ISSN 2320-5407 International Journal of Advanced Research (2015), Volume 3, Issue 5, 1376-1386
1376
Journal homepage: http://www.journalijar.com INTERNATIONAL JOURNAL
OF ADVANCED RESEARCH
RESEARCH ARTICLE
Antifungal Susceptibility Pattern and Species Distribution of Candida Isolates from Patients with Vulvovaginal Candidiasis
Mona Fattouh1, Asmaa Nasr El-din1, Ashgan Badawy1, Mohamed Nour El-din2, Hydi Ahmed3
1. Department of Medical Microbiology and Immunology, Faculty of medicine, Sohag University
2. Department of Obstetrics & Gynecology, Faculty of medicine, Sohag University
3. Department of Clinical pathology, Faculty of medicine, Sohag University
Manuscript Info Abstract
Manuscript History: Received: 15 March 2015 Final Accepted: 19 April 2015 Published Online: May 2015 Key words: Vulvovaginal candidiasis; Candida species; Antifungal sensitivity *Corresponding Author
Mona Fattouh Department of Medical Microbiology & Immunology, Faculty of Medicine, Sohag University.
E-mail: monarahman2002@yahoo.co.uk
The objectives of this study were to determine the prevalence of Candida in patients suffering from vaginitis, to assess predisposing factors, to identify Candida species and to determine the susceptibility profile of different Candida species to antifungal agents. This study was carried out in Sohag University Hospital during the period from August 2013 to August 2014. High vaginal swabs (HVS) were collected from 300 women with age range from (18-50 years) clinically diagnosed as vulvovaginitis. HVS were processed by wet mount microscopy, Gram stain, culture onto Sabouraud’s dextrose agar and CHROM agar plates. Isolates were identified and speciated using conventional methods, by the color of the colonies on the CHROM agar and by VITEK 2 System. Susceptibility testing to Clotrimazole, Miconazole, Fluconazole and Voriconazole by disc diffusion method was performed. Out of 300 HVS, Candida was isolated in 90 (30%) of cases. C.albicans was the predominant species isolated; 49 isolates (54.4%) followed by non albicans species like C. glabrata 22 (24.4%), C. tropicalis 13 (14.4%), C. famata 3 (3.3%), Candida dubliniensis1 (1.1%), C. Lusitania 1 (1.1%) and C. parapsilosis 1 (1.1%). Mixed infection with two species of Candida was seen in 5.6% of patients. The most common mixed cause was the combination of Candida albicans and Candida tropicalis (3.3%). Recurrent vulvovagintis has been the most frequently implicated risk factor (45.56%). Susceptibility tests revealed that; highest resistance was observed against Miconazole (14.3%) among the Candida albicans isolates and was observed against Clotrimazole (19.5%) among the non albicans isolates.
Copy Right, IJAR, 2015,. All rights reserved
INTRODUCTION
Vulvovaginal candidiasis (VVC) refers to a disorder characterized by signs and symptoms of vulvovaginal inflammation in the presence of Candida species. It is the second most common cause of vaginitis symptoms (after bacterial vaginosis) and accounts for approximately one-third of vaginitis cases (Workowski and Berman, 2010). Most cases of VVC is caused by Candida albicans (80 to 90%), while non-albicans Candida (NAC) including Candida tropicalis, Candida glabrata, Candida krusei and Candida parapsilosis account for 10 to 20% (Achkar and Fries ,2010). the virulence and antifungal drug susceptibility of Candida isolates differ according to the species, with Candida glabrata and Candida krusei being innately resistant to commonly prescribed antifungal drugs e.g. fluconazole (Farooqi et al.,2013).The clinical diagnosis of vaginal candidiasis is unreliable, and laboratory
ISSN 2320-5407 International Journal of Advanced Research (2015), Volume 3, Issue 5, 1376-1386
1377
confirmation is needed (Bello et al., 2012). Also; the clinical manifestation caused by NAC spp. is indistinguishable from those caused by C.albicans but they differ in their susceptibility to antifungal agents and often demonstrate low sensitivity to commonly used antifungal drugs (Deorukhkar et al., 2012). On the other hand, a vaginal sample may contain mixed species of Candida and the isolation and separation process of those different species seems complicated and time consuming using the usual culture media. Identification of the infecting Candida to the species level is of utmost importance for clinical microbiological services for prediction of likely drug susceptibility and to guide treatment. A number of identification systems have been developed and are commercially available. Enzymatic reaction methods using chromogenic substrates in Chromogenic agar (CHROMagar) medium has high sensitivity and specificity in the differentiation of Candida species in clinical samples. Using this medium, it is possible to identify Candida species based on color and other morphologic characteristics (Mahmoudi et al., 2011). VITEK 2 System is a rapid automated microbiological system used for bacteria and yeast identification, and antimicrobial susceptibility testing (AST) would allow clinical laboratories to perform both fungal identification and antifungal susceptibility testing simultaneously using a fully automated and completely standardized format (Pfaller et al., 2007). Failure to correctly diagnose and treat appropriately has led to prolonged distress in women who end up suffering from recurrent VVC defined as four or more proven infections per year (Sobel, 2007).Therefore, the aim of this study was to find out the prevalence of VVC, along with speciation of Candida, with special reference to its antifungal susceptibility pattern and evaluation of the risk factors responsible for VVC in patients attending our tertiary care hospital.
PATIENTS AND METHODS
Study design and patients
This study was carried out in the Department of Medical Microbiology and Immunology, Faculty of Medicine, Sohag University during the period from August 2013 to August 2014. A total of 300 women attending the out-patient clinic of the Department of Obstetrics and Gynaecology; clinically diagnosed as vulvovaginitis cases were included in this study. The study was approved by the ethical committee of Sohag Faculty of Medicine.
Inclusion criteria: women between 18- 50 years of age who presented with self-reported symptoms of vaginal discharge, pruritis, irritation, soreness, dysuria, and/or dyspareunia, not using any vaginal medication in the previous 48 hrs.
Exclusion criteria: Women of age group below 18 and above 50 years, catheterized patients, patients with genital neoplasms, and Post menopausal women.
Collection of High Vaginal Swabs (HVS)
Two sterile, cotton tipped swabs were collected from each patient according to the laboratory procedures of the WHO (2009); a sterile vaginal speculum was inserted into the vagina to examine and appreciate the state of the cervix. A sterile cotton tipped swab was then inserted 20-30 mm into the endocervical canal and gently rotated against the endocervical wall to pick a high vaginal swab. The swab was immediately replaced in its casing and labeled appropriately with the patient’s information. In addition to the vaginal discharge sampling, participants were asked to complete a questionnaire with information covering complaints, nature of vaginal discharge, personal history, marital history, and predisposing factors for VVC.
Processing of samples
One swab was used for microscopy: Direct wet mount examination with normal saline solution (Figure 1) and Gram stained film examination (Figure 2). Presence of psuedohyphae was specially examined because it is an indicator for invasion and active infection. Demonstration of pseudohyphae along with yeast cells (Figure 1 & 2) is an important diagnostic feature to distinguish infection from normal colonization (Segal and Elad, 2005).
The second swab was inoculated onto Sabouraud's dextrose agar (SDA) (Oxoid, UK) and incubated at 37°C for 48 h. It permits the growth of Candida and suppresses the growth of many but not all bacteria due to its low pH. As some strains of C. krusei, C. tropicalis and C. parapsilosis are sensitive to cycloheximide, we used SDA without cycloheximide (Segal and Elad, 2005). The colonies of candida are opaque white to creamy on SDA plates. (Figure 3)
ISSN 2320-5407 International Journal of Advanced Research (2015), Volume 3, Issue 5, 1376-1386
1378
HVS also; was inoculated onto CHROM agar plates (BBL™ CHROMagar™ Candida; Becton Dickinson GmbH) and incubated at 37°C for 48 h. These media contains chromogenic substrates that react with enzymes secreted by yeast cells, resulting in various pigmentations. These enzymes are species specific and allow species identification on the basis of colony color and characteristics (Horvath et al., 2003). (Figure 4)
Species identification procedures
Candida species was identified based on colony morphology and pigmentations on CHROM agar (Nadeem et al., 2010), germ tube testing, and VITEK 2 Compact system results (bioMerieux, Marcy l’ Etoile, France).
Germ Tube Test:
A rapid method for identifying C.albicans and C. dubliniensis by its ability to produce short, slender, tube like structures called germ tubes when it is incubated in serum at 37°C for 2 hours (Deorukhkar and Saini, 2014). In this test we must be able to differentiate between germ tubes and pseudohyphae. The elongated daughter cells from the mother cell without constriction at their origin are referred to as germ tubes (Figure 5) whereas constriction at the origin of mother cells is called pseudohyphae (Figure 1) (Kim et al., 2002). A criterion for germ tube positivity is observation of minimum five germ tubes in entire wet mount preparation. Negative results are confirmed by examining at least 10 high power fields for the presence of germ tubes (Deorukhkar et al., 2012).
VITEK 2 Compact System:
Fresh subcultures of each of the Candida isolates were obtained for identification using the VITEK 2 Compact ID YST cards as per manufacturer’s instructions. Prior to testing, a suspension of each isolate was inoculated at least twice onto chromogenic agar plates and onto Sabouraud dextrose agar slants to ensure the purity and the viability of the cultures. The inoculum suspensions for the VITEK 2 were prepared in sterile saline at turbidity equal to a 2.0 McFarland standard. The individual test cards were automatically filled with the prepared culture suspension, sealed, and incubated by the VITEK 2 instrument. The cards were incubated at 35.5 °C for 18 h, and optical density readings were taken automatically every 15 min. The final profile results were compared with the database, and the identification of the unknown organism was obtained. A final identification of “excellent,” “very good,” “good,” “acceptable,” or “low- discrimination” was considered to be correct. Both the conventional tests and the tests performed using the VITEK 2 was considered to correctly identify the clinical isolates at the species level.
Antifungal susceptibility testing:
All isolates were subjected to antifungal susceptibility testing for Clotrimazole (50 μg), Miconazole (10 μg), Fluconazole (25μg), and Voriconazole (1 μg) (Liofilchem diagnostic, Italy) by disc diffusion method according to M44-A Clinical and Laboratory Standards Institute(CLSI) guidelines (Clinical and Laboratory Standards Institute, 2009).
Disk diffusion method:
Mueller Hinton Agar (MHA) (Oxoid, UK) supplemented with 2% glucose and 0.5 μg/ ml methylene blue dye was used. The agar surface was inoculated by using a swab dipped in a Candida cell suspension adjusted to the turbidity of a 0.5 McFarland standard. Antifungal discs were dispensed on agar surface and the plates were incubated aerobically at 35°C for 24 h. Zone diameter endpoints were read manually with calipers and interpreted according to standard sizes. The interpretive categories include susceptible (S), resistant (R) and susceptible dose-dependent (SDD). By maintaining blood levels with higher doses of antifungal, an isolate with an SDD endpoint may be successfully treated with a given azole (Wayne, 2004). Criteria for susceptibility to used antifungal drugs have been summarized in Table 1. (Gianni, 2010) & (Al-mamari et al., 2014).
ISSN 2320-5407 International Journal of Advanced Research (2015), Volume 3, Issue 5, 1376-1386
1379
Statistical Analysis
Data was analyzed using STATA intercooled version 9.2. Quantitative data was analyzed using analysis of variance (ANOVA). Qualitative data was compared using either Chi square test or fisher exact test. . P values < 0.05 were considered significant. P values < 0.01 and P values < 0.001 were considered highly significant.
RESULTS
Of the 300 symptomatic women studied, vaginal candidiasis was found in 90 (30%) patients. In the present study we were able to characterize seven species of Candida by conventional, CHROM agar and by the VITEK 2 system method. No isolates failed to grow in the VITEK 2 system. C. albicans (apple green colonies on CHROM agar) 49 (54.4%) was the predominant species isolated followed by non albicans species 41, with an overall rate of (45.6%): C.glabrata (pale pink to violet colonies on CHROM agar) 22(24.4%), C.tropicalis (dull blue colonies on CHROM agar) 13(14.4%), C.famata (White to light pink colonies on CHROM agar) 3(3.3%) and one strain (1.11%) of each of C. Lusitania (pink gray purple on CHROM agar), and C. parapsilosis (white to pale pink colonies on CHROM agar) were isolated (Nadeem et al., 2010). The seventh species which is the Candida dubliniensis was identified by the VITEK 2 system. Mixed infection with two species of Candida was seen in 5 (5.6%) of patients. The most common mixed cause was the combination of Candida albicans and Candida tropicalis in 3 (3.3%) cases followed by Candida albicans and Candida glabrata in 2 (2.2%) cases. (Table 2)
Figure 1: Wet smear showing oval budding yeast cells with pseudohyphae (Candida spp.).
Figure 2: Gram stained smear showing Gram positive oval budding yeast cells with pseudohyphae (Candida spp.).
ISSN 2320-5407 International Journal of Advanced Research (2015), Volume 3, Issue 5, 1376-1386
1380
Figure 3: Creamy white opaque colonies of Candida albicans on SDA.
a) b)
c) d)
Figure 4: Differentiation of Candida spp. on CHROM agar candida medium showing: a) Apple green Colonies of candida albicans, b) Violet colonies of Candida glabrata and c) Dull blue colonies of Candida tropicalis. d) Greenish, Bluish, and violet colonies of candida albicans, tropicalis, and glabrata respectively.
ISSN 2320-5407 International Journal of Advanced Research (2015), Volume 3, Issue 5, 1376-1386
1381
Figure 5: Germ tube formation by Candida albicans.
Table 1: Criteria of Susceptibility and Resistance of Antifungal Drugs.
Zone Diameter in mm
Sensitive
Dose Dependant
Resistant
Clotrimazole (50 μg)
CLO
≥ 20
12-19
≤ 11
Miconazole (10 μg)
MCL
≥ 20
12-19
≤ 11
Fluconazole (25 μg)
FLU
≥ 19
15-18
≤ 14
Voriconazole (1μg)
VO
≥ 17
14-16
≤ 13
Table 2: Species distribution of candida isolates.
Species of candida
No.
Percent %
C. albicans
49
54.44
C. glabrata
22
24.44
C. tropicalis
13
14.44
C. famata
3
3.33
C. dubliniensis
1
1.11
C. lusitaniae
1
1.11
C. parapsilosis
1
1.11
Total
90
100
Age wise distribution showing that; the highest prevalence of vaginal candidiasis (43.3%) was in the age group of (36-45 years). (Table 3)
Table 3: Distribution of candida species in different age groups.
Age range
Candida albicans
Candida glabarata
Candida tropicalis
Candida famata
Candida parapsilosis
Candida dubliniensis
Candida lusitaniae
NO.
%
18-25
4
0
2
0
0
0
0
6
6.7
26-35
12
7
3
0
0
0
0
22
24.4
36-45
20
10
6
2
0
0
1
39
43.3
45-50
13
5
2
1
1
1
0
23
25.6
total
49
22
13
3
1
1
1
90
100
ISSN 2320-5407 International Journal of Advanced Research (2015), Volume 3, Issue 5, 1376-1386
1382
Risk factors associated with candida vulvovaginitis among the study group:
Recurrent vulvovagintis has been the most frequently associated risk factor (45.56%) followed by pregnancy (25.56%), use of antibiotics (24.44%), the use of Intrauterine devices (IUD) (24.44%), then the use oral contraceptive pills (OCPs) (23.33%), and lastly the presence of associated diabetes mellitus (18.89%). There was a highly statistically significant association between all the studied risk factors and occurrence of candida vulvovaginitis in the study group (p value < 0.001). (Table 4)
Table 4: The association between vulvovaginal candidiasis and identified risk factors among the study participants
Variable
Cases positive for Candida
N= 90
Cases negative for Candida
N=210
P value
Recurrent vulvovaginitis
No
Yes
49 (54.44%)
41 (45.56%)
190 (90.48%)
20 (9.52%)
<0.001*
Pregnancy
No
Yes
67 (74.44%)
23 (25.56%)
200 (95.24%)
10 (4.76%)
<0.001*
Use of antibiotic
No
Yes
68 (75.56%)
22 (24.44%)
190 (90.48%)
20 (9.52%)
<0.001*
IUD
No
Yes
68 (75.56%)
22 (24.44%)
195 (92.86%)
15 (7.14%)
<0.001*
OCP
No
Yes
69 (76.67%)
21 (23.33%)
195 (92.86%)
15 (7.14%)
<0.001*
Diabetes
No
Yes
73 (81.11%)
17 (18.89%)
202 (96.19%)
8 (3.81%)
<0.001*
* Highly significant p<0.001
Antifungal susceptibility pattern of candida isolates:
C. albicans were (100%) susceptible to Voriconazole; (91.8%) susceptible to Fluconazole, (89.8%) susceptible to Clotrimazole, and (85.7%) were susceptible to Miconazole. As regards the non-albicans Candida; (95.1%) were susceptible to voriconazole; (87.8%) susceptible to Fluconazole, (80.5%) susceptible to Clotrimazole, and (90.2%) were susceptible to Miconazole. In spite of the higher resistance rates in non-albicans candida compared to Candida albicans isolates to antifungal drugs tested (except for Miconazole) the difference was only statistically significant with p value < 0.05 for Voriconazole and Clotrimazole but was not statistically significant (p value > 0.05) for Fluconazole and Miconazole. (Table 5)
All NAC isolates were (100%) susceptible to Voriconazole except C. glabrata isolates where (9%) of them were resistant to Voriconazole (Table 6).
Table 5: Antifungal susceptibility of candida isolates.
Antifungal
Drug
Candida albicans
N=49
Non-albicans candida
N= 41
P- value
S
R
S
R
Voriconazole
49 (100%)
- (0%)
39 (95.1%)
2 (4.9%)
0.02
ISSN 2320-5407 International Journal of Advanced Research (2015), Volume 3, Issue 5, 1376-1386
1383
Fluconazole
45 (91.8%)
4 (8.2%)
36 (87.8%)
5 (12.2%)
0.35
Clotrimazole
44 (89.8%)
5 (10.2%)
33 (80.5%)
8 (19.5%)
0.04
Miconazole
42 (85.7%)
7 (14.3%)
37 (90.2%)
4 (9.8%)
0.38
Table 6: Antifungal susceptibility pattern of Candida species
Variable
C. albicans
N=49
C. tropicalis
N=13
C. glabrata
N=22
Others
N=6
Voriconazole
Susceptible
Resistant
49 (100%)
0
13 (100%)
0
20 (91%)
2 (9%)
6 (100%)
0
Fluconazole
Susceptible
Resistant
45 (91.8%)
4 (8.2%)
10 (76.9%)
3(23.1%)
21 (95.5%)
1 (4.5%)
5 (83.3%)
1 (16.7%)
Clotrimazole Susceptible
Resistant
44 (89.8%)
5 (10.2%)
11(84.6%)
2(15.4%)
17(77.27%)
5(22.73%)
5 (83.3%)
1(16.7%)
Miconazole Susceptible
Resistant
42 (85.7%)
7 (14.3%)
12 (92.3%)
1(7.7%)
20 (91%)
2 (9%)
5 (83.3%)
1(16.7%)
DISCUSSION
Vulvovaginal candidiasis is a common fungal infection that affects healthy women of all ages. At least 75% of women develop one or more infections once during their lifetime with 5% of those developing recurrent type (Consolaro et al., 2004). In the present study, vaginal candidiasis was found in 90 (30%) of symptomatic women with a higher prevalence rate than that reported by other studies as; Bauters et al., 2002; who reported a prevalence of 20.1% and by Ragunathan et al., 2014 who reported 22.2% prevalence of vulvovaginal candidiasis in their study.74.4% of our patients were in the age group of 18-45 years. Our results were in consistent with other studies done by various authors saying that; the incidence of reproductive tract infections in women is highest in the age group of 15-45 years and followed by a decline. The reason for the high incidence in this age group includes low levels of protective cervical antibodies, increased sexual activity, and new influence of reproductive hormones that may lead to increased susceptibility to reproductive tract infections (Sobel, 1993). In addition, many related problems are associated with Candida vaginitis such as; reduced immunity, prolonged antibiotic therapy, use of contraceptives, malnutrition, pregnancy, diabetes, obesity, use of immunosuppressive agents and neutropenia (Babin et al.,2013). In our study; we analyzed the predisposing factors associated with vaginal candidiasis. Recurrent vulvovagintis has been the most frequently associated risk factor (45.56%); followed by pregnancy (25.56%). We found a highly statistically significant difference in incidence of VVC between pregnant and non pregnant women. This may be probably due to high level of reproductive hormones during pregnancy which provides an excellent carbon source for growth of Candida, inducing higher glycogen contents in the vaginal epithelial cells and also some studies say that estrogens have a direct effect on the growth of Candida and its adherence to the vaginal epithelium (Lakshmi and Lakshmi, 2014). Our results agreed with those of Ragunathan et al., (2014) where there was highly statistically significant difference in incidence of VVC between pregnant and non pregnant women; and 55% of the females who presented with the complaints were pregnant. Also; we observed highly statistically significant difference in incidence of VVC between patients used OCPs (23.33%) and those did not. Many investigators continue to identify OCPs as a predisposing factor. This might be because of similarity between the mechanism operating in pregnancy and high estrogen OCP in increasing vaginal colonization of Candida (Neerja et al., 2006). In addition, we reported highly statistically significant influence during use of broad spectrum antibiotics by increasing the incidence of VVC (24.44%). VVC is a common occurrence after systemic use of broad spectrum antibiotics. Antibiotic usage will cause the elimination and alteration of normal bacterial flora and allows the colonization of Candida (Ahmad and Khan, 2009). Antibiotic agents are thought to act by eliminating lactobacilli, thereby facilitating Candida to grow, adhere and germinate. The concept of interaction between lactobacilli and
ISSN 2320-5407 International Journal of Advanced Research (2015), Volume 3, Issue 5, 1376-1386
1384
Candida includes competition for nutrients and stearic interference of adherence to vaginal epithelial cells (Mohanty et al., 2007). In our study; out of 90 candida species isolated, C.albicans accounted for 54.4%, followed by C.glabrata in 24.5%, then C. tropicalis in 14.4%, and C. famata in 3.3%. Non albicans species were found to present in 45.5% of infections. Recently several authors have also reported an increase in the incidence of VVC caused by nonalbicans species of candida (Bankar et al., 2012). Our results agreed with those of different studies conducted in various countries revealed C. albicans to be the most common species in women with VVC (76 to 89%), followed by C. glabrata (7 to 16%) (Corsello et al., 2003). The percentage of non C. albicans species associated with VVC in these countries ranged from 11% to 24%. Candida glabrata was the second most common isolated species (24.5%) in our study, which was consistent with the results of prior studies with a prevalence of 18–37% (Mahmoudi et al., 2011) & (Ahmad and Khan ,2009). However in the study of Mohanty et al., (2007); C. glabrata was the most common species among the vaginal isolates (50.4%) followed by C. albicans (35.1%). Thus, the prevalence of nonalbicans species (especially Candida glabrata) seems to increase steadily and that raises the concern regarding the increase of antifungal drug resistance. We speculated this increasing detection of non-albicans species are probably related to the widespread and inappropriate use of antimycotic treatments (self medication, topical use, and long-term treatments). Hence, the reliable and rapid identification method of Candida species is a fundamental goal of microbiology laboratories. In the present study, our results were obtained regarding speciation of Candida by conventional and CHROM agar methods. CHROM agar has the advantage of being rapid, simple and cost effective as compared to conventional methods which are slow, technically demanding and expensive (Vijaya et al., 2011). However, one of the NAC species; Candida dubliniensis we could not be able to identify it by CHROM agar methods only and it was identified by the VITEK 2 system. VITEK 2 system correctly identified all of the 90 isolates at the species level. In fact, the VITEK 2 system performed satisfactorily in the identification of non-Candida yeast species. In addition, we confirmed a clinically important advantage of the VITEK 2 system over conventional identification methods: species were identified within 18 h compared to 48-72 h for the other methods. There is an important point which must be taken into consideration that; with the increasing rate of infections due to non-albicans Candida and varying susceptibilities to commonly used empirical antifungal agents like fluconazole, early and accurate species identification would help the clinician in befitting therapeutic management (Tellapragada et al.,2014). For example; Candida glabrata is known to have acquired resistance to fluconazole and other azole drugs (Melhem et al., 2013). So, in the present study we performed antifungal susceptibility testing by CLSI M44-A disc diffusion method with glucose methylene blue Muller Hinton agar for Clotrimazole, Miconazole, Fluconazole, and Voriconazole. It was easy to perform and inexpensive for routine laboratories. Previously; it was stated that, given the rarity of VVC caused by resistant C.albicans strains, susceptibility testing was rarely indicated (Nabhan, 2006); but in the present study highest resistance was observed against Miconazole (14.3%), followed by Clotrimazole (10.2%) and Fluconazole (8.2%). The resistance of C.albicans to azoles has also been reported by others (Zomorodian et al., 2011). Most non-albicans Candida species in our study had higher azole resistance: (4.5%) of C. glabrata isolates were resistant to fluconazole while; (23.1%) of C. tropicalis isolates were resistant to fluconazole. Our results agreed with those of Babin et al., (2013) who reported a higher resistance rates. In our study, C.albicans and majority of NAC species were found susceptible to voriconazole as compared to fluconazole. So, Voriconazole can be preferred over fluconazole as it has broad spectrum activity against Candida species which are inherently resistant to fluconazole. Therefore vaginal culture is valuable not only for identifying the species of vaginal candida but also for monitoring the changing trends in the microbiology of vulvovaginal candidiasis which is essential for the complete and prolonged treatment. As without rapid and accurate diagnostic tools, safe and effective drug treatment, prevention of resistance to antimicrobial therapy, and monitoring of resistance are not possible.
CONCLUSION
VVC cannot be definitely identified by clinical criteria alone. Culture is valuable not only for the accurate diagnosis of VVC but also to avoid indiscriminate use of antifungal agents. In our setting we found an increase in non albicans Candida infections and the emergence of azole resistant C.albicans and non albicans species in vaginal candidiasis cases. Resistance to various commonly used antifungal agents among non-albicans Candida suggests the need for species identification in routine laboratories for initiation of appropriate antifungal therapy. Surveillance of antifungal susceptibility profiles provides a useful tool for hospitals to validate empiric treatment regimens.
ISSN 2320-5407 International Journal of Advanced Research (2015), Volume 3, Issue 5, 1376-1386
1385
REFERENCES
Achkar JM and Fries BC. (2010): Candida infections of the genitourinary tract. Clinical Microbiology Reviews; 23(2):253-273.
Ahmad A and Khan AU. (2009): Prevalence of Candida species and potential risk factors for vulvovaginal candidiasis in Aligarh, India. Eur J Obstet Gynecol Reprod Biol; 144(1):68–71.
Al-mamari A, Al-buryhi M, Al-heggami M A and Al-hag S. (2014): Identify and sensitivity to antifungal drugs of Candida species causing vaginitis isolated from vulvovaginal infected patients in Sana’a city. Der Pharma Chemica, 6 (1):336-342.
Babin D, Kotigadde S, Rao P S, and Rao T V. (2013): Clinico-mycological profile of vaginal candidiasis in a tertiary care hospital in Kerala. International Journal of Research in Biological Sciences; 3(1): 55-59
Bankar SM, Powar RM, Patil SA, and Kalthur SG. (2012): Prevalence of non-albican candida infection in Maharashtrian women with leucorrhea. Ann Trop Med Public Health; 5:119-23.
Bauters, TG, Dhont MA, Temmerman MI, and Nelis HJ. (2002): Prevalence of Vulvovaginal Candidiasis and susceptibility to Fluconazole in women. Am. J. Obstet. Gynecol. ; 187:569–7
Bello O O, Mabekoje O O, Efuntoye M O and Bello T K. (2012): Prevalence of Vaginal Pathogens Associated with Genital Tract Infections in Ogun State, Nigeria, British Microbiology Research Journal 2(4): 277-289.
Clinical and Laboratory Standards Institute. (2009): Method for antifungal disk diffusion susceptibility testing of yeasts; Approved guidelines, 2nd ed, M‑44‑A2; 29. Wayne: CLSI; 2009.
Consolaro M E L , Albertoni T A , Yoshida C S, Mazucheli J, Peralta R M and Svidzinski T I E.(2004): Correlation of Candida species and symptoms among patients with vulvovaginal candidiasis in Maringá, Paraná, Brazil; Rev Iberoam. Micol; 21; 202-205.
Corsello S, Spinillo A, Osnengo G, Penna C, Guaschino S, and Beltrame A. (2003): An epidemiological survey of Vulvovaginal Candidiasis in Italy. Eur. J. Obstet. Gynecol. Reprod. Biol.; 110(1):66–72.
Deorukhkar S and Saini S. (2014): Virulence markers and antifungal susceptibility profile of Candida glabrata: An emerging pathogen. British Microbiol. Res. J. 4:35-45.
Deorukhkar S, Saini S, and Jadhav P. (2012): Evaluation of different media for germ tube production of Candida albicans and Candida dubliniensis. International Journal of Biomedical and Advance Research. 3:704-707.
Farooqi JQ, Jabeen K, Saeed N, Iqbal N, Malik B, and Lockhart SR . (2013): Invasive candidiasis in Pakistan: clinical characteristics, species distribution and antifungal susceptibility. Journal of Medical Microbiology; 62(Pt 2):259-268.
Gianni C. (2010): Update on antifungal therapy with terbinafine. G ItalDermatolVenereol, 145(3), 24-415.
Horvath L, Hospenthal D, Murray C, and Dooley D. (2003): Direct isolation of Candida spp. from blood cultures on chromogenic medium CHROMagar Candida. J Clin Microbiol. 41:2629-2632.
Kim D, Shin W, Lee K, Park J, and Koh C. (2002): Rapid differentiation of Candida albicans and Candida species using its unique germ tube formation at 39°C. Yeast. 19:957-962.
Lakshmi M S and Lakshmi G J. (2014): Vulvo Vaginal Candidiasis: Importance Of Species Identification. Journal of Evolution of Medical and Dental Sciences, 3(4): 788-793.
Mahmoudi R M, Zafarghandi S, Abbasabadi B, and Tavallaee M. (2011): The epidemiology of Candida species associated with vulvovaginal candidiasis in an Iranian patient population European Journal of Obstetrics & Gynecology and Reproductive Biology 155: 199–203.
Melhem MSC, Bertoletti A, Lucca HRL, Silva RBO, Meneghin FA, and Szeszs MW. (2013): Use of the VITEK 2 system to identify and test the antifungal susceptibility of clinically relevant yeast species. Brazilian Journal of Microbiology 44, 4, 1257-1266.
Mohanty S, Xess I, Hasan F, Kapil A, Mittal S, and Tolosa JE. (2007): Prevalence & susceptibility to fluconazole of Candida species causing vulvovaginitis. Indian J Med Res.; 126(3): 216-9.
Nabhan A. (2006): Vulvovaginal candidiasis. Ain Shams J Obstet Gynecol; 3:73‑8.
Nadeem S G, Hakim S T and Kazmi S U. (2010): Use of CHROMagar Candida for the presumptive identification of Candida species directly from clinical specimens in resource-limited settings. Libyan J Med, 5: 2144.
Neerja J, Aruna A, and Paramjeet G. (2006): Significance of Candida culture in women with vulvovaginal symptoms. J Obstet Gynecol India; 56:139‑41.
Pfaller MA, Diekema DJ, Procop GW, and Rinaldi MG. (2007): Multicenter comparison of the VITEK 2 yeast susceptibility test with the CLSI broth microdilution reference method for testing fluconazole against Candida spp. J Clin Microbiol 45:796-802.
ISSN 2320-5407 International Journal of Advanced Research (2015), Volume 3, Issue 5, 1376-1386
1386
Ragunathan L, Poongothai G K, Sinazer A R, Kannaiyan K, Gurumurthy H, Jaget N, and Kuthalaramalingam S. (2014): Phenotypic Characterization and Antifungal Susceptibility Pattern to Fluconazole in Candida species Isolated from Vulvovaginal Candidiasis in a Tertiary Care Hospital. Journal of Clinical and Diagnostic Research, 8(5): 01-04.
Segal E and Elad D. (2005): Candidiasis. In Topley and Wilson s Medical Mycology. 10th edn. Edward Arnold Publishers. 579-623.
Sobel JD. (1993): Candidal vulvovaginitis. Clin Obstet Gynecol.; 36:153-65.
Sobel JD. (2007): Vulvovaginal candidosis. Lancet; 369(9577):1961-1971.
Tellapragada C , Eshwara V K, Johar R , Shaw T, Malik N, Bhat P V, Kamath A , and Mukhopadhyay C .(2014): Antifungal Susceptibility Patterns, In Vitro Production Of Virulence Factors, And Evaluation Of Diagnostic Modalities For The Speciation Of Pathogenic Candida From Blood Stream Infections And Vulvovaginal Candidiasis. Journal of Pathogens; 1-8.
Vijaya D, Harsha TR, and Nagarathnamma T. (2011): Candida speciation using chrom agar. J Clin Diagn Res; 5:755‑7.
Wayne P A. (2004): Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts, CLSI M44-A.
World Health Organization (WHO). (2009): Laboratory manual for diagnosis of fungal opportunistic infections in HIV/AIDS patients. World Health Organization, New Delhi, Assia India 1-83.
Workowski KA and Berman S. (2010): Centers for Disease Control and Prevention (CDC). Sexually transmitted diseases treatment guidelines, 2010. MMWR Recomm Rep; 59:1.
Zomorodian K, Rahimi MJ, Pakshir K, Motamedi M, Ghiasi MR, and Rezashah H. (2011): Determination of antifungal susceptibility patterns among the clinical isolates of Candida species. J Glob Infect Dis; 3:357‑60.