Journal of Medical Society

ORIGINAL ARTICLE
Year
: 2021  |  Volume : 35  |  Issue : 3  |  Page : 98--102

E-test antifungal susceptibility profile of Candida spp. associated with vulvovaginal candidiasis in patients of Manipur


Sudipta Naorem1, Ajit Kumar Singh Yanglem2, Randhir Babu Singh Yendrembam3, Ranjit Singh Laiphrakpam2, Ranjana Devi Khuraijam4,  
1 Microbiologist, Manipur Health Services, State Medical Services, Imphal, Manipur, India
2 Department of Obstetrics and Gynaecology, Institute of Bioresources and Sustainable Development, Imphal, Manipur, India
3 Department of Microbial Resources Division, Institute of Bioresources and Sustainable Development, Imphal, Manipur, India
4 Department of Microbiology, Regional Institute of Medical Sciences, Institute of Bioresources and Sustainable Development, Imphal, Manipur, India

Correspondence Address:
Randhir Babu Singh Yendrembam
Microbial Resources Division, Institute of Bioresources and Sustainable Development, Takyelpat, Imphal - 795 001, Manipur
India

Abstract

Background and Objectives: Vulvovaginal candidiasis (VVC) is one of the most common vaginal infections during fertile period of women. An increase in the prevalence of non-albicans Candida which are resistant to commonly used antifungals has been documented. Therefore, studying the antifungal susceptibility pattern of the causative agents is of great significance in successfully treating the ailment and understanding the local data. Materials and Methods: Forty-six Candida spp. isolated from VVC patients were subjected to antifungal minimum inhibitory concentration testing for itraconazole, fluconazole, and voriconazole using E-test method. Results: Candida albicans and Candida glabrata showed 87.8% and 60% sensitivity, respectively, to itraconazole. Twenty percent of Candida parapsilosis and 40% of C. glabrata were resistant to fluconazole. Voriconazole showed higher sensitivity with 0.013 μg/ml as the minimum concentration to inhibit all Candida spp. C. glabrata noted higher minimum inhibitory concentrations against itraconazole, fluconazole, and voriconazole. Interpretation and Conclusion: Voriconazole is the drug of choice in case of fluconazole treatment failure among VVC.



How to cite this article:
Naorem S, Singh Yanglem AK, Singh Yendrembam RB, Laiphrakpam RS, Khuraijam RD. E-test antifungal susceptibility profile of Candida spp. associated with vulvovaginal candidiasis in patients of Manipur.J Med Soc 2021;35:98-102


How to cite this URL:
Naorem S, Singh Yanglem AK, Singh Yendrembam RB, Laiphrakpam RS, Khuraijam RD. E-test antifungal susceptibility profile of Candida spp. associated with vulvovaginal candidiasis in patients of Manipur. J Med Soc [serial online] 2021 [cited 2022 Nov 30 ];35:98-102
Available from: https://www.jmedsoc.org/text.asp?2021/35/3/98/347640


Full Text



 Introduction



Vulvovaginal candidiasis (VVC) is a frequent gynecologic ailment, affecting three out of four women in their lifetime according to Das-Neves et al.,[1] and 40% of the affected women have two or more VVC episodes as per studies from Ferrer[2] and Eschenbach.[3] Similarly, Fidel and Sobel estimated that 70%–75% of women of childbearing age worldwide experience at least one episode during their lifetime and 5%–10% of women with a primary episode of VVC experience frustrating recurrent VVC.[4] Risk factors associated with VVC include diabetes mellitus, pregnancy, immunosuppression, contraception, antibiotic therapy, genetic factors, dietary factors, sexual and behavioral factors, immunological factors, and microbial factors as shown in a study by Leema.[5] A wide species of Candida can cause VVC. Etiological agents, namely Candida albicans, Candida tropicalis, Candida krusei, Candida parapsilosis, Candida glabrata, Candida stellatoidea, and Candida guilliermondii, have been forwarded by Vijaya et al.[6] and Candida africana and Candida dubliniensis have been reported by Hazırolan et al.[7] For treatment of VVC, accurate diagnosis of the causative agent is warranted so as to choose the best antifungal agent for effective treatment and to avoid Recurrent vulvovaginal candidiasis (RVVC). Antifungal agents employed for VVC and RVCC management comprises fluconazole, clotrimazole, itraconazole, flucytosine, econazole, metronidazole, miconazole, tioconazole, voriconazole, and isoconazole as documented by Lema[5] and Himmat et al.[8] Apart from species identification, knowledge of antifungal sensitivity pattern of the regional isolates is strongly advocated for successful treatment of VVC. In this context, the study was performed to isolate and identify the causative agents of VVC from patients attending a tertiary care hospital in Northeast India. Furthermore, minimum inhibitory concentration assay of commonly used antifungal agents against the etiological agents was determined so as to aid the clinician in choosing the best antifungal drug for effective treatment.

 Materials and Methods



Specimen collection

Vaginal swabs were collected from patients with clinical and symptomatic indications of VVC, attending the Obstetrics and Gynaecology Department of the study center between November 2013 and September 2015. Two high vaginal swabs collected with sterile cotton swabs were immediately transferred to the microbiology laboratory without delay for further processing.

Processing and culture of specimens

Direct microscopy of specimens for detection of fungal elements was performed through 10% KOH wet mount and Gram staining using one of the swabs. The second swab was used for inoculation in Sabouraud dextrose agar (SDA) and incubated at 25°C and examined for growth every day. Whenever there was growth, it was confirmed by Gram stain and subjected to further processing.

Identification and speciation

Isolation and identification of Candida spp. were done by phenotypic characterization, namely germ tube test, growth in corn meal agar, and sugar assimilation assay according to standard operating procedure. In addition, Candida speciation was performed using CHROMagar (HiMedia, Mumbai, India) and VITEK 2 compact (bioMérieux, France).

Minimum inhibitory concentration assay

Antifungal susceptibility testing was done by E-test method using Ezy minimum inhibitory concentration (MIC) strip (HiMedia, Mumbai, India) according to CLSI (M27-A2) guidelines for the antifungals itraconazole, fluconazole, and voriconazole.[9] Briefly, fungal suspension was prepared to achieve an optical density of 0.20 at 620 nm using sterile distilled water. The suspension was inoculated onto SDA plates through cotton swab, and onto the center of the plates, MIC strips were placed aseptically. MICs were interpreted as susceptible (S), susceptible dose dependent (S-DD), and resistant (R) according to manufacturer's instruction. C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 were included as quality control strains for MIC determination.

Interpretation of results

  • Itraconazole – ≤0.125 μg/ml (S), 0.25–0.5 μg/ml (S-DD), ≥1 μg/ml (R)
  • Fluconazole – ≤8 μg/ml (S), 16–32 μg/ml (S-DD), ≥64 μg/ml (R)
  • Voriconazole – ≤1 μg/ml (S), 2 μg/ml (S-DD), ≥4 μg/ml (R).


 Results and Discussion



A total of 46 vaginal swabs were collected and processed. All the specimens were 10% KOH and Gram staining positive of fungal elements. In addition, 100% of the specimens showed growth of Candida spp. Based on phenotypic characters, CHROMagar and VITEK 2 compact analysis, 33 (71.7%) C. albicans, 5 (10.8%) C. glabrata, 5 (10.8%) C. parapsilosis, and 3 (6.5%) C. tropicalis were isolated and identified. Similarly, Vijaya et al. reported C. albicans 35 (66.0%) as the most common species isolated, followed by C. tropicalis 14 (26.4%), C. krusei 2 (3.8%), and C. parapsilosis and C. glabrata in 1 (1.9%) case each.[6]

The tested Candida spp. showed varying susceptibility patterns [Table 1] and [Table 2]. With respect to itraconazole, C. albicans and C. glabrata showed 87.8% and 60% sensitivity, respectively. Nevertheless, C parapsilosis and C. tropicalis were 100% sensitive. Although C. albicans and C. tropicalis were sensitive to fluconazole, 20% and 40% of C. parapsilosis and C. glabrata were resistant. Interestingly, voriconazole noted higher sensitivity compared to itraconazole and fluconazole, with 0.013 μg/ml as the minimum concentration to inhibit Candida spp. Of all the Candida spp. tested, C. glabrata noted higher MICs against itraconazole, fluconazole, and voriconazole. An isolate of C. glabrata required as high as 4 μg/ml of itraconazole, whereas the rest of the Candida spp. were inhibited at ≤0.75 μg/ml.{Table 1}{Table 2}

The potential clinical importance of species-level identification has been recognized as Candida spp. varies in the expression of virulence factors and antifungal susceptibility. Foxman et al. stated that most non-albicans Candida spp. have higher MICs than the azole antifungal agents and infections they cause are often difficult to treat.[10] Hence, the present finding of non-albicans Candida having higher MIC is a cause of concern for clinicians and patients. With respect to itraconazole, the MIC range of C. albicans was 0.016–0.75 μg/ml, however, a previous study from Pfaller et al. noted 0.008–≥8 μg/ml which is higher to the present finding.[11] According to Pfaller et al., itraconazole was quite active against most Candida spp. (modal MICs, 0.03–0.12 μg/ml), with the exception of C. glabrata (modal MIC, 0.5–1 μg/ml), C. krusei (modal MIC, 0.25–0.5 μg/ml), and C. guilliermondii (modal MIC, 0.5 μg/ml).[11] Interestingly, the present study determined C. glabrata MIC range with 0.047–4 μg/ml which is relatively higher.

Dror et al. highlighted that the definition of in vitro non-albicans Candida susceptibility to fluconazole in the context of vaginal infections remains unvalidated in contrast to systemic infections.[12] In addition, Rex et al. also highlighted that previous determination of Candida species breakpoints was based on studies conducted on blood and oropharyngeal isolates, ignoring specific pharmacokinetic considerations applicable to the vagina.[13]

Srujana et al. acknowledged that 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 μg/ml.[14] Even though there is no fluconazole resistance C. albicans in this study, a previous study by Dror et al. reported and highlighted the importance of fluconazole resistance C. albicans.[12] In addition, an Indian population study by Srujana et al. reported no fluconazole resistance non-albicans Candida which is in contrast to the present finding imparting the difference of MIC pattern depending upon region.[14] Arikanand and Rex underscored increased dose-dependent resistance non- albicans Candida spp. may change in the optimal therapy of non-albicans Candida vaginitis.[15] This emphasized the need to determine the antifungal susceptibility of local Candida isolates at regular intervals for effective treatment.

Voriconazole is a synthetic triazole derived from fluconazole. It is also effective on fluconazole-resistant Candida strains. Nevertheless, Fahriye et al. stated that a significant number of fluconazole-resistant Candida isolates also become resistant to voriconazole as a result of cross-resistance besides being resistant to ketoconazole and itraconazole.[16] Although the study found all isolates to be sensitive to voriconazole, a previous study from Espinel-Ingroff et al. reported voriconazole resistance in 1.72% of C. tropicalis strains and 1.72% of C. glabrata as the dose-dependent susceptible strain.[17] Espinel-Ingroff et al. found that C. albicans were sensitive, yet 4.3% of non-albicans isolates were resistance to voriconazole.[17] In congruence with the present study, an earlier study from Aydin et al. did not detect any voriconazole resistance in 166 Candida strains.[18] These findings yet again proved that sensitivity pattern varies from place to place. Hence, determination of MIC of regional isolates at regular interval plays a vital role in effective management of microbial infections.

 Conclusion



Candidal vaginitis remains to be a significant burden globally including the state of Manipur, India. The MIC findings are of great importance not only for prompt and effective treatment of the cases but also to understand the changing species spectrum and antifungal sensitivity. The study advocates voriconazole as the drug of choice if VVC does not recover from fluconazole medication.

Financial support and sponsorship

This study was financially supported by the Department of Biotechnology, Government of India.

Conflicts of interest

There are no conflicts of interest.

References

1das Neves J, Pinto E, Teixeira B, Dias G, Rocha P, Cunha T, et al. Local treatment of vulvovaginal candidosis: General and practical considerations. Drugs 2008;68:1787-802.
2Ferrer J. Vaginal candidosis: Epidemiological and etiological factors. Int J Gynaecol Obstet 2000;71 Suppl 1:S21-7.
3Eschenbach DA. Chronic vulvovaginal candidiasis. N Engl J Med 2004;351:851-2.
4Fidel PL Jr., Sobel JD. Immunopathogenesis of recurrent vulvovaginal candidiasis. Clin Microbiol Rev 1996;9:335-48.
5Lema VM. Recurrent vulvo-vaginal candidiasis: Diagnostic and management challenges in a developing country context. Obstet Gynecol Int J 2007;7:00260.
6Vijaya D, Dhanalakshmi TA, Kulkarni S. Changing trends of vulvovaginal candidiasis. J Lab Physicians 2014;6:28-30.
7Hazirolan G, Altun HU, Gumral R, Gursoy NC, Otlu B, Sancak B. Prevalence of Candida africana and Candida dubliniensis, in vulvovaginal candidiasis: First Turkish Candida africana isolates from vulvovaginal candidiasis. J Mycol Med 2017;27:376-81.
8Himmat SJ, Tarun G, Goutam R, Goyal AK. Advanced topical drug delivery system for the management of vaginal candidiasis. Drug Deliv 2016;23:550-63.
9Reference method for broth dilution antifungal susceptibility testing of yeasts. In: Approved Standard – Second Edition. 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA: NCCLS; 2002
10Foxman B, Barlow R, D'Arcy H, Gillespie B, Sobel JD. Candida vaginitis: Self-reported incidence and associated costs. Sex Transm Dis 2000;27:230-5.
11Pfaller MA, Espinel-Ingroff A, Canton E, Castanheira M, Cuenca-Estrella M, Diekema DJ, et al. Wild-type MIC distributions and epidemiological cutoff values for amphotericin B, flucytosine, and itraconazole and Candida spp. as determined by CLSI broth microdilution. J Clin Microbiol 2012;50:2040-6.
12Marchaim D, Lemanek L, Bheemreddy S, Kaye KS, Sobel JD. Fluconazole-resistant Candida albicans vulvovaginitis. Obstet Gynecol 2012;120:1407-14.
13Rex JH, Rinaldi MG, Pfaller MA. Resistance of Candida species to fluconazole. Antimicrob Agents Chemother 1995;39:1-8.
14Mohanty S, Xess I, Hasan F, Kapil A, Mittal S, Tolosa JE. Prevalence & susceptibility to fluconazole of Candida species causing vulvovaginitis. Indian J Med Res 2007;126:216-9.
15Arikan S, Rex JH. Antifungal agents. In: Murray PR, Baron EJ, Landry ML, Jorgensen JH, Pfaller MA, editors. Manual of Clinical Microbiology. Washington, DC, USA: ASM Press; 2007. p. 1949-60.
16Eksi F, Gayyurhan ED, Balci I. In vitro susceptibility of Candida species to four antifungal agents assessed by the reference broth microdilution method. ScientificWorldJournal 2013;2013:236903.
17Espinel-Ingroff A, Canton E, Gibbs D, Wang A. Correlation of neo-sensitabs tablet diffusion assay results on three different agar media with CLSI broth microdilution M27-A2 and disk diffusion M44-A results for testing susceptibilities of Candida spp. and Cryptococcus neoformans to amphotericin B, caspofungin, fluconazole, itraconazole, and voriconazole. J Clin Microbiol 2007;45:858-64.
18Aydin F, Bayramoglu G, Guler NC, Kaklikkaya N, Tosun I. Bloodstream yeast infections in a university hospital in Northeast Turkey: A 4-year survey. Med Mycol 2011;49:316-9.