.69
0.63
50
23.9
206
98.6
98.74
0.70
208
99.5
10
4.8
95.19
0.82
Culture testing 207
100
76
36.7
63.29
n.s.
C. albicans
Th therapy, p p values (Chi-square test for distribution), ns change was not statistically significant
Th therapy, P p values (Chi-square test for distribution)
summary parameter for each group of complaints as the presence of any complaint from the treatment group.
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 (??, ???).
Results 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 satisfied 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 enrollment 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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Table 5 Clinical indicator improvements (%) among women in the first and second treatment groups before and after therapy Clinical parameters
Clinical complaints
Group I n = 207
Group II n = 209
Improvement after Th
Improvement after Th
n
n
%
%
30
15.38
79
29.06
Vaginal fluorine (??, ???) 119
94.44
137
98.55
149
72.66
193
94.61
87
97.73
85
98.66
Vaginal tissue changes pH (alkaline) Th therapy
Table 6 Microbiological parameter differences (%) among women in the first and second treatment groups before and after treatment Microbiological parameters
Group I n = 207
Group II n = 209
Improvement after Th
Improvement after Th
n
%
n
%
Spores, filaments
139
67.96
194
94.17
Lactobacilli
118
91.54
156
99.37
Native microscope slide
Gram-stained microscope slide Hyphae, spores
129
62.32
193
93.69
Lactobacilli
123
91.79
156
98.74
131
63.29
198
95.19
Culture testing C. albicans Th therapy
Therapy Efficacy Measured by Clinical Examination Table 3 shows the clinical efficacy based on all measured clinical parameters, showing a higher efficacy in second treatment group compared with the first, with combined azole monotherapy. Therapy Efficacy Measured by Microbiological Testing Table 4 shows the treatment microbiological efficacy for all measured microbiological parameters, with values higher in this second treatment group compared with the first. Comparison of clinical and microbiological parameters between the first and second treatment groups. Table 5 shows that the treatment schedule followed by patients in the second group had higher clinical efficacies compared with the first group based on clinical parameters typical to vaginal dysbacteriosis in C. albicans-predominant infections. Measured differences in improvement of clinical parameters after treatment was low in both groups.
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Table 6 shows microbiological parameter differences before and after therapy in the first and second group as well as the relative microbiological efficacies (percentages) of combined azole as well as topical probiotic with conventional azole therapies (first group) for treatment of predominantly C. albicans-induced vaginal dysbacteriosis.
Discussion Fungal resistance to the most common azole agents and frequent recurrence of infections determine the failure of C. albicans treatments [9, 13, 14]. Modern azole antifungal therapy failed in 10–20 % of patients in Bulgaria due to resistant fungal strains [13]. Similar resistance prevalence has been reported in other countries [9, 14]. C. albicans resistance to azoles, the commonly used agents and most accessible in everyday life, has been characterized at a genetic level. Research has shown resistance to be due to expression of specific genes that control transport and accumulation of azole antifungal agents [2, 8, 15]. Recent studies have shown that the predominant C. albicans species genotype and mutations in the ERG11 gene determines azole susceptibility [8]. Most reports have studied the in vitro susceptibility of C. albicans to various therapeutic agents [14–17]. Although Candida strain susceptibility in vitro does not always mean successful treatment, in vitro resistance almost always predicts therapy failure [6, 9]. Therefore, this study evaluated in vivo response to antifungal agents used in VVC treatment. Some researchers have hypothesized that immune response to a recurrent pathogen (C. albicans) is the basis of recurrent C. albicans infections [18, 19]. The inability of antifungal drugs to influence immunity in such cases results contributes to their inefficacy [20]. Wagner et al. [21] reported in 2012 that C. albicans infection induces a pro-inflammatory immune response in vaginal epithelial cells. Vaginal lactobacilli in the same study inhibited NF-jB-associated inflammatory genes and also induced IL-1a and IL-1b expression through an alternative signal transduction pathway [21]. Lactobacilli activation of an alternative signaling mechanism modulates vaginal epithelial cell cytokine production [21]. This is the most likely mechanism for probiotic modulation of C. albicans infections [21]. Lactobacilli can modulate the immune response and protect the vagina from predominantly Candida chronic dysbacteriosis, an important feature for control of chronic and recurrent fungal infections [19, 20, 22]. Moreover, lactic acid bacteria competitively block adhesion of Candida strains to vaginal epithelial cells and produce antimicrobial substances that inhibit C. albicans growth and development [23–25]. These lactobacilli features recommend their use as antifungal agents either alone
Probiotics & Antimicro. Prot. (2015) 7:38–44
or in combination with other therapeutic agents [16, 17]. Clinical studies have evaluated the abilities of oral or intravaginal 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 probiotics 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 probiotics 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 efficacy 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 fenticonazole to a single oral dose of fluconazole for increased and more rapid antimycotic and clinical efficacies [6]. Fenticonazole is an imidazole derivative with broad-spectrum 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 complaints, 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 microbiological 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 comparative testing of combination treatment schedules. However,
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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 fluconazole 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, administration of a vaginal probiotic increased clinical and microbiological efficacies [10]. The clinical and microbiological 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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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 complaints 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: [email protected] R. S. Vatcheva-Dobrevska Department of Microbiology and Virology, University Hospital Queen Joanna- ISUL, Bialo More Str. 8, 1527 Sofia, Bulgaria
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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 socioeconomic 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 isolated 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. guilliermondii), 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 membrane [5, 6]. Imidazoles include miconazole, ketoconazole, and clotrimazole. Triazole agents most commonly used to treat fungal infections include fluconazole, itraconazole,
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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 effective 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 recommend 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 multicenter studies have reported primary and secondary resistance to standard treatment regimens [8, 9]. Due to therapeutic and microbiological failures of standard therapy, 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.
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Chlamydia trachomatis, or human immunodeficiency virus (HIV) infections were excluded. Pregnant women and those who took corticosteroids, antibiotics, azoles, or probiotics 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 malignancies 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, consistency, 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 transparent, 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)
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 provided, 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 predominantly C. albicans vaginal infections were enrolled in the study. Patients with Neisseria gonorrhoeae, herpes simplex virus (HSV), human papillomavirus (HPV),
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
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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, pseudomycelium (hyphae), and blastospores. Culture Vaginal discharge samples from all women enrolled in this study were applied to selective culture media for microbiological 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-DextroseAgar, 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 CandidaBD, 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.
•
on the same day as a single vaginal globule of fenticonazole (600 mg Lomexin). Second treatment schedule: a single oral dose of fluconazole (150 mg Mycomax or Mycosyst) administered 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 menstrual 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. albicans 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]. Primary resistance of C. albicans to fluconazole was not seen in previous studies [14]. In vitro cross-resistance was reported between fluconazole and other azoles (ketoconazole and itraconazole), but to a lesser extent [14].
Random Group Assignment Statistical Analysis After obtaining informed consent, 436 patients with identified 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 fluconazole (150 mg Mycomax or Mycosyst) administered
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Clinical and microbiological data obtained at the follow-up examination were analyzed using Chi-square test. Statistical 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 laboratory or microbiological tests) due to treatment were compared. A two-factor dispersion analysis with repeated observations (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
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Table 1 Clinical parameters of women in the first treatment group before and after therapy
Table 3 Clinical parameters of women in the second treatment group before and after therapy
Clinical parameters
Before Th
After Th
Improvement
Clinical parameters
Before Th
After Th
Improvement
N—%
n
%
n
%
N—%
n
%
n
%
65
31.1
29.06
0.005
2
1
98.55
0.308
%
p
Clinical complaints
195
94.2
165
79.7
15.38
0.001
Clinical complaints
144
68.9
Vaginal fluorine (??, ???)
126
60.9
7
3.4
94.44
0.031
Vaginal fluorine (??, ???)
138
66
Vaginal tissue changes
205
99
pH (alkaline)
88
%
p
56
27
72.66
0.387
Vaginal tissue changes
204
97.6
11
5.3
94.61
0.594
2
1
97.73
0.098
pH (alkaline)
88
42.1
3
1.4
98.66
0.757
42.5
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 group before and after therapy
Table 4 Microbiological parameters of women in the second treatment group before and after therapy
Microbiological parameters N—%
Microbiological Before Th parameters N—% n % Native microscope slide
Before Th
After Th
Improvement
n
n
%
%
%
p
After Th
Improvement
p
n
%
%
12 206
5.7 98.6
94.17 99.37
0.67 0.00
Native microscope slide Spores, filaments Lactobacilli
206
99.5
67
32.4
67.96
0.17
77
37.2
195
94.2
91.54
0.03
Lactobacilli Culture testing C. albicans
207 73
100 35.3
206 50
98.6 23.9
Gram-stained microscope slide
Gram-stained microscope slide Hyphae, spores
Spores, filaments Lactobacilli
78 196
37.7 94.7
62.32 91.79
n.s.
Hyphae, spores
0.02
Lactobacilli
206
98.6
13
6.2
93.69
0.63
50
23.9
206
98.6
98.74
0.70
208
99.5
10
4.8
95.19
0.82
Culture testing 207
100
76
36.7
63.29
n.s.
C. albicans
Th therapy, p p values (Chi-square test for distribution), ns change was not statistically significant
Th therapy, P p values (Chi-square test for distribution)
summary parameter for each group of complaints as the presence of any complaint from the treatment group.
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 (??, ???).
Results 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 satisfied 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 enrollment 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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Table 5 Clinical indicator improvements (%) among women in the first and second treatment groups before and after therapy Clinical parameters
Clinical complaints
Group I n = 207
Group II n = 209
Improvement after Th
Improvement after Th
n
n
%
%
30
15.38
79
29.06
Vaginal fluorine (??, ???) 119
94.44
137
98.55
149
72.66
193
94.61
87
97.73
85
98.66
Vaginal tissue changes pH (alkaline) Th therapy
Table 6 Microbiological parameter differences (%) among women in the first and second treatment groups before and after treatment Microbiological parameters
Group I n = 207
Group II n = 209
Improvement after Th
Improvement after Th
n
%
n
%
Spores, filaments
139
67.96
194
94.17
Lactobacilli
118
91.54
156
99.37
Native microscope slide
Gram-stained microscope slide Hyphae, spores
129
62.32
193
93.69
Lactobacilli
123
91.79
156
98.74
131
63.29
198
95.19
Culture testing C. albicans Th therapy
Therapy Efficacy Measured by Clinical Examination Table 3 shows the clinical efficacy based on all measured clinical parameters, showing a higher efficacy in second treatment group compared with the first, with combined azole monotherapy. Therapy Efficacy Measured by Microbiological Testing Table 4 shows the treatment microbiological efficacy for all measured microbiological parameters, with values higher in this second treatment group compared with the first. Comparison of clinical and microbiological parameters between the first and second treatment groups. Table 5 shows that the treatment schedule followed by patients in the second group had higher clinical efficacies compared with the first group based on clinical parameters typical to vaginal dysbacteriosis in C. albicans-predominant infections. Measured differences in improvement of clinical parameters after treatment was low in both groups.
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Table 6 shows microbiological parameter differences before and after therapy in the first and second group as well as the relative microbiological efficacies (percentages) of combined azole as well as topical probiotic with conventional azole therapies (first group) for treatment of predominantly C. albicans-induced vaginal dysbacteriosis.
Discussion Fungal resistance to the most common azole agents and frequent recurrence of infections determine the failure of C. albicans treatments [9, 13, 14]. Modern azole antifungal therapy failed in 10–20 % of patients in Bulgaria due to resistant fungal strains [13]. Similar resistance prevalence has been reported in other countries [9, 14]. C. albicans resistance to azoles, the commonly used agents and most accessible in everyday life, has been characterized at a genetic level. Research has shown resistance to be due to expression of specific genes that control transport and accumulation of azole antifungal agents [2, 8, 15]. Recent studies have shown that the predominant C. albicans species genotype and mutations in the ERG11 gene determines azole susceptibility [8]. Most reports have studied the in vitro susceptibility of C. albicans to various therapeutic agents [14–17]. Although Candida strain susceptibility in vitro does not always mean successful treatment, in vitro resistance almost always predicts therapy failure [6, 9]. Therefore, this study evaluated in vivo response to antifungal agents used in VVC treatment. Some researchers have hypothesized that immune response to a recurrent pathogen (C. albicans) is the basis of recurrent C. albicans infections [18, 19]. The inability of antifungal drugs to influence immunity in such cases results contributes to their inefficacy [20]. Wagner et al. [21] reported in 2012 that C. albicans infection induces a pro-inflammatory immune response in vaginal epithelial cells. Vaginal lactobacilli in the same study inhibited NF-jB-associated inflammatory genes and also induced IL-1a and IL-1b expression through an alternative signal transduction pathway [21]. Lactobacilli activation of an alternative signaling mechanism modulates vaginal epithelial cell cytokine production [21]. This is the most likely mechanism for probiotic modulation of C. albicans infections [21]. Lactobacilli can modulate the immune response and protect the vagina from predominantly Candida chronic dysbacteriosis, an important feature for control of chronic and recurrent fungal infections [19, 20, 22]. Moreover, lactic acid bacteria competitively block adhesion of Candida strains to vaginal epithelial cells and produce antimicrobial substances that inhibit C. albicans growth and development [23–25]. These lactobacilli features 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 intravaginal 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 probiotics 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 probiotics 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 efficacy 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 fenticonazole to a single oral dose of fluconazole for increased and more rapid antimycotic and clinical efficacies [6]. Fenticonazole is an imidazole derivative with broad-spectrum 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 complaints, 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 microbiological 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 comparative testing of combination treatment schedules. However,
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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 fluconazole 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, administration of a vaginal probiotic increased clinical and microbiological efficacies [10]. The clinical and microbiological 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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