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ORIGINAL ARTICLE
Year : 2018  |  Volume : 7  |  Issue : 2  |  Page : 94-97

Speciation and antifungal susceptibility profiles of Candida isolates from vaginitis patients attending STD Clinic at a Tertiary Care Hospital


Department of Microbiology, Osmania Medical College, Hyderabad, Telangana, India

Date of Web Publication6-Jun-2018

Correspondence Address:
Dr. G Sasikala
Department of Microbiology, Osmania Medical College, Hyderabad, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JDRNTRUHS.JDRNTRUHS_33_17

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  Abstract 


Back ground: Candidiasis is the most common vaginal infection affecting approximately 50–72% of women. Rapid identification of yeast isolates to species level is essential to optimize antifungal treatment.
Aim: To determine the prevalence of various Candida species among vaginal candidiasis and to determine the antifungal susceptibility pattern of the isolates.
Materials and Methods: A total of 471 women who were clinically diagnosed to have vaginal candidiasis were included in the study. Out of 471 vaginitis patients, 91 were positive for Candida species. All the isolates were speciated comprising five species – C. albicans 42 (46.1%), C. krusei 5 (5.5%), C. glabrata 40 (43.9%), C. tropicalis 3 (3.3%), and C. gullermondi 1 (1.1%). Antifungal susceptibility testing result of all Candida isolates are 100% susceptible to amphotericin B, nystatin, flucytosine, econazole, ketoconazole, miconazole, fluconazole. C. krusei isolates are showing 100% resistance to fluconazole.
Discussion: In the present study, C. albicans is most common species 46.1% followed by C. glabarata. C. albicans adhere to vaginal, epithelial cells in significantly higher number than other Candida species. This could explain relative higher frequency of C. albicans in vaginal candidiasis.
Conclusion: Presumptive identification followed by confirmation of Candida species helps to initiate early appropriate antifungal treatment, thereby reducing the morbidity and mortality.

Keywords: Antifugal susceptibility2, candida, candifast, vaginitis


How to cite this article:
Sasikala G, Udayasri B. Speciation and antifungal susceptibility profiles of Candida isolates from vaginitis patients attending STD Clinic at a Tertiary Care Hospital. J NTR Univ Health Sci 2018;7:94-7

How to cite this URL:
Sasikala G, Udayasri B. Speciation and antifungal susceptibility profiles of Candida isolates from vaginitis patients attending STD Clinic at a Tertiary Care Hospital. J NTR Univ Health Sci [serial online] 2018 [cited 2018 Aug 19];7:94-7. Available from: http://www.jdrntruhs.org/text.asp?2018/7/2/94/233843




  Introduction Top


Candidiasis is the most common vaginal infection in most countries affecting approximately 50–72% of women.[1],[2] Vaginal candidiasis is second to bacterial vaginosis as most common mucosal infections that affect large number of otherwise healthy women of childbearing age group.[3],[4],[5] Up to 75% of reproductive age women are infected with vaginal candidiasis at least once and about half of these women experience recurrence.[6],[7] In addition to discomfort and the cost associated with medication and health care visits, several studies have suggested that vaginal candidiasis may increase a woman's risk of contracting other sexually transmitted diseases such as human immunodeficiency virus (HIV).[8] Vaginal candidiasis if untreated can lead to chorioamnionitis with subsequent abortion, prematurity and congenital infection of the neonate in pregnant women, and pelvic inflammatory disease resulting in infertility in nonpregnant women.[9]

The lack of specificity of symptoms and signs in vulvovaginal candidiasis explains the need for laboratory confirmation by culture.[10]Candida albicans is the most commonly isolated species. More recently, non-albicans Candida (NAC) species have been recovered with increasing frequency, which are known for their variable resistance to azoles. To avoid selection of less susceptible NAC species by empirical antifungal treatment or prophylaxis, speciation of Candida isolates is essential in routine specimen processing.[11],[12]

Consequently, the present study was undertaken to determine the prevalence of various Candida species among vaginal candidiasis and to determine the antifungal susceptibility pattern of the isolates. Such data will provide important information in developing effective strategies for prevention and possible treatment option for vaginal candidiasis.


  Materials and Methods Top


The present study was conducted over a period of 1 year from April 2014 to March 2015 at an STD clinic, OGH, Hyderabad. A total of 471 women of childbearing age group of 19–45 years with complaints of vaginal discharge, itching, dyspareunia, low backache, and pain in the lower abdomen and clinically diagnosed as vaginal candidiasis were included in the study group. Prior consent was taken from every patient before sample collection. Secretions from posterior vaginal fornix were collected with the help of sterile disposable pipette from every patient under aseptic precautions with the help of sperculum and posterior vaginal wall retractor.

These secretions were inoculated on Sabourauds dextrose agar (SDA) tubes with chloramphenicol and CHROM agar plates. Direct smears were prepared from the sample and Gram's staining was done according to the standard protocol. The inoculated culture tubes and plates were incubated at 37°C for 48 hours. Colonies suggestive of Candida species were further identified and speciated by Gram's staining, Germ tube test, Chlamydospore formation on corn meal agar, and reactions based on candifast kit (ELITech MICROBIO 83870 SIGNES (FRANCE).[13],[14]

Candifast test is a combined test that allows the identification of Candida species with an antifungal resistant test adapted against amphotericin B, nystatine, flucytosine, econazole, ketoconazole, miconazole, fluconazole.

Principle of the Candifast: Identification of yeast is based on the sensitivity of the strain to the actidione, visualized by the turn to yellow or yellow-orange. The study of fermentation of seven sugars visualized by the turn to yellow or yellow-orange of the indicator colored due to acidification of the medium. The detection of a urease activity that alkalinizes the medium and turns the colored indicator to fuchsia. Determination of yeast resistance to antifungal agents is based on the growth or not of these yeasts in the presence of different antifungal agents. This growth is visualized by a color change of the medium. The fermentation of glucose by the yeasts results it an acidification of the medium that turns the phenol red present in the middle of orange red to yellow or yellow-orange.

The kit is provided with R1 solution (4 mL vial of buffered medium for dilution and identification), R2 solution [2 mL vial of YNB (yeast nitrogen base) medium containing urea and for resistance testing phenol red], TC (4 mL vial of barium sulphate solution for the control of inoculum standardization), and candifast tray (20-well tray individually packed in an aluminum sachet each tray allows the testing of one sample identification + resistance).

It is a 20-well tray with two rows, one for identification and the other for susceptibility testing. The determination of resistance of yeasts to antifungal agents is based on the growth or absence of growth of yeasts in the presence of various antifungal agents. An isolated colony of Candida was inoculated into reagent-1 (R1) bottle and mixed well. The turbidity of the suspension was compared with the turbidity control. One hundred microliter of inoculated R1 was added to R2, and 100 μl of R2 was then added to each of the wells; 2 drops of paraffin oil was added to each well and the test tray was sealed and incubated at 37°C for 24 hours. Readings were taken once the yeast grew in the control well. The indicator used is phenol red. A yellow or orange-yellow color in the susceptibility test row due to glucose fermentation indicated that the yeast was able to grow in the presence of the antifungal agent and hence was resistant to that drug. If the color in the well was red or pink, the isolate was inhibited by the drug in that well and so was sensitive to that drug. The susceptibility testing by Candifast kit can be done for the following antifungal drugs: amphotericin B (4 μg/ml), fluconazole (16 μg/ml), ketoconazole (16 μg/ml), nystatin (200 units/ml), flucytosine (35 μg/ml), econazole (16 μg/ml), and miconazole (16 μg/ml).

Candida species identified according to biochemical reactions and antifungal susceptibility patterns were recorded.


  Results Top


A total of 471 women were included in the study. The age distribution of participants less than 19 years of age 30 (6.4%), 19–24 years of age 57 (12.2%) cases, 25–40 years of age 352 (74.73) cases, and more than 40 years of age 27 (5.73) cases [Table 1].
Table 1: Age distribution

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Out of 471 vaginitis patients, 91 were positive for Candida species. Age distribution of confirmed cases of vaginal discharge was less than 19 years age group 1 (1.1%), 19–24 years age group 15 (16.48%) cases, 25–40 years age group 70 (77.1%) cases, and more 40 years age group 5 (5.5%) cases.

All the isolates were speciated comprising five species C. albicans 42 (46.1%), C. krusei 5 (5.5%), C. glabarata 40 (43.9%), C. tropicalis 3 (3.3%), C. gullermondi 1 (1.1%) [Table 2].
Table 2: Distribution of Candida species

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Antifungal susceptibility testing result of all Candida isolates were 100% susceptible to amphotericin B, nystatine, flucytosine, econazole, ketoconazole, miconazole, fluconazole. Except C. krusei isolates which showed 100% resistance to fluconazole [Figure 1].
Figure 1: Diagram showing antifungal susceptibility of isolates

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  Discussion Top


Bacterial vaginosis, candidiasis, and trichomoniasis are responsible for 90% vaginal infections. In the present study, vaginal candidiasis was found in 91 (19.8%) symptomatic women.[1],[10],[15] in the present study, the highest incidence of vaginal candidiasis was seen in 25–40 years age group.

Vaginal discharge culture is the most sensitive method compared to Gram stain and other tests.[9] In the present study, similar results were obtained by chrome agar and Candifast test. In the present study, C. albicans is the most common species (46.1%) followed by C. glabrata. C. albicans adhere to vaginal, epithelial cells in significantly higher number than other Candida species. This could explain relative higher frequency of C. albicans in vaginal candidiasis. Current trends show an increased prevalence of non-albicans Candida which was significantly higher in present study. The possible reason for this may be the indiscriminate use of antifungals which eliminates more sensitive C. albicans and selects azole resistant non-albicans Candida.[15]

In the present study, C. glabrata is the most common non-albicans Candida.[3],[10],[16] It has been stated, that given the variety of vaginal candidiasis caused by resistant Candida strains, susceptibility testing was rarely indicated. However, in the present study, C. krusei isolates are found to be resistant to fluconazole. Hence, there is a need for routine antifungal susceptibility testing of all Candida isolates. In the present study, C. albicans and majority of non-albicans Candida species are susceptible to voriconazole and fluconazole.


  Conclusion Top


Changing trends in the antifungal susceptibility testing towards fluconazole recommends routine antifungal susceptibility testing of Candida isolates in clinical microbiology laboratories. Presumptive identification followed by confirmation of Candida species helps to initiate early appropriate antifungal treatment there by reducing the morbidity and mortality.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Esmaeilzadeh S, Omran SM, Rahmani Z. Frequency and etiology of vulvovaginal candidiasis in women referred to a gynecological center in Babol, Iran. Int J Fertil Steril 2009;3:74-7.  Back to cited text no. 1
    
2.
Nabhan A. Vulvovaginal candidiasis. Ain Shams J Obstet Gynecol 2006;3:73-8.  Back to cited text no. 2
    
3.
Vijaya D, Dhanalakshmi TA, Kulkarni S. Changing trends of vulvovaginal candidiasis. J Lab Physicians 2014;6:28-30.  Back to cited text no. 3
[PUBMED]  [Full text]  
4.
Kumari V, Banerjee T, Kumar P, Pandey S, Tilak R. Emergence of non-albicans Candida among candidal vulvovaginitis cases and study of their potential virulence factors, from a tertiary care center, North India. Indian J Pathol Microbiol 2013;56:144-7.  Back to cited text no. 4
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Nwadioha SI, Bako IA, Onwuezobe I, Egah DZ. Vaginal trichomoniasis among HIV patients attending primary health care centers of Jos, Nigeria. Asian Pacific J Trop Dis 2012;2:337-41.  Back to cited text no. 5
    
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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. Infectious Dis Obstet Gynecol 2012;2012.  Back to cited text no. 6
    
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Sobel JD, Faro S, Force RW, Foxman B, Ledger WJ. Vulvovaginal candidiasis: Epidemiologic, Diagnostic and therapeutic considerations. Am J Obstet Gynecol 1998:178:203-11.  Back to cited text no. 7
    
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Hester RA, Kennedy SB. Candida infection as a risk factor for HIV transmission. J Women's Health 2003;12:487-94.  Back to cited text no. 8
    
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Omar AA. Gram stain versus culture in the diagnosis of vulvovaginal Candidiasis. East Mediterr Health J 2001;7:925-34.  Back to cited text no. 9
    
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Neerja J, Aruna A, Paramjeet G. Significance of Candida culture in women with vulvovaginal symptoms. J Obstet Gynecol India 2006;56:139-41.  Back to cited text no. 10
    
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Vijaya D, Harsha TR, Nagarathnamma T. Candida speciation using chrom agar. J Clin Diagn Res 2011;5:755-7.  Back to cited text no. 11
    
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Agarwal S, Manchanda V, Verma N, Bhalla P. Yeast identification in routine clinical microbiology laboratory and its clinical relevance. Indian J Med Microbiol 2011;29:172-7.  Back to cited text no. 12
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Milne LJ. Fungi. In: Collee JG, Frazer AG, Marmion BP, Simmons A, editors. Mackie McCartney-Practical Medical Microbiology. 14th ed. Edinburgh: Churchill Living Stone; 1996. p. 697-717.  Back to cited text no. 13
    
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Chander J. Text book of Medical Mycology. 2nd ed. New Delhi: Mehtha Publishers; 2002. p. 212-27.  Back to cited text no. 14
    
15.
Nabhan A. Vulvovaginal candidiasis. Ain Shams J Obstet Gynecol 2006;3:73-8.  Back to cited text no. 15
    
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Oviasogie FE, Okungbowa FI. Candida species amongst pregnant women in Benin city, Nigeria: Effect of predisposing factors. Afr J Clin Expert Microbiol 2009;10:92-8.  Back to cited text no. 16
    


    Figures

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    Tables

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