Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Prevalence of Vaginal Infection by Multidrug Resistant Candida Species among Different Ages in Egypt

Mohammad. H. A. Hassan, Mady. A. Ismail, Ahmed. M. Moharram Ahmed. M. Shoreit*

Department of Botany and Microbiology, Faculty of Science, Assiut University, Egypt

Abstract: During 12 months’ the period of study from December 2015 to November 2016, one hundred and sixty women were clinically examined for vaginal yeast infection. Result revealed that 100 cases (62.5%) proved to have vaginal infection. The mean age (±SD) of the participants was 27.70±5.79 years. Most of the positive cases with high count belonged to the age group1 (19-25 year) followed by group 2 (26-30 year) representing 39 and 32 cases respectively (71 % of the total cases). Phenotypic and genotypic characterization of the yeast isolates showed that C. albicans were the predominant Candida species 258 cfu out of 410 cfu represent (62.9%) followed by C. lusitaniae (56 cfu, 13.7%), C. krusei (44 cfu 10.7%), C. glabrata (26 cfu, 6.3%), C. tropicalis (18 cfu, 4.4%) and C. Parapsilosis (8 cfu, 2.0%) respectively. In-vitro sensitivity test showed high resistance to the tested antifungals; whereas 73 isolates out of 128 (57.3%) were resistant to more than one type of tested antifungal agents, including 14 isolates showed resistance to all antifungal agents (Multidrug resistant strains). C. albicans represented by 67 isolates, showed resistance to one or more of the tested antifungal agents such as Fluconazole (61.2 %), Ketoconazole (56.7 %), Amphotericin B (52.2), Itraconazole and Nystatin (43.3 %). In case of non- albicans isolate (61 isolate) the most effective antifungal agent was Nystatin were it susceptible 65 % form C. lusitaniae isolates, 62.5 % from C. tropicalis and 60 % from C. Krusei isolates. The relation between risk factors, such as pregnancy, diabetes mellitus, history of antibiotic uses, and contraceptive methods, was recorded.

Keywords: vulvovaginal candidiasis, antifungal agents, multidrug resistant, phenotypic and genotypic characterization

Citation: Mohammad. H. A. Hassan, Mady. A. Ismail, Ahmed. M. Moharram Ahmed. M. Shoreit, Prevalence of Vaginal Infection by Multidrug Resistant Candida Species among Different Ages in Egypt. American Journal of Microbiological Research, 5 (4). 78-85. January 2017. doi:10.12691/ajmr-5-4-2

1. Introduction

Vulvovaginal candidiasis (VVC) or Candida vaginitis; is a common fungal infection of the vagina attributed to an overgrowth of Candida species. It is one of the most common medical problem throughout the world which affecting a large proportion of healthy women during their life time [1].

Candida species considered as a part of the normal microbiota of the mucous membrane of the respiratory, gastrointestinal and female genital tracts [2]. overgrowth of Candida in the vagina produces symptoms include vaginal and vulvar pruritus, itching, pain, sexual dysfunctions, dryness, cracks, and odorless thick, white vaginal discharge [3]. Vaginal symptoms are one of the most common reasons for consulting gynecologist for approximately 10 million visits each year [4]. In recent years, the number of vaginal yeast infections has been dramatically increasing. It is estimated that about 75% of all women infected by Candida spp. at least once during their reproductive years, and approximately between 40-50% during the childbearing years suffer from recurrent chronic infection [5,6]. Candida albicans is indicated as the most common causative pathogen of vulvovaginal candidiasis which represent about 80–95% [4,7,8,9]. However, recently there are many reports from different countries indicate that about 10-30% of the patients infected with VVC are due to non-albicans Candida species as C. glabrata, C. parapsilosis, C. tropicalis and C. krusei [10,11,12,13,14]. In recent years, number of resistant vaginal yeast pathogen has been dramatically increasing, and several reports indicated differences in the distribution of Candida spp. associated with VVC and their antifungal drug susceptibility patterns from different geographic locations [15,16,17,18]. Mårdh et al. (2002) reported that up to 7.5% of vaginal Candida isolates have been resistant to one or more of the commonly used azoles [19]. In addition, recently non-albicans species have shown resistance to the antifungals commonly used to treat vulvovaginal candidiasis [20,21,22]. The appearance of resistant candida to certain antifungal may play a role in cases of recurrent vulvovaginal candidiasis. In a study conducted by Richter et al. (2005), fluconazole resistance was observed among 15.2% and 41.7% of vaginal isolates of C. glabrata and C. krusei, respectively [23]. Another study conducted by Consolaro et al. (2005), reported that vaginal isolates of non-albicans species, as C. glabrata, C. parapsilosis, C. krusei, and S. cerevisiae were susceptible to nystatin, ketoconazole and Itraconazole [24]. There are many reasons for the emergence of resistances; prolonged exposure and increased use of antifungals for recurrent vulvovaginitis candidiasis are the most common risk factors for resistance [20,25,26]. Furthermore, the distribution of these infections may depend on numerous risk factors that have been associated with VVC such as, pregnancy, uncontrolled diabetes mellitus, obesity, poor personal hygiene, poor dietary habits, and uses contraceptives [27,28].

This study was carried out to determine the prevalence of clinical multidrug resistant Candida species in different age groups of women diagnosed as having Vulvovaginal candidiasis. In addition, the possible risk factors in relation to resistance profile was also considered.

2. Materials and Methods

2.1. Patient

This study was conducted on 160 women age ranged from (19-40 years) with signs and symptoms of vulvovaginal candidiasis; who visited three private women's health centers in Assiut city, Egypt during the period from December 2015 to November 2016.

2.2. Cases of VVC

Typical symptoms of VVC includes vulvovaginal irritation, soreness, purities, change in vaginal discharge to thick white adherent vaginal discharge and growth of Candida spp. [29].

2.3. Sampling and Data Collection

Vaginal swabs were taken by a physician from women with a symptom of Vulvovaginal candidiasis. Clinical information was also recorded in questionnaire. this information comprised a date of sampling, age of patients, possible risk factors such as pregnancy, diabetes mellitus, contraceptive methods and history of antimicrobial uses. Samples were obtained by a sterile cotton swab, and directly placed onto 10 ml sterile normal saline (0.85% NaCl, pH 7), transported in cooler boxes to the Lab, for analysis. This study included only the patients who accepted to participate in the study.

2.4. Mycological Analyses

2.4.1. Culturing of Specimens

Under aseptic conditions, the collected specimen swabs were directly placed into 90 ml sterile normal saline (0.85% NaCl, pH 7), vortexed, then 1 ml of the suspension was streaked onto sterile Petri plates containing Sabouraud Dextrose Agar (SDA)- Difco, supplemented with chloramphenicol (250 mg/ L) -Sigma, in replicate; and incubated at 37°C, for 48-96 h. The growing colonies were counted, recorded and kept in SDA slants for further study.

2.4.2. Direct Microscopic Examination (DME)

Smears from the collected sample stained with lactophenol cotton blue (LPCB) were prepared. Appearance of budding cells with or without pseudohyphae under microscope indicates positive results [30].

2.5. Identification of Isolates

Phenotypic and genotypic characteristics were used to identify the different isolates of Candida.

2.5.1. Phenotypic Identification

Growth on HiCrome Candida Differential Agar

Is a selective and differential medium, allows differentiation of Candida species. According to the manufacturer C. albicans appears as light green colored smooth colonies, C. tropicalis blue to metallic blue, C. glabrata cream to white smooth colonies, C. krusei purple fuzzy colonies and C. lusitaniae pale to pink colonies [31].

2.5.2. API Candida (BioMérieux).

A commercial Analytical Profile Index (API) yeast identification kit, namely API Candida (bioMérieux) was used to conduct the biochemical tests on the isolated Candida spp. The API Candida strip consists of 12 biochemical tests. Five carbohydrate-acidification tests (glucose, galactose, saccharose, trehalose, and raffinose) and seven enzymatic tests (β-maltosidase, α-amylase, β-xylosidase, β-glucuronidase, urease, Nacetyl-β-glucosaminidase, and β-galactosidase). Inoculum yeast suspension was prepared, inoculated to the strip and incubated for (24 hours at a temperature of 35°C), visual color reaction was observed. The results were compared with those given in the profile list in the package insert. One of the strips was used as negative control where sterile distilled water was used as inoculum.

2.5.3. Genotypic Identification

Some isolates which have revealed high resistance to the tested antifungal agents were selected. Nucleotide sequencing of rRNA gene was done at Solgent Company, Daejeon, South Korea. Primers used for gene amplification have the following composition: ITS1 (5' - TCC GTA GGT GAA CCT GCG G - 3'), and ITS4 (5'- TCC TCC GCT TAT TGA TAT GC -3'). Then the amplification was carried out in a thermal cycler under the following conditions: one round of denaturation at 95°C for 15 sec followed by 30 cycles of denaturation at 95°C for 20 sec, annealing at 50°C for 40 sec and extension at 72°C for 1 min, with a final extension step at 72 °C for 5 min. The PCR products were then purified with the SolGent PCR Purification Kit-Ultra (SolGent, Daejeon, South Korea) prior to sequencing. Purified PCR products were reconfirmed (using size marker) by electrophoreses on 1% agarose gel. Then the bands were eluted and sequenced with the incorporation of dideoxynucleotides (dd NTPs) in the reaction mixture. Each sample was sequenced in the sense and antisense directions using ITS1 and ITS4 primers [32]. Sequences were further analyzed using BLAST from the National Center of Biotechnology Information (NCBI) website. Phylogenetic analysis of sequences was done with the help of software DNA Star version 14.

2.6. Antifungal Susceptibility Testing

A total of 128 isolate of Candida species obtained from patients diagnosed with VVC were tested for their susceptibility to eight standard antifungal agents namely; Fluconazole (10 μg), Amphotericin B (100 units), Nystatin (100 units), Ketoconazole (10 μg), Clotrimazole (10 μg), Itraconazole (10 μg), Miconazole (30 μg) and Voriconazole (1 μg) (Himedia, India) by disk diffusion method according to the procedure described in the Clinical and Laboratory Standards Institute (CLSI) [33]. Briefly, each isolate was sub-cultured on SDA and an inoculum was suspended in sterile normal saline and the turbidity was adjusted to 0.5 McFarland standard and streaked on the surface of sterilized Mueller-Hinton agar (MHA) (Oxoid). The antifungal agent discs were placed onto seeded agar plates and incubated at 37°C, for 24 h. The diameter of inhibition zone was measured and recorded. The antifungal agent, concentration, abbreviation and interpretative breakpoints are shown in Table 1.

Table 1. Antifungal agent, Abbreviation, Potency and Interpretative breakpoints

3. Results and Discussion

3.1. Patient Age in relation to VVC

During the period study (12 months), a total of 160 samples were collected from different age groups with signs and symptoms of vulvovaginal candidiasis. The direct microscopic examination; revealed that only 59 out of 160 cases (36.9 %) were positive showing budding yeast cells and/or pseudohyphae. However, the number of positive culture on SDA was higher (100 patients representing 62.5 % of total samples). The mean age of the studied patient was 27.70±5.79 and the average total count of yeast colony isolated were 410 cfu for all samples. 78 % from the culture positive cases showed a single yeast growth whereas other 22 % showed mixed growth. Most of the positive cases with high count belonged to the age group 1 (19-25 year) and group 2 (26-30 year) given 39 and 32 cases respectively representing 71 % of total cases as shown in Table 2 & Figure 1. This result is slightly more than those obtained by Nurat et al. (2015) who reported that, the prevalence of VVC was 25% and the highest numbers of VVC were in age group 21-25 years represent (40.44%) followed by 26-30 years with (32.58%) [34]. Benchellal et al. (2011) reported that the commonest age group to have positive VVC was from 26-30 [35]. Holland et al. (2003) reported that the mean age of patients with C. albicans and non-albicans species was 33±13.5 and 43±15.9 years old, respectively [36]. Kent (1991) reported that, the women who are younger and sexually active are more vulnerable to the infected with Candida species [37].

Table 2. Total number of the collected sample (positive and negative) classified according to age group

Figure 1. Incidence of vaginal yeast infection in relation to age group

3.2. Risk factors

The risk factors, including pregnancy, diabetes mellitus, uses of contraceptives, and history of antimicrobial uses were assessed in all positive cases (Table 3). Out of all cases, 82 % of patients were non-pregnant status.

Table 3. Distribution of risk factors among positive cases of patients with vulvovaginal candidiasis infection

Only 7 cases have diabetes mellitus. About 39 % of patients doesn’t use any contraception tools whereas others use either oral contraceptive (10 cases) or an intrauterine contraceptive device (51 cases). In a total, 36 % of the patients had a history of antibiotic therapy, and 14 % using of antifungal during or two months before sample collection. A history of previous VVC episodes was reported by 25 patients all of them had previous history of using antimicrobial agents. In the respect, Kanagal et al. (2014) highlights that there are several factors associated with increased rates of Candida vaginitis including diabetes, previous candidiasis infection, use of antibiotics and different forms of contraceptive [38]. Nelson et al. (2013) reported also that in pregnant women, vaginal candidiasis has been related to emotional stress and suppression of immune system. This high prevalence of vaginal candidiasis may lead to pregnancy complications like abortions, premature birth, low birth weight and other morbidities. [39,40]. Another main factor of prevalence of VVC include the misuse of antibiotics for treatment of such infections. This misuse of drugs results in resistance especially to the common antifungal agents.

3.3. Strain Identification and Distribution

3.3.1. Phenotypic Identification

In the current study, six species of Candida were identified; C. albicans were the predominant Candida species (258 cfu out of 410 representing 62.9%) followed by C. lusitaniae (56 cfu, 13.7%), C. krusei (44 cfu, 10.7%). C. glabrata (26 cfu, 6.3%), C. tropicalis (18 cfu, 4.4%) and C. Parapsilosis (8 cfu, 2.0%) respectively. Rhodotorula sp. was represented with (23 cfu, 5.6 %) Table 4 and Figure 2, Figure 4.

Table 4. Incidence of the different isolated Candida species in relation to age group

Figure 2. HiCrome agar plate showing different colors of Candida species. (A) purple fuzzy colonies for Pichia kudriavzevii (anamorph Candida krusei) (B) Light pink for Clavispora lusitaniae, (teleomorph of Candida lusitaniae) (C) dark green colored for C. Parapsilosis, (D) light green colored smooth colonies, for C. albicans, (E) blue to metallic blue colored raised colonies for C. tropicalis (F) cream to white smooth colonies for C. glabrata

3.3.2. API Candida (BioMérieux).

A group of API Candida strips were employed to confirm the identification of yeast isolates as shown in Table 5.

Table 5. API candida identification kits, used to conduct the biochemical test

3.3.3. Genotypic identification

Molecular characterization rRNA gene sequencing and establishment of the phylogenetic tree confirmed the phenotypic identification of Candida species Figure 3.

Figure 3. Phylogenetic relationship of the multidrug resistant Candida isolates recovered from vulvovaginal candidiasis and closely related species based on 18S rRNA gene sequencing. Each isolate given AUMC No. and the GenBank accession number in parentheses

Figure 4. Incidence of the different isolated Candida species in relation to age group

Many investigations from different countries had shown that C. albicans was the most common species identified in patients with VVC; varying from 46.9% to 76.3% [41,42,43,44]. In the same context, the present study highlights that C. albicans (62.9 %) is still the predominant pathogen causing VVC. and the remaining 37.1% were other non-albicans species. The distribution of Candida species varies greatly depending on the geographical areas as well as the population habits. Recently, there has been an increasing trend in the isolation rate of non-albicans species reported from some countries, such as C. glabrata was the most common species isolated from the vaginal swabs with isolation rate ranged from 30 to 71 % from non- albicans isolates [45,46,47,48,49]. In contrast to most studies, our study revealed that the non- albicans, C. lusitaniae is the second most common species, and C. krusei disbanded in third place. According to Nyirjesyet al. (1995) an increase of non-albicans species has been observed, particularly in recurrent cases [50].

3.4. Sensitivity of Yeasts to Antifungal Agents

In the present study, 8 different standard antifungal agents were evaluated against 128 isolates of candida species (C. albicans 67 isolates, C. lusitaniae 23 isolates, C. krusei 20 isolates, C glabrata 8, C. tropicalis 2, and C. Parapsilosis 2). The results showed a high frequency of resistant isolates to the tested antifungals; It was found that only 4 isolates (3.1%) sensitive to all antifungal agents, 51 isolates (39.8%) showed intermediate effect, whereas 73 isolates (57.3%) showed resistant to more than one type of antifungal agent, including 14 isolates showed resistance to all tested antifungal agents (Multidrug resistant strain). Candida from the age group 1 (19-25) year (24 out of 39 cases, 61.5%) were the most resistant. Followed by the second highest group 2 (31-35) with 16 out of 32 cases (50.0 %) Table 7 and Figure 5.

Table 6. Resistant prevalence for positive sample

Table 7. In vitro susceptibility profile of Candida spp. isolates against 8 standard antifungal agents

Figure 5. Susceptibility profile of 8 antifungal agents

Results in the Table 7 revealed that C. albicans (represented by 67 isolates) showed resistance to one or more of the frequently prescribed antifungal agents such as Fluconazole (61.2 %), Ketoconazole (56.7 %), Amphotericin B (52.2%), Itraconazole and Nystatin (43.3 %). In case of non- albicans isolates (61 isolate) the most effective antifungal agent was Nystatin, it susceptible 65 % of C. lusitaniae isolates, 62.5 % of C. tropicalis and 60 % of C. Krusei isolates.

Many studies have found that the resistance rates of C. albicans are 8.6% for voriconazole, 19.1% for Itraconazole, 9.7% for fluconazole, 2.6% for ketoconazole and 12.7% for miconazole [17,18]. In 2002, a study from Belgium reported an alarmingly high fluconazole resistance rate of 21% in 84 vaginal C. albicans isolates [12]. An earlier study reported fluconazole non-susceptibility in 67% of vaginal Candida isolates [23]. Liu et al (2014) showed that the resistance rates to C. albicans were 1.1, 2.2 and 4.2% for fluconazole, Itraconazole and miconazole, respectively [51]. Candida infection treatments, which fail to respond to conventional antifungal drug treatments, have become increasingly reported. Recently there are many infected cases don’t respond to treatment using azole class anymore, such as Fluconazole. This is due to the widespread, long-term use of antifungal for treating and preventing yeast infection.

In conclusion, the current results indicate that C. albicans was the predominant Candida species isolated from cases of VVC, while non-albicans species came second. The high frequency of the resistant strains found in our study give an alarming to demonstrate further studies to evaluate in vitro susceptibility of clinical yeast isolates. Successful treatment of vaginal yeast infections can be helpful in precise diagnosis of VVC and control the prevalence of resistant candida.

References

[1]   | Sobel, JD., Vazquez JA. Symptomatic vulvovaginitis due to fluconazole-resistant Candida albicans in a female who was not infected with human immunodeficiency virus. Clinical infectious diseases 1996, 22(4):726-727.
[2]   | Ghannoum, MA., Radwan SS. Candida adherence to epithelial cells: CRC Press; 1990.
[3]   | Falagas, ME., Betsi GI., Athanasiou S. Probiotics for prevention of recurrent vulvovaginal candidiasis: a review. Journal of Antimicrobial Chemotherapy 2006, 58(2):266-272.
[4]   | Akinbiyi, A., Watson R., Feyi-Waboso P. Prevalence of Candida albicans and bacterial vaginosis in asymptomatic pregnant women in South Yorkshire, United Kingdom. Archives of gynecology and obstetrics 2008, 278(5):463-466.
[5]   | Mayer, FL., Wilson D., Hube B. Candida albicans pathogenicity mechanisms. Virulence 2013, 4(2):119-128.
[6]   | Gandhi, TN., Patel MG., Jain MR. Prospective study of vaginal discharge and prevalence of Vulvovaginal candidiasis in a tertiary care hospital. International Journal of Current Research and Review 2015, 7(1):34.
[7]   | Roshan, R., Sabokbar A., Badali H. Identification of Candida species isolated from vulvovaginal candidiasis using PCR-RFLP. International Journal of Molecular and Clinical Microbiology 2014, 4(2): 406-410.
[8]   | Achkar, JM., Fries BC. Candida infections of the genitourinary tract. Clinical microbiology reviews 2010, 23(2):253-273.
[9]   | Foxman, B., Muraglia R., Dietz J-P., Sobel JD., Wagner J. Prevalence of recurrent vulvovaginal candidiasis in 5 European countries and the United States: results from an internet panel survey. Journal of lower genital tract disease 2013, 17(3):340-345.
[10]   | Sobel, JD. Genital candidiasis. In: Sexually Transmitted Infections and Sexually Transmitted Diseases. Springer; 2011: 613-624.
[11]   | Nyirjesy, P., Sobel JD. Genital mycotic infections in patients with diabetes. Postgraduate medicine 2013, 125(3): 33-46.
[12]   | Bauters, TG., Dhont MA., Temmerman MI., Nelis HJ. Prevalence of vulvovaginal candidiasis and susceptibility to fluconazole in women. American journal of obstetrics and gynecology 2002, 187(3):569-574.
[13]   | Corsello, S., Spinillo A., Osnengo G., Penna C., Guaschino S., Beltrame A., Blasi N., Festa A. An epidemiological survey of vulvovaginal candidiasis in Italy. European journal of obstetrics & gynecology and reproductive biology 2003, 110(1):66-72.
[14]   | [Wang, F-J., Zhang D., Liu Z-H., Wu W-X., Bai H-H., Dong H-Y. Species distribution and in vitro antifungal susceptibility of vulvovaginal Candida isolates in China. Chinese medical journal 2016, 129(10):1161.
[15]   | Organization, WH. Antimicrobial resistance: 2014 global report on surveillance: World Health Organization; 2014.

[16]  
Sievert, DM., Ricks P., Edwards JR., Schneider A., Patel J., Srinivasan A., Kallen A., Limbago B., Fridkin S. Antimicrobial-resistant pathogens associated with healthcare-associated infections summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2009-2010. Infection Control & Hospital Epidemiology 2013, 34(01): 1-14.

[17]   | Zhang, JY., Liu JH., Liu FD., Xia YH., Wang J., Liu X., Zhang ZQ., Zhu N., Ying Y., Huang XT. Vulvovaginal candidiasis: species distribution, fluconazole resistance and drug efflux pump gene overexpression. Mycoses 2014, 57(10): 584-591.
[18]   | Nagashima, M., Yamagishi Y., Mikamo H. Antifungal susceptibilities of Candida species isolated from the patients with vaginal candidiasis. Journal of Infection and Chemotherapy 2016, 22(2): 124-126.
[19]   | Mårdh, P-A., Rodrigues AG., Genç M., Novikova N., Martinez-de-Oliveira J., Guaschino S. Facts and myths on recurrent vulvovaginal candidosis--a review on epidemiology, clinical manifestations, diagnosis, pathogenesis and therapy. In.: SAGE Publications Sage UK: London, England; 2002.
[20]   | Pappas, PG., Kauffman CA., Andes DR., Clancy CJ., Marr KA., Ostrosky-Zeichner L., Reboli AC., Schuster MG., Vazquez JA., Walsh TJ. Clinical practice guideline for the management of candidiasis: 2016 update by the Infectious Diseases Society of America. Clinical Infectious Diseases 2015: civ933.

[21]  
Theill, L., Dudiuk C., Morano S., Gamarra S., Nardin ME., Méndez E., Garcia-Effron G. Prevalence and antifungal susceptibility of Candida albicans and its related species Candida dubliniensis and Candida africana isolated from vulvovaginal samples in a hospital of Argentina. Revista Argentina de Microbiología 2016, 48(1): 43-49.

[22]   | Papon, N., Courdavault V., Clastre M., Bennett RJ. Emerging and emerged pathogenic Candida species: beyond the Candida albicans paradigm. PLoS Pathog 2013, 9(9): e1003550.
[23]   | Richter, SS., Galask RP., Messer SA., Hollis RJ., Diekema DJ., Pfaller MA. Antifungal susceptibilities of Candida species causing vulvovaginitis and epidemiology of recurrent cases. Journal of Clinical Microbiology 2005, 43(5):2155-2162.
[24]   | Consolaro, M., Albertoni T., Svidzinski A., Peralta R., Svidzinski T. Vulvovaginal candidiasis is associated with the production of germ tubes by Candida albicans. Mycopathologia 2005, 159(4): 501-507.

[25]  
Alexander, BD., Johnson MD., Pfeiffer CD., Jiménez-Ortigosa C., Catania J., Booker R., Castanheira M., Messer SA., Perlin DS., Pfaller MA. Increasing echinocandin resistance in Candida glabrata: clinical failure correlates with presence of FKS mutations and elevated minimum inhibitory concentrations. Clinical infectious diseases 2013, 56(12): 1724-1732.

[26]  
Fekkar, A., Dannaoui E., Meyer I., Imbert S., Brossas J., Uzunov M., Mellon G., Nguyen S., Guiller E., Caumes E. Emergence of echinocandin-resistant Candida spp. in a hospital setting: a consequence of 10 years of increasing use of antifungal therapy? European journal of clinical microbiology & infectious diseases 2014, 33(9):1489-1496.

[27]   | Apalata, T., Longo-Mbenza B., Sturm A., Carr W., Moodley P. Factors associated with symptomatic vulvovaginal candidiasis: A study among women attending a primary healthcare clinic in Kwazulu-Natal, South Africa. Annals of medical and health sciences research 2014, 4(3):410-416.
[28]   | Deorukhkar, SC., Saini S., Mathew S. Virulence factors contributing to pathogenicity of Candida tropicalis and its antifungal susceptibility profile. International journal of microbiology 2014, 2014.
[29]   | Sobel, JD., Faro S., Force RW., Foxman B., Ledger WJ., Nyirjesy PR., Reed BD., Summers PR. Vulvovaginal candidiasis: epidemiologic, diagnostic, and therapeutic considerations. American journal of obstetrics and gynecology 1998, 178(2): 203-211.
[30]   | Ellis, D., Davis S., Alexiou H., Handke R., Bartley R. Descriptions of medical fungi. Nexus Print Solutions, Adelaide, South Australia, Australia 2007.
[31]   | Shettar, SK., Patil AB., Nadagir SD., Shepur T., Mythri B., Gadadavar S. Evaluation of HiCrome differential agar for speciation of candida. Journal of Academy of Medical Sciences 2012, 2(3):101.
[32]   | White, TJ., Bruns T., Lee S., Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications 1990, 18(1): 315-322.
[33]   | Wayne, P. Clinical and Laboratory Standards Institute: Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard-; CLSI document M27-A3. CLSI 2008a 2008, 28: 6-12.
[34]   | Nurat, AA., Babalola GO., Shittu MO., Tijani MA., Adekola SA. Detection and epidemiology of vulvovaginal candidiasis among asymptomatic pregnant women attending a tertiary hospital in Ogbomoso, Nigeria. International Journal of Biomedical Research 2015, 6(7):518-523.
[35]   | Benchellal, M., Guelzim K., Lemkhente Z., Jamili H., Dehainy M., Moussaoui DR., El Mellouki W., Idrissi KS., Lmimouni B. La candidose vulvo-vaginale à l’hôpital militaire d’instruction Mohammed V (Maroc). Journal de Mycologie Médicale/Journal of Medical Mycology 2011, 21(2): 106-112.
[36]   | Holland, J., Young M., Lee O., Chen SC. Vulvovaginal carriage of yeasts other than Candida albicans. Sexually transmitted infections 2003, 79(3): 249-250.
[37]   | Kent, HL. Epidemiology of vaginitis. American journal of obstetrics and gynecology 1991, 165(4): 1168-1176.
[38]   | Kanagal, D., Vineeth V., Kundapur R., Shetty H., Rajesh A. Prevalence of Vaginal Candidiasis in Pregnancy among Coastal South Indian Women. J Womens Health, Issues Care 3 2014, 6:2.
[39]   | Nelson, M., Wanjiru W., Margaret MW. Prevalence of vaginal candidiasis and determination of the occurrence of Candida species in pregnant women attending the antenatal clinic of Thika District Hospital, Kenya. Open Journal of Medical Microbiology 2013, 2013.
[40]   | Aslam, M., Hafeez R., Ijaz S., Tahir M. Vulvovaginal Candidiasis in pregnancy. Biomedica 2008, 24: 54-56.
[41]   | Paulitsch, A., Weger W., Ginter‐Hanselmayer G., Marth E., Buzina W. A 5‐year (2000–2004) epidemiological survey of Candida and non‐Candida yeast species causing vulvovaginal candidiasis in Graz, Austria. Mycoses 2006, 49(6): 471-475.
[42]   | Vermitsky, J-P., Self MJ., Chadwick SG., Trama JP., Adelson ME., Mordechai E., Gygax SE. Survey of vaginal-flora Candida species isolates from women of different age groups by use of species-specific PCR detection. Journal of clinical Microbiology 2008, 46(4): 1501-1503.
[43]   | Goswami, D., Goswami R., Banerjee U., Dadhwal V., Miglani S., Lattif AA., Kochupillai N. Pattern of Candida species isolated from patients with diabetes mellitus and vulvovaginal candidiasis and their response to single dose oral fluconazole therapy. Journal of Infection 2006, 52(2): 111-117.
[44]   | Shi, X-Y., Yang Y-P., Zhang Y., Li W., Wang J-D., Huang W-M., Fan Y-M. Molecular identification and antifungal susceptibility of 186 Candida isolates from vulvovaginal candidiasis in southern China. Journal of medical microbiology 2015, 64(4): 390-393.
[45]   | Yang, Z-T., Wu L., Liu X-Y., Zhou M., Li J., Wu J-Y., Cai Y., Mao E-Q., Chen E-Z., Lortholary O. Epidemiology, species distribution and outcome of nosocomial Candida spp. bloodstream infection in Shanghai. BMC infectious diseases 2014, 14(1): 241.

[46]  
Gunther, LSA., Martins HPR., Gimenes F., Abreu ALPd., Consolaro MEL., Svidzinski TIE. Prevalence of Candida albicans and non-albicans isolates from vaginal secretions: comparative evaluation of colonization, vaginal candidiasis and recurrent vaginal candidiasis in diabetic and non-diabetic women. São paulo medical journal 2014, 132(2):116-120.

[47]   | Kumari, V., Banerjee T., Kumar P., Pandey S., Tilak R. Emergence of non-albicans Candida among candidal vulvovaginitis cases and study of their potential virulence factors, from a tertiary care center, North India. Indian Journal of Pathology and Microbiology 2013, 56(2): 144.
[48]   | Deorukhkar, SC., Saini S., Mathew S. Non-albicans Candida infection: an emerging threat. Interdisciplinary perspectives on infectious diseases 2014, 2014.
[49]   | Ng, KP., Kuan CS., Kaur H., Na SL., Atiya N., Velayuthan RD. Candida species epidemiology 2000–2013: a laboratory‐based report. Tropical Medicine & International Health 2015, 20(11): 1447-1453.
[50]   | Nyirjesy, P., Seeney SM., Grody MHT., Jordan CA., Buckley HR. Chronic fungal vaginitis: the value of cultures. American journal of obstetrics and gynecology 1995, 173(3): 820-823.
[51]   | Liu, X., Fan S., Peng Y., Zhang H. Species distribution and susceptibility of Candida isolates from patient with vulvovaginal candidiasis in Southern China from 2003 to 2012. Journal de Mycologie Médicale/Journal of Medical Mycology 2014, 24(2): 106-111.