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Probiotic and Tea Tree Oil Treatments Improve Therapy of Vaginal Candidiasis: A Preliminary Clinical Study.

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Abstract

Recent studies have documented that 29-49% of women has been affected by vulvovaginal candidiasis (VVC) at least once in the lifetime and about 10% of them has history of recurrent VVC. Tea tree oil (TTO), an essential oil extracted by steam distillation from the leaves of Melaleuca alternifolia tree, has been known for many years for its antiseptic properties. Currently, TTO is used in vaginal suppositories (VS) for the treatment of vaginal candidiasis. TTO-VS exhibits an in vitro fungicidal activity towards Candida spp., only slightly affecting some vaginal lactobacilli population isolated from patients with vaginitis (Di Vito et al. 2015). However, preclinical and clinical studies addressing efficacy and safety profile are still few and not exhaustive, especially with regard to the complex vaginal microbial environment. On this basis, we have investigated the efficacy and safety profile of a combined therapy with probiotics and TTO-VS on female volunteers with vaginitis in order to find alternatives to fight the growing antimicrobial resistance to the most common synthetic antifungal remedies.
Central
Medical Journal of Obstetrics and Gynecology
Cite this article: Di Vito M, Fracchiolla G, Mattarelli P, Modesto M, Tamburro A, et al. (2016) Probiotic and Tea Tree Oil Treatments Improve Therapy of
Vaginal Candidiasis: A Preliminary Clinical Study. Med J Obstet Gynecol 4(4): 1090.
*Corresponding authors
Maura Di Vito, Department of Infectious, Parasitic
and Immune-mediated Diseases, Istituto Superiore di
Sanità (National HealthInstitute), Viale Regina Elena
299,00161 Rome, Italy, Tel: +39-06-49902808; Fax: +39-
06-49902808; E-mail:
Submitted: 29 July 2016
Accepted: 05 Novemeber 2016
Published: 08 Novemeber 2016
ISSN: 2333-6439
Copyright
© 2016 Di Vito et al.
OPEN ACCESS
Short Communication
Probiotic and Tea Tree Oil
Treatments Improve Therapy
of Vaginal Candidiasis: A
Preliminary Clinical Study
Maura Di Vito1,2*, Giuseppe Fracchiolla3, Paola Mattarelli4,
Monica Modesto4, Annunziata Tamburro2, Francesco Padula5,
Luigi Agatensi6, Francesca Romana Giorlandino5, Antonietta
Girolamo1, Giuseppe Gerardo Carbonara4, Antonio Carrieri4,
Filomena Corbo4, Francesca Mondello1
1Department of Infectious, Parasitic and Immune-mediated Diseases, National Health
Institute, Italy
2U.O.C. of Microbiology and Virology, San Filippo Neri Hospital- ASL-RME, Italy
3Department of Pharmacy-Drug Sciences, University of Bari, ‘‘Aldo, Italy
4Moro’’ Department of Agricultural Science, Bologna University, Italy
5Altamedica, Fetal-MaternalMedical Centre, Italy
6Presidium San Filippo Neri, Sant’AndreaAmbulatory, Italy
Keywords
•Candidosis
•Tea tree oil
•Probiotics
•Vaginal suppositories
ABBREVIATIONS
TTO: Tea Tree Oil; VS: Vaginal Suppositories; TTO-VS:
Vaginal Suppositories based on Tea Tree Oil; VVC: Vulvovaginal
Candidiasis; RVVC: Recurrent Vulvovaginal Candidiasis
INTRODUCTION
Recent studies have documented a percentage of women
comprised between 29% and 49% who have been affected by
vulvovaginal candidiasis (VVC), 10%of them with history of
recurrent VVC (RVVC) – four or more episodes in a 12-month
period [1-3]. In Italy, the prevalence of RVVC is estimated at
about 5% out of 16% [4].
Lactobacillus spp. is the dominant bacterium of the vaginal
tract in healthy women and an imbalance of the local microbiota

antifungals, there is still a high incidence of recurrence with
associated increase of microbial resistance. Moreover, clinical
and epidemiological data suggest that topical treatment for VVC

microenvironment. Thus, the administration of probiotics
incorporating selected Lactobacillus strains as an effective
strategy for preventing vaginal infections has been suggested [5].
As known, the vaginal biotais mainly formed of Lactobacillus
spp. mainly Lactobacillus crispatus, Lactobacillus gasseri,
Lactobacillus jenseniiand Lactobacillus iners producing
lactic acid from sugar substrates and making the vaginal
microenvironment typically acidic. Furthermore, resident
Lactobacillus  
of their metabolism, hence creating a barrier against pathogen
invasion [6,7]. Recent studies have shown that the presence of
Lactobacillus spp. in vaginal microbiota is able to counteract the
colonization of Candida spp. through mechanisms of exclusion,
competition and displacement [8].
Abstract
Recent studies have documented that 29-49% of women has been affected by vulvovaginal candidiasis (VVC) at least once in the lifetime and about 10%
of them has history of recurrent VVC.
Tea tree oil (TTO), an essential oil extracted by steam distillation from the leaves of Melaleuca alternifolia tree, has been known for many years for
its antiseptic properties. Currently, TTO is used in vaginal suppositories (VS) for the treatment of vaginal candidiasis. TTO-VS exhibits an in vitro fungicidal
activity towards Candida spp., only slightly affecting some vaginal lactobacilli population isolated from patients with vaginitis (Di Vito et al. 2015). However,
preclinical and clinical studies addressing efcacy and safety prole are still few and not exhaustive, especially with regard to the complex vaginal microbial
environment.
On this basis, we have investigated the efcacy and safety prole of a combined therapy with probiotics and TTO-VS on female volunteers with vaginitisin
order to nd alternatives to ght the growing antimicrobial resistance to the most common synthetic antifungal remedies.
Central
Di Vito et al. (2016)
Email:
Med J Obstet Gynecol 4(4): 1090 (2016) 2/6
Other recent studies have evaluated the in vitro microbicidal
action of some essential oils, such as that of Melaleuca alternifolia,
against fungal and probiotic strains [9-11].
TTO is an essential oil extracted by steam distillation from the
leaves of Melaleuca alternifolia (Myrtaceae), native of Australia
and well-known for its use in traditional medicine. Currently,
TTO is used in some herbal preparations and medicinal products,
including VS for the treatment of candidiasis.
However, preclinical and clinical studies are still inconclusive

environment like that of the vaginal microbiota.
The purpose of this short communication is to identify a
possible treatment based on probiotics and TTO-VS potentially
able to decontaminate the vaginal canal and counteract the fungal
colonization by combining the known fungicidal action of TTO
with the competitive and immune stimulating one of probiotics.
The objective is to lay the groundwork for future clinical study
on integrated treatments based on probiotics, administered per
       
antimicrobial resistance to the most common synthetic antifungal
remedies.
MATERIALS AND METHODS
Patients screening and recruiting
Patients with presence of fungal cells in vaginal swabs were
recruited from January 2015 to March 2016 in the department
of Gynaecology at the Sant’Andrea Hospital (UOC San Filippo
NeriASL RME-Rome) and in the department of Altamedica
(Rome).
All microbiological tests were performed by the Laboratory
of Clinical Microbiology of the San Filippo Neri Hospital (Rome).
Inclusion criteria for patients were, besides fungal positivity,
the presence of at least one of the symptoms that characterize
candidiasis (vaginal discharge, vaginal itching, erythema,
dyspareunia, bleeding). Among 147 eligible women, 13 – with
different ages and with or without previous vaginal infection
were recruited and treated. The screening was performed using
vaginal swabs containing 1 ml of Amies medium, suitable for both
microbiological and molecular analysis (ESwab, COPANITALIA,
Brescia, Italy). The research of fungi was performed using the

carried out with MALDI-TOF technology (Vitek MS, Biomerieux,

having isolated single colonies of fungal strains on Sabouraud
Dextrose Agar. The treatment received a positive opinion from
the Ethical Review Board Committee (Prot. EC 396/16) and all
patients gave informed consent. A number of 4 out of 13 women
left the study because they did not return for the control, thus 9
completed the treatment.
The treatment included 15 days of oral treatment with 2
cps/day of probiotics made of Lactobacillus acidophilus and
S. boulardii at 2.5 and 4 CFU/cps respectively (Candinorm
Capsule® PegasoSrl Verona - Italy). If the patient was still
        
above treatment was followed by further 15 days with VS applied
1/day before bedtime (Candinorm ® VS, Pegasosrl Verona –
Italy. Composition: 0.5% TTO, Aloe vera, GOS, colloidal silica and
        

obtained from vaginal washing with 1 ml of saline; the recovered
     

Voluntary recruitment
In addition to the screening and treatment of patients, 5
female healthy volunteers were recruited to study the kinetics
of VS based on TTO (TTO-VS).The healthy female volunteers
were treated for 15 days with probiotics and, starting from the
10th day, also with TTO-VS for 5 days (1 TTO-VS/day minimum
treatment recommended by manufacturer). During the vaginal
  

(T1), 15 min (T2), 3 days (T3), and 5 days (T4).Sera obtained
        
used to study both the presence of TTO major components by
GC/MS and the nitric oxide variation by ELISA test.

The samples obtained from vaginal washes were examined
      
samples obtained from female volunteers were studied for the
nitric oxide plasma levels. All the analyses were performed using
colorimetric ELISA kits (respectively, ab 46034, ab 46052 and ab
65328 from abcam Cambridge Science Park, UK). As said above,
vaginal washings were centrifuged at 1000 x g for 10 minutes to
remove debris, while serum samples were tested directly without
other treatments. The samples were analysed following the
manufacturer’s instructions and each analysis was in triplicate.
Lactobacillus spp
Total DNA was extracted by using the DNeasy Blood & Tissue Kit
(Qiagen, Hilden, Germany) following the protocol “Pretreatment

       
  
       

 
 
         


          
Measurements were performed in triplicate and repeated when
variation between measurements exceeded 0.5 Ct. Data obtained
        
bacterial cells per ml of transport medium, expressed as colony
forming unit (CFU)/ml according to the ribosomal RNA (rRNA)
copy number available in the rRNA copy number database
[13,14]. Standard curves were made by plotting cycle threshold
(CT) values, against dilutions of the quantitative standard for
which the number of gene copies was known.
Standard curves were constructed using PCR product of the
Central
Di Vito et al. (2016)
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16S rRNA gene of Lactobacillus plantarum ATCC 14917. The
        
(Macherey-Nagel GmbH & Co. KG, Germany) according to the
    
         
1.28 ng, 128 pg, 12.8 pg, 1.28 pg, 128 fg, 12.8 fg, 1.28 pg, 0.128
pg DNA) were used as a template for the standard curve. One
nanogram of PCR product corresponded to 2.47x109 copies of
the fragment gene. The qPCR assays were replicated three times
independently.

(GC/MS)
The pure TTO (Lot 140/0000324) and the biological samples
were analysed by GC/MSMS following a previously reported
     
from biological samples, we added pure TTO ranging from 0.01%
to 1% in sera samples obtained before the treatment with TTO-VS
(T0). The calibration curves of two TTO components (Terpinen-
         
percentage.
All the sera samples obtained from female healthy volunteers
were extracted with ethyl acetate in order to assess the presence
of TTO-VS compounds.
The value of the GC signal related to the standard Terpinene-
4-ol at 3 ppm v/v concentration was measured and compared
with the value of the same signal of all the sera samples. We found
that the signal values of the sera samples were always lower than
the corresponding signal control value of 3 ppm. The solvents

GC/MS analyses were performed on HP GC/MS 6890N-5973N
MSD HP ChemStation, equipped with autosampler and HP-5MS


measured in the range 35–360 amu. Qualitative analysis was
carried out by comparing the retention indices and MS spectra
for the obtained peaks with the analogous data from NIST2011
databases.
Statistical analysis
The data obtained in triplicate from each experiment were
presented as means ± standard deviation (SD). The data obtained
from PCR assay for cytokines were analysed with t test for paired
data, whereas ELISA assay for NO expression was analysed
with the variance test for repeated measures. The data were

RESULTS AND DISCUSSION
Patients with positivity to the fungal vaginal search were
treated with a mixture of L.acidophilus and S.boulardii. These
probiotics were selected in an attempt to strengthen both vaginal
and gut microbiota, usually reservoir of fungal strains [16-
18] because a healthy microbiota is able to contrast the fungal
colonization and, consequently, the RVVC.
S.boulardii        
has generated interest among researchers for its properties
in preventing the colonization of fungal strains, altering their
adhesion to the substrate [19-21].
Colonization means the presence of a microorganism in or
on a host, with growth and multiplication but without any overt
    
patients that, in routine microbiological analysis, were colonized
by fungi showing only some characteristics present in overt
fungal infections (Table 1). Furthermore, only 4 out of 9 patients
remembered having already suffered from fungal infections prior
to recruitment.
After verifying the in vitro 

on 5 volunteers, during 5 days of treatments, the possibility to
detect TTO residues in the bloodstream and/or variations of
       
results indicate that nocomponentswere noticed correlated to
the formulation in any of the 5 blood samples taken during the
         
      
present during the treatment if compared to time zero (Figure 2).
In contrast, 112 TTO components, of which 90% represented by
12 major components (terpinen-4-ol equal to 38% of the total),
were detected starting directly from the VS formulation.
Data obtained from the study on patients indicate that, after
   
were no longer colonized by fungi, and that this percentage
reached 77.7% (7/9) when patients, not showing a negativization

additional treatment characterized by vaginal suppository
applications.
Data collected through both questionnaire and medical
examination show that all patients at the end of the treatments
reported the disappearance of symptoms and a general
improvement in well-being. No adverse effects due to treatment
were reported.
The ability of L. acidophilus to colonize the vaginal canal
was evaluated by RT-qPCR analysis performed on vaginal
swabs taken before and after treatment. As shown in Table (2),
the higher number of lactobacilli found in 7 out of 9 (77.8%)
patients analysed after the treatment can be explained by
their colonization ability of the vaginal canal. This variation is
         
the L.acidophilus ability to colonize the vaginal canal when
administered orally [23].
It is well-known that both vaginal microbiota and fungal


a physiological immune response also in pregnant women: the
IL-10 is expressed 100 times more in healthy women than in
those with VB [24-26].

in vaginal washing collected at the beginning and at the end of
treatment. Data obtained by ELISA analysis show that all patients
turning negative for fungal colonization during the treatment had

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Table 1: The table shows the physiological data of each patient enrolled and microbiological data obtained from routine tests after each control.
Number Patient’s age   


at enrolment
Microbial

control

at second control
1 40 NO VD, VI, E  CP, EC, EF None None
2 50 NO VD, VI, D, B DNA CP, EC, EF None None
4 43 NO VD, VI, E, D  CG, EC, EF CG, EC, EF CG, EC, EF
5 21 NO VD, VI, E NO CA None None
6 46 NO VI, E, D NO CA, EF None None
7 52 NO VI, E, D DNA CA, EC, EF CA, EC, EF None
9 58 SI VI, E, D NO SC, EC SC None
10 34 NO VD, VI, E, D  CA, EF CA CA
14 42 NO VD, VI, E, D  CA None None
Abbreviations: CA: Candida Albicans; CG: Candida Glabrata; CP: Candida Parapsilosis; EC: Escherichia Coli; EF: Enterococcus Faecali; SC:
Saccharomyces Cerevisiae; DNA: Data Not Available; VD: Vaginal Discharge; VI: Vaginal Itching; E: Erythema; D: Dyspareunia; B: Bleeding
Figure 1 Intensity of terpinen-4-ol GC signal in sera sample of TTO-VS treated volunteers. Each bar represents mean ± SD of terpinen-4-ol GC signal
of the 5 volunteer’s sera samples before the treatment (T0) and 5 min (T1), 15 min (T2), 3 days (T3) and 5 days (T4) after TTO-VS administration,
respectively. Ctrl represents the 3ppm v/v terpinen-4-ol standard signal intensity value.
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
T0 T1 T2 T3 T4
nmol (mean values)
Figure 2         
application (T0) of TTO-VS and after 5 min (T1), 15 min (T2), 3 days (T3) and 5 days (T4). The bars indicate the standard deviation. The variation

While, the two patients with persistence of fungal colonization
after the treatments (patients 4 and 10 in Table 2) experienced
a decrease in the expression of IL-10 associated with a marked
increase of IL-1both after the treatment with probiotics and after

experimental models and pre-clinical literature studies: a right
     
with preserved vaginal microbiota might be essential for proper
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Med J Obstet Gynecol 4(4): 1090 (2016) 5/6
immune response in defence against colonization and infection
by fungi [24-28].
CONCLUSION
The most common anti-fungal treatments are based on azoles.
Even if azoles reduce the frequency of RVVC with no toxicity
when applied topically, they are generally not able to eradicate
the fungal infection because they are fungistatic. These remedies,
although transiently, improve the quality of life of women and
require cyclical treatments which gradually generate resistance
to the active substances. In contrast, fungicidal treatments are
generally resolutive but with important adverse effects especially
when administered per os.
        
shows the development of fungal resistance to the most
common synthetic remedies. It is known that the development
of antimicrobial resistance is an urgent problem that must be
overcome both by identifying new molecules with cytocidal
activity and by reducing the widespread use of active molecules.
RVVC, in addition to being a problem for about 10% of the female
population causing great discomfort, can potentially generate
resistors. This requires the use of alternative approaches to
common anti-fungal remedies to improve the quality of life of
VVC patients and prevent, at the same time, the development of
resistances.
The combined treatment with probiotics administered per os

able to eradicate the fungal colonization. It appears to be easy to
perform, generally well-tolerated unless there is hypersensitivity
to the formulation components, and safe.
Compared to conventional therapies, the proposed treatment
combines the action of a natural substance, a known fungicide,
with the competitive and immune stimulantone of probiotics
belonging to the vaginal microbiota. This type of approach is
reasonably safe and allows the battle against fungi through
two mechanisms. On the one hand, TTO destabilizes the fungal
cell by altering the structure of the cell wall [29]; on the other,
probiotics, in addition to “undermining” the fungal colonization
(by restoring the vaginal microbiota competing for territory
and reducing the fungal adhesion to the substrate),stimulate the
physiological defensive response, depressed by the action of the
fungus itself. Therefore, the idea is to weaken “the enemy” with
a safe and effective natural substance and restore, at the same
        
At the same time, this treatment is at low risk for development
of resistance since, to date, no fungal strains have developed
resistance to TTO. Although the small number of patients studied
is a limitation, our work could be an important support for future
in vivo          
and safety in a combined therapy based on probiotics and VS for
women with VVC.
In conclusion, our data – although preliminary – are new

tea tree oil combination for an effective vaginal decontamination
from fungal organisms has been demonstrated.
ACKNOWLEDGEMENTS
The project was partially funded by the Fellowship Program,
2013, from Gilead and partially by the Giuliana Gilletti 2015
award from Pegasus. We thank Dr Heide De Togni, Technical
Director of Pegasosrl, for organizing the production and the
delivery of the studied products. We thank Dr Fabiana Giorgi for
the proofreading.
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