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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 24  |  Issue : 2  |  Page : 71-75

Vulvovaginal candidiasis: Clinical profile, species distribution and antifungal susceptibility pattern


1 Department of Clinical Microbiology and Infection Control, Gleneagles Global Hospitals, Hyderabad, Telangana, India
2 Department of Clinical Microbiology and Infection Control, Gleneagles Global Hospitals; Department of Clinical Microbiology and Infection Control, Rainbow Children's Hospital and Birthright, Hyderabad, Telangana, India
3 Department of Obstetrics and Gynaecology, Rainbow Children's Hospital and Birthright, Hyderabad, Telangana, India

Date of Submission09-Nov-2022
Date of Acceptance19-Nov-2022
Date of Web Publication13-Dec-2022

Correspondence Address:
Rekha Rao Jangam
Department of Clinical Microbiology and Infection Control, Gleneagles Global Hospitals, 6-1-1070/1 to 4, Lakdikapul, Hyderabad - 500 004, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jacm.jacm_24_22

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  Abstract 


INTRODUCTION: Vulvovaginal candidiasis (VVC) is a common problem seen in women and 70%–75% of women experience VVC at least once in their lifetime. The relative incidence of vaginitis caused by non-albicans Candida (NAC) species is increasing which poses a problem as NAC is less susceptible to fluconazole which is commonly used for the treatment of VVC. In this study, we determined the species distribution of Candida isolates causing VVC and the antifungal susceptibility pattern of these isolates to fluconazole.
MATERIALS AND METHODS: A prospective study was done in a tertiary care hospital in Hyderabad, South India (August 2019–December 2020), on 500 women with suggestive clinical features and culture-confirmed VVC. Five hundred Candida strains isolated from the vaginal swab of these women were speciated and fluconazole antifungal susceptibility testing (AFST) was done by broth microdilution technique (CLSI guidelines) for 103 isolates. The following fluconazole concentrations were tested: 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml, 32 μg/ml and 64 μg/ml which encompassed the clinical breakpoint concentrations and the expected results for the quality control strains.
RESULTS: The majority of the women (93%) with VVC were in the reproductive age group (21–40 years). The most common presenting symptoms were vaginal discharge (66.4%) and pruritus (26.6%). The most common predisposing factor for VVC was pregnancy (44.8%). Candida albicans (62.8%) was the most common species isolated, followed by Candida glabrata (27.2%). Overall fluconazole susceptibility was 57%. About 72.2% of C. albicans were fluconazole susceptible.
CONCLUSION: VVC has a wide clinical presentation with non-specific signs and symptoms. Diagnosis based only on signs and symptoms leads to over treatment. Thus, culture confirmation is crucial for the accurate diagnosis and to detect VVC caused by NAC which is less susceptible to fluconazole. Fluconazole AFST on these isolates would help in developing local antibiograms and to monitor resistance trends.

Keywords: Candida albicans, fluconazole, non-albicans Candida, vulvovaginal candidiasis


How to cite this article:
Mariyah S, Iyer RN, Jangam RR, Kesireddy S. Vulvovaginal candidiasis: Clinical profile, species distribution and antifungal susceptibility pattern. J Acad Clin Microbiol 2022;24:71-5

How to cite this URL:
Mariyah S, Iyer RN, Jangam RR, Kesireddy S. Vulvovaginal candidiasis: Clinical profile, species distribution and antifungal susceptibility pattern. J Acad Clin Microbiol [serial online] 2022 [cited 2023 Feb 9];24:71-5. Available from: https://www.jacmjournal.org/text.asp?2022/24/2/71/363476




  Introduction Top


Vulvovaginal candidiasis (VVC) is a common problem seen in women of the reproductive age group. 70–75% of women experience VVC at least once in their lifetime, of which 40%–50% will have a recurrence and 5%–10% will have recurrent VVC, defined as four or more episodes every year. 85% to 95% of yeast strains isolated from the vagina belong to the species Candida albicans with the remaining being non-albicans Candida (NAC), especially Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei.[1],[2] The relative incidence of vaginitis caused by NAC is increasing. NAC infections are associated with recurrent disease.[1],[2]

The treatment of VVC is approached by classifying the patient as having either an uncomplicated or complicated infection. For uncomplicated cases, short courses of topical imidazole creams are used for 3–7 days in the vast majority of patients. However, a single dose of oral fluconazole can also be used. Complicated VVC (caused by NAC, recurrent VVC, severe infection with C. albicans, VVC in diabetic women or VVC in immunosuppressed) requires treatment with topical imidazole preparations for a longer duration (7–14 days) or oral fluconazole every 72 h for 2–3 doses. Following treatment of the acute episode in recurrent VVC, often maintenance therapy with oral fluconazole or topical imidazole preparations is recommended for six months. Only topical azole therapies, applied for seven days, are recommended for use amongst pregnant women.[1],[2],[3]

Topical imidazole preparations and oral fluconazole are available over the counter in India as well as many developed countries without prescription resulting in the indiscriminate use of these agents in self-diagnosed VVC. In addition, the diagnosis of VVC is often made clinically based on non-specific symptoms and treated with antifungal agents without culture confirmation. This inadvertent use of antifungal agents can lead to the development of resistance of Candida isolates to antifungal agents.[1] Furthermore, there is widespread use of oral fluconazole for prophylaxis, especially in patients with HIV disease and solid organ transplant recipients resulting in the development of fluconazole resistance. The increasing immunocompromised population has led to a gradual increase in infections with NAC which are less susceptible to fluconazole.[1],[4]

Therefore, it is important to speciate Candida isolates causing VVC and perform antifungal susceptibility testing (AFST) for optimal treatment and to develop local antibiograms to aid in the empirical selection of antifungals. Fluconazole is widely used in India for the treatment of VVC but there are very few studies in India which have done fluconazole AFST by broth microdilution (BMD) for Candida isolated from VVC patients. Hence, we conducted our study to determine the species distribution of Candida isolates causing VVC and to determine the antifungal susceptibility pattern of these isolates to fluconazole.


  Materials and Methods Top


A prospective study was done in a tertiary care hospital in Hyderabad, South India, on women with symptoms and signs suggestive of VVC. A total of 500 women were enrolled in the study conducted from August 2019 to December 2020. The study was approved by the institutional ethics committee. Each patient was included only once in the study after taking informed consent. Inclusion criteria were as follows: women with at least one of the following signs and symptoms of VVC along with isolation of Candida spp. from vaginal swabs with no alternative pathogens. Symptoms of VVC: vulvar pruritus, pain, swelling, redness and external dysuria.[5] Signs of VVC: vulvar oedema, erythema, fissures, excoriations and thick curdy vaginal discharge.[5] Exclusion criteria: age <18 years. Demographic details and clinical and laboratory findings were recorded for all patients included in the study.

Microbiological methods

Specimen collection

Vaginal discharge from patients enrolled in the study was collected using sterile cotton swabs (two swabs from each patient included in the study).

Specimen processing

One vaginal swab was inoculated onto the following culture media – 5% Sheep blood agar, MacConkey agar, Chocolate agar and Sabouraud dextrose agar (SDA) and incubated aerobically at 37°C for 48 h. Gram-stained smear was prepared from the second vaginal swab by Preston Morrell's modification to demonstrate budding yeasts and/or pseudohyphae. Candida isolates growing on SDA were speciated by the following phenotypic tests – Germ tube test, growth on chromogenic agar (CHROMagar 75,006 Paris-France), morphology on cornmeal agar, growth at 42°C, growth in presence of cycloheximide, sugar assimilation and fermentation reactions (EliTech Microbio 83,870 Signes France).[6] AFST of Candida isolates to fluconazole was done by BMD technique.[7]

Fluconazole susceptibility testing by broth microdilution

Candida isolates were tested for fluconazole susceptibility by BMD according to CLSI M27-Ed4 guidelines.[7] BMD was performed in sterile, disposable, untreated, 96-well polystyrene microdilution plates with U-shaped wells (Tarsons, 902, Martin Burn Business Park, BP-3, Saltlake, Kolkata) using RPMI 1640 (Sigma-Aldrich Co.,3050 Spruce Street, St Louis, MO 63103 USA) with L-glutamine (but without bicarbonate), buffered to a pH of 7 with 3-(N-morpholino) propane sulfonic acid and a final glucose concentration of 0.2%. Fluconazole powder (Sigma-Aldrich, Co., 3050 Spruce Street, St Louis, MO 63103 USA 314,171-5765) with 98% potency was used and it was diluted in analytical-grade dimethyl sulfoxide (Sigma-Aldrich CHEMIEGmbH, Reid's tr2D-89,555 Steinheim 497329970, Product of France). The following fluconazole concentrations were tested: 0.125 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml, 32 μg/ml and 64 μg/ml which encompassed the clinical breakpoint concentrations and the expected results for the quality control strains. C. parapsilosis ATCC 22,019 with expected fluconazole MIC between 0.5 and 4 μg/mL and C. krusei ATCC 6258 with expected fluconazole MIC between 8 and 64 μg/mL were used for quality control.

Testing procedure

One hundred microliters of the two times antifungal drug concentrations (128-0.25 μg/mL) were dispensed from Well1 to Well 10 in each row followed by the addition of 100 μL of two-fold inoculum (1 × 103–5 × 103 CFU/mL). The (two-fold) inoculum was diluted 1:1 when the wells were inoculated and the desired final inoculum size was achieved (0.5 × 103–2.5 × 103 CFU/mL). Well 11 was used as broth control i.e., drug-free medium (only RPMI 200 μL was added) and Well 12 was used for the growth control (100 μL RPMI + 100 μL of two-fold inoculum). Microdilution plates were incubated at 35°C for 24 h.

MIC for fluconazole was the lowest concentration, in which a prominent decrease in turbidity was observed corresponding to approximately 50% inhibition in growth as compared to the drug-free well. Species-specific interpretative fluconazole breakpoints were used.[8] For species with no established clinical breakpoints, the epidemiological cutoff values (ECVs) were used to differentiate the wild and non-wild strains.[9]


  Results Top


Patient's age ranged from 18 years to 64 years (median age: 29 years; mean age: 30.54 years). The age distribution of the study patients is as follows: <20 years (5 [1%]), 21–30 years (285 [57%]), 31–40 years (180 [36%]), 41–50 years (21 [4.2%]), 51–60 years (6 [1.2%]) and >60 years (3 [0.6%]). A majority of the patients were in the 21–30 year age group (57%), followed by the 31–40 year age group (36%). The most common symptoms were curdy white discharge (66.4% [332/500]) and pruritis (133 [26.6%]), followed by dysuria (24 [4.8%]) and pain (9 [1.8%]). The most common sign was curdy white discharge (405 [81%]) with others being excoriation (22 [4.4%]) and inflammatory signs (12 [2.4%]). The most common predisposing factor was pregnancy (44.8% [224/500]) with the majority of them in the second (41%) and third trimesters (39%). Six patients had diabetes mellitus (DM), of which three patients had gestational DM and three patients were using oral contraceptive pills (OCPs).

C. albicans (314/500 [62.8%]) was the most frequently isolated species in our study, followed by C. glabrata (136/500 [27.2%]), C. tropicalis (24/500 [4.8%]), C. kefyr (11/500 [2.2%]), C. utilis (5/500 [1%]), C. krusei (5/500 [1%]), C. parapsilosis (2/500 [0.4%]), C. dubliniensis (2/500 [0.4%]) and C. melibiosa (1/500 [0.2%]). Eighteen (3.6%) patients had recurrent VVC, of which nine were due to C. glabrata.

AFST for fluconazole was done by the BMD method for 103 isolates and the results are depicted in [Table 1].
Table 1: Fluconazole susceptibility of Candida strains (n=103)

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Thirteen of 72 (18.1%) C. albicans isolates, two of the 22 C. glabrata isolates and one of the 6 C. tropicalis isolates were fluconazole-resistant. Due to the lack of clinical breakpoints for C. kefyr, ECVs were taken into consideration and were categorised as wild type. Neither the breakpoints nor the ECV is available for C. utilis so only the MIC was recorded for C. utilis.


  Discussion Top


VVC is a common gynaecological finding amongst women worldwide and is the isolation of Candida species from vaginal secretions of patients with signs and symptoms of vulvovaginitis. VVC shows acute or chronic courses and different disease patterns which can strongly affect the quality of life of women.[5]

In our study, we found that women in the reproductive age group (21–30 years: 57% and 31–40 years: 36%) had the highest prevalence of VVC. Similar findings were reported by Dharmik et al.[10] in a study done in Nagpur, India. The reason for the high prevalence in the younger age group of women includes low levels of protective cervical antibodies, increased sexual activity, pregnancy and use of OCP that may lead to increased susceptibility to reproductive tract infections.

The knowledge of clinical signs and symptoms of VVC helps in early diagnosis and treatment. The clinical profile of VVC comprises pruritus, curdy white vaginal discharge, pain, dysuria, dyspareunia, redness, excoriations and fissures.[5] In our study, the most common complaints reported were vaginal discharge (66.4%) and pruritus (26.6%) and on examination, vaginal discharge (81%) was most common. Rathod et al.[11] in their study done in Mysore, India, also found pruritus (17.8%) and vaginal discharge (15.3%) as the most common presenting feature in non-pregnant sexually active women between 16 and 30 years of age. Khan et al.[12] reported pruritus (31%) and vaginal discharge (29.4%) as the major complaints in pregnant women presenting with VVC. However, VVC has a wide clinical presentation with no pathognomonic signs or symptoms. Diagnosis based only on signs and symptoms leads to an overestimation of the prevalence of VVC and over treatment. Only a minority of women with vaginal discharge have VVC.[1],[2],[11] Thus, culture confirmation is crucial for accurate diagnosis.

Understanding the risk factors for VVC helps in identifying at-risk or vulnerable cases. The increased risk of VVC in pregnancy is likely sustained by pregnancy-related factors, such as immunologic alterations, increased oestrogen levels and increased vaginal glycogen production.[13] In our study, as many as, 224 (44.8%) women with VVC were pregnant. Ragunathan et al.[14] reported 22 pregnant patients (55%) of the 40 patients with VVC in their study in Puducherry, India. In a study by Dias et al.[15] in Brazil, 90 (56.25%) of the 160 patients with VVC were pregnant. A majority of the patients in our study were in the second trimester (41%). This is consistent with the findings of Khan et al.[12] who reported 65 (60.2%) patients in the second trimester with VVC. Patients with DM are more susceptible to fungal infections, including those caused by species of Candida.[16] However, only 6/500 (1.2%) women in our study were diabetic, out of which 3 had GDM (gestational DM). Ragunathan et al.[14] reported 6 diabetic patients (15%) of the 40 patients with VVC in their study.

C. albicans is the most frequently isolated species, followed by C. glabrata.[1],[2] In our study, the frequency of isolation of C. albicans was 62.4% (314/500) and 37.2% (186/500) for NAC. C. glabrata was the second-most common species isolated (136/500 [27.2%]) and also the most common NAC species isolated (136 of the 186 NAC isolates – 73.1%). Our findings are similar to those of Dharmik et al.[10] A retrospective study was done by Wang et al.[17] in China from 2006 to 2013. A total of 2204 Candida spp. were isolated from 2122 patients with VVC. C. albicans (80%) was most commonly isolated, followed by C. glabrata (18%). However, from 2006 to 2013, there was a considerable drop in isolation of C. albicans from 90% in 2006 to 77% in 2013 and the C. glabrata cases doubled from 10% in 2006 to 20% in 2013 which was attributed to factors such as indiscriminate use of broad-spectrum antimicrobials and increase in immunocompromised or immunodeficient population. With an ever-increasing number of cases of VVC caused by NAC species which are known to have high MICs to fluconazole, it is important to speciate Candida isolates causing VVC.

Of the various methods for AFST, BMD remains the standard method. While agar-based methods (disk diffusion and gradient diffusion) and molecular methods are easier alternatives, BMD can analyse the potential antifungal activity of the target compound using small quantities of the compound and at the same time test it in a range of concentrations.[18] In our study, we tested 103 Candida isolates for fluconazole susceptibility by BMD and 55.3% were susceptible to fluconazole. About 18.05% of C. albicans isolates were found resistant to fluconazole. The study by Wang et al.[17] over an eight-year period on 2204 Candida isolates from patients with VVC showed increasing resistance of C. albicans against fluconazole from 2.4% in 2006 to 8.9% in 2013. The increased use of over-the-counter antifungal drugs, self-treatment, misdiagnosis and prolonged treatments for recurrent candidiasis are risk factors for the emergence of azole resistance amongst C. albicans isolated from vulvovaginitis patients.[1],[2]


  Conclusion Top


The majority of the women with VVC were in the reproductive age group (21–40 years). The most common presenting features were vaginal discharge and pruritus. The most common predisposing factor for VVC was pregnancy followed by DM. C. albicans was the most common species isolated from VVC followed by C. glabrata. The frequency with which C. albicans was recovered in our study and its susceptibility to fluconazole supports the continued use of azole agents for empirical therapy of uncomplicated vaginal candidiasis in our region. However, more prospective studies are needed to evaluate the species distribution of yeasts in cases of VVC and perform AFST to determine the optimal therapy for VVC especially those caused by NAC species which are known to have high MIC to azole antifungals.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Sobel JD. Vulvovaginal candidosis. Lancet 2007;369:1961-71.  Back to cited text no. 1
    
2.
Sobel JD. Recurrent vulvovaginal candidiasis. Am J Obstet Gynecol 2016;214:15-21.  Back to cited text no. 2
    
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British Association for Sexual Health and HIV (BASHH). British Association for Sexual Health and HIV National Guideline for the Management of Vulvovaginal Candidiasis. British Association for Sexual Health and HIV; 2019. Available from: www.bashhguidelines.org/media/1223/vvc-2019.pdf. [Last accessed on 2022 Oct 09].  Back to cited text no. 3
    
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Ngouana TK, Toghueo RM, Kenfack IF, Lachaud L, Nana AK, Tadjou L, et al. Epidemiology and antifungal susceptibility testing of non-Albicans Candida species colonizing mucosae of HIV-infected patients in Yaoundé (Cameroon). J Mycol Med 2019;29:233-8.  Back to cited text no. 4
    
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Clinical and Laboratory Standards Institute. Epidemiological Cutoff Values for Antifungal Susceptibility Testing. 3rd ed. CLSI Supplement M59. Wayne, PA: Clinical and Laboratory Standards Institute; 2020.  Back to cited text no. 9
    
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Dharmik PG, Gomashe AV, Upadhyay VG. Susceptibility pattern of various azoles against Candida species causing vulvovaginal candidiasis. J Obstet Gynaecol India 2013;63:135-7.  Back to cited text no. 10
    
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Rathod SD, Klausner JD, Krupp K, Reingold AL, Madhivanan P. Epidemiologic features of vulvovaginal candidiasis among reproductive-age women in India. Infect Dis Obstet Gynecol 2012;2012:859071.  Back to cited text no. 11
    
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Khan M, Ahmed J, Gul A, Ikram A, Lalani FK. Antifungal susceptibility testing of vulvovaginal Candida species among women attending antenatal clinic in tertiary care hospitals of Peshawar. Infect Drug Resist 2018;11:447-56.  Back to cited text no. 12
    
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Aguin TJ, Sobel JD. Vulvovaginal candidiasis in pregnancy. Curr Infect Dis Rep 2015;17:462.  Back to cited text no. 13
    
14.
Ragunathan L, Poongothai GK, Sinazer AR, Kannaiyan K, Gurumurthy H, Jaget N, et al. Phenotypic characterization and antifungal susceptibility pattern to fluconazole in Candida Species isolated from vulvovaginal candidiasis in a tertiary care hospital. J Clin Diagn Res 2014;8:C01-4.  Back to cited text no. 14
    
15.
Dias LB, de Souza Carvalho Melhem M, Szeszs MW, Filho JM, Hahn RC. Vulvovaginal candidiasis in Mato Grosso, Brazil: Pregnancy status, causative species and drugs tests. Braz J Microbiol 2011;42:1300-7.  Back to cited text no. 15
    
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Wang FJ, Zhang D, Liu ZH, Wu WX, Bai HH, Dong HY. Species distribution and in vitro antifungal susceptibility of vulvovaginal candida isolates in china. Chin Med J (Engl) 2016;129:1161-5.  Back to cited text no. 17
    
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Pfaller MA, Diekema DJ. Progress in antifungal susceptibility testing of Candida spp. by use of clinical and laboratory standards institute broth microdilution methods, 2010 to 2012. J Clin Microbiol 2012;50:2846-56.  Back to cited text no. 18
    



 
 
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