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Prevalence & susceptibility to fluconazole of Candida species causing vulvovaginitis.

Background & objectives: Vulvovaginal candidiasis is an important cause of morbidity in women of reproductive age. This study was carried out to determine the species prevalence and susceptibility pattern to fluconazole of yeasts isolated from the vagina of symptomatic women.

Methods: This prospective study was conducted in a rural primary health care center of north India from May 2003 to April 2004 and included 601 married, sexually active women (18-49 yr) with the self reported symptoms of vaginal discharge and/or genital itching and/or genital burning. Specific aetiology of the genitourinary symptoms including candidal infection were determined. Specimens from the lateral wall of vagina were subjected to direct wet mount microscopy and fungal culture on Sabouraud's dextrose agar. Susceptibility testing to fluconazole was carried out using broth microdilution method.

Results: Yeasts were isolated in 111 (18.5%) women and these consisted of Candida glabrata (56, 50.4%), C. albicans (39, 35.1%), C. tropicalis (12, 10.8%), C. krusei (3, 2.7%) and C. parapsilosis (1, 0.9%). Susceptibility testing carried out on 30 representative isolates (15 C. glabrata, 10 C. albicans, 4 C. tropicalis and 1 C. parapsilosis) revealed that 21 isolates (70%) were susceptible (MIC, < 8 [micro]g/ ml) to fluconazole while 9 (30%) were susceptible-dose dependent (S-DD, MIC 16-32 [micro]g/ml).

Interpretation & conclusion: Our findings suggest a low prevalence of fluconazole resistance in vaginal candida isolates in our population. However, a high prevalence of non-albicans candida species and increased dose-dependent resistance in these isolates necessitates vigilance since this may warrant a change in the optimal therapy of non-albicans candida vaginitis.

Key words Antifungal susceptibility--Candida--candida vaginitis--fluconazole--non-albicans species

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Approximately three-quarters of all women experience at least one episode of vulvovaginal candidiasis during their lifetime and nearly half of them suffer from multiple episode (1). In about 5 per cent of cases, the disease has a chronic course, showing frequent and refractory episodes (1,2). The majority of cases of vulvovaginal candidiasis are caused by Candida albicans; however, episodes due to non-albicans species of Candida appear to be increasing (1,3). Most nonalbicans Candida species have higher minimum inhibitory concentrations (MICs) to the azole antifungal agents, and infections they cause are often difficult to treat (3,4). This phenomenon emphasizes the importance of identification and surveillance of the Candida species in the clinical settings. This study was carried out to determine the species prevalence and fluconazole susceptibility among yeast isolates from women with candidal vulvovaginitis.

Material & Methods

This prospective study was conducted from May 2003 to April 2004 at a rural primary health care center at Ballabhgarh, Haryana, a rural field practice area in north India, under the All India Institute of Medical Sciences (AIIMS), New Delhi. It was done as part of a larger study of evaluation of the use of syndromic management of reproductive tract infections. The ethical review committees of AIIMS, New Delhi, Thomas Jefferson University, Global Network for Perinatal and Reproductive Health (GNPRH), USA, and Population Council approved the protocol.

Patient population: The inclusion criteria for the study were married and sexually active women between 18-49 yr of age who presented to the health care center with self reported symptoms of vaginal discharge and/ or genital itching and/or genital burning during the study period. Pregnant women, women with severe medical disorders requiring immediate referral to higher level of healthcare, women who were currently menstruating, never been sexually active, who had a hysterectomy, had taken a course of antibiotics within preceding three weeks and who had been previously enrolled in this study were excluded. Written informed consent was obtained from each participant woman.

An attempt was made to determine the specific aetiology of the genitourinary symptoms and determination of candidal infection was one of them. Two sterile, cotton tipped swabs were used to collect specimens from lateral wall of vagina of each woman. One of the two swabs was used to determine the presence of yeast by direct wet-mount microscopy using a drop of 10 per cent potassium hydroxide solution. The other swab was used for fungal culture on Sabouraud's dextrose agar (Hi-Media, Mumbai, India) supplemented with 0.06 [micro]g/ml gentamicin, with and without cycloheximide (0.5%) (5,6).

Identification: Species identification of yeast isolates was done by standard procedures including morphology, germ tube test, cornmeal agar test (Hi-Media, Mumbai, India), triphenyl tetrazolium chloride reduction (Hi-Media Mumbai, India), assimilation of various sugars, and growth in presence of actidione (5,6).

Antifungal susceptibility testing: Susceptibility testing to fluconazole was performed using a broth microdilution method (M27-A2) according to the Clinical Laboratory Standards Institute (CLSI) guidelines (7). The microtitre plates were incubated at 35[degrees]C for 24-48 h. The amount of growth in a well containing the antifungal agent was compared with the amount of growth in an antifungal-free growth control well. The minimum inhibitory concentration (MIC) was read as the lowest concentration of antifungal that inhibited 50 per cent growth of the organism detected visually. Quality control was ensured by testing the CLSI recommended quality control strains Candida parapsilopsis ATCC 22019 (MIC range 2-8 [micro]g/ ml) and Candida krusei ATCC 6258 (MIC range 16-64 [micro]g/ml).

Isolates with MIC [less than or equal to] 8 [micro]g/ml were considered to be susceptible to fluconazole, whereas isolates with MIC [greater than or equal to] 64 [micro]g/ml were considered to be resistant(7). Isolates with MICs between 16-32 [micro]g/ml were fluconazole susceptible-dose dependant (S-DD) (7).

Results & Discussion

A total of 710 women were screened, of whom only 611 were found eligible for enrollment. Ten women declined internal examination. Thus, a total of 601 women were included in the study and yeasts were isolated in 111 (18.5%) of them. Candida glabrata was the most common species among the isolates (56, 50.4%) followed by C. albicans (39, 35.1%). Other Candida species isolated were C. tropicalis (12, 10.8%), C. krusei (3, 2.7%) and C. parapsilosis (1, 0.9%). Thus, the overall prevalence of non-albicans candida species was 64.8 per cent (72/111). Susceptibility testing to fluconazole was carried out on 30 representative isolates (15 C. glabrata, 10 C. albicans, 4 C. tropicalis and 1 C. parapsilosis). Of these, 21 isolates (70%) were susceptible to fluconazole while 9 (30%) were susceptible-dose dependent (Table). Complete resistance was not detected in any of the isolates tested. Of the 9 isolates with elevated MICs, 7 were C. glabrata, 1 was C. tropicalis and 1 was C. albicans.

In this study, the overall prevalence of vulvovaginal candidiasis in a community setting was found to be 18.5 per cent which is similar to studies from India (8,9) and elsewhere (10,11) with rates ranging from 20.8-23 per cent. However, the overall percentage of non-albicans vaginitis (64.8%) was much higher than in previous reports (3,4,10,11). In a study conducted on 1498 women who attended the Edinburgh Family Planning Centre over a 18 month period (10), fungi other than C. albicans comprised 27.7 per cent of all fungal species obtained. An Australian study found vulvovaginal yeast carriage among 21 per cent of 5,802 women in a primary care setting; non-albicans species were isolated in only 11 per cent of the positive cultures (11). Spinillo et al (3) reported that 17 per cent of 209 isolates from symptomatic patients referred to an Italian vulvovaginitis clinic were non-albicans species. Richter et al (4) reported an overall percentage of 24 per cent of non-albicans vaginitis among 564 vaginal yeast cultures collected from 429 symptomatic candidal vuvovaginitis patients.

Antifungal susceptibility testing in our study revealed that none of the Candida isolates tested were resistant to fluconazole, though 9 of 30 (30%) isolates were fluconazole S-DD and maximum number of these S-DD isolates were C. glabrata. However, we could not perform susceptibility testing for C. krusei which is intrinsically resistant to fluconazole. Fluconazole resistance in vaginal C. albicans isolates is an uncommon occurrence (12-14). A recent US study found no fluconazole resistance among 401 C. albicans isolates recovered from women with recurrent vulvovaginitis (12). No fluconazole resistance was identified among 75 C. albicans vaginal isolates from symptomatic women in England (13). A study in Brazil also reported no fluconazole resistance among 56 C. albicans vaginal isolates (14). However, in another US study, fluconazole resistance has been reported in 14 of 393 (3.6 %) C. albicans isolates collected from complicated vaginitis patients prior to 2001 (15). As regards the antifungal susceptibility of non-albicans Candida species in a study by Richter et al (4), a large proportion (67%) of the C. glabrata vaginal isolates was non-susceptible 51.8% susceptible-dose dependent, 15.2% resistant).

Thus in recent years, there has been a significant increase in infections caused by non-albicans species of Candida, particularly, C. glabrata and C. tropicalis. We speculate this increasing detection of non-albicans Candida species is probably related to the widespread and inappropriate use of antimycotic treatments (self-medication, long-term maintenance treatments and repeated treatments for candidosis episodes) (1). C. albicans eradication by these means causes a selection of species (such as C. glabrata) that are resistant to commonly used agents (1).

In conclusion, our study provides information on antifungal susceptibility of vaginal yeast isolates in a rural community in India. Since the majority of C. albicans isolates were susceptible to fluconazole, its use may be continued for empirical therapy of uncomplicated candidal vulvovaginitis in the community. Use of alternative agents (like boric acid, flucytosine) (16) may be considered when treating vulvovaginitis caused by non-albicans species (especially C. glabrata and C. krusei). As only a limited number of Candida isolates could be tested in this study, further clinical studies need to be performed involving more number of isolates to confirm the findings.

Acknowledgment

This work was supported by the European Commission through a grant, provided to the HIV/STI Prevention and Care Research Programme of the Population Council India and with support from the Global Network for Perinatal and Reproductive Health and its donors, INCLEN, the Rockefeller Foundation. Authors thank Dr Sarah Hawkes from the London School of Hygiene and Tropical Medicine; Dr Christopher Elias from Population Council (Thailand) for technical support; Dr Heiner Grosskurth, Dr Sabine Flessenkaemper and Dr Gurumurthy Rangaiyan from Population Council, India for technical support and co-ordination of the study.

Received October 3, 2006

References

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(2.) Sobel JD. Candida vulvovaginitis. Sem Dermatol 1996; 15 : 17-28.

(3.) Spinillo A, Capuzzo E, Gulminetti R, Marone P, Colonna L, Piazzi G. Prevalence of and risk factors for fungal vaginitis caused by non-albicans species. Am J Obstet Gynecol 1997; 176 : 138-41.

(4.) Richter SS, Galask RP, Messer SA, Hollis RJ, Diekema DJ, Pfaller MA. Antifungal susceptibilities of Candida species causing vulvovaginitis and epidemiology of recurrent cases. J Clin Microbiol 2005; 43 : 2155-62.

(5.) Moore GS, Jaciow DM. General microscopic and cultural techniques used in medical mycology. In: Mycology for the Clinical Laboratory. USA: Preston Publishing Company Inc; 1979 p. 11-49.

(6.) Segal E, Elad D. Candida species and Blastoschizomycescapitatus. In: Collier L, Balows A, Sussman M, editors. Topley & Wilson's microbiology and microbial infections, vol IV, 9th ed. New York: Oxford University Press, Inc; 1998 p. 423-60.

(7.) Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard M27-A2, 2nd ed. Wayne, PA: Clinical Laboratory Standards Institute; 2002.

(8.) Solanki A, Mathur DR, Joshi KR. Bacterial, fungal and parasitic flora in vaginitis. J Indian Med Assoc 1983; 81 : 151-3.

(9.) Goswami R, Dadhwal V, Tejaswi S, Datta K, Paul A, Haricharan RN, et al. Species-specific prevalence of vaginal candidiasis among patients with diabetes mellitus and its relation to their glycaemic status. J Infect 2000; 41 : 162-6.

(10.) Goldacre MJ, Milne LJR, Watt B, Loudon N, Vessey MP. Prevalence of yeasts and fungi other than Candida albicans in the vagina of normal young women. Br J Obstet Gynecol 1981; 88 : 596-600.

(11.) Holland J, Young ML, Lee O, C-A Chen S. Vulvovaginal carriage of yeasts other than Candida albicans. Sex Transm Infect 2003; 79 : 249-50.

(12.) Sobel JD, Wiesenfeld HC, Martens M, Danna P, Hooton TM, Rompalo A, et al. Maintenance fluconazole therapy for recurrent vulvovaginal candidiasis. N Engl J Med 2004; 351 : 876-83.

(13.) El-Din SS, Reynolds MT, Ashbee HR, Barton RC, Evans EGV. An investigation into the pathogenesis of vulvovaginal candidiasis. Sex Trans Infect 2001; 71 : 179-83.

(14.) Ribeiro MA, Dietze R, Paula CR, Da Matta DA, Colombo AL. Susceptibility profile of vaginal yeast isolates from Brazil. Mycopathologia 2000; 15 : 5-10.

(15.) Sobel JD, Zervos M, Reed BD, Hooton T, Soper D, Nyirjesy P, et al. Fluconazole susceptibility of vaginal isolates obtained from women with complicated Candida vaginitis: clinical implications. Antimicrob Agents Chemother 2003; 47 : 34-8.

(16.) Sobel JD, Chaim W, Nagappan V, Leaman D. Treatment of vaginitis caused by Candida glabrata: use of topical boric acid and flucytosine. Am J Obstet Gynecol 2003; 189 : 1297-300.

Reprint requests: Dr Immaculata Xess, Associate Professor, Department of Microbiology, All India Institute of Medical Sciences New Delhi 110029, India e-mail: i_xess@yahoo.com

Srujana Mohanty, Immaculata Xess, Fahmi Hasan, Arti Kapil, Suneeta Mittal * & Jorge E. Tolosa **

Departments of Microbiology & * Obstetrics & Gynaecology, All India Institute of Medical Sciences New Delhi, India & ** Global Network for Perinatal & Reproductive Health, Thomas Jefferson University Philadelphia, PA, USA
Table. Susceptibility to fluconazole of vaginal Candida isolates

 Number of isolates (%)

MIC C. C. C. C.
([micro]g/ml) albicans glabrata tropicalis parapsilosis

0.25 1 (10) 0 1 (25) 0
0.50 4 (40) 0 0 0
1 2 (20) 2 (13.3) 0 0
2 1 (10) 0 1 (25) 1 (100)
4 0 3 (20) 0 0
8 1 (10) 3 (20) 1 (25) 0
16 1 (10) 2 (13.3) 0 0
32 0 5 (33.3) 1 (25) 0
Total 10 15 4 1

 Number of isolates (%)

MIC
([micro]g/ml) Total

0.25 2 (6.7)
0.50 4 (13.3)
1 4 (13.3)
2 3 (10)
4 3 (10)
8 5 (16.7)
16 3 (10)
32 6 (20)
Total 30

MIC, minimum inhibitory concentration
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Author:Mohanty, Srujana; Xess, Immaculata; Hasan, Fahmi; Kapil, Arti; Mittal, Suneeta; Tolosa, Jorge E.
Publication:Indian Journal of Medical Research
Article Type:Clinical report
Date:Sep 1, 2007
Words:2357
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