Resistance to Fluconazole of Candida albicans in Vaginal Isolates: a 10-Year Study in a Clinical Referral Center

ABSTRACT

Review of vaginal isolates of Candida albicans that caused clinical failure in a 10-year collection of vaginal C. albicans specimens obtained in a university vaginitis referral clinic indicated an increase in fluconazole resistance. Factors contributing to azole resistance are discussed, including treatment choice associated with fluconazole-resistant C. albicans vaginal infection.

INTRODUCTION

The global emergence of antifungal drug resistance among fungal genera is of growing concern (1–5). Resistance in Candida species is attracting attention, especially in invasive Candida infections by Candida auris (6). Less attention has been directed at clinically resistant, often-refractory Candida vaginitis (VVC), which is responsible for considerable suffering in women. Resistance to fluconazole is widely recognized in Candida krusei and Candida glabrata; however, problematic vaginitis due to Candida albicans has escaped attention, with few epidemiologic studies (5–7). Reports include a growing clinical problem of fluconazole resistance, especially in women with recurrent VVC (RVVC) (8–14). The present manuscript reports a single-site, large longitudinal study of fluconazole-resistant VVC caused by C. albicans over a 10-year period.

The Vulvovaginitis Clinic of Wayne State University in Detroit sees approximately 1,200 patients per year, including 300 new referrals. Antibiotic susceptibility tests (ASTs) are only performed when requested by a single practitioner (J.D.S.), when antifungal drug resistance is suspected because of either refractory clinical disease suggested by continuing acute patient symptoms and positive microscopy in spite of appropriate antifungal therapy or breakthrough symptomatic VVC while receiving maintenance prophylactic fluconazole therapy.

AST methodology included determination of MICs using broth dilution according to the CLSI M27ed4 method (15–17). MICs were read at 24 h. ATCC strains of C. parapsilosis (22,019) and C. krusei (6,258) were used as quality controls. It has been the local laboratory practice for several years to determine the MIC at pH 7.0 and 4.5 (18). Clinical breakpoint standards to determine susceptibility at pH 7.0 are based upon CLSI recommendations, with the resistant level being ≥8 μg/mL, susceptible dose dependent (SDD) level at 4 μg/mL, and susceptible level at ≤2 μg/mL, with identical breakpoint values extrapolated to pH 4.5.

Results of all C. albicans susceptibility tests performed over 10 years (2012 to 2021) are shown in Table 1. Each year, approximately 1,000 vaginal cultures were obtained, of which approximately 20% were positive for Candida and other yeast species. Of the isolates collected, 1,578 (76.3%) were positive for C. albicans. Susceptibility testing was performed on 193 (12.2%) C. albicans isolates based upon practitioner request. Fluconazole resistance at pH 7.0 was observed in 44 (23%) isolates. Forty isolates were defined as SDD, with a MIC estimation of 4 μg/mL (21%), and 109 (56%) isolates were determined to be susceptible (MIC, ≤2 μg/mL). The percentage of fluconazole-resistant C. albicans isolates varied by year (Table 1), with a dramatic increase in fluconazole resistance detected in 2021. Results indicated that while the number of patients seen and examined remained fairly constant (except for COVID pandemic years 2020 and 2021), the number of patients with clinically suspected resistance increased, together with an increase in the number of patients with fluconazole-resistant C. albicans.

TABLE 1.

Antifungal susceptibility studies, 2012 to 2021

Yr	No. of total cultures	No. (%) positive for any yeast	No. (%) positive for C. albicans	No. with AST result	No. (%) of isolates resistant to fluconazole (MIC ≥ 8 μg/mL)
2012	1,202	201 (16.7)	155 (77.1)	14	4 (28.6)
2013	1,132	215 (19.0)	157 (73.0)	22	5 (22.7)
2014	1,069	195 (18.2)	147 (75.3)	19	3 (15.8)
2015	1,177	247 (21.0)	192 (77.7)	23	4 (17.4)
2016	1,092	213 (19.5)	153 (71.8)	19	2 (10.5)
2017	1,044	246 (23.6)	191 (77.6)	21	7 (33.3)
2018	961	204 (21.2)	165 (80.8)	18	3 (16.6)
2019	1,027	233 (22.6)	171 (73.4)	18	3 (16.6)
2020	667	127 (19.0)	102 (80.3)	11	3 (27.3)
2021	933	186 (19.9)	145 (77.9)	28	10 (35.7)
Total	10,304	2,067 (20.1)	1,578 (76.3)	193	44 (22.8)

Comparison of AST results obtained over the last 4 years (n = 65) and performed at both pH 7.0 and 4.5 in C. albicans isolates further revealed a dramatic increase in C. albicans fluconazole resistance at pH 4.5, from 20% to 52% (Table 2). Most change in status occurred for isolates for which MICs were previously determined at pH 7.0 and the classification was SDD (4 μg/mL). Overall resistance frequency at pH 4.5 more closely correlated with the original clinical suspicion of resistance, with only 32% of C. albicans isolates deemed susceptible to fluconazole.

TABLE 2.

Comparison of ASTs analyzed by pH of median (2018 to 2021)

Susceptibility category	% vaginal C. albicans isolates in category when tested at:
	pH 7.0	pH 4.5
Susceptible (MIC ≤ 2 μg/mL)	52%	38%
Sensitive dose dependent (MIC 4 μg/mL)	23%	10%
Resistant (MIC ≥ 8 μg/mL)	25%	52%

This report reflects the experience in a single center only, with acknowledged accrual bias, and does not measure the overall prevalence of resistance in C. albicans causing VVC in the region. The true prevalence of fluconazole resistance both locally and worldwide is largely unknown (14, 19, 20). Unfortunately, reports in the literature of antifungal drug susceptibility of C. albicans causing VVC are compromised by difficulties based upon highly variable patient characteristics, yeast as commensal or pathogens, susceptibility test variables, broth dilution versus disc measurement, or commercial test methods. Overall estimates of in vitro fluconazole resistance in C. albicans vaginal isolates in general is below 5%, and as indicated in the present study, was 3% of total C. albicans isolates but 20% of tested isolates at pH 7.0 and 52% at pH 4.5, supporting the recommendation for suspecting resistance in women with clinical suspicion of fluconazole therapy failure (Table 2) (8, 14). In some geographic areas, considerably higher levels of fluconazole resistance of >20% have been reported in unselected isolates (21).

The present review describes a considerable number of symptomatic women with refractory and recurrent VVC caused by fluconazole-resistant C. albicans, whereas at the same time there have been no reports of increased azole resistance in blood isolates or of invasive candidiasis due to C. albicans (2, 3, 19). The differences between blood and vaginal isolates of C. albicans are numerous, including duration of fluconazole exposure, days in patients with candidemia and weeks and months in RVVC patients, analogous to patients with untreated HIV immunodeficiency in whom C. albicans fluconazole resistance was first reported (22). Repeated short courses of inexpensive fluconazole are also encouraged by practitioners.

The implications of refractory vaginitis caused by fluconazole-resistant C. albicans are legion, with chronic and often severe vulvovaginal symptoms aggravated by the paucity of alternative therapeutic options (23). Physicians are largely unaware of drug resistance in vaginal yeast infections, except with non-albicans Candida species. New antifungals oteseconazole and ibrexafungerp are now available, with in vitro data indicating potent activity against azole-resistant Candida species (24, 25). However, there are few data of clinical efficacy against fluconazole-resistant organisms in infected patients. The new agents are contraindicated in pregnancy, with other use restrictions.

Breakpoints used universally are based upon extrapolation of values derived from studies only in invasive candidiasis. Pharmacokinetics, especially fluconazole vaginal tissue concentrations and the vaginal pH circumstances, are capable of influencing breakpoint values applied in VVC. Considerable experience utilizing simultaneous in vitro testing at both pH 7.0 and pH 4.5 indicates that C. albicans isolates determined to have a MIC of 4 μg/mL at pH 7.0 frequently demonstrate an increased fluconazole MIC of ≥8 μg/mL when tested at pH 4.5 and that failure to test vaginal isolates at the lower pH results in underestimation of clinically relevant drug resistance (26–30).