Journal de Mycologie Médicale (2015) 25, 23—28
Available online at
ScienceDirect www.sciencedirect.com
ORIGINAL ARTICLE/ARTICLE ORIGINAL
Species spectrum and antifungal susceptibility profile of vaginal isolates of Candida in Kuwait ` ces et profil de sensibilite ´ aux Spectre des espe antifongiques des isolats vaginaux de Candida au Koweı¨t W. Alfouzan a,*,b, R. Dhar b, H. Ashkanani b, M. Gupta c, C. Rachel a, Z.U. Khan a a
Department of Microbiology, Faculty of Medicine, Kuwait University, P.O. Box 760, 51007 Fintas, Kuwait Microbiology Unit, Department of Laboratories, Farwania Hospital, Kuwait c Department of Obstetrics and Gynecology, Farwania Hospital, Kuwait b
Received 2 July 2014; received in revised form 27 October 2014; accepted 30 October 2014 Available online 7 November 2014
KEYWORDS Vulvovaginitis; Candida spp.; Antifungal susceptibility
Summary Objective. — The study was undertaken to determine the prevalence of vulvovaginal candidiasis (VVC) among patients with vaginitis, frequency of different Candida species, and their susceptibility profile. Patients and methods. — Over six months period, high vaginal swabs were cultured on Sabouraud’s dextrose agar and isolates were identified by culture on CHROMagar Candida and Vitek2 yeast identification system or/and API 20C (BioMerieux, France). Antifungal susceptibility of the Candida isolates was determined by E-test against amphotericin B, flucytosine, fluconazole, voriconazole, posaconazole and caspofungin. Results. — One thousand seven hundred and fifty-two women with vaginitis were screened for the prevalence of Candida spp. Vaginal swab cultures of 231 (13.2%) women yielded Candida spp. The isolation rates of different species were as follows: Candida albicans (73.9%), Candida glabrata (19.8%), Candida kefir (1.94%), Candida tropicalis (0.96%), Candida parapsilosis (0.96%), Candida krusei (0.96%), Candida guilliermondii (0.96%), and Saccharomyces cerevisiae (0.52%). All strains of C. albicans and non-C. albicans were susceptible to most of the antifungal agents tested. Conclusion. — The high frequency with which C. albicans was recovered and its azole susceptibility support the continued use of azole agents for empirical therapy of uncomplicated VVC.
* Corresponding author. E-mail address: [email protected] (W. Alfouzan). http://dx.doi.org/10.1016/j.mycmed.2014.10.021 1156-5233/# 2014 Elsevier Masson SAS. All rights reserved.
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W. Alfouzan et al. However, a larger controlled study is required to determine the role of non-C. albicans in recurrent VVC. # 2014 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Vulvovaginitis ; Candida spp. ; Antifongiques susceptibilité
Re ´sume ´ Objectif. — L’étude a été menée afin de déterminer la prévalence de candidose vulvovaginale (CVV) chez les patientes atteintes de vaginite, la fréquence des différentes espèces de Candida, et leur profil de sensibilité. Patients et me´thodes. — Sur une période de six mois, 1752 femmes avec vaginite ont fait l’objet d’un dépistage de Candida spp. Les échantillons vaginaux sur écouvillon ont été cultivés sur agar de Sabouraud dextrose et les isolats ont été identifiés par culture sur CHROMagar pour Candida, Vitek2 pour levure et/ou sur API 20C (Bio Mérieux, France). La sensibilité du Candida isolé aux antifongiques a été déterminée par E-test contre amphotéricine B, flucytocine, fluconazole, voriconazole, posaconazole et caspofungine. Re ´sultats. — Les échantillons vaginaux de 231 (13,2 %) femmes ont produit des cultures positives pour Candida spp. Le taux d’isolement des différentes espèces était : Candida albicans (73 %), Candida glabrata (19,5 %), Candida kefir (1,95 %), Candida tropicalis (0,9 %), Candida parapsilosis (0,9 %), Candida krusei (0,9 %), Candida guilliermondii (0,9 %),et Saccharomyces cerevisiae (0,5 %). Presque toutes les souches de C. albicans et non C. albicans ont été sensibles à la plupart des agents antifongiques testés. Conclusion. — La haute fréquence avec laquelle C. albicans a été isolé et la sensibilité aux azolés sont en faveur de continuer l’utilisation des agents azolés en tant que traitement empirique de la CVV non compliquée. Toutefois, une plus grande étude contrôlée est nécessaire pour déterminer le rôle des Candida non-albicans dans la CVV récidivante. # 2014 Elsevier Masson SAS. Tous droits réservés.
Introduction Both Candida albicans and non-C. albicans species are known to colonize the skin, the gastrointestinal and the reproductive tracts in humans [1]. Among genital tract infections in women of child-bearing age, vaginitis is the commonest infection necessitating a visit to their gynecologists. Although bacteria are the more prevalent causative agents for this infection, 20—25% of the cases are due to Candida species [12]. It is estimated that approximately three-quarters of all healthy women will experience at least one episode of vulvovaginal candidiasis (VVC) during their productive lives and that 5% of these suffer from recurrent, chronic or refractory episodes of the infection [21,24]. There are several reports, which indicate that 80%-90% of cases of VVC are caused by C. albicans. However, other Candida species are now emerging as identifiable causes of VVC and differ considerably with regard to epidemiology, virulence and antifungal drug susceptibility [17,25]. Although clinical experience has shown that these isolates have lower virulence in the lower genital tract infections, presence of potential risk factors in the host, such as, pregnancy, uncontrolled diabetes mellitus, antibiotic use, immunosuppression and hormone replacement therapy predispose them to the development of VVC [30]. Among non-C. albicans isolates the most commonly identified species in women with VVC are Candida glabrata and Candida tropicalis followed by Candida parapsilosis, Candida krusei, Candida kefir, Candida guilliermondii and others, which have been reported from different countries [1,25,2,26]. Several advances have been made for rapid clinical and laboratory diagnosis of VVC such as antigen detection by
latex agglutination test, immunochromatography, rapid PCR-based assays for specific detection of Candida species and culture on CHROMagar Candida [12,21,30,4,19]. However, all these laboratory diagnostic methods have limitations of sensitivity and specificity. Only 22% of female with VVC will have a positive microscopy [14]. Likewise, isolation of yeast alone does not always suggest that it is responsible for vaginal symptoms [23]. Microscopic examination of discharge together with measurement of vaginal pH (< 4.7) maybe a practical diagnostic approach in symptomatic cases of vaginitis particularly in low resources. Generally, antifungal susceptibility is not performed on Candida isolates from vaginal cultures since susceptibility of C. albicans to azoles is well known leading to continued use and misuse of these agents over the counter due to free availability. However, it is important to identify the Candida non-albicans species in order to initiate appropriate therapy as C. krusei exhibits innate resistance to fluconazole and C. glabrata gives variable results or may present dose-dependent susceptibility [22]. There are several reports indicating differences in the distribution of Candida spp. associated with VVC and their antifungal drug susceptibility patterns from different geographic locations [22,15]. In view of the widespread use of antifungal agents for the treatment of VVC, there is a definite need to monitor its possible impact on species spectrum distribution and antifungal susceptibility of Candida spp [1]. With this objective in mind, the present study was undertaken to determine the prevalence of VVC among patients with vaginitis, frequency of different Candida species, and their susceptibility to antifungal drugs.
Species spectrum and antifungal susceptibility profile of vaginal isolates
Patients and methods This prospective study was conducted from November 2010 through April 2011 at a tertiary care hospital and its satellite clinics in Farwania area of Kuwait. The ethical review committees of Ministry of Public Health and Kuwait Institute of Medical Specialization approved this study.
Patients and clinical data Women with signs and symptoms of vaginitis, presenting at the out-patient clinic of obstetrics and gynecology department of Farwania Hospital as well as at the peripheral clinics, were recruited for this study. The collection of vaginal specimen was performed by the attending physician who submitted the specimen along with required clinical information on a pre-distributed questionnaire. Recurrent VVC was defined as four or more episodes annually.
Isolation and identification of pathogenic microorganisms At least one cotton-tipped swab (Copan innovation, Italy) was used to collect the sample from each patient and submitted to our laboratory, where a Gram-stained smear and a wet mount were prepared before being cultured on to 5% sheep blood agar, chocolate agar, and Sabouraud dextrose agar (SDA) containing chloramphenicol (50 mg/L). The agar plates were incubated at 37 8C for 48 h and then examined for bacterial pathogens on blood and chocolate agars and for yeast colonies on SDA, which were identified as C. albicans or non-C. albicans by performing germ tube production test after 4 h of incubation in serum at 35 8C. All the SDA plates with positive yeast growth were collected and stored at 4 8C for further subculture on to CHROMagar Candida (Mast Diagnostics, UK) for presumptive identification of Candida species. For further confirmation of these strains and for those, which were not identifiable by CHROMagar Candida, Vitek2 yeast identification system (BioMerieux, France) and or API 20 C AUX/ID32 C (BioMerieux, France) were employed.
Antifungal drug susceptibility testing All Candida isolates were tested for susceptibility against amphotericin B, flucytosine, fluconazole, voriconazole, posaconazole and caspofungin by E-test (AB Biodisk, Solna,
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Sweden) according to manufacturer’s recommendation. Briefly, each test isolate was freshly sub-cultured on SDA and five isolated colonies were suspended in sterile normal saline and turbidity adjusted to 0.5 McFarland standard. Using sterile cotton swab the suspension was inoculated on to RPMI agar (supplemented with 2% glucose and buffered with MOPS, 0.165 M, pH 7.0) plates (150 mm diameter) and allowed to dry for 10 to 15 minutes before applying the E-test strips. The plates were incubated at 35 8C and minimum inhibitory concentration (MIC) was recorded after 48 h. The interpretive susceptibility breakpoints as recommended by Clinical Laboratory Standards Institute (CLSI) were used for fluconazole, flucytosine and voriconazole. Due to lack of defined breakpoints for amphotericin B, an isolate showing MIC < 1.0 mg/L were considered as susceptible. In case of caspofungin an isolate with MIC < 2 mg/L was recorded as susceptible. Quality control ensured by testing C. parapsilosis ATCC 22019, C. albicans ATCC 90028, and C. tropicalis ATCC 750, as recommended by CLSI.
Results Culture During a period of six months, 1752 high vaginal swabs were received for culture in our laboratory. Of these 231 (13%) were found to be positive for yeast by culture on SDA; 153 (66.2%) C. albicans and 78 (33.8%). Candida species were identified by germ tube test, CHROMagar Candida, Vitek2 and or API 20 C AUX/ID32 C. Since 26 of the strains failed to subculture, 205 samples were evaluated. Of these 205 samples, single isolate of yeast was found in 203 and > 1 strains were isolated from two of the high vaginal specimens. The final numbers of the isolates evaluated were 207 for a total of 205 patients. Identification of the species as follow C. albicans (n = 153), C. glabrata (n = 41), C. tropicalis (n = 2), and C. parapsilosis (n = 2), 2 C. krusei (n = 2), C. kefir (n = 4), C. guilliermondii (n = 2), and Saccharomyces cerevisiae (n = 1).
Epidemiological investigation The clinical information obtained from 199 questionnaires is presented in Table 1; six questionnaires were excluded due to incomplete data. It revealed that the majority of the women with positive high vaginal swab culture for Candida species belonged to 21—30 year age group. Of these 86 (43%)
Table 1 Age distribution, risk factors and symptoms in vulvovaginal candidiasis. ˆ ge, les facteurs de risque et les sympto ˆ mes de la candidose vulvovaginale. ´ partition selon l’a Re Age (year) distribution (%)
Risk factors
Symptoms
15—20 = 5 (2.5%) 21—25 = 47 (24%) 26—30 = 58 (29.0%) 31—35 = 34 (17.0%) 36—40 = 30 (15.0%) 41—45 = 18 (9.0%) 46—50 = 6 (3.0%) 51—55 = 1 (0.5%)
Pregnant = 86 (43.3%) Non-pregnant = 113 (56.7%) Diabetes mellitus = 4 (2.0%) Antibiotic use = 2 (1.0%) Oral contraceptive = 6 (3.0%)
Vaginal discharge = 183 (91.5%) Itching = 127 (63.5%) Dyspareunia = 39 (19.5%) Soreness = 37 (18.5%) > 1 symptom = 64 (32.0%) Recurrent vaginitis and previous Antifungal treatment= 27 (13.5%)
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W. Alfouzan et al.
Table 2A Susceptibility of C. albicans and C. glabrata to antifungal agents. Comparison of MIC50, MIC90 range and mean values of antifungal agents against C. albicans isolated from pregnant and non-pregnant women with vaginitis. ´ de C. albicans et C. glabrata aux agents antifongiques. Comparaison des CMI50 et des CMI90 chez les femmes enceintes Sensibilite et non enceintes. Pregnant (n = 68a)
Amphotericin B Fluconazole Caspofungin Voriconazole Posaconazole Flucytosine a
Non-Pregnant (n = 84)
MIC 50
MIC 90
Range
Mean
STDEV
MIC 50
MIC 90
range
Mean
STDEV
0.064 0.25 0.094 0.008 0.012 0.047
0.25 0.5 0.19 0.032 0.032 0.25
0.004—0.75 0.094—4 0.004—0.25 0.003—0.19 0.003—2 0.012—32
0.113 0.400 0.101 0.017 0.064 0.675
0.126 0.556 0.067 0.031 0.269 4.228
0.064 0.25 0.094 0.008 0.012 0.047
0.19 1 0.19 0.047 0.064 0.25
0.002—0.5 0.016—24 0.004—0.5 0.003—0.125 0.003—3 0.004—1.5
0.102 1.020 0.110 0.017 0.061 0.143
0.094 3.684 0.087 0.024 0.327 0.269
1 strain failed to grow.
Table 2B Susceptibility of C. albicans and C. glabrata to antifungal agents. Comparison of MIC50, MIC90 range and mean values of antifungal agents against C. glabrata isolated from pregnant and non-pregnant women with vaginitis. ´ de C. albicans et C. glabrata aux agents antifongiques. Comparaison des CMI50 et des CMI90 chez les femmes enceintes Sensibilite et non enceintes. Pregnant (n = 19)
Amphotericin B Fluconazole Caspofungin Voriconazole Posaconazole Flucytosine
Non-pregnant (n = 22)
MIC 50
MIC 90
Range
Mean
STDEV
MIC 50
MIC 90
range
Mean
STDEV
0.19 4 0.125 0.125 1 0.012
0.38 8 0.25 0.19 4 0.032
0.047—0.5 0.19—16 0.003—0.094 0.012—0.38 0.006—32 0.004—0.064
0.215 5.219 0.121 0.142 4.551 0.019
0.156 3.872 0.082 0.086 10.053 0.018
0.19 4 0.125 0.125 1 0.008
0.5 32 0.38 0.5 32 0.032
0.004—0.5 0.032—256 0.004—0.75 0.023—1.5 0.094—32 0.003—0.19
0.229 17.728 0.211 0.250 5.684 0.020
0.174 55.052 0.178 0.348 10.799 0.039
were pregnant. The commonest symptom was vaginal discharge and was present in over 90% of cases. The risk factors, such as, diabetes mellitus, antibiotic use and use of oral contraceptives were seen in minority of cases, (Table 1) Recurrent vulvovaginal candidiasis with previous antifungal use was recorded in 27 women (13.5%).
Susceptibility testing Susceptibility test results of 192 isolates of C. albicans and C. glabrata to six antifungal agents are shown in (Tables 2A and 2B); excluding one strain of C. albicans that failed to subculture. Generally, C. albicans strains were uniformly susceptible to most of the agents. Among non-C. albicans strains, C. glabrata presented MIC50 and MIC90 of 4 mg/mL and 8 mg/mL against fluconazole and 1 mg/mL and 32 mg/mL against posaconazole, respectively. There were six strains of C. glabrata, which presented with high MICs to posaconazole (MIC 6— 32 mg/mL). There were no significant differences between the mean MIC values of isolates recorded for pregnant and nonpregnant women with vaginitis (Tables 2A and 2B).
Discussion There are several global studies, which indicate that the distribution of Candida species identified in women with VVC varies greatly depending on the location as well as the population studied. In both, complicated and uncomplicated VVC, usually a single species is isolated but two or more
species have also been reported from a small number (2—5%) of cases [22,10]. Our study 95% of women harbored a single species and only 1% had more than two yeast isolates. Reports from most of the countries have shown that C. albicans is the commonest species identified in patients with VVC accounting for 69—89% cases, whereas 11—31% of non-C. albicans species are associated with the infection [1,3,16,26,26]. Our results corroborate these findings since 66% of our isolates were C. albicans and the remaining 34% were other Candida species. Recently, there has been an increasing trend in the isolation rate of non-C. albicans species reported from some countries [2,13]. Among these, C. glabrata has been more commonly associated with VVC with isolation rate of 30% - 37% in some Asian and African countries. Among our non-C. albicans strains, C. glabrata (71%) was the most common species isolated from the vaginal swabs. C. albicans is considered a commensal organism of the gastrointestinal tract and often of the genital tract in humans resulting in specific immune response early in life [8,18]. Disruption of the balance between the host and the commensals, caused by various risk factors, can lead to the disease process [11]. Experimental studies in rodent models have shown that these animals require estrogen induction in order to produce vaginal candidiasis. The estrogen administration results in thicker and keratinized vaginal epithelium thus allowing fungal attachment, growth and biofilm formation [14,7,9]. This observation strongly correlates with VVC in women during child-bearing age and pregnancy, which are phases
Species spectrum and antifungal susceptibility profile of vaginal isolates influenced by estrogen. Furthermore, it has been observed that non-C. albicans are more often isolated from women after menopause, women with uncontrolled diabetes and those infected with HIV [1]. Among our selected patients 86 (43%) were pregnant while only 4, 2, and 6 had diabetes mellitus, received antibiotics and used oral contraceptives, respectively. Others have also reported similar findings [14,20]. With regards to age, the commonest age group to have positive VVC was from 26-30, matching a study by Benchellal et al. [6]. Several earlier studies have reported no fluconazole resistance among C. albicans isolates [29,27]. In an earlier US study, 100% susceptibility to fluconazole recorded for C. albicans isolates from vulvovaginitis cases [29]. In addition, no fluconazole resistance identified among C. albicans vaginal isolates in studies from Brazil and England [17,5]. In 1995, there was a case report of a fluconazole-resistant (MIC > 64 mg/L) C. albicans vaginal isolate from a patient with VVC [8]. Subsequently, similar case reported from the United Kingdom [26]. A US study reported fluconazole resistance in 3.6% C. albicans strains isolated from patients with the diagnosis of complicated vaginitis [27]. In contrast to this finding, a 2002 study from Belgium reported an alarmingly high fluconazole resistance rate of 21% in 84 vaginal C. albicans isolates, although eight of these strains proved to be susceptible on retesting by the EUCAST method [5]. This illustrates the difficulty that can be encountered in interpreting the susceptibility test reading with azoles and the importance of retesting the isolates at a reference laboratory when unexpected results are obtained. In the current study, fluconazole resistance was observed in 0.6% of C. albicans isolates. Since the susceptibility test was done in a Mycology Reference Laboratory in Kuwait, retesting the strains was not considered necessary. In contrast to the findings in C. albicans, higher fluconazole MICs are found among some of the non-C. albicans isolates. An earlier study reported fluconazole non-susceptibility in 67% (51.8% susceptible-dose dependent; 15.2% resistant) of vaginal C. glabrata isolates [22]. However, in the current study only a solitary isolate demonstrated resistance to fluconazole (MIC 256 mg/L), and three strains exhibited dose-dependent susceptibility (MIC 16—256 mg/L). The innate fluconazole resistance seen in C. krusei influences and supports the use of alternative agents for treatment of VVC due to these species. Patients with recurrent C. glabrata vulvovaginitis show improvement following treatment with boric acid [25,28]. Successful therapy with topical flucytosine in 90% of 27 women with C. glabrata vulvovaginitis achieved following failed therapy with azoles and boric acid [27]. As reported in an earlier study we also found the in vitro activity of flucytosine to be superior to all other agents tested against C. glabrata [22]. The conclusions that can be drawn from the epidemiologic data from the current study are limited because of the lack of a control group. It has been implied that recurrent VVC is caused by a host defect in local vaginal mucosa immunity and not by impaired systemic cell-mediated immunity. Except for the state of pregnancy other risk factors, which are known to aid in the development of VVC (antibiotic therapy, diabetes mellitus, and use of oral contraceptives) were present in a small number of our cases; 1%, 2% and 3% respectively. Furthermore, predominance of antifungal susceptible C. albicans in our study supports the
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theory of host defect in vaginal mucosal immunity as a key factor in the pathogenesis of VVC. Also, our recurrent cases had no higher MIC than the non-recurrent ones. The high frequency with which C. albicans was recovered in this study and its azole susceptibility support the continued use of azole agents for empirical therapy of uncomplicated VVC. However, a larger controlled study is required to determine the role of non-C. albicans in recurrent VVC since a previous study from Kuwait reported emergence of C. glabrata vaginal isolate resistant to amphotericin B and triazoles in a case of recurrent vaginitis [15].
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgments We would like to thank Dr. Albaraheem N, Consultant Pathologist, Previous Head of Clinical Laboratory, Farwania hospital for his support.
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Species spectrum and antifungal susceptibility profile of vaginal isolates of Candida in Kuwait ` ces et profil de sensibilite ´ aux Spectre des espe antifongiques des isolats vaginaux de Candida au Koweı¨t W. Alfouzan a,*,b, R. Dhar b, H. Ashkanani b, M. Gupta c, C. Rachel a, Z.U. Khan a a
Department of Microbiology, Faculty of Medicine, Kuwait University, P.O. Box 760, 51007 Fintas, Kuwait Microbiology Unit, Department of Laboratories, Farwania Hospital, Kuwait c Department of Obstetrics and Gynecology, Farwania Hospital, Kuwait b
Received 2 July 2014; received in revised form 27 October 2014; accepted 30 October 2014 Available online 7 November 2014
KEYWORDS Vulvovaginitis; Candida spp.; Antifungal susceptibility
Summary Objective. — The study was undertaken to determine the prevalence of vulvovaginal candidiasis (VVC) among patients with vaginitis, frequency of different Candida species, and their susceptibility profile. Patients and methods. — Over six months period, high vaginal swabs were cultured on Sabouraud’s dextrose agar and isolates were identified by culture on CHROMagar Candida and Vitek2 yeast identification system or/and API 20C (BioMerieux, France). Antifungal susceptibility of the Candida isolates was determined by E-test against amphotericin B, flucytosine, fluconazole, voriconazole, posaconazole and caspofungin. Results. — One thousand seven hundred and fifty-two women with vaginitis were screened for the prevalence of Candida spp. Vaginal swab cultures of 231 (13.2%) women yielded Candida spp. The isolation rates of different species were as follows: Candida albicans (73.9%), Candida glabrata (19.8%), Candida kefir (1.94%), Candida tropicalis (0.96%), Candida parapsilosis (0.96%), Candida krusei (0.96%), Candida guilliermondii (0.96%), and Saccharomyces cerevisiae (0.52%). All strains of C. albicans and non-C. albicans were susceptible to most of the antifungal agents tested. Conclusion. — The high frequency with which C. albicans was recovered and its azole susceptibility support the continued use of azole agents for empirical therapy of uncomplicated VVC.
* Corresponding author. E-mail address: [email protected] (W. Alfouzan). http://dx.doi.org/10.1016/j.mycmed.2014.10.021 1156-5233/# 2014 Elsevier Masson SAS. All rights reserved.
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W. Alfouzan et al. However, a larger controlled study is required to determine the role of non-C. albicans in recurrent VVC. # 2014 Elsevier Masson SAS. All rights reserved.
MOTS CLÉS Vulvovaginitis ; Candida spp. ; Antifongiques susceptibilité
Re ´sume ´ Objectif. — L’étude a été menée afin de déterminer la prévalence de candidose vulvovaginale (CVV) chez les patientes atteintes de vaginite, la fréquence des différentes espèces de Candida, et leur profil de sensibilité. Patients et me´thodes. — Sur une période de six mois, 1752 femmes avec vaginite ont fait l’objet d’un dépistage de Candida spp. Les échantillons vaginaux sur écouvillon ont été cultivés sur agar de Sabouraud dextrose et les isolats ont été identifiés par culture sur CHROMagar pour Candida, Vitek2 pour levure et/ou sur API 20C (Bio Mérieux, France). La sensibilité du Candida isolé aux antifongiques a été déterminée par E-test contre amphotéricine B, flucytocine, fluconazole, voriconazole, posaconazole et caspofungine. Re ´sultats. — Les échantillons vaginaux de 231 (13,2 %) femmes ont produit des cultures positives pour Candida spp. Le taux d’isolement des différentes espèces était : Candida albicans (73 %), Candida glabrata (19,5 %), Candida kefir (1,95 %), Candida tropicalis (0,9 %), Candida parapsilosis (0,9 %), Candida krusei (0,9 %), Candida guilliermondii (0,9 %),et Saccharomyces cerevisiae (0,5 %). Presque toutes les souches de C. albicans et non C. albicans ont été sensibles à la plupart des agents antifongiques testés. Conclusion. — La haute fréquence avec laquelle C. albicans a été isolé et la sensibilité aux azolés sont en faveur de continuer l’utilisation des agents azolés en tant que traitement empirique de la CVV non compliquée. Toutefois, une plus grande étude contrôlée est nécessaire pour déterminer le rôle des Candida non-albicans dans la CVV récidivante. # 2014 Elsevier Masson SAS. Tous droits réservés.
Introduction Both Candida albicans and non-C. albicans species are known to colonize the skin, the gastrointestinal and the reproductive tracts in humans [1]. Among genital tract infections in women of child-bearing age, vaginitis is the commonest infection necessitating a visit to their gynecologists. Although bacteria are the more prevalent causative agents for this infection, 20—25% of the cases are due to Candida species [12]. It is estimated that approximately three-quarters of all healthy women will experience at least one episode of vulvovaginal candidiasis (VVC) during their productive lives and that 5% of these suffer from recurrent, chronic or refractory episodes of the infection [21,24]. There are several reports, which indicate that 80%-90% of cases of VVC are caused by C. albicans. However, other Candida species are now emerging as identifiable causes of VVC and differ considerably with regard to epidemiology, virulence and antifungal drug susceptibility [17,25]. Although clinical experience has shown that these isolates have lower virulence in the lower genital tract infections, presence of potential risk factors in the host, such as, pregnancy, uncontrolled diabetes mellitus, antibiotic use, immunosuppression and hormone replacement therapy predispose them to the development of VVC [30]. Among non-C. albicans isolates the most commonly identified species in women with VVC are Candida glabrata and Candida tropicalis followed by Candida parapsilosis, Candida krusei, Candida kefir, Candida guilliermondii and others, which have been reported from different countries [1,25,2,26]. Several advances have been made for rapid clinical and laboratory diagnosis of VVC such as antigen detection by
latex agglutination test, immunochromatography, rapid PCR-based assays for specific detection of Candida species and culture on CHROMagar Candida [12,21,30,4,19]. However, all these laboratory diagnostic methods have limitations of sensitivity and specificity. Only 22% of female with VVC will have a positive microscopy [14]. Likewise, isolation of yeast alone does not always suggest that it is responsible for vaginal symptoms [23]. Microscopic examination of discharge together with measurement of vaginal pH (< 4.7) maybe a practical diagnostic approach in symptomatic cases of vaginitis particularly in low resources. Generally, antifungal susceptibility is not performed on Candida isolates from vaginal cultures since susceptibility of C. albicans to azoles is well known leading to continued use and misuse of these agents over the counter due to free availability. However, it is important to identify the Candida non-albicans species in order to initiate appropriate therapy as C. krusei exhibits innate resistance to fluconazole and C. glabrata gives variable results or may present dose-dependent susceptibility [22]. There are several reports indicating differences in the distribution of Candida spp. associated with VVC and their antifungal drug susceptibility patterns from different geographic locations [22,15]. In view of the widespread use of antifungal agents for the treatment of VVC, there is a definite need to monitor its possible impact on species spectrum distribution and antifungal susceptibility of Candida spp [1]. With this objective in mind, the present study was undertaken to determine the prevalence of VVC among patients with vaginitis, frequency of different Candida species, and their susceptibility to antifungal drugs.
Species spectrum and antifungal susceptibility profile of vaginal isolates
Patients and methods This prospective study was conducted from November 2010 through April 2011 at a tertiary care hospital and its satellite clinics in Farwania area of Kuwait. The ethical review committees of Ministry of Public Health and Kuwait Institute of Medical Specialization approved this study.
Patients and clinical data Women with signs and symptoms of vaginitis, presenting at the out-patient clinic of obstetrics and gynecology department of Farwania Hospital as well as at the peripheral clinics, were recruited for this study. The collection of vaginal specimen was performed by the attending physician who submitted the specimen along with required clinical information on a pre-distributed questionnaire. Recurrent VVC was defined as four or more episodes annually.
Isolation and identification of pathogenic microorganisms At least one cotton-tipped swab (Copan innovation, Italy) was used to collect the sample from each patient and submitted to our laboratory, where a Gram-stained smear and a wet mount were prepared before being cultured on to 5% sheep blood agar, chocolate agar, and Sabouraud dextrose agar (SDA) containing chloramphenicol (50 mg/L). The agar plates were incubated at 37 8C for 48 h and then examined for bacterial pathogens on blood and chocolate agars and for yeast colonies on SDA, which were identified as C. albicans or non-C. albicans by performing germ tube production test after 4 h of incubation in serum at 35 8C. All the SDA plates with positive yeast growth were collected and stored at 4 8C for further subculture on to CHROMagar Candida (Mast Diagnostics, UK) for presumptive identification of Candida species. For further confirmation of these strains and for those, which were not identifiable by CHROMagar Candida, Vitek2 yeast identification system (BioMerieux, France) and or API 20 C AUX/ID32 C (BioMerieux, France) were employed.
Antifungal drug susceptibility testing All Candida isolates were tested for susceptibility against amphotericin B, flucytosine, fluconazole, voriconazole, posaconazole and caspofungin by E-test (AB Biodisk, Solna,
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Sweden) according to manufacturer’s recommendation. Briefly, each test isolate was freshly sub-cultured on SDA and five isolated colonies were suspended in sterile normal saline and turbidity adjusted to 0.5 McFarland standard. Using sterile cotton swab the suspension was inoculated on to RPMI agar (supplemented with 2% glucose and buffered with MOPS, 0.165 M, pH 7.0) plates (150 mm diameter) and allowed to dry for 10 to 15 minutes before applying the E-test strips. The plates were incubated at 35 8C and minimum inhibitory concentration (MIC) was recorded after 48 h. The interpretive susceptibility breakpoints as recommended by Clinical Laboratory Standards Institute (CLSI) were used for fluconazole, flucytosine and voriconazole. Due to lack of defined breakpoints for amphotericin B, an isolate showing MIC < 1.0 mg/L were considered as susceptible. In case of caspofungin an isolate with MIC < 2 mg/L was recorded as susceptible. Quality control ensured by testing C. parapsilosis ATCC 22019, C. albicans ATCC 90028, and C. tropicalis ATCC 750, as recommended by CLSI.
Results Culture During a period of six months, 1752 high vaginal swabs were received for culture in our laboratory. Of these 231 (13%) were found to be positive for yeast by culture on SDA; 153 (66.2%) C. albicans and 78 (33.8%). Candida species were identified by germ tube test, CHROMagar Candida, Vitek2 and or API 20 C AUX/ID32 C. Since 26 of the strains failed to subculture, 205 samples were evaluated. Of these 205 samples, single isolate of yeast was found in 203 and > 1 strains were isolated from two of the high vaginal specimens. The final numbers of the isolates evaluated were 207 for a total of 205 patients. Identification of the species as follow C. albicans (n = 153), C. glabrata (n = 41), C. tropicalis (n = 2), and C. parapsilosis (n = 2), 2 C. krusei (n = 2), C. kefir (n = 4), C. guilliermondii (n = 2), and Saccharomyces cerevisiae (n = 1).
Epidemiological investigation The clinical information obtained from 199 questionnaires is presented in Table 1; six questionnaires were excluded due to incomplete data. It revealed that the majority of the women with positive high vaginal swab culture for Candida species belonged to 21—30 year age group. Of these 86 (43%)
Table 1 Age distribution, risk factors and symptoms in vulvovaginal candidiasis. ˆ ge, les facteurs de risque et les sympto ˆ mes de la candidose vulvovaginale. ´ partition selon l’a Re Age (year) distribution (%)
Risk factors
Symptoms
15—20 = 5 (2.5%) 21—25 = 47 (24%) 26—30 = 58 (29.0%) 31—35 = 34 (17.0%) 36—40 = 30 (15.0%) 41—45 = 18 (9.0%) 46—50 = 6 (3.0%) 51—55 = 1 (0.5%)
Pregnant = 86 (43.3%) Non-pregnant = 113 (56.7%) Diabetes mellitus = 4 (2.0%) Antibiotic use = 2 (1.0%) Oral contraceptive = 6 (3.0%)
Vaginal discharge = 183 (91.5%) Itching = 127 (63.5%) Dyspareunia = 39 (19.5%) Soreness = 37 (18.5%) > 1 symptom = 64 (32.0%) Recurrent vaginitis and previous Antifungal treatment= 27 (13.5%)
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W. Alfouzan et al.
Table 2A Susceptibility of C. albicans and C. glabrata to antifungal agents. Comparison of MIC50, MIC90 range and mean values of antifungal agents against C. albicans isolated from pregnant and non-pregnant women with vaginitis. ´ de C. albicans et C. glabrata aux agents antifongiques. Comparaison des CMI50 et des CMI90 chez les femmes enceintes Sensibilite et non enceintes. Pregnant (n = 68a)
Amphotericin B Fluconazole Caspofungin Voriconazole Posaconazole Flucytosine a
Non-Pregnant (n = 84)
MIC 50
MIC 90
Range
Mean
STDEV
MIC 50
MIC 90
range
Mean
STDEV
0.064 0.25 0.094 0.008 0.012 0.047
0.25 0.5 0.19 0.032 0.032 0.25
0.004—0.75 0.094—4 0.004—0.25 0.003—0.19 0.003—2 0.012—32
0.113 0.400 0.101 0.017 0.064 0.675
0.126 0.556 0.067 0.031 0.269 4.228
0.064 0.25 0.094 0.008 0.012 0.047
0.19 1 0.19 0.047 0.064 0.25
0.002—0.5 0.016—24 0.004—0.5 0.003—0.125 0.003—3 0.004—1.5
0.102 1.020 0.110 0.017 0.061 0.143
0.094 3.684 0.087 0.024 0.327 0.269
1 strain failed to grow.
Table 2B Susceptibility of C. albicans and C. glabrata to antifungal agents. Comparison of MIC50, MIC90 range and mean values of antifungal agents against C. glabrata isolated from pregnant and non-pregnant women with vaginitis. ´ de C. albicans et C. glabrata aux agents antifongiques. Comparaison des CMI50 et des CMI90 chez les femmes enceintes Sensibilite et non enceintes. Pregnant (n = 19)
Amphotericin B Fluconazole Caspofungin Voriconazole Posaconazole Flucytosine
Non-pregnant (n = 22)
MIC 50
MIC 90
Range
Mean
STDEV
MIC 50
MIC 90
range
Mean
STDEV
0.19 4 0.125 0.125 1 0.012
0.38 8 0.25 0.19 4 0.032
0.047—0.5 0.19—16 0.003—0.094 0.012—0.38 0.006—32 0.004—0.064
0.215 5.219 0.121 0.142 4.551 0.019
0.156 3.872 0.082 0.086 10.053 0.018
0.19 4 0.125 0.125 1 0.008
0.5 32 0.38 0.5 32 0.032
0.004—0.5 0.032—256 0.004—0.75 0.023—1.5 0.094—32 0.003—0.19
0.229 17.728 0.211 0.250 5.684 0.020
0.174 55.052 0.178 0.348 10.799 0.039
were pregnant. The commonest symptom was vaginal discharge and was present in over 90% of cases. The risk factors, such as, diabetes mellitus, antibiotic use and use of oral contraceptives were seen in minority of cases, (Table 1) Recurrent vulvovaginal candidiasis with previous antifungal use was recorded in 27 women (13.5%).
Susceptibility testing Susceptibility test results of 192 isolates of C. albicans and C. glabrata to six antifungal agents are shown in (Tables 2A and 2B); excluding one strain of C. albicans that failed to subculture. Generally, C. albicans strains were uniformly susceptible to most of the agents. Among non-C. albicans strains, C. glabrata presented MIC50 and MIC90 of 4 mg/mL and 8 mg/mL against fluconazole and 1 mg/mL and 32 mg/mL against posaconazole, respectively. There were six strains of C. glabrata, which presented with high MICs to posaconazole (MIC 6— 32 mg/mL). There were no significant differences between the mean MIC values of isolates recorded for pregnant and nonpregnant women with vaginitis (Tables 2A and 2B).
Discussion There are several global studies, which indicate that the distribution of Candida species identified in women with VVC varies greatly depending on the location as well as the population studied. In both, complicated and uncomplicated VVC, usually a single species is isolated but two or more
species have also been reported from a small number (2—5%) of cases [22,10]. Our study 95% of women harbored a single species and only 1% had more than two yeast isolates. Reports from most of the countries have shown that C. albicans is the commonest species identified in patients with VVC accounting for 69—89% cases, whereas 11—31% of non-C. albicans species are associated with the infection [1,3,16,26,26]. Our results corroborate these findings since 66% of our isolates were C. albicans and the remaining 34% were other Candida species. Recently, there has been an increasing trend in the isolation rate of non-C. albicans species reported from some countries [2,13]. Among these, C. glabrata has been more commonly associated with VVC with isolation rate of 30% - 37% in some Asian and African countries. Among our non-C. albicans strains, C. glabrata (71%) was the most common species isolated from the vaginal swabs. C. albicans is considered a commensal organism of the gastrointestinal tract and often of the genital tract in humans resulting in specific immune response early in life [8,18]. Disruption of the balance between the host and the commensals, caused by various risk factors, can lead to the disease process [11]. Experimental studies in rodent models have shown that these animals require estrogen induction in order to produce vaginal candidiasis. The estrogen administration results in thicker and keratinized vaginal epithelium thus allowing fungal attachment, growth and biofilm formation [14,7,9]. This observation strongly correlates with VVC in women during child-bearing age and pregnancy, which are phases
Species spectrum and antifungal susceptibility profile of vaginal isolates influenced by estrogen. Furthermore, it has been observed that non-C. albicans are more often isolated from women after menopause, women with uncontrolled diabetes and those infected with HIV [1]. Among our selected patients 86 (43%) were pregnant while only 4, 2, and 6 had diabetes mellitus, received antibiotics and used oral contraceptives, respectively. Others have also reported similar findings [14,20]. With regards to age, the commonest age group to have positive VVC was from 26-30, matching a study by Benchellal et al. [6]. Several earlier studies have reported no fluconazole resistance among C. albicans isolates [29,27]. In an earlier US study, 100% susceptibility to fluconazole recorded for C. albicans isolates from vulvovaginitis cases [29]. In addition, no fluconazole resistance identified among C. albicans vaginal isolates in studies from Brazil and England [17,5]. In 1995, there was a case report of a fluconazole-resistant (MIC > 64 mg/L) C. albicans vaginal isolate from a patient with VVC [8]. Subsequently, similar case reported from the United Kingdom [26]. A US study reported fluconazole resistance in 3.6% C. albicans strains isolated from patients with the diagnosis of complicated vaginitis [27]. In contrast to this finding, a 2002 study from Belgium reported an alarmingly high fluconazole resistance rate of 21% in 84 vaginal C. albicans isolates, although eight of these strains proved to be susceptible on retesting by the EUCAST method [5]. This illustrates the difficulty that can be encountered in interpreting the susceptibility test reading with azoles and the importance of retesting the isolates at a reference laboratory when unexpected results are obtained. In the current study, fluconazole resistance was observed in 0.6% of C. albicans isolates. Since the susceptibility test was done in a Mycology Reference Laboratory in Kuwait, retesting the strains was not considered necessary. In contrast to the findings in C. albicans, higher fluconazole MICs are found among some of the non-C. albicans isolates. An earlier study reported fluconazole non-susceptibility in 67% (51.8% susceptible-dose dependent; 15.2% resistant) of vaginal C. glabrata isolates [22]. However, in the current study only a solitary isolate demonstrated resistance to fluconazole (MIC 256 mg/L), and three strains exhibited dose-dependent susceptibility (MIC 16—256 mg/L). The innate fluconazole resistance seen in C. krusei influences and supports the use of alternative agents for treatment of VVC due to these species. Patients with recurrent C. glabrata vulvovaginitis show improvement following treatment with boric acid [25,28]. Successful therapy with topical flucytosine in 90% of 27 women with C. glabrata vulvovaginitis achieved following failed therapy with azoles and boric acid [27]. As reported in an earlier study we also found the in vitro activity of flucytosine to be superior to all other agents tested against C. glabrata [22]. The conclusions that can be drawn from the epidemiologic data from the current study are limited because of the lack of a control group. It has been implied that recurrent VVC is caused by a host defect in local vaginal mucosa immunity and not by impaired systemic cell-mediated immunity. Except for the state of pregnancy other risk factors, which are known to aid in the development of VVC (antibiotic therapy, diabetes mellitus, and use of oral contraceptives) were present in a small number of our cases; 1%, 2% and 3% respectively. Furthermore, predominance of antifungal susceptible C. albicans in our study supports the
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theory of host defect in vaginal mucosal immunity as a key factor in the pathogenesis of VVC. Also, our recurrent cases had no higher MIC than the non-recurrent ones. The high frequency with which C. albicans was recovered in this study and its azole susceptibility support the continued use of azole agents for empirical therapy of uncomplicated VVC. However, a larger controlled study is required to determine the role of non-C. albicans in recurrent VVC since a previous study from Kuwait reported emergence of C. glabrata vaginal isolate resistant to amphotericin B and triazoles in a case of recurrent vaginitis [15].
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgments We would like to thank Dr. Albaraheem N, Consultant Pathologist, Previous Head of Clinical Laboratory, Farwania hospital for his support.
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