Antifungal susceptibility profile of Candida species isolated from women with vulvovaginal candidiasis

INTRODUCTION

Vaginitis is the most common syndrome in women presenting to the gynecology outpatient department (OPD).^[1] Patients usually show signs of inflammation of the vaginal mucosa, such as abnormal vaginal discharge, itching, soreness, burning, and pain during coitus.^[1] The bacterial vaginosis accounts for 40–50% of cases, followed by vulvovaginal candidiasis (VVC) accounting for 20–25% and trichomoniasis for 15–20%.^[2] Nearly, 75% of women of reproductive age will experience VVC at least once in their lifetime, and about 40%–50% will have one additional episode. About 5–8% of women will have four or more episodes of VVC, which is referred to as recurrent VVC (RVVC).^[3] Among VVC, Candida albicans accounts for 80–95%. Recently, infection due to non-albicans Candida spp. (NAC) is on the rise (10–30%).^[4]

Candida spp. is part of the normal vaginal flora in about 20% of asymptomatic women. It becomes an opportunistic pathogen when the immunity is lowered.^[5] The risk factors for VVC include pregnancy, the use of antibiotics, age, uncontrolled diabetes, oral contraceptives, hormone replacement therapy, immunosuppression, and frequent sex.^[1] Recurrent vulvovaginal candidiasis (RVVC) can occur in women colonized with Candida spp in the oral cavity and lower gastrointestinal tract.^[4,5]

VVC is now considered a global public health problem associated with direct or indirect economic loss.^[6,7] It not only causes morbidity in women but also poses a negative impact on work and social life.^[8] Hence, the present study was conducted with the aim (i) to study the occurrence of VVC among women with vaginitis attending the Gynecology OPD at a tertiary care teaching hospital in South India and (ii) to speciate and determine the antifungal susceptibility profile of the Candida spp. isolated from them.

MATERIALS AND METHODS

A prospective study was conducted on women attending the gynecology outpatient clinic with symptoms of vaginitis from April 2020 to March 2021. After obtaining informed consent, two high vaginal swabs (HVSs) were collected from each patient by a Gynecologist. One HVS was used for Gram stain and wet mount, while the other was used for culture on sheep blood agar (SBA) and Sabouraud dextrose agar (SDA) with chloramphenicol. Epithelial cells, pus cells, Gram-positive budding yeast cells with or without pseudohyphae, and Gram-positive bacilli resembling lactobacilli were recorded on Gram stain. For wet mount preparation, the swab was placed in 0.5 mL sterile saline and gently mixed and examined under the microscope for the presence or absence of motile trophozoites of Trichomonas vaginalis, pus cells, and red blood cells.

For culture, the swab was inoculated onto 5% SBA and SDA (HiMedia, Mumbai, India) incubated at 37°C for 18–24 h in an ambient air incubator. Any growth of white cream colonies on the SDA was further characterized.

RESULTS

A total of 961 patients with vaginitis were included during the study period. The Candida spp. was isolated in 108 vaginal samples making the overall prevalence 11.2%. The median age of women suffering from VVC was 29 years (range 20–73 years). The majority belonged to the 20–30 years age group (65/108; 60.2%), followed by 31–40 years (30/108; 27.8%) and above 40 years (13/108; 12%). Age <40 years was the significant risk factor for VVC (P = 0.016).

Vaginal discharge was the most common presenting complaint (n = 89; 82.4%), followed by itching (n = 60; 55.6%), vulval edema (n = 21; 19.4%), dyspareunia (n = 16; 14.8%), and dysuria (n = 13, 12%). Out of 108 cases, 30 (27.8%) women were pregnant at the time of the study. Twelve percentages of women had a history of prior antibiotic usage, and 6.5% (n = 7) had diabetes mellitus.

One hundred and eight isolates from the vaginal samples represented eight different species. C. albicans was the predominant species (50/108, 46.3%), followed by Candida glabrata (39/108, 36.1%), C. krusei (7/108, 6.5%), and Candida tropicalis (7/108, 6.5%). The phenotypic method could correctly identify 99 (91.7%) isolates to the species level. The nine isolates which had discrepant results between the two methods were re-tested with MALDI-TOF MS, and results consistently showed a MALDI score of >2. The phenotypic method identified Candida inconspicua, Candida nivariensis, and Candida viswanathii as guilliermondii, C. glabrata, and C. albicans, respectively. The correct identification rates of C. albicans, C. glabrata, C. krusei, and C. tropicalis by the phenotypic method were 98% (49/50 isolates), 92.3% (36/39 isolates), 100% (7/7 isolates), and 100% (7/7 isolates), respectively.

Prior use of antibiotics (P = 0.017) and pregnancy (P = 0.028) was significantly associated with NAC infection. The association between different symptoms and the species type was not statistically significant.

The antifungal susceptibility testing results by minimum inhibitory concentration (MIC) are shown in Table 1. All the isolates were susceptible to most of the antifungals tested except C. glabrata and C. krusei. Ninety-six percentages (48/50) of C. albicans isolates were sensitive to fluconazole, while only 13/58 (22.4%) NAC were sensitive to fluconazole. If we exclude the C. krusei and C. glabrata, which are intrinsically resistant, all other NAC were susceptible to fluconazole.

The MICs of azole and echinocandin antifungals were higher for C. glabrata and C. krusei than those of C. albicans. The C. albicans showed 8% resistance to itraconazole (>0.25 μg/mL). C. glabrata showed 100% and 92.3% dose-dependent susceptible MICs for fluconazole (>2 μg/mL) and itraconazole (>0.25 μg/mL), respectively. Persistent symptoms were observed among 25 women (23.1%) infected with C. krusei and C. glabrata. Such patients were treated with prolonged fluconazole therapy.

DISCUSSION

Despite advances in diagnosis and treatment, VVC remains a significant health problem among immunocompetent women. Minimal epidemiological information is available from India, and most of the data available are from observational studies. The present study estimated the prevalence of VVC as 11.2%, which was comparable to the results of Pereira et al.^[11] The previous data from India showed a varied prevalence of 14– 21%.^[12] The signs and symptoms of VVC cases in our study were almost comparable to the previous studies.^[13] The most common age group affected was between 20 and 40 years (88%). Yano et al.^[13] and Bitew and Abebaw^[6] had similar findings in their study. The risky sexual behavior, age at first sexual intercourse <20, hormonal effects, educational status, menstrual hygiene, eating sweet foods, and wearing tight undergarments were found to be the risk factors for VVC.^[5,14]

Limited data are available regarding the distribution of yeasts causing the VVC in India and their drug susceptibility. Eight different species of Candida were isolated from vaginal samples. C. albicans was the most common isolate (46.3%), but the NAC was more frequent (53.7%). The isolation rates of NAC species from various studies ranged from 19.8% to 65%.^[6] In the present study, C. glabrata (36.1%) was the most common NAC species, followed by C. krusei (6.5%) and C. tropicalis (6.5%).

Other studies also report C. glabrata as the most common NAC species in VVC.^[6] NAC species cause milder disease compared to C. albicans.^[15] However, inherent or acquired resistance to azole antifungals can complicate the management of VVC caused by NAC species.^[6] For infections with NAC species, prolonged antifungal therapy is required to clear the infection. Most studies reporting high proportions of NAC species were from tertiary care hospitals, focusing on patients who failed the conventional treatment.^[16] The data from these studies may overestimate NAC prevalence if extrapolated to the general population. The other reason for increased NAC prevalence is the use of over-the-counter azole antifungals, which leads to the selection of NAC species. Hence, accurate identification up to the species level is essential for successful therapy, especially in the age group between 20 and 40 years and in women in the perimenopausal and postmenopausal stages.

C. albicans, C. glabrata, C. krusei, and C. tropicalis were the four most common species of VVC cases. The phenotypic method identified C. albicans (98%) with greater accuracy, while it had a lower correct identification rate for NAC (86.2%). Although MALDI-TOF MS has excellent sensitivity and specificity in identifying the species, the equipment is expensive, and not always accessible for diagnosis.

The C. glabrata and C. krusei had higher azole MIC and were difficult to treat, often requiring prolonged therapy. Fluconazole resistance was 4% with C. albicans isolates, whereas C. glabrata exhibited higher MIC for azoles and echinocandins than those for C. albicans. Similar findings were also reported earlier by Shi et al.^[17] and Castanheira et al.^[18]

All the patients were clinically diagnosed by obstetricians and gynecologists and treated with a prescription of antifungal drugs. Almost 60% of patients reported control or relief of symptoms on standard treatment with topical and oral azoles. Relapse was reported among the women infected with NAC. Hence, speciation and antifungal susceptibility testing will help clinicians decide on the antifungal drug and the duration of therapy.

The limitations of the present study were relatively of a small sample size. Moreover, follow-up of cases could not be done, and relief and cure were based on self-reported data. A large and multicentric study to identify the species distribution and antifungal susceptibility testing (AFST) data is the need of the hour. This is mainly to manage this important public health problem affecting many young women in India.

CONCLUSIONS

The age group between 20 and 40 years was a significant risk factor for VVC. Compared to the previous studies from India, the prevalence of NAC was higher in the present study. In addition, antibiotic use in the past, pregnancy, and diabetes was all strongly linked to NAC infection. Therefore, the management of VVC should be guided by culture, accurate species identification, and antifungal susceptibility testing, particularly for recurrence or relapses.