A Protocol for the Role of Fractional CO2 Laser in Consolidation Treatment of Recurrent Vulvovaginal Candidiasis
A Protocol for the Role of Fractional CO2 Laser in Consolidation Treatment of Recurrent Vulvovaginal Candidiasis
Background: Recurrent vulvovaginal candidiasis (RVVC) is a common obstinate vulvovaginal
 inflammation in gynecology. At present, the pathogenesis of RVVC is not clear. In recent
 years, it is emphasized that the changes of vaginal microecological environment play an
 important role in the occurrence and development of RVVC. The treatment of RVVC can be
 divided into intensive treatment and consolidation treatment. Currently, local antifungal
 treatment is the main treatment regimen. While it is difficult and had an insufficient effect
 on prevention from recurrence of clinical signs and the improvement of vaginal mycological
 status in long term. Besides, there is no mature consolidation treatment regimen at home and
 abroad. Through the current application of the fractional CO2 laser in the treatment of
 atrophic vaginitis, it is confirmed that local irradiation of the fractional CO2 laser on
 vulva and vagina can regenerate and repair mucosal tissue, enhance the function of vaginal
 epithelial cells, gradually restore the normal pH value of vagina, improve vaginal
 microecological environment, restore the proportion of vaginal flora and reduce the
 recurrence rate of RVVC. This trial aims to explore the efficacy of two consolidation therapy
 schemes: the fractional CO2 laser and traditional antifungal drugs.

 Methods/Design: The ongoing study will include 200 RVVC patients who voluntarily joined the
 study and signed the informed consent form the Second Affiliated Hospital of WMU during
 December 2019 to March 2022. After patients were cured by transvaginal local drug intensive
 therapy, participants will be randomly divided into control group (n = 100) and experimental
 group (n = 100). The control group continue to receive a monthly course of vaginal local drug
 consolidation therapy for 6 months, while the experimental group will be given monthly local
 vaginal CO2 laser consolidation therapy for 3 months. The vaginal secretions samples of RVVC
 patients will be collected before intensive therapy, after intensive therapy and at the end
 of consolidation therapy, and the species abundance and structure of vaginal flora were
 detected and analyzed. In the same period, 5 healthy women of childbearing age were recruited
 as the normal control group and the volunteers do not take any treatment. The vaginal
 secretions samples of 5 volunteers will be collected when signing the informed consent to
 detect and analyze the species abundance and structure of vaginal flora.

 The patients with RVVC will be followed up for 3, 6 and 12 months after the treatment and
 vaginal secretions samples will be collected for vaginal flora structure detection to compare
 the vaginal microecological environment. For recurrent patients, the investigators will carry
 out targeted treatment, and again collect vaginal secretions sample to detect and analyze the
 species abundance and structure of vaginal flora.

 Objectives: The aim of this study is to compare the effects of fractional CO2 laser
 consolidation therapy and traditional antifungal consolidation therapy for RVVC, and assess
 the role of fractional CO2 laser in changes of vaginal microecological environment and
 recurrence rate of VVC after consolidation treatment.
1. Study design and general procedures

 200 RVVC patients will be selected according to the above eligibility criteria form the
 Second Affiliated Hospital of WMU during December 2019 to March 2022. Women will be informed
 about the study protocol, procedures, investigational product and potential risks of
 treatment. After 200 RVVC patients were cured (mycological cured) by traditional intensive
 antifungal drug treatment, they will be randomly divided into control group (n=100) and
 experimental group (n=100). The control group continued to receive one course of Clotrimazole
 vaginal tablets for consolidation therapy once a month for 6 months, and the experimental
 group will be given the fractional CO2 laser therapy once a month for 3 months. In the same
 period, 5 healthy women of childbearing age will be recruited as the normal control group,
 and the normal control group do not receive any treatments.

 2.Treatment

 2.1 Intensive treatment: Clotrimazole tablets 500mg PV biw Q3d, with or without injector, the
 tablets are placed deep in the vagina for 2 weeks.

 2.2 Consolidation treatment

 2.2.1 Control group: Clotrimazole tablets 500mg PV biw q3d in consolidation therapy, with or
 without injector, the tablets are placed deep in the vagina once a month for 6 months.
 Treatment is prohibited during the menstrual period.

 2.2.2 Experimental group: The fractional CO2 laser therapeutic machine will be used with
 wavelength 10.6 μ m, output power 40 w, action time 2000 μ s, lattice spacing 700 ~ 1000 μ m,
 gradually retreated from the vagina to the vaginal orifice according to the 5mm distance, and
 irradiated the vagina at 360 °once a month for 3 consecutive months as a course of treatment.
 Treatment is prohibited during menstrual period.

 3. Sample collection and detection

 3.1 Collection: Vaginal secretion samples will be collected from patients with RVVC before
 and after intensive therapy, at the end of consolidation therapy, and at 3, 6 and 12 months
 after consolidation therapy. The vaginal secretions samples of 5 volunteers will be collected
 when signing the informed consent. After emptying the urine, the participants take the
 lithotomy position in the gynecological examination bed, fully exposed the vulva and
 perineum. The gynecologists expose the cervix with a disposable vaginal dilator, collect the
 vaginal posterior fornix secretion with two disposable vaginal aseptic cotton swabs. Then the
 investigators send one sample to the Second Affiliated Hospital of WMU for vaginal discharge
 laboratory examination, put another sample into the 1.5ml cryopreservation tube and seal it
 in a liquid nitrogen tank at -80 ℃. These samples will be submitted to Shanghai Pudong
 Decoding Life Institutes for vaginal flora structure detection within 3 months.

 3.2 Vaginal discharge laboratory examination: The Second Affiliated Hospital of WMU will take
 up the laboratory examination, including vaginal cleanliness, pH value of vagina, vaginal
 microbiological conditions. The use of a microscope to examine vaginal secretions wet smears
 and staining smears to observe their cleanliness and the presence of special bacteria and
 cells to confirm vaginal cleanliness and vaginal microbiological conditions. Vaginal pH value
 will be determined by the precise pH test paper method.

 4. Follow-up visit: The investigatorswill take follow-up visit with RVVC patients at 3,6 and
 12 months after consolidation therapy, and vaginal secretions samples will be collected for
 fungal culture to compare the vaginal microecological environment, evaluate clinical cure
 rate and recurrences of infection. Besides, the investigators will carry out targeted
 treatment for recurrent patients and again collect vaginal secretions sample to detect and
 analyze the species abundance and structure of vaginal flora.

 5.Data analysis: ①By comparing the vaginal microecological environment (vaginal cleanliness,
 pH value of vagina, vaginal microbiological conditions and vaginal flora structure) between
 the normal control group (n=5) and 200 RVVC patients before the intensive therapy, the
 investigators will verify the vaginal microecological environment of RVVC patients has
 changed, and find out the difference between healthy women and RVVC patients. ②By comparing
 the vaginal microecological environment between control group (n=100) and experimental group
 (n=100) in different period (before and after intensive therapy, at the end of consolidation
 therapy, and at 3, 6 and 12 months after consolidation therapy), the investigators will find
 out the change of vaginal microecological environment in all treatment and development stages
 of RVVC, verify whether the fractional CO2 laser could restore the normal pH value of vagina,
 improve vaginal microecological environment, restore the proportion of vaginal flora or not.
 ③By comparing the cure rate and recurrence rate of VVC between control group (n=100) and
 experimental group (n=100) at 3, 6 and 12 months after consolidation therapy, the
 investigators can draw which treatment effect is better between fractional CO2 laser
 consolidation therapy and traditional antifungal consolidation therapy for RVVC.
200 RVVC patients will be eligible for the trial if they fulfil the following criterion:

 1. women aged 18 to 60;

 2. chief complaint: vulvar itching, burning pain, sexual intercourse pain and urine pain,
 leucorrhea increased, occurring 4 or more times a year;

 3. gynecological examination: vulvar erythema, edema, often accompanied by scratches,
 vaginal mucosa red and swollen, the inside of the labia minor and vaginal mucosa with
 white lumps, curd or bean curd-like, red and swollen mucous membrane exposed after
 erasure;

 4. laboratory examination: microscopic examination of vaginal secretions to find the
 spores or hyphae of Candida albicans;

 5. sign the informed consent form voluntarily.

 5 healthy volunteers as the normal control group will be eligible for the trial if they
 fulfil the following criterion:

 1. women aged 18 to 60;

 2. no clinical symptoms;

 3. vaginal cleanliness grade Ⅰ ~ Ⅱ;

 4. laboratory examination: Candida spores or hyphae were not found in vaginal secretions;

 5. menstruation was normal;

 6. voluntary informed consent was signed.

 Exclusion criteria:

 1. those who did not meet the above inclusion criteria;

 2. those with autoimmune or immunodeficiency diseases;

 3. those who received antibiotics or antifungal therapy (including systemic and topical);

 4. and special drugs and foods within 1 month;

 5. women during lactation and pregnancy;

 6. patients with liver insufficiency, neutropenia, adrenocortical dysfunction and
 diabetes;

 7. those who are mentally abnormal and can not cooperate normally;

 8. those who are allergic to clotrimazole vaginal tablets or other imidazole drugs.