Vulvovaginal candidiasis infection affects 75% of women at least once in their lifetime.1,2 More than 90% of vulvovaginal candidiasis infections are caused by Candida albicans.2 Recurrent vulvovaginal candidiasis infection, defined as four or more episodes of vulvovaginal candidiasis infection in 1 year, affects 5–8% of women during their childbearing age, which translates into disease affecting millions of women worldwide.1–6 Recurrent vulvovaginal candidiasis infection is associated with considerable suffering, costs, and interference with sexual relations.2–4 Acute episodes of vulvovaginal candidiasis infection are frequently self-diagnosed, self-treated with over-the-counter antimycotics, or treated empirically by physicians without obtaining cultures.2 As a result of low cure and high recurrence rates, a long-term fluconazole maintenance therapy regimen for recurrent vulvovaginal candidiasis infection has been advocated.3,5,7 A low-dose regimen, of 150 mg fluconazole taken once weekly for at least 6 months, has been shown in a randomized placebo-controlled trial to be associated with reduced rate of recurrences5 and is now considered the standard of care.5–8 The frequent empiric prescription of fluconazole for sporadic vulvovaginal candidiasis infection, coupled with over-the-counter availability of topical azole agents and the widespread use of a low-dose weekly fluconazole regimen for recurrent vulvovaginal candidiasis infection, combines to create ideal conditions for fluconazole-resistant C albicans strains causing recurrent vulvovaginal candidiasis infection to evolve, emerge, and spread. To date, reports of vulvovaginal candidiasis infection caused by fluconazole-resistant C albicans have been rare.5,9–11
In contrast to systemic infections, the definition of in vitro C albicans nonsusceptibility to fluconazole in the context of vaginal infections remains unvalidated. Previous determination of Candida species breakpoints was based on studies conducted on blood and oropharyngeal isolates, ignoring specific pharmacokinetic considerations applicable to the vagina.12,13 The breakpoint or measurement of in vitro fluconazole resistance in C albicans had recently been reduced by the Clinical and Laboratory Standards Institution from 64 to 8 micrograms/mL.14,15 There are limited clinical data, however, based on studies in women with Candida vaginitis suggesting that fluconazole susceptibility be defined as 1 microgram/mL or less instead of 2 micrograms/mL or less.16
The consequences of emergence of fluconazole-resistant recurrent vulvovaginal candidiasis infection are substantial, because few alternative therapeutics agents are available. Study aims were to: 1. describe the epidemiologic characteristics of the cohort of patients with fluconazole-resistant recurrent vulvovaginal candidiasis infection; 2. analyze the risk factors for clinical or mycologic failures or both among patients with fluconazole-resistant recurrent vulvovaginal candidiasis infection; and 3. review therapeutic options to treat refractory recurrent vulvovaginal candidiasis infection.
PATIENTS AND METHODS
The outpatient Vaginitis Clinic at Wayne State University admits approximately 400 new patients and a total of 1,200 visits annually. Patients are referred with complicated and difficult to manage vaginal infections. The study cohort consisted of patients with recurrent vulvovaginal candidiasis infection, who were admitted to the clinic from 2000 to 2010 who presented on admission or follow-up visits with refractory Candida vaginitis and with a C albicans isolate with fluconazole minimum inhibitory concentration (MIC) 2 micrograms/mL or greater. Fluconazole refractory vulvovaginal candidiasis infection was defined as culture positive symptomatic vulvovaginal candidiasis infection, either breakthrough infection while receiving 150 mg weekly fluconazole or unresponsive to current multidose fluconazole administration. The study was retrospective and was approved by the Wayne State University institutional review board before its initiation.
Patients with fluconazole-resistant recurrent vulvovaginal candidiasis infection were initially contacted by phone, and after obtaining permission, an informed consent and a questionnaire were mailed to study participants accompanied by stamped returning envelope. When the patient could not be located by phone or had not returned the signed consent, questionnaire, or both by mail, data were extracted from charts. The epidemiologic questionnaire consisted of data pertaining to patients’ demographics, comorbidities, behavioral characteristics, exposure to antimicrobials and antifungals, fluconazole consumption in defined daily doses in the previous 6 months, management received for the fluconazole-resistant recurrent vulvovaginal candidiasis infection, and outcomes.
Vaginal cultures were processed at the Wayne State University infectious diseases research laboratory. Swabs were inoculated on Sabouraud dextrose agar plates and incubated at 30°C for 48 hours. When growth of yeast was detected, germ-tube and chlamydospore tests were performed. If both tests were positive, the isolate was identified as C albicans. If either one of the tests was negative, API 20C AUX yeast identification strips were used to confirm identification of C albicans. Minimum inhibitory concentrations to various antifungals were tested by using the broth microdilution method conducted in accordance with Clinical and Laboratory Standards Institution criteria and breakpoints.17
All analyses were performed by using IBM SPSS 19. Univariate analyses were performed using the Fisher’s exact and χ2 tests for categorical variables and the independent samples t test, analysis of variance, and Mann–Whitney test for (for nonnormally distributed parameters for continuous variables). The χ2 test for trend was used to evaluate the prevalence and incidence of fluconazole-resistant C albicans isolates over the study period. All P values were two-sided.
Twenty-five patients were diagnosed with refractory vulvovaginal candidiasis infection caused by fluconazole-resistant C albicans between 2000 and 2010 at the Wayne State University Vaginitis Clinic. As indicated in Figure 1, although cases were identified throughout the decade, 16 of 25 (64%) occurred in the previous 5 years. Both the prevalence and incidence (per 1,000 admissions) of cases increased over the years, although insignificantly (P for trend=.16).
Twenty-one of the patients were contacted by phone, and 16 returned questionnaires by mail and signed consent forms. Patient characteristics are displayed in Table 1. The study cohort consisted primarily of married, insured white women with more than 12 years of formal education and with an average or above average socioeconomic status. All women had recent extensive exposure to fluconazole and 16 of 25 (64%) women had received low-dose maintenance weekly fluconazole in the 12 months before isolation of the fluconazole-resistant strain. The level of symptoms and disability reported by patients was extremely high (Table 1).
The median fluconazole MIC was 8 micrograms/mL (range 2–128 micrograms/mL) (Table 2). Eight patients (32%) had fluconazole MICs of 2 micrograms/mL, and 17 patients had MICs greater than 2 micrograms/mL (range 4–128 micrograms/mL). Cross-resistance to itraconazole (0.5 micrograms/mL or greater) was present in five patients and in four isolates, cross-resistance to ketoconazole was evident. Voriconazole resistance (2 micrograms/mL or greater) was detected in five fluconazole-resistant isolates (20%). There was no correlation between the fluconazole MIC and pan-azole resistance phenotype.
Until susceptibility test results became available, symptomatic patients initially seen with refractory vulvovaginal candidiasis infection were most frequently prescribed 600-mg intravaginal boric acid vaginal suppositories per day for 2 weeks. Invariably, patients became asymptomatic and microscopically negative and maintenance antifungal therapy was recommended at the next follow-up visit based on fluconazole MIC results. In the event of low-level fluconazole resistance of 2–4 micrograms/mL, patients were restarted on fluconazole but at a higher dose of 150–200 mg twice weekly. A total of 11 patients received a higher dose fluconazole maintenance regimen (median MIC of strains was 4 micrograms/mL) and remained asymptomatic with negative vaginal yeast cultures. Of the 11 women, maintenance fluconazole was eventually discontinued in five, and all patients remained asymptomatic and culture-negative.
Five women were treated with 100 mg ketoconazole daily based on in vitro susceptibility and a high level of fluconazole resistance. Four women remained asymptomatic and culture-negative with successful discontinuation of therapy in two. Three patients were successfully controlled on 200 mg maintenance itraconazole daily and two additional patients did not tolerate therapy. One woman failed to tolerate itraconazole and ketoconazole and was maintained in clinical and mycologic remission using a daily dose of 100 mg fluconazole therapy. Three patients with a high level of resistance to multiple azole drugs were treated effectively with 600-mg boric acid capsules three times weekly. One patient with high-level pan azole resistance and clinically uncontrolled disease while on boric acid, nystatin, and topical azoles was finally controlled using daily gentian violet for 14 days. The patient remained asymptomatic and culture-negative in follow-up visits during the next 2 years.
Fifteen of the patients had additional C albicans isolation at one of their follow-up visits, ie, mycologic failures. Risk factors for mycologic failure included older age (45 compared with 37 years, P=.05), older age at first vulvovaginal candidiasis infection attack (37 compared with 25 years, P=.04), and number of fluconazole treatment courses in the 6 months preceding the isolation of resistant strain (median of six courses, interquartile range=1.3–11 compared with a median of one, interquartile range=1–3.25, P=.03). As a result of low numbers, no multivariate analysis was conducted.
After diagnosis of fluconazole resistance, it is noteworthy that three patients initially controlled with the aforementioned suppressive regimens did subsequently recur with symptomatic vulvovaginal candidiasis infection caused by a fluconazole-susceptible strain of C albicans. None of the 25 patients had vaginal coinfection or sequential infections with a non-albicans Candida species or had concomitant bacterial infection.
Fluconazole was introduced into clinical use in 1993 and its use for recurrent vulvovaginal candidiasis infection followed over the next few years.18,19 By the end of the 1990s, only one case of fluconazole-resistant C albicans vulvovaginitis had been isolated at the Wayne State University clinic and reports elsewhere were rare.9,10 In the past 10 years, an increase in women with fluconazole-resistant recurrent vulvovaginal candidiasis infection was observed in our center although as yet not significantly different (Fig. 1).
To correlate fluconazole consumption with recurrent vulvovaginal candidiasis infection caused by a fluconazole-resistant strain, a matched case–control study design is mandatory.20,21 This case series analysis revealed that all fluconazole-resistant patients consumed high amounts of systemic fluconazole in the previous 6 months with median defined daily doses of 22 per patient. Increased fluconazole consumption was especially evident in patients with longitudinal mycologic failures. Matching a control group to reflect the true background population from which the cases arose, as per established criteria to study the epidemiology of resistant strains, would be challenging for this clinical entity.20,21 In the absence of a matched case–control study to quantify fluconazole consumption as a risk factor for fluconazole-resistant recurrent vulvovaginal candidiasis infection, our data are highly suggestive that frequent use of fluconazole in some form or regimen can lead to emergence of recurrent vulvovaginal candidiasis infection caused by a fluconazole-resistant strain.
Primary or intrinsic azole resistance is rare in C albicans isolates.13–15 Historical details available indicate that only a minority of patients receiving low-dose, long-term weekly fluconazole developed secondary resistance presenting as breakthrough symptomatic infections. In a previous prospective controlled study, susceptibility profiles of C albicans isolates in women who received weekly fluconazole showed no increase in MIC values after 6 months of fluconazole use.5 Nevertheless, in the present series, women received considerably longer fluconazole regimens and hence exposure often lasting several years. Although not focusing on refractory vulvovaginal candidiasis infection or fluconazole resistance, two previous studies conducted in the same clinic reviewing azole susceptibility reported a detectable and steady creep or increase in MIC values over the past two decades.22,23 It is noteworthy that in reviewing Candida species responsible for candidemia, Lortholary et al24 reported that recent exposure to antifungals, especially fluconazole, profoundly influenced species selection and hence antifungal drug susceptibility.
At present, no information is available regarding the biologic mechanism of fluconazole resistance in the vaginal isolates and these studies are ongoing. Previous studies in nonvaginal isolates indicate multiple steps are involved usually resulting in activation of both CDR and MDR efflux pumps as well as ERG 11 mutation resulting in an increase in target 14-α-demethylase enzyme activity.25,26 Although new Clinical and Laboratory Standards Institution guidelines consider a fluconazole MIC of 2 micrograms/mL or less to reflect a drug-sensitive isolate of C albicans, this determination did not consider any vaginitis clinical data nor did it take into consideration the pharmacokinetics of fluconazole achieving a peak value of no more than 4 micrograms/mL in vaginal tissue and secretions.27 Moreover, the low pH of vaginal tissue and secretions during episodes of vulvovaginal candidiasis infection is known to further reduce azole activity.28,29 A previous clinical study suggested that a breakpoint MIC for Candida vaginal isolates of 1 microgram/mL or less should be considered.16 In the present study, 8 of 25 isolates of women failing fluconazole therapy had organisms with MIC values of 2 micrograms/mL supporting the lower breakpoint value.
The treatment of women with refractory vulvovaginal candidiasis infection, especially those with recurrent episodes, is especially problematic given the paucity of drug classes available.2,30 Long-term therapeutic decisions should be based on in vitro susceptibility tests. In the short term, initially faced with an acutely symptomatic patient with active vaginitis and unknown azole susceptibility, topical boric acid or nystatin suppositories prescribed for 10–14 days are likely to provide rapid relief and provide time to obtain susceptibility data.29 This allows for planning of long-term therapy when indicated in women with recurrent vulvovaginal candidiasis infection. Long-term maintenance therapy with boric acid or nystatin is possible but rarely used because of inconvenience, unknown efficacy together with a paucity of safety data. In considering azole use as maintenance prophylactic therapy, decisions should be based entirely on MIC data. Patients in the present study with fluconazole MIC values of 2 and 4 micrograms/mL could be treated successfully by increasing fluconazole dosage to 200 mg twice weekly. Clinical experience indicated that with MIC value of 8 micrograms/mL or greater, fluconazole use is precluded. At this juncture, cross-resistance to itraconazole and ketoconazole should be excluded, and, as shown in the present study, seven patients were treated effectively with long-term maintenance daily imidazole therapy. The latter regimens do, however, require intermittent hepatic function testing, not required with fluconazole use. Not infrequently, all maintenance antimycotic regimens were successfully discontinued. Nevertheless, in the event of severe recurrent vulvovaginal candidiasis infection, when high-level pan azole resistance is present, therapeutic measures are limited and achieving control of symptoms was extremely difficult.
The current retrospective study has several limitations. Given the duration of 11 years collecting data, we were not able to contact or receive a response in one-third of patients, hence the need for chart review. However, in a clinic seeing 400 new cases annually with less than 10% presenting with recurrent vulvovaginal candidiasis infection, numbers did not allow creation of a matched control group or estimation of percentage of C albicans isolates resistant to fluconazole. Clinical and in vitro resistance is still uncommon, but as this study reveals, it is certainly not rare. Any further epidemiologic claims would be distorted by the accrual bias of referred cases. In conclusion, fluconazole resistance and reduced susceptibility C albicans strains causing refractory vulvovaginal candidiasis infection are increasingly emerging and responsible for considerable suffering, especially when high-level resistance exists. Given the likely causal role of fluconazole exposure, unnecessary and inappropriate prescription should be avoided and long-term use must be based on diagnostic confirmation. Similarly, any movement toward liberalizing public availability of fluconazole should be discouraged. Fluconazole susceptibility trends require monitoring and new antimycotic agents are urgently needed.
1. Sobel JD. Epidemiology and pathogenesis of recurrent vulvovaginal candidiasis. Am J Obstet Gynecol 1985;152:924–35.
2. Sobel JD. Vulvovaginal candidosis. Lancet 2007;369:1961–71.
3. Donders GG, Bellen G, Mendling W. Management of recurrent vulvo-vaginal candidosis as a chronic illness. Gynecol Obstet Invest 2010;70:306–21.
4. Foxman B, Marsh JV, Gillespie B, Sobel JD. Frequency and response to vaginal symptoms among white and African American women: results of a random digit dialing survey. J Womens Health 1998;7:1167–74.
5. 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.
6. Sobel JD. Management of patients with recurrent vulvovaginal candidiasis. Drugs 2003;63:1059–66.
7. Donders G, Bellen G, Byttebier G, Verguts L, Hinoul P, Walckiers R, et al.. Individualized decreasing-dose maintenance fluconazole regimen for recurrent vulvovaginal candidiasis (ReCiDiF trial). Am J Obstet Gynecol 2008;199:613.e1–9.
8. Workowski KA, Berman SM. Sexually transmitted diseases treatment guidelines, 2006. MMWR Recomm Rep 2006;55:1–94.
9. Sobel JD, Vazquez JA. Symptomatic vulvovaginitis due to fluconazole-resistant Candida albicans in a female who was not infected with human immunodeficiency virus. Clin Infect Dis 1996;22:726–7.
10. Dorrell L, Edwards A. Vulvovaginitis due to fluconazole resistant Candida albicans following self treatment with non-prescribed triazoles. Sex Transm Infect 2002;78:308–9.
11. 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.
12. Rex JH, Pfaller MA, Galgiani JN, Bartlett MS, Espinel-Ingroff A, Ghannoum MA, et al.. Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro-in vivo correlation data for fluconazole, itraconazole, and candida infections. Subcommittee on Antifungal Susceptibility Testing of the National Committee for Clinical Laboratory Standards. Clin Infect Dis 1997;24:235–47.
13. Rex JH, Rinaldi MG, Pfaller MA. Resistance of Candida species to fluconazole. Antimicrob Agents Chemother 1995;39:1–8.
14. Pfaller MA, Andes D, Diekema DJ, Espinel-Ingroff A, Sheehan D; CLSI Subcommittee for Antifungal Susceptibility Testing. Wild-type MIC distributions, epidemiological cutoff values and species-specific clinical breakpoints for fluconazole and Candida: time for harmonization of CLSI and EUCAST broth microdilution methods. Drug Resist Updat 2010;13:180–95.
15. Pfaller MA, Espinel-Ingroff A, Boyken L, Hollis RJ, Kroeger J, Messer SA, et al.. Comparison of the broth microdilution (BMD) method of the European Committee on Antimicrobial Susceptibility Testing with the 24-hour CLSI BMD method for testing susceptibility of Candida species to fluconazole, posaconazole, and voriconazole by use of epidemiological cutoff values. J Clin Microbiol 2011;49:845–50.
16. 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.
17. CLSI. Performance standards for antimibrobial susceptibility testing. Nineteenth informational supplement. Approved standard M100–S19. Wayne (PA): Clinical and Laboratory Standards Institute; 2009.
18. Brammer KW, Feczko JM. Single-dose oral fluconazole in the treatment of vaginal candidosis. Ann N Y Acad Sci 1988;544:561–3.
19. Herzog RE, Ansmann FB. Treatment of vaginal candidiasis with fluconazole. Mycoses 1988;32:204–8.
20. Harris AD. Control group selection is an important but neglected issue in studies of antibiotic resistance. Ann Intern Med 2000;132:925.
21. Kaye KS, Harris AD, Samore M, Carmeli Y. The case-case-control study design: addressing the limitations of risk factor studies for antimicrobial resistance. Infect Control Hosp Epidemiol 2005;26:346–51.
22. Bulik CC, Sobel JD, Nailor MD. Susceptibility profile of vaginal isolates of Candida albicans prior to and following fluconazole introduction—impact of two decades. Mycoses 2011;54:34–8.
23. Shahid Z, Sobel JD. Reduced fluconazole susceptibility of Candida albicans isolates in women with recurrent vulvovaginal candidiasis: effects of long-term fluconazole therapy. Diagn Microbiol Infect Dis 2009;64:354–6.
24. Lortholary O, Desnos-Ollivier M, Sitbon K, Fontanet A, Bretagne S, Dromer F, et al.. Recent exposure to caspofungin or fluconazole influences the epidemiology of candidemia: a prospective multicenter study involving 2,441 patients. Antimicrob Agents Chemother 2011;55:532–8.
25. Sanglard D, Odds FC. Resistance of Candida species to antifungal agents: molecular mechanisms and clinical consequences. Lancet Infect Dis 2002;2:73–85.
26. MacCallum DM, Coste A, Ischer F, Jacobsen MD, Odds FC, Sanglard D. Genetic dissection of azole resistance mechanisms in Candida albicans and their validation in a mouse model of disseminated infection. Antimicrob Agents Chemother 2010;54:1476–83.
27. Houang ET, Chappatte O, Byrne D, Macrae PV, Thorpe JE. Fluconazole levels in plasma and vaginal secretions of patients after a 150-milligram single oral dose and rate of eradication of infection in vaginal candidiasis. Antimicrob Agents Chemother 1990;34:909–10.
28. Marr KA, Rustad TR, Rex JH, White TC. The trailing end point phenotype in antifungal susceptibility testing is pH dependent. Antimicrob Agents Chemother 1999;43:1383–6.
29. Danby CS, Boikov D, Rautemaa R, Sobel JD. Effect of pH on in vitro susceptibility of Candida glabrata and Candida albicans to eleven antifungal agents—implications for clinical use. Antimicrob Agents Chemother 2012;56:1403–6.
© 2012 by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.
30. Fan SR, Liu XP. In vitro fluconazole and nystatin susceptibility and clinical outcome in complicated vulvovaginal candidosis. Mycoses 2011;54:501–5.