The global incidence of mucocutaneous and invasive fungal infections have increased in the last two decades due to an expanding population of susceptible patients, and increasing use of antifungal prophylaxis [1, 2, 3, 4, 5, 6]. Candida spp. play a crucial role in causing mucocutaneous and invasive fungal infections around the world [7, 8, 9, 10, 11]. The problem is further complicated by the emergence of drug-resistant and multidrug-resistant fungal pathogens in recent years [4, 12, 13, 14, 15, 16]. For instance, the recently recognized multidrug-resistant Candida auris (C. auris) is now a major fungal pathogen in some countries [3]. C. auris has also caused outbreaks in healthcare facilities, which have been difficult to control [17, 18]. While Candida albicans (C. albicans) is the most frequently isolated yeast species from clinical specimens and is considered the most pathogenic, non-albicans Candida species (NACS) currently constitute over 50% of all Candida infections [19, 20, 21]. Candida glabrata (C. glabrata) is also one of the most frequently isolated yeast species from bloodstream and other anatomical sites, especially from the critically ill patients and those over 65 years [7, 19, 20, 21].

Vulvovaginal candidiasis (VVC) represents a worldwide problem, affecting approximately 75% of adult females of childbearing-age at least once in their lifetime [8, 10, 22, 23]. The management of VVC becomes challenging when caused by some NACS, given their reduced susceptibility to fluconazole [8, 10, 22, 23]. Furthermore, the ready availability of topical azoles and their widespread usage in certain countries for the treatment of VVC has resulted in the emergence of resistant strains [22, 23]. As an example, C. glabrata isolates exhibit reduced susceptibility to fluconazole, and instances of resistance to echinocandins and amphotericin B have also been documented among clinical isolates [24, 25, 26, 27, 28]. Recurrent VVC (defined as at least three confirmed infections within a 12-month period) affects nearly 10% of women and presents a challenging treatment scenario [10, 29]. In addition to oral fluconazole, various topical treatments such as probiotics, boric acid, honey, and other remedies have been employed, with variable success [30, 31, 32, 33]. Studies have also shown that the vaginal environment in patients with VVC has a pH of 3.8 to 4.5, which negatively affects the activity of some antifungal drugs commonly used to treat VVC [34, 35, 36]. In this report, we have explored the effect of pH on in vitro susceptibility of vaginal isolates of C. albicans, C. glabrata, Candida tropicalis (C. tropicalis) and Candida parapsilosis (C. parapsilosis) to fluconazole and flucytosine.

A total of 40 Candida spp. isolates cultured from vaginal specimens comprising C. albicans (n = 15), C. glabrata sensu stricto (n = 15), C. tropicalis (n = 5) and C. parapsilosis sensu stricto (n = 5) were tested. All isolates were correctly identified to the species level by their colony characteristics on CHROMagar Candida. Subsequently, their assimilation profiles were confirmed by Vitek2 yeast identification system. All C. albicans isolates (n = 15) yielded an amplicon of ~120 bp in duplex PCR, which facilitated the differentiation from C. dubliniensis. The presence of C. africana among C. albicans isolates was ruled out by the PCR assay targeting the HWP1 gene. All 15 isolates yielded an amplicon of ~941 bp which is specific for C. albicans [42]. All C. glabrata isolates (n = 15) yielded an amplicon of ~360 bp in multiplex PCR which is specific for C. glabrata sensu stricto strains. Similarly, PCR amplicons of ~260 bp and ~170 bp were successfully obtained from C. tropicalis (n = 5) and C. parapsilosis (n = 5) isolates, respectively, consistent with the expected results. The DNA sequences of the ITS region of rDNA from the isolates exhibited 1% difference with the sequence from the reference strains of the corresponding species, as expected.

The mean fluconazole MICs of 14.96 $\pm$ 7.71 µg/mL (geometric mean $\pm$ standard deviation (SD)) were obtained at pH 7.0 for 15 C. glabrata isolates. When the same 15 isolates were tested at pH 4.5, the mean fluconazole MICs increased significantly (82.55 $\pm$ 100.32 µg/mL, p = 0.001). The increase in MICs led to change in the classification of some C. glabrata isolates from ‘susceptible dose-dependent’ to ‘resistant’ to fluconazole (Table 1). Similarly, the mean fluconazole MICs for C. albicans isolates (n = 15) were also higher at pH 4.5 (1.32 $\pm$ 7.98 µg/mL) compared to the values at pH 7.0 (0.96 $\pm$ 1.35 µg/mL), and this disparity was also statistically significant (p = 0.017), as shown in Table 1. However, the mean fluconazole MICs for C. tropicalis (n = 5) and C. parapsilosis(n = 5) showed only slight increases when tested at pH 4.5 compared to pH 7.0, and these differences were not found to be statistically significant (Table 1). Unlike fluconazole MIC results, the mean flucytosine MICs for C. glabrata, C. albicans, C. tropicalis and C. parapsilosis isolates were lower at pH 4.5 compared to the MIC values at pH 7.0. Notably, the differences were statistically significant for C. glabrata (p = 0.042) and C. parapsilosis (p = 0.041), though not for C. albicans (p = 0.066) and C. tropicalis (p = 0.068) isolates (Table 2).

The VVC represents a serious public health issue globally which causes considerable morbidity. Approximately 75% women of childbearing age experience at least one episode of VVC in their life-time, with nearly 10% experiencing recurrent VVC [8, 10, 22, 23]. While recurrent VVC may not be a life-threatening condition, it affects the quality of life, causing both physical discomfort and psychological symptoms in the affected individuals. Several common antifungal drugs, including both conventional and newer drugs, as well as various topical remedies, have been used with varying degrees of success in the treatment of VVC [31, 32, 33, 36, 47, 48]. The causative agents of VVC mainly include C. albicans and C. glabrata [8, 22]. C. africana, a rare yeast species mostly associated with VVC, is genotypically very closely related to C. albicans and so, it is often misidentified as C. albicans by routine molecular testing [8, 42, 49]. Despite previous isolations of C. africana in Iran and Turkey within the Middle East [49, 50], extensive attempts to identify it among clinical C. albicans isolates in Kuwait have not been successful (Z. Khan and S. Ahmad, Unpublished data). Moreover, C. africana was also not detected among the 15 C. albicans isolates examined in this study.

Considering the challenges associated with the successful treatment of VVC, likely due to the acidic vaginal environment, the effect of pH on in vitro susceptibility of vaginal isolates of C. albicans and C. glabrata to fluconazole and flucytosine was investigated. Additionally, two other less commonly encountered Candida spp. from vaginal specimens, C. tropicalis and C. parapsilopsis, were also included.

Consistent with an earlier study [34], our in vitro susceptibility data also showed significantly higher mean fluconazole MICs at normal vaginal pH (4.0–4.5) compared to the values obtained at normal physiological pH (pH 7.0) for both C. glabrata and C. albicans isolates. Our findings are thus in agreement with earlier studies indicating reduced therapeutic efficacy of fluconazole for VVC caused by the two most common agents, i.e., C. glabrata and C. albicans [22, 33, 34, 35]. Although fluconazole MICs were also increased at lower pH for C. tropicalis and C. parapsilopsis, the differences were not statistically significant, most likely due to small number of isolates tested. Similar observations have also been made in another recent study. Spitzer et al. [35], in a study using first yeast isolates collected from 217 patients with VVC, showed that in vitro potency of four (terconazole, clotrimazole, miconazole and fluconazole) azole drugs was reduced when these antifungals were tested at pH 4.0 compared to their activity at pH 7.0. Furthermore, the reduced potency of these drugs was more pronounced for C. glabrata than for C. albicans isolates [35]. For terconazole, mean MIC values increased nearly 40-fold (from 0.17 µg/mL at pH 7.0, to 6.17 µg/mL at pH 4.0) for C. albicans isolates. However, the increase in mean MICs for C. glabrata was nearly 65-fold as all isolates yielded an MIC of $\le$1 µg/mL at pH 7.0, which increased to $>$64 µg/mL at pH 4.0 for nearly all the isolates [35].

Our findings also show that mean flucytosine MICs for C. glabrata were significantly lower at pH 4.5 (p = 0.042), and although not statistically significant (p = 0.066) for C. albicans, they were lower compared to the mean MICs at pH 7.0. These results suggest that topical flucytosine can be used as an effective therapeutic option for VVC caused by C. glabrata as well as for fluconazole-resistant and -susceptible strains of C. albicans. The mean flucytosine MICs at pH 4.5 were also lower for C. tropicalis and significantly lower for C. parapsilosis compared to the mean MICs at pH 7.0 for these two species. Furthermore, two previous studies have also reported favorable outcome with therapy regimens, including flucytosine in patients with VVC. Sobel et al. [30] reported 90% success following two weeks of intra-vaginal application of 17% flucytosine cream in patients with VVC who failed boric acid therapy. Similarly, White et al., [48] used lubricating jelly base containing flucytosine and amphotericin B for treatment of refractory vaginal candidiasis due to highly azole-resistant C. glabrata. These studies along with our preliminary results, indicate the importance of confirming these observations by testing a larger and more diverse collection of vaginal isolates from various dominant genotypes of C. glabrata, C. albicans, C. africana, C. tropicalis and C. parapsilosis for better management of VVC [30, 48].

Clinical trials have recently shown that Ibrexafungerp exhibits an efficacy similar to fluconazole in the treatment of VVC [51]. In a recent study, susceptibility testing of fluconazole-resistant (n = 52) and susceptible (n = 30) C. albicans and C. glabrata (n = 25) isolates from women with VVC against the new antifungal agent, Ibrexafungerp, also showed that the MIC values were not affected for C. albicans and were only slightly elevated for C. glabrata isolates when tested at pH 4.0 compared to pH 7.0 [36]. These findings suggest that antifungal drugs with excellent activity at pH 4.0 to pH 4.5 will have a better therapeutic outcome for patients with VVC and recurrent VVC. Taken together, these studies and our data, suggest that flucytosine either alone or in combination with newer drugs like Ibrexafungerp, could be considered as an attractive alternative for the treatment of patients with VVC or recurrent VVC [30, 36, 48].

Our study has few limitations. (1) Only a small number of vaginal isolates of C. glabrata, C. albicans, C. tropicalis and C. parapsilosis were tested for their in vitro susceptibility to fluconazole and flucytosine at the two pH values. The study needs to be repeated with a larger number of Candida spp. isolates recovered from patients with VVC. (2) The antifungal susceptibility testing was performed by ETEST and not by the reference broth microdilution method.

Consistent with other published reports, our data also showed that the antifungal activity of fluconazole is reduced at pH 4.5 compared to its activity at pH 7.0 for Candida spp. that cause VVC, particularly for C. glabrata isolates. In contrast, flucytosine MICs against C. glabrata and C. albicans isolates were lower at normal vaginal pH value (pH 4.5) compared to the MICs at pH 7.0. These observations, along with recent reports on the excellent activity of the newer antifungal drug Ibrexafungerp at reduced pH, suggest that topical flucytosine, either used alone or in combination with Ibrexafungerp, may present itself as an attractive alternative for patients with VVC or recurrent VVC.

All the data are available in the manuscript file. Other details will be provided by the corresponding author upon reasonable request.

ZK: conceived the idea, project development, data collection and analysis, manuscript writing; MA, SA: data collection and analysis, manuscript writing. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work. All authors contributed to revisions of the manuscript and approved the final manuscript.

The study was approved by the Health Sciences Center Ethics Committee (vide approval no. VDR/EC/3724), Kuwait University and all the methods and investigations were carried out according to their guidelines. The need for informed consent was waived by the Health Sciences Center Ethics Committee as no direct contact was made with the patients yielding Candida spp. isolates.

We thank Rachel Chandy and Leena Joseph for technical assistance.

This research received no external funding.

The authors declare no conflict of interest.