Clinical Application And Research Of Vitamin B Complex As An Adjuvant Therapy For The Treatment Of Patients With Complicated Vulvovaginal Candidiasis

Object:1.To explore an economic and effective way to treat RVVC.2.To study the efficacy of vitamin B complex as an adjuvant therapy for the treatment of RVVC.3.To study the important adjuvant therapy effect on VVC by establishment VVC mouse model,vaginal epithelial cell culture and establishment of VVC epithelial cell model and fluconazole antifungal susceptibility test.4.To provide a new treatment theories and explore the improvement of clinical treatment of RVVC.Methods1.Questionnaire survey and drug susceptibility tests164 people filled out the questionnaire in this study to know the basic information of the patients,recorder the following-up table content,the time of the following-up visit are one to two weeks,one month,three months and six months after the end of treatment.We advised the patient to do a vaginal secretions fungal culture and drug sensitive test at the first visit and the following three or six months.2.Clinical study According to the random control principle,158 patients met the inclusion criteria.They were randomly divided into three groups:Group A included 49 patients treated with local vaginal suppository for 1 week and oral antifungal agents for 4 weeks?1 tablet miconazole every night+150 mg fluconazole once for a week?;Group B enrolled 54 patients treated with local vaginal suppository for 1 week and vaginal ointment for 4weeks?1 tablet miconazole every night+compound ketoconazole ointment every night?;Group C included 55 patients treated with local vaginal suppository for 1 week,vaginal ointment for 4 weeks,and oral vitamin B complex for 2 weeks?1 tablet miconazole every night+compound ketoconazole ointment every night+2 tablets vitamin B complex twice a day?.Sexual action was prohibited during the first treatment week.After treatment,patients would come back regularly to the Department of Gynecology in the First Affiliated Hospital of Anhui Medical University for an outpatient review or telephone follow-up review.In order to facilitate follow-up and timely feedback,phone numbers of follow-up worker will be given to patients so that psychological counseling can be provided at any time.Recovery meant that all symptoms and signs disappeared within 6 months after the treatment and the test results of Candida albicans was negative.Marked effectiveness meant that symptoms and signs were significantly improved within 6 months after treatment and the test result of Candida albicans was negative.Improvement indicated that symptoms and signs were somewhat improved within 6months after treatment,but the test results of Candida albicans was still positive.Ineffectiveness meant that there was no improvement in symptoms and signs with 6months after treatment and the test results of Candida albicans were still positive.3.Laboratory research3.1 One hundred and five female mice of ICR strains?aging 8¹0 weeks and weighing22.31±3.26 g?were fed with standard feed for more than a week in order to reduce the stress response caused by environmental change.Subcutaneous injection of estradiol benzoate was conducted to cause fake estrous state of mice?one injection in every two days with 0.1 mg each time?.On the 6th day after injection,inoculated fugal solution was injected into absorbable hemostatic sponge and the sponge was inverted into the vagina of mice.On the 2nd,4th,7th and 21 st day after inoculation,5 mice were selected for vaginal lavage for identification of VVC mouse model and vaginal tissues were obtained for study.3.2 About 21 d after successful establishment of model?first day for treatment?,all mice were treated through intragastric administration with fluconazole 150?l/time on the 1st,4th,and 7th day,and later once for 1 week,with totally 3 weeks.At the same time,vitamin B complex was injected as adjuvant treatment.Vitamin B complex injection?specification:20mg 2ml vitamin B1,2 mg riboflavin sodium phosphate?by vitamin B2?,2 mg vitamin B6,and 30 mg nicotinamide?.80 mice were randomly divided into 4groups according to the dosage of vitamin B complex?n=20?.Mice in V1 group were injected with 150?l normal saline;V2 group,50?l vitamin B complex solution+100?l normal saline;V3 group,100?l vitamin B complex solution+50?l normal saline;and V4 group,150?l vitamin B complex solution.After 4 weeks of treatment,vaginal secretions of mice were obtained for smear microscopic examination and vaginal tissue HE staining.3.3 Vaginal epithelial cell culture and establishment of VVC epithelial cell model and:vaginal epithelial cell were cultured by keratinocyte serum-free medium and cell passage.The passaged cell and Candida spore were selected to construct the VVC cell model.After 24 h culture in incubator,50?L fluconazole was added for further culture.Cells were devided into 4 groups.V1 control?15?L PBS?,V2?10?l PBS+5?l vitamin B complex?,V3?5?L PBS+10?l vitamin B complex?,V4?15?l vitamin B complex?groups.After 48 h,the supernatant was collected and the levels of IL-1?,IL-6 and IL-8were detected.3.4 Antifungal susceptibility test:To dilute the Candida into 10³ CFU/ml and seeded into the 96-well plate and was divided into the V1 control?15?l RPMI1640 solution?,V2?10?l RPMI 1640 solution+5?l vitamin B complex solution?,V3?5?l RPMI1640solution+10?l vitamin B complex solution?,V4?15?l vitamin B complex solution?.Different concentrations of fluconazole were set and the final concentration were as follows:2.0?g/ml,1.8?g/ml,1.6?g/ml,1.4?g/ml,1.2?g/ml,1.0?g/ml,0.8?g/ml,0.6?g/ml,0.4?g/ml,0.2?g/ml),minimum inhibitory concentration(MIC₈₀)was analyzed.3.5 Statistical analysis All the data were analyzed with SPSS 21.0 statistical analysis?SPSS,Chicago,IL,USA?.When assessing the curative effect,recovery,marked effectiveness and improvement were counted as effective rate.Enumeration data were presented by mean±standard deviation?SD?????±S?.Differences between groups were analyzed using independent-sample t-test.P<0.05 was considered statistically different.Results1.Questionnaire The youngest patient was 21 years old and the oldest one was 50 years old.,the main incidence of the disease was 25-45 years,accounting forthe incidence of disease is mainly concentrated in 25-45 years old,was 84.2%;82.3%patients had self diagnosis and treatment during the course of the disease;76.8%patients had the signs of anxiety and tension;61.6%patients were viginal delivery.All the patients had different treatment plan.The high risk factors are unstandard clinical treatment,misuse of antibiotics,misconceptions about women's health knowledge etc.condoms and intrauterine contraceptives accounted for 34.8%and 37.2%of these patients;the habit of vaginal washing accounts for 22%;18.9%patients had been using antibiotics recently;14.6%patients had fungal infection in other parts;oral sex and anal sex accounted for 12.8%,12.8%sexual partner had similar symptoms.2.Clinical study2.1 129 cases?81.6%?of 158 Candida positive patients were infected with Candida albicans,non-Candida albicans accounted for 29 cases?18.4%?;118 cases?91.5%?of Candida albicans sensitivity to fluconazole,18?62.1%?non-Candida albicans were sensitive to fluconazole;the susceptibility,mediator and drug resistance rate of Candida albicans to fluconazole were 91.5%,5.3%,3.2%respectively;the total sensitivity rate of fluconazole and itraconazole to RVVC were 86.1%and 62%respectively,while resistance rate were 6.1%and 7.2%respectively;the incidence of fluconazole resistance in non-Candida albicans was significantly higher than that in Candida albicans,which has statistical significance?P?27?0.01?.2.2.Clinical curative effect among three groups By comparison,we found that there were significantly difference in curative effect and cure rate among VVC patients in three groups?all P<0.05?.effective rate of patients in the group C was 92.73%,which is significantly higher than in the A?73.47%?and B?79.63%?groups.Compared with the A?61.22%?and group B?62.96%?,recovery rate of the group C?72.73%?was higher than group A and B,which had no statistical difference?P>0.05?.3.Laboratory research3.1 VVC mouse model The rate of the successful establishment of VVC mouse model is 95.23%.from the second day and genital swelling was also observed.On the 4th day the smear was the most intensive with pelotons and symptoms such as severe infection,hyperemia and edema and festering.From the second day of the infection,high level of CFU was2.99±0.32 and the level peaked on the 4th day?7.28±0.55CFU/ml?,then it gradually decreased to 4.96±0.24 CFU/ml and to 3.13±0.19 on the 21 st.3.2 Vaginal secretions among four groups after intragastric administration After a period of treatment,pseudohypha in cluster with rich blastospores can be seen in the vaginal secretion of mice in V1 group under the microscope.In the V2 group,a lot of pseudohypha with rich blastospores were visible in the vaginal secretion of mice,but the pseudohypha were not in cluster.In the V3 group,there were a few pseudohypha and a small amount of blastospores in vaginal secretion.In the V4 group,it was almost impossible to see pseudohypha under the microscope,and blastospores were occasionally observed.3.3 Vaginal tissues histopathological change In the V1 group,vaginal tissues of mice manifested severe hyperemia,edema,severe inflammatory infiltration,and many inflammatory cells can be seen in vaginal mucosa and submucosa.V2 group were significantly reduced in lesions severity and the degree of inflammatory infiltration with obvious edema and congestion.V4 group had a milder edema than those in the V3 group and no inflammatory infiltration was observed?P<0.01?.3.4 Curative effects in VVC mouse model treated with four different regimens The effective rate of V1 group treated with fluconazole alone was 60%and the recovery rate was 30%,While V4 group was 90%and 65%respectively.As to V2 and V3 groups,the effective rate were 70%and 85%,the recovery rate were 40%,50%?P<0.05?.3.5 IL-1?,IL-6 and IL-8 levels in vaginal tissues of the four group mice ELISA was used to analyze the expression of inflammatory factors in vaginal tissues of the four group mice.The expressions of IL-1?,IL-6,and IL-8 in mice of V1 group were 308.15±23.65 ng/l,93.85±4.92 ng/l,135.65±14.13 ng/l,V2 group were199.65±21.43ng/l,71.05±3.95ng/l,96.55±16.27ng/l,V3 group were 205.31±22.18ng/l,68.34±3.25ng/l,89.09±13.05ng/l,V4 group were 118.16±21.52ng/l,55.49±2.41ng/l,66.0±12.92 ng/l.V1 group were significantly higher than those of mice in other groups?all P<0.05?.V4 group were significantly decreased?P<0.05?.3.6 IL-1?,IL-6 and IL-8 levels in VVC vaginal epithelial cell model of the four different regimens.ELISA was used to analyze the expression of inflammatory factors in VVC vaginal epithelial cell model of the four different regimens.The levels of IL-1?,IL-6 and IL-8 in the V1 group were 418.25±22.75 ng/l,99.48±4.43 ng/l,155.74±11.11ng/l,V2 group were 269.65±19.53ng/l,86.18±4.05ng/l,107.44±17.29ng/l,V3 group were 223.34±22.18ng/l,71.09±4.67ng/l,96.01±13.52ng/l.V4 group were108.89±22.19ng/l,50.32±3.11ng/l,60.99±11.12 ng/l,V1 group were significantly higher than those of mice in other groups?all P<0.05?.V4 group were significantly decreased?P<0.05?.with the increase of vitamin B complex,the inflammation was effectively alleviated.3.7 Effects of vitamin B complex on MIC₈₀0 of fluconazole treated with four different regimens.Without vitamin B complex,the MIC₈₀0 of fluconazole was 2.0?g/ml.with the addition of vitamin B complex,the MIC₈₀0 of fluconazole significantly decreased.When the dosage of vitamin B complex reached 15?l,the MIC₈₀of fluconazole was only1.2?g/ml.it was indicated that vitamin B complex enhanced the inhibitive ability of fluconazole on Candida growth.Conclusion:1.Clinical study1.1 The susceptible group of RVVC was mainly Candida albicans,accounting for 81.6%,secondly,non-Candida albicans,accounting for 18.4%.The sensitivity of Candida albicans to fluconazole was 91.5%,and the sensitivity of non-Candida albicans to fluconazole was 62.1%.The resistance of non-Candida albicans to fluconazole was significantly higher than that of Candida albicans.Combined with the Chinese Medical Association's obstetrics and Gynecology branch infection cooperation group's guidelines for diagnosis and treatment of recurrent vulvovaginal candidiasis?RVVC?,We believed fluconazole can be used as one of the first choice drugs for the treatment of RVVC.At the same time,we suggested that those who are dissatisfied with the effect of treatment should pay attention to the possibility of non-Candida albicans infection and Candida albicans resistance to the drug and these patients should take drug sensitive experiment and change treatment plan in time.1.2 The clinical treatment plan of VVC is had no unified treatment method,the clinical efficacy was not satisfactory.Miconazole 1 tablets every night x 1 weeks+Compound Ketoconazole Cream uses×4 weeks around the vulva every night+take vitamin B 2tablets at a time,two times a day for×2 weeks.It is a better method for efficiency and cure rate,Less expensive,easy for patients to accept and it is worth promoting.2.Laboratory research:VVC mice were successfully modeled with different regimens,Vitamin B adjuvant therapy can significantly improve the efficiency of treatment and cure rate,It can be proved from the following aspects:Vaginal smear after treatment,pathological features of vaginal tissue,expression levels of inflammatory factors and the expression of inflammatory factors in vaginal epithelium cultured in vitro with Candida.In addition,fluconazole susceptibility test indicated Vitamin B complex can strengthen fluconazole antifungal effect.