Three out of four women have at least one episode of vaginal candidiasis during their reproductive years, 40% of whom will experience a second episode and 5% of them will have recurrent vaginitis that is characterized by four or more episodes of vaginitis during one year, with clinical diagnosis and confirmation by culture    . Vulvovaginitis caused by Candida spp can be classified as uncomplicated (CVV), when they are manifested by sporadic or infrequent episodes, with mild or moderate symptoms, and complicated (CVVC), such as recurrent vulvovaginitis (CVVR)    .
Vulvovaginal infection by Candida spp is diagnosed by clinical and epidemiological conditions, without which colonization cannot be distinguished from infection. About 20% to 30% of women have vaginal colonization by Candida spp and are asymptomatic  . It is accepted that C. albicans is responsible for 80 to 92% of cases of candidiasis vulvovaginal  . The clinical syndrome caused by non-albicans species can be indistinguishable from that caused by C. albicans  . Increased frequency of other species such as C. glabrata, C. tropicalis, C. parapsilosis, C. guilliermondii and C. krusei, among others, has occurred   . Recurrences have been attributed to factors such as sexual transmission, hygiene habits, intestinal colonization, allergic and idiopathic causes. However, the epidemiological profile of women with recurrent idiopathic vulvovaginitis has not yet been established   .
Conventional treatments for fungal vulvovaginitis include antifungals such as ketoconazole, fluconazole and other azoles, but therapeutic failures are common, particularly in patients with C. glabrata vaginitis  . Fluconazole is a broad spectrum antifungal, with excellent therapeutic results for several Candida species, except for C. glabrata and C. krusei, as they have intrinsic resistance to this medication. C. glabrata vaginitis is considered the greatest challenge to the antifungals used   . The indiscriminate use of broad spectrum antifungal agents has contributed to the increase in recurrent vaginitis, due to the increasing proportion of events caused by non-albicans species and often resistant to azole derivatives  . The continued and progressive intolerance to antifungals is a problem of extreme gravity and concern that encourages the design of new antifungal drugs and new therapeutic techniques.
Photodynamic therapy (PDT) consists of the use of a photo-sensitive component and an appropriate light source, representing an effective, promising and safe alternative in the treatment of fungal diseases, among others      . Studies have shown the susceptibility of Candida spp to PDT, however, studies on the susceptibility of strains of Candida resistant to fluconazole are restricted      . Some aspects related to the results of PDT on Candida spp in vitro still need to be better elucidated  -  . The efficacy of PDT associated with sensitizers such as methylene blue and toluidine blue is reported in the treatment of patients with candidiasis caused by fluconazole-resistant C. glabrata and C. albicans     .
Photodynamic therapy using methylene blue at a concentration of 250 to 400 mg/ml promotes a reduction in the growth of C. albicans, occurring its complete eradication, in the oral cavity, when concentrations of 450 to 500 mg/ml of methylene blue are used  . A study evaluated in vitro, the action of 1% gentian violet dye associated with laser and observed an important effect of photodynamic therapy against Candida albicans  . The gentian violet solution has a potent antifungal activity against several species of Candida, such as C. albicans, C. glabrata and C. parapsilosis  . Martins et al. (2009), evaluated the antifungal activity of Curcumin in 23 strains of fungi in vitro and an inhibitory effect on several species of Candida spp, much more efficient than fluconazole  . Results from another study showed that blue LED associated with Curcumin proved to be effective in the photodynamic inactivation of C. albicans in vitro  .
The present study aims to verify in vitro the synergistic effect of the combination of photodynamic light associated with sensitizers such as methylene blue, gentian violet or curcumin to inhibit the growth of Candida spp from complicated VVC and asymptomatic women.
2. Materials and Methods
2.1. Study Population
Microbial isolates were from of women with clinical suspicion of vulvovaginitis, coming from Basic Public Health Units, Private Offices and other Gynecology Services, and the study in question was developed in four years (2017-2020), together with the Laboratory of Mycology and the Department of Gynecology of Paulista School of Medicine/Federal University of São Paulo under the scientific opinion of the Research Ethics Committee N˚ 5505. All participants signed an informed consent form (FICF). Financial resources and responsibility for research costs were borne by the researcher.
Were included in the study adult participants aged 18 to 65 years with chronic and/or recurrent vaginitis and asymptomatic women who attend for the collection of gynecological routine and oncotic colpocytology examination. Were excluded from the study pregnant women with diabetes mellitus, on corticotherapy, antibiotic therapy or hormone therapy, with immunosuppression, carriers of the human immunodeficiency virus (HIV), using an intrauterine device (IUD), or who used vaginal douches or spermicides.
2.2. Collection, Isolation and Identification of Yeasts
Vaginal secretion was collected with the help of a non-lubricated speculum from the ectocervix regions and from the bottom of the vagina using a swab moistened in a sterile saline solution. A total of 630 participants were evaluated, 230 clinically compatible with vulvovaginal candidiasis and 400 asymptomatic. The samples were seeded on Sabouraud dextrose agar media supplemented with chloramphenicol (100 mg/mL) and Chromagar Candida, obtaining 140 positive cultures from symptomatic women and 94 from asymptomatic women. From pure cultures, yeasts were identified using non-specific primers from the ITS1 and ITS4 regions, CALB1 and CALB2 for C. albicans, CGLAB1 and CGLAB2 for C. glabrata, CTR1 and CTR2 for C. tropicalis, and CPAR3 and CPAR2 for C. parapsilosis.
For antifungal susceptibility tests, fluconazole and ketoconazole from the standard E-Test Kit (AB Biodisk, Solna, Sweden) were used. The isolates were cultivated on Sabouraud-dextrose agar (DIFCO), at a temperature of 37˚C, for a period of 48 hours, suspended in sterile saline solution according to a 0.5 McFarland scale and cultured in Petri dishes containing the solidified medium. The E-Test antimycotic strips, after a period of 15 minutes at room temperature, were added in contact with the seeding in agar. The reading of the MICs was made after 24 and 48 hours of incubation at 35˚C following the edge of the ellipse until the lowest concentration on the E-Test strip. Inconsistent and contradictory readings or high minimum inhibitory concentration (MIC) values of the antifungal fluconazole were repeated, as instructed by AB Biodisk (1993). In addition to the isolated fungi, C. parapsilosis ATCC 22019 was included in each experiment for quality control in the verification of drug activity, since fluconazole MICs for this microorganism are previously known, according to Rex et al., 1996  and NCCLS, CLSI     . For ketoconazole there are no established breakpoints. Susceptibility criteria to fluconazole adopted: ≤8 μg/ml (Sensitive, S), 16 and 32 μg/ml (susceptibility-dependent-dose, SDD), ≥64 μg/ml (Resistant, R)  .
2.4. Use of Photodynamic Light (PL) and Photosensitizing Dyes
Irradiation source consisting of LED devices (Light Emitting Diode) type adapted photopolymerizers, with blue light emitting diodes, wavelengths between 450 and 470 nm, at the power of 260 mW, dosimetry 270 J/cm2, during periods of time of 15 minutes was used in the research. The incident light was oriented perpendicularly at a distance of 15 cm and directed from top to bottom, over all Candida spp colonies, before and after antifungigram, seeking to evaluate in vitro the effectiveness of (PL) in the viability of Candida spp inactivation. Photosensitizers such as methylene blue, gentian violet and curcumin, associated or not with LED light irradiation, were used at concentrations of 450 mg/ml (MB), 2% (GV) and 50 µM (CR), respectively. Thus, the different groups were classified: single treatment: LED, GV, MB, CR; treatment in combination: LED + GV, LED + MB, LED + CR; and control (K) without treatment.
Candida spp cultures, after tests with LED and photosensitizing dyes, were inoculated in 5 mL of sterilized peptone saline (SPS), being homogenized in a vortex, to obtain concentrations of 106 CFU/mL, measured in a spectrophotometer, with 76% transmittance at 530 nm. Suspensions from different groups were introduced separately into 96-well microplates and then subjected to different treatments. Such Candida spp growths, at concentrations of 10−1 and 10−2 mL, were seeded by the spread-plate method in Petri dishes containing Sabouraud-dextrose agar and incubated under aerobic conditions for 48 hours at a temperature of 35˚C. Subsequently, the counts of the respective Candida spp colonies were performed.
Means resulting from the measurements of the procedures performed with all isolates and treatments were then calculated. The interpretation of the results was based on the MIC values recommended by CLSI M27 A2, CLSI M27 S3, CLSI M27 S4 and NCCLS  -  Documents. All tests were developed in triplicate. Fluconazole susceptibility criteria: ≤8 μg/ml (Susceptible, S), 16 and 32 μg/ml (Susceptible-dose-dependent, DDS), ≥64 μg/ml (Resistant, R)  .
From the material collected from patients with complicated vaginitis, 120 cultures were obtained (111 chronic and 9 recurrent), 10 from patients with sporadic vaginitis and 94 from asymptomatic women, making a total of 224 positive cultures for Candida spp (Table 1). Of these isolates, 181 (80.8%) were identified as C. albicans, 14 (6.25%) C. glabrata, 13 (5.8%) C. tropicalis and 16 (7.15%) C. parapsilosis. Among the 9 patients with recurrent vaginitis, the prevalence of non-albicans species was observed, being 5 C. glabrata (55.5%), 2 C. albicans (22.25%) and 2 C. tropicalis (22.25%).
Referring to the microbial growth, in Table 2 we observe a marked reduction in the number of colonies of all Candida species after treatment with LED + GV, LED + MB, LED + CR and little inhibition with LED, MB, CR used alone. Gentian violet at a concentration of 2% substantially inhibited the growth of Candida spp.
After analyzing the susceptibility profile of Candida species to fluconazole
Table 1. Number of Candida species identified from chronic and recurrent vaginitis, asymptomatic and sporadic participants.
Table 2. Average of CFU/ml Candida spp counts 48 h after using photodynamic light with GV, MB, CR, or LED, GV, MB and CR photosensitizers alone. Percentages of colony reduction compared to the control group (K) are superscripted in parentheses.
Mean values of CFU/ml Candida spp counts (n = 224) expressed in (log10). Groups: without treatment administration (control group, K) and all groups, after using photodynamic light plus photosensitizers and isolated agents (LED, GV, MB and CR). Ca = C. albicans, Ct = C. tropicalis, Cp = C. parapsilosis, Cg = C. glabrata.
in vitro (Table 3) compared to the use of photodynamic light and MB, GV and CR sensitizers (Tables 4-6), a reduction in the number of resistant isolates was observed to the antifungal, mainly in resistant species and susceptible-dose-dependent (SDD) from women with chronic, recurrent and sporadic vulvovaginitis.
After treatment with photodynamic light and GV photosensitizing, there was a reduction in the percentage of resistant isolates from women with vulvovaginitis (C. albicans, 42.8%; C. tropicalis, 100%; C. glabrata, 75%) and asymptomatic (C. glabrata, 50%), as shown in Table 4. There was also a reduction in the percentage of SDD isolates, originating from vulvovaginitis (C. parapsilosis, 33.3%; C. glabrata, 100%) and asymptomatic (C. albicans, 40%; C. tropicalis, 100%).
Treatment with photodynamic light and methylene blue intensified the susceptibility to fluconazole, decreasing the percentage of resistant isolates from women with vulvovaginitis (C. albicans, 28%; C. glabrata, 75%) and asymptomatic (C. glabrata, 50%), according to Table 5. The DDS isolates also had a reduction in isolates from women with vulvovaginitis (C. glabrata, 100%) and asymptomatic (C. albicans, 20%; C. tropicalis, 100%; C. parapsilosis, 100%).
The use of curcumin and photodynamic light caused a decrease in the resistant profiles among isolates of vulvovaginites (C. albicans, 57.7%; C. tropicalis, 100%, C. glabrata, 100%) and asymptomatic (C. glabrata, 100%), Table 6.
Table 3. In vitro fluconazole susceptibility profile of Candida spp isolated from asymptomatic women (a) and vulvovaginitis with chronic, recurrent and sporadic (v).
Table 4. Susceptibility to fluconazole in vitro of Candida spp isolated from asymptomatic (a) and with vulvovaginitis (v) after photodynamic light with VG. Percentages of reduction in resistant isolates are expressed in italics, compared to the values expressed in Table 3.
Reduction in the percentage of DDS isolates from vulvovaginitis (C. parapsilosis, 66.7%; C. glabrata, 100%) and asymptomatic (C. albicans, 60%; C. tropicalis, 100%; C. glabrata, 100%) was also observed.
Regarding ketoconazole, six isolates, two C. albicans, two C. tropicalis and two C. glabrata, four from patients with chronic or recurrent vaginitis and two from asymptomatic women, showed high MICs > 16 μg/mL before submitted to the LED associated with photosensitizers. We did not obtain MIC > 16 μg/mL in C. parasilosis. After undergoing LED + MB, we observed that there was a 50% reduction in MICs > 16 μg/mL in C. tropicalis, C. glabrata.
Treated under the action of LED + GV, we found that there was a 50% reduction in MICs > 16 μg/mL in C. albicans, C. tropicalis, C. glabrata. After LED +
Table 5. Susceptibility to fluconazole in vitro from Candida spp isolated from asymptomatic (a) and with vulvovaginitis (v) after PL with MB. Percentages of reduction in resistant and SDD isolates compared to the values expressed in Table 3.
Table 6. Susceptibility to fluconazole in vitro of Candida spp isolated from asymptomatic (a) and with vulvovaginitis (v) after PL with CR. Percentages of reduction in resistant and SDD isolates compared to the values expressed in Table 3.
CR, here was a 100% reduction in MICs > 16 μg/mL in C. tropicalis, C. glabrata.
Two C. albicans isolates were resistant to fluconazole after the three photodynamic procedures administered (LED + MB, LED + GV and LED + CR) and with high MICs > 16 μg/mL for ketoconazole. Both of these isolates originated from secretions collected from patients with recurrent vaginitis. The trailing phenomenon was also registered in these two C. albicans isolates and in two C. tropicalis.
Among women participating in this study, asymptomatic and affected by chronic or recurrent vaginitis, we found high percentages of non-albicans species, in acordance with what was also observed in other studies   . We identified isolates with a resistant and dose-dependent profile to fluconazole, both among C. albicans and non-albicans species, coming from materials collected from patients with chronic and recurrent vaginitis such as Candida albicans (13, 11.92%), C. tropicalis (1, 33.3%), C. parapsilosis (3, 60%), C. glabrata (5, 50%), and those obtained from asymptomatic women, such as Candida albicans (5, 6.94%), C. tropicalis (1, 10%), C. parapsilosis (1, 12.5%), C. glabrata (3.75%). We also recorded MIC > 16 μg/mL in relation to ketoconazole between C. albicans and non-albicans species. Resistance to antifungal treatments is also frequently observed in other studies     . Non-albicans species, in addition to being frequent in complicated vulvovaginitis, have shown high levels of resistance to conventional antifungal agents, which was also verified in this research.
Our work has evidenced C. glabrata as an etiological agent resistant to antifungal agents ordinarily listed in the treatment of vaginitis, emphasizing that PDT reduced or even blocked in vitro the development of colonies of this agent, which is considered yeast associated with chronic vaginitis and/or recurrence of vaginitis and nowadays, it constitutes a considerable obstacle to the therapeutic efficacy of widely used antifungals, an event also registered in other studies     . Candida glabrata is often associated with a chronic evolution, recurrence in vulvovaginitis and high resistance to antifungal agents. LED alone did not show homogeneous results for yeast in this study.
With respect to C. albicans, studies on photodynamic (PD) light and methylene blue also report similar results regarding the substantial blockage in the growth of colonies of this particular yeast    . In this study, the synergistic use of PD light and methylene blue with increased efficacy in blocking the growth of colonies of the tested yeast species such as C. glabrata, C. parapsilosis, C. tropicalis and C. albicans.
The PD light associated with methylene blue showed important reductions in the number of colonies resistant to fluconazole or ketoconazole (MICs > 16 μg/mL) and also in the reduction of resistant phenotypes, when compared to values recorded before treatment. A study with PD light and methylene blue found its use viable to inhibit C. albicans species from infection  , which was also observed in this research, including in relation to other Candida species. These findings suggest PD light with methylene blue as a promising therapeutic alternative for fungal infections, including vulvovaginitis, especially in relation to non-albicans species, and further studies should be prospected to prove their in vivo efficacy. Other research on PD light and methylene blue treatments used for fluconazole resistant yeasts such as C. glabrata and C. albicans also describes similar results     . Non-albicans species, many times, show resistance to conventional antifungal treatments   .
Investigations have shown the efficiency of gentian violet used alone, in terms of blocking the growth of C. albicans and non-albicans colonies. Gentian violet in 1% concentration was effective with regard to the total blockade in the growth of C. albicans, both in its use as a single agent and in association with light  . In our research, we also found the inhibitory activity C. albicans and non albicans species with PD light by LED and gentian violet solution 2% in both the separate use or as jointly.
Martins et al. in 2009 and Freitas in 2015 observed that curcumin, when compared to fluconazole, showed antifungal activity with greater inhibitory potential, on C. albicans and non-albicans species, such as C. tropicalis, C. parapsilosis and C. glabrata   . In our work, however, it was only when we used curcumin together with LED light irradiation that we obtained antifungal efficacy, blocking the growth of C. albicans and non-albicans yeast colonies. We recorded only a mild antifungal effect of curcumin when used alone. However, its use associated with PD light revealed a good synergism in vitro, markedly inhibiting the growth of C. albicans and non-albicans colonies. Similar findings were also obtained in another investigation   .
We emphasize in our study, when using only LED or photosensitizers alone, we did not obtain reduction in the growth of Candida colonies, both C. albicans and non-albicans, resistant to fluconazole and with MIC > 16 μg/mL for ketoconazole, except for the gentian violet solution 2%, which actually showed antifungal activity, both in PDT and as a single agent. Other investigations have also found antifungal activity of gentian violet used in isolation      .
We call attention that associations of photosensitizers with LED seem to be impactful with regard to blocking the growth of colonies of C. albicans or non-albicans species and the PDT procedure, after more studies and clinical trials, may constitute in future an important basis for the treatment of vulvovaginitis, particularly in chronic or recurrent cases, considering that it can represent efficient, low-cost treatment, without major side effects. In the specific case of gentian violet, we would still consider the practicality of representing possibly effective antifungal agent, potentially effective, having greater synergism when used with LED irradiation.
We emphasize that all 224 Candida spp isolates were submitted to photodynamic light with methylene blue, gentian violet and curcumin photosensitizers. We observed a reduction in the percentage of UFC/mL and of resistant and SDD phenotypes to fluconazole and to ketoconazole MICs > 16 μg/mL. Photodynamic therapy proved to be promising and effective in vitro, with regard to the significant reduction of fungal colonies of C. albicans and non-albicans species, including C. tropicalis and mainly C. glabrata, yeast species associated with severe cases of chronic recurrent vulvovaginitis. Our investigation was developed “In Vitro”, but its results may serve as parameters in future to in vitro and in vivo researches. New therapeutic solutions, more efficient, less aggressive, with minor or without organic toxicity, with treatments based on a short period of time, and that do not provoke the selection of new resistant and potentially aggravating microbial strains, are required in fungal vulvovaginitis mainly for complicated cases. In these aspects, PDT could be an interesting tool, used as a single treatment or in addition to antifungal agents, for future studies involving Candida vulvovaginitis chronicity and recurrence especially to ways for gynecological clinic practice improvement.
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