Recurrent Vulvovaginal Candidiasis: Could It Be Related
to Cell-Mediated Immunity Defect in Response
to Candida Antigen?
Recurrent vulvovaginal candidiasis (RVVC) is a common cause of morbidity affecting millions of women worldwide. Patients with RVVC are thought to have
an underlying immunologic defect. This study has been established to evaluate cell-mediated immunity defect in response to
Materials and Methods
Our cross-sectional study was performed in 3 groups of RVVC patients (cases), healthy individuals (control I) and known cases of chronic mucocutaneous candidiasis (CMC) (control II). Patients who met the inclusion criteria of RVVC were selected consecutively and were allocated in the case group. Peripheral blood mononuclear cells were isolated and labeled with CFSE and proliferation rate was measured in exposure to candida antigen via flow cytometry.
T lymphocyte proliferation in response to
Our findings revealed that T cells do not actively proliferate in response to Candida antigen in some RVVC cases. So it is concluded that patients with cell-mediated immunity defect are more susceptible to recurrent fungal infections of vulva and vagina. Nonetheless, some other cases of RVVC showed normal function of T cells. Further evaluations showed that these patients suffer from atopy. It is hypothesized that higher frequency of VVC in patients with history of atopy might be due to allergic response in mucocutaneous membranes rather than a functional impairment in immune system components.
Vulvovaginal candidiasis (VVC) is the second
most common cause of genital tract infections (1).
It has been shown that VVC affects 75% of female
population at least once during their lives and
5-10% at higher frequencies (2). In more than 85%
of the cases, VVC is primarily caused by
There are several ways to evaluate cellular immunity
Materials and Methods
Our cross-sectional study was performed from January 2014 till May 2015. Patients with 4 or more episodes of VVC infection during the past year who were initially visited by gynecologists and referred to immunology clinics were enrolled in RVVC case group. All episodes of RVVC were confirmed with vaginal swab smear and culture. Control I subjects were healthy individuals without history of vulvovaginitis during the past year and also had less than 3 episodes of vulvovaginitis in a year during the previous years. Age and educational level did not vary among cases and individuals in control I group. Patients with chronic, persistent or recurrent non-invasive mucocutaneous candidiasis associated with organ infections, autoimmunity, vasculopathy and absence of predisposing conditions such as diabetes or HIV were enrolled in the study as control II group (CMC patients) (17).
Patients with pregnancy, history of using any antibiotic, corticosteroid, hormone therapy, antifungal within the past 30 days and medical history of diabetes mellitus were excluded. Also patients with refractory VVC were excluded because RVVC means episodes of candida infection, with complete response to treatment each time. Required information including age, education status, family history of primary immunodeficiency diseases (PID) (in 1st, 2nd or 3rd degree relatives), history of allergy (confirmed by the clinical immunology and allergy specialist), history of hypothyroidism, history of using antifungal and frequency of VVC within last year was collected using a questionnaire. PID is defined as a heterogeneous group of diseases with higher susceptibility to infections as a result of immunity defect. International Union of Immunological Societies classifies PIDs in 8 large categories according to the impaired components of immune system (18). Severity of vaginitis was measured with a semiquantitative basis scoring from 0-3: 0 (absent), 1 (mild), 2 (moderate), 3 (severe). Sign and symptoms like pruritus, erythema, burning, edema and excoriation/fissure have been scored according to the patient’s statement. The sum-score of <4 is considered as asymptomatic/mild vulvovaginitis and excluded from our study and total score of >7 is defined as severe vulvovaginitis (19). Written and signed informed consents were obtained from all participants. The study was approved by Ethical Committee of Isfahan University of Medical Sciences (reference number: 283457). All enrolled patients were assisted by only one clinician.
At first, prior to blood sampling, phytohemagglutinin (PHA)-induced skin test was done in patients and control II group as an index of cell-mediated immunocompetence. By this test the mitogen PHA is injected subcutaneously and the swelling is measured 24 hours later. Blood samples were taken from the subjects. White blood cell count, immunoglobulin level and basic immunological markers were measured. Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-hypaque gradient separation (Amersham Biosciences, Germany). The number of PBMCs was set at 5-10×106 million cells per milliliter in PBS (Cayman Kit, Canada), were labeled with CFSE (Cayman Kit, Canada), and incubated for 30 minutes at 37°C with 5% CO2. Cells were then centrifuged and the supernatant was discarded. Cell pellet was re-suspended in RPMI-1640 culture medium containing 10% fetal calf serum (FCS) and incubated again at 37°C with 5% CO2 (Cyman kit, Canada). Triplicate cultures of 2×105 cells in 200 μl medium per well were established in 96-well round-bottomed cell culture plates. The candida antigen (HollisterStier, Germany) was diluted at a ratio of 1 to 10 V/V in RPMI-1640 culture medium containing 10% FBS and added to the wells containing the cell suspension. After addition of the antigen, cell plate was incubated at 37°C and 5% CO2. After 5 days, the cells were transferred to microtubes and washed with PBS. Finally, Proliferation was evaluated with flow cytometery (Partec, Denmark) using the FloMax software. The assays were done in totally blinded manner. Statistical analyses were done by Student’s t test, ANOVA and chi square test using SPSS16 software program (SPSS Inc., Chicago, IL, USA).
Twenty-eight patients with RVVC, 28 healthy
individuals (control I) and 7 patients with chronic
mucocutaneous candidiasis (control II) entered the
study. Four RVVC cases and 8 healthy subjects
were excluded; hence 24 cases, 20 controls and
7 CMC cases enrolled the study. Characteristics
of individuals in each group are shown in Table
1. Age and educational level were not different
among the 3 groups. Immunoglobulin level, white
blood cell (WBC) counts, immunological biomarkers
and PHA skin test were all normal among
controls and patients. Mean proliferation of T lymphocytes
in response to
|RVVC casen=24||Control I n=20||Control II (CMC) n=7||P value|
|Age (Y)||33.3 ± 8.6||32.8 ± 7.9||29.1 ± 8.4||0.5|
|Lower than high school||15 (62.5%)||8 (40%)||4 (57.1)|
|Higher than high school||9 (37.5%)||12 (60%)||3 (42.9%)|
|Family history of PID||0.013|
|Yes||7 (29.2%)||0||3 (42.9%)||0.008 (post hoc case-control I )|
|No||17 (70.8%)||20 (100%)||4 (57.1%)||0.49 (post hoc case-control II)|
|History of atopy||NS|
|Yes||10 (41.7%)||3 (15%)||1(14.3%)||0.054 (post hoc case-control II)|
|No||15 (58.3%)||17 (85%)||6 (86.6%)|
|Drug history (antifungal)|
|Yes||15 (60%)||0||2 (28.6%)|
|No||10 (40%)||20 (100%)||5 (71.4%)|
|Clinical symptom severity (mean ± SD)||5.8 ± 1.5||-||6.8 ± 1.3||0.1|
PID; Primary immunodeficiency diseases.
Although T cell response was significantly different among the 3 groups (P<0.001), it did not differ statistically between cases and CMC patients (P>0.05). Family history of PID was seen in 29.2% of cases, 42.9% of CMC patients and none of the healthy individuals. History of PID in family members was significantly different among the groups (P=0.01). The prevalence of allergy in RVVC cases was higher than control II group (P=0.054, 41.3 vs. 15%). The median of recurrence in patients during last year was 5.5 times with 4 times as minimum and 8 times as maximum episodes of recurrences. Vaginal symptoms in RVVC cases were not different from CMC group (P>0.05). T cell proliferation was negatively correlated with frequency of RVVC and clinical symptom severity respectively (r=-0.7, P<0.001, r=-0.4, P=0.013). T cell activation was greater in RVVC cases who had allergy compared to the ones without allergy (P=0.01) and also in patients who had used antifungals in comparison with patients who did not have the history of using antifungals (P=0.057). Vaginal clinical symptoms were different in cases with or without allergy and cases with or without history of using antifungal agents (Table 2,).
|n||T cell proliferation (mean ± SD)||P value||Clinical symptom severity (mean ± SD)||P value|
|Atopy in RVVC cases||0.01||0.02|
|Yes||10||2.90 ± 1.5||5.0 ± 1.1|
|No||14||1.27 ± 1.31||6.4 ± 1.5|
|Yes||16||1.53 ± 0.89||5.3 ± 1.2|
|No||15||0.93 ± 0.78||6.8 ± 1.5|
RVVC; Recurrent vulvovaginal candidiasis.
VVC is a fungal infection predominantly
There is considerable conflict about susceptibility
to RVVC in the literature, whether it is
mainly due to impairment in T cell function. A
study done by Corrigan et al. (23) revealed that
subjects with RVVC have decreased T cell proliferation
and IFN-γ secretion in stimulation with
Early clinical studies have shown that defects
in CMI by Th1 cells lead to recurrent fungal infections
(26-28). It has been revealed that mutations
affecting Th17/IL17 increase susceptibility
to CMC and was confirmed with results of the
study assessing vaginal yeast problems in response
to inhibition of Th17 (29). These results
are consistent with our findings while they are in
contrast with some other studies, which reported
normal cellular immunity in evaluation of RVVC
patients (15, 30, 31). Controversial results have
been published about contribution of Th2 cells in
Some of the studies conducted on the relationship between patient’s immunity and the incidence of VVC have focused on the evaluation of local safety and allergic reactions in the vaginal environment. Several evidences exist suggesting that RVVC has strong correlation with atopy (36- 39). Treatment with zafirlukast, cetirizine or other allergy immunotherapy medicines induce remission and are sometimes considered as maintenance therapy in patients who failed to get resolution of symptoms by variant antifungal treatments (40, 41). Weissenbacher et. al. (42) have studied immunological factors including IL-4, IL-5, IL- 13 and PGE2 in vaginal discharge in women with RVVC proposing that infected cases had a specific local immune deficiency in that area. Another study evaluating patients with hypersensitivity to their spouse’s seminal plasma proteins suggests that IgE-mediated immune responses may be involved in this process (43). We concluded that the etiology of RVVC in allergic patients is an inflammatory response to allergens in different mucosal membranes (oropharynx, sinus and vagina), which provide vaginal environment susceptible to fungal growth.
Other studies have demonstrated that maintenance
therapy with antimycotic drugs is effective
in lowering sign and symptom severity and
frequency of recurrence (44, 45). We found that
patients with history of using antifungal agents
did not have significantly higher lymphoproliferative
activity but presented milder symptoms than
the groups not having received such drugs. In our
studies patients taking antifungal medicine in the
past 30 days were excluded and only patients who
had the history of receiving antimycotics prior to
that were analyzed. Previous
As opposed to this publication, a study done
by Leigh et al. (48) indicated that the incidence
of mucosal infection was not different between
HIV+ patients and healthy persons. However,
another study evaluating HIV+ patients with oral
and vaginal candidiasis, suggested that immunity
The result of this study showed that the patients
with RVVC could have had a pre-existing defect
in their CMI or a proven history of allergy, which
could increase the susceptibility of mucosa to get
Special thanks to Acquired Immunodeficiency Research Center for supporting different stages of the project. Financial support for this study was from Cellular and Molecular Immunology Research Center, Isfahan University of Medical Sciences. We also thank all those who helped us in this study including Dr. Mohammad Talaei for criticizing, revising the manuscript and statistical consultation. There was no conflict of interest among authors.