VULVOVAGINAL CANDIDIASIS: PREVALENCE, SPECIES DISTRIBUTION AND RISK FACTORS AMONG NON-PREGNANT WOMEN, IN SANA'A, YEMEN

Background and objectives: Vulvovaginal candidiasis is known to be a global issue of concern due to its association with economic costs, sexually transmitted diseases, and the escalation of genital tract infections.  This study aimed to determine the prevalence, species distribution and risk factors associated with Candida species causing vulvovaginal candidiasis. 
Subjects and Methods:  Non-pregnant women attending routine antenatal visits at Al-Olaifi-Family Center in Sana'a were enrolled in a cross-sectional study conducted from June 2018 to March 2019. Laboratory work was carried out at the National Center of Public Laboratories (NCPHL). Vaginal swabs were sampled from participants after oral consent was obtained. The swabs were inoculated in Sabouraud glucose agar supplemented with chloramphenicol and incubated at 37°C for 24 to 48 h under aerobic conditions in order to perform a fungal culture. Candida species were determined by culturing on HiCrome Candida differential Agar at 35°C for 48 h to produce species-specific colours. Data on demographic, clinical, and risk factors were collected in a pre-designed questionnaire. 
Results:  A total of 190 non-pregnant women were included. The prevalence of VVC was 22.1%. Candida albicans accounted for 16.3% and non-Candida albicans accounted for 5.8% of the isolates, mainly C. glabrata (3.2%), C. rugosa (1.05%), C. lipolytica (1.05%), and C. dubliniensis (0.53%). When VVC risk factors were considered, there were significant risk factors with age group 30-34 years (33.3%, odds ratio=2.1) and age group  ≥35 years (62.5%, odds ratio=10.3), residence in a rural area (39.5 %, OR=3.3), negative emotions (30.2%, OR=2.3), underwear replacement over 1 day (29.3%, OR=4.2), impure cotton underwear (29.4%, OR=4.9), while Condom use and vulvar cleaning before or after sexual life were found to be highly significant protective factors against VVC (p=0.008, 0.03, respectively). 
Conclusions: Guidelines for the management of VVC syndrome in Yemen should be revised to include a protocol specifically for women over 30 years of age. VVC undoubtedly poses a significant threat to women's reproductive health. Risk factors for VVC are varied, and include ages, health habits, history of the disease, and other aspects. It is necessary to take appropriate measures to avoid risk factors and to help reduce the prevalence of VVC among women of childbearing age. 
                   
Peer Review History: 
Received: 3 May 2021; Revised: 23 June; Accepted: 30 June, Available online: 15 July 2021 
Academic Editor: Dr. Ali Abdullah Al-yahawi, Al-Razi university, Department of Pharmacy, Yemen, alyahawipharm@yahoo.com 
UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency.  
Received file:                Reviewer's Comments: 
Average Peer review marks at initial stage: 6.5/10 
Average Peer review marks at publication stage: 7.0/10 
Reviewer(s) detail: 
Dr. Michael Otakhor Erhunmwunse, St. Philomena Catholic Hospital, Nigeria,dedoctor4life@yahoo.com 
Dr. A.A. Mgbahurike, University of Port Harcourt, Nigeria, amaka_mgbahurike@yahoo.com 
Dr. Wadhah Hassan Ali Edrees, Hajja University, Yemen, edress2020@gmail.com 
  
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INTRODUCTION
Candidiasis is a fungal infection caused by any species of Candida. It affects the mouth and vagina, while the penis is less common and very rare, Candida infection may become invasive, spreading to other parts of the body 1-7 . Vulvovaginal candidiasis (VVC) is the second most widespread type of vaginal infection among women of childbearing age, involve mainly the vulva and vagina. It is expected that about 70-75% of women of childbearing age will experience at least one episode of VVC during their lifetime and that 40-50% will ISSN: 2456- 8058 31 CODEN (USA): UJPRA3 experience recurrence 8 . Approximately 80-90% of VVC is produced by Candida albicans excluding that only a minor of cases (10-20%) are caused as a result of non-C. albicans, commonly Candida glabrata 9 . Part of the normal vaginal microflora is Candida albicans.
It is known to have become a powerful opportunistic fungal pathogen and is the main causative agent of VVC in case the body lacks protective immunity and has a rebellion of purification insurgence to clearance 10 . Furthermore, the anus is anatomically adjacent to the vagina, which tenders much appropriateness for the migration of intestinal organisms, including Candida, into the vagina. VVC causes untold inconvenience to many patients, poses a serious problem for clinicians, and produces significant direct and indirect economic costs associated with medications and healthcare visits. Infection of the vagina or vulva may be associated with symptoms and signs such as pain, severe itching, pruritus inflammation, irritation, foul odor, dysuria and burning during urination 8 . It has been found in the Arab countries that the epidemic of VVC infection varies in different countries 11-13 . Thus, local studies are important to obtain relevant epidemiological data and sensitivity characteristics of Candida to antifungal drugs in order to aid the management and treatment of patients with Candida infection. Vaginitis in Yemen is one of the most common conditions to seek medical care. In Sana'a city, vaginal infections were prevalent in Yemen among 37.6% of women of reproductive age, 50% among pregnant women and where VVC was significantly associated with younger age female and intrauterine contraceptive use 4, 5,14 . Also in Hadhramaut 372 (39.2%) of 950 pregnant women had abnormal vaginal discharge and were positive for bacterial vaginosis 15 . Currently, the high rate of VVC infection was 70.45% recorded among pregnant women in Hajjah governorate 16 . According to the current epidemiological studies, the prevalence and incidence of genital tract infections differ between countries and ethnicities 4,5,17,18 . Even in similar populations, the epidemiological features of low reproductive tract infection differ 19 . It is therefore important to analyze the risk factors for VVC among women of reproductive age in Sana'a city and then provide a reference for clinical prevention and treatment of VVC.

SUBJECTS AND METHODS
Non-Pregnant women attending routine antenatal visits in Al-Olify -family Center in Sana'a city were enrolled in a cross-sectional study was conducted from June 2018 to March 2019 which is the time given by the NCPHL to complete the study. Inclusion criteria for subject selection were healthy individuals with no systemic disease. In addition, non-pregnant women who currently taking antifungal, steroids, antibiotics, or immunosuppressive drugs in the past 6 months were excluded. The sample included 190 non-pregnant mothers who met the inclusion criteria and agreed to participate in the study. All non-pregnant females at reproductive age was examined clinically by a specialist and vaginal swabs were taken. The vaginal swabs were sent to the National Center of Public Laboratories (NCPHL) where the laboratory works were carried out. Vaginal swabs samples were taken from participants after obtaining oral consent. The swabs were inoculated into Sabouraud's glucose agar supplemented with chloramphenicol and incubated at 37°C for 24 to 48 hours under aerobic conditions in order to perform a fungal culture. The identification of the Candida species was done by culture on HiCrome Candida Differential Agar at 35°C for 48 h for the production of species-specific colors. Data of demographic, clinical and risk factors were collected in a pre-designed questionnaire.

Data analysis
The data was statistically analyzed using EPI-Info version 6. The difference in the distribution of Candida types among groups was based on a comparison of repeat distributions by chi-square test. The odds ratio associated with VVC risk factors was performed by 2x2 tables to obtain an OR, 95% CI, Chi squared and p value by uncorrected static tests where the value of p <0.05 was considered significant.

Ethical approval
The written consent in all cases were obtained. Approval was obtained from the participants prior to collection of samples. Ethical approval was obtained from the Medical Research and Ethics Committee of the Faculty of Medicine and Health Sciences, Sana'a University with reference number (95) on 11/05/2018.

RESULTS
A total of 190 non-pregnant women were included. The prevalence of VVC was 22.1%. Candida albicans accounted for 16.3% and non-Candida albicans accounted for 5.8% of the isolates, mainly C. glabrata (3.2%), C. rugosa (1.05%), C. lipolytica (1.05%), and C. dubliniensis (0.53%). When VVC risk factors were considered, there were significant risk factors with age group 30-34 years (33.3%, odds ratio=2.1) and age group, 35 years (62.5%, odds ratio=10.3), residence in a rural area (39.5%), OR=3.3), negative emotions (30.2%, OR=2.3), underwear replacement over 1 day (29.3%, OR=4.2), impure cotton underwear (29.4%, OR=4.9), while Condom use and vulvar cleaning before or after sexual life were found to be highly significant protective factors against VVC (p=0.008, 0.03, respectively). Detailed results of the study are presented in Table 1 to Table 5. This result with respect to VVC-causing species, is similar to that reported by other researchers where Candida albicans is the prodrome and increased prevalence of non-Candida albicans species, especially C. glabrata, C. krusei and C. parapsilosis 29 . Some researchers have also found that the incidence of infection with Candida albicans is reduced and that of other Candida species including C. glabrata. C. tropical, C. parapsilosis, C. kefyr, C. africana, C. dubliniensis, C. Famata, C. Guilliermondii and C. lusitaniae mainly associated with vulvovaginitis of immunocompromised patients or in pregnant women 30 . This study showed that women in the age group 30-34 years and >35 years have a double and 10-fold risk of developing VVC, respectively, compared to younger age groups ( Table 2, and Table 3). However, age at first sexual intercourse <20 years increased the risk of VVC by two times (OR=2). The current study differs from that reported by Zeng et al., in which younger women were at double the risk of developing VVC compared to the elderly 21 . The results of the current study are also in line with the facts described that chance was the cause of increased Candida vaginitis in women of childbearing age and not in menopause 30 . The reason why VVC is more common in the third and fourth decade of life may lie in the fact that they are easy to suffer from negative factors such as risky sexual behaviors 31 .  In addition to some physiological and histological changes caused by reproductive hormones that occur in women during this stage of life (third and fourth decade), which increase the susceptibility to Candida infection. In this study, area of rural residence showed a significant effect on the incidence of VVC (OR=3.3, CI=1.5-7, p=0.0001) (Table 4). However, a previous study indicated that episodes of VVC symptoms were not significantly associated with residency 21,32 . The difference can be explained by the fact that there was difference between rural and urban areas with greater awareness of self-care and better medical conditions in current times. At the same time, people from different educational backgrounds cannot get the same avenues to get the vast information related to healthcare through the internet 21,33,34 . In the current study, educational background showed no effect on the incidence of VVC (p=0.29) (Table 4). However, a previous study indicated that higher education can protect against infection 33 . In the current study, use of pads during non-menstruation, trend of wiping after toilet, and frequency of wearing tights clothes did not show any effect on the incidence of VVC. However, previous studies indicated that episodes of VVC symptoms were significantly associated with these factors 21, 32 . The overall crude prevalence of diabetes among women of reproductive age in Yemen is 6.3% 35  statistically associated with frequent VVC 40 and this may be attributed to the fact that wearing synthetic underwear appears to enhance friction and maceration, thereby increasing local acidity and thus fungal infection. The study discovered that condoms were a protective agent helping to prevent VVC (OR=0.2, p=0.008) ( Table 5). It is unfortunate that this study classified ligation, intrauterine device (IUD), oral contraceptives (OCP), etc. into one category. It was therefore difficult to determine the effect of a particular species on the incidence of VVC (Table 5). Likewise, Du et al., 33 Zheng et al., 21 also found no relationship between OCP and VVC. However, a study reported that there is a link between IUD use and VVC 41 . Therefore, the role of the IUD in the development of VCC remains unclear, and needs further investigation. At the same time, this study showed that the frequency of vulvar cleaning did not affect the incidence of VVC significantly (OR=1.7, p=0.13), while vulvar cleaning before or after sexual life was a beneficial factor for VVC prophylaxis (OR=0.4, p=0.03) ( Table 5). There have been a limited number of studies exploring the relationship between the two before 21 . Also pad use while not menstruating may increase the risk of VVC, but in this study, pad use while not menstruating was not a risk factor for VVC (Table 5). Often, sexual life can reduce the risk of suffering from VVC. An observational study reported that sexual contact more often was only associated with asymptomatic colonization 42 . The result did not find a clear association between prior history of vaginitis and suffering from VVC (OR=1.5, p=0.24) (

CONCLUSION
The study has demonstrated a high incidence of VVC among non-pregnant women and this highlights the need for health authorities to develop strategies for diagnosing VVC, including vaginal swabs for