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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2013  |  Volume : 56  |  Issue : 2  |  Page : 144-147
Emergence of non-albicans Candida among candidal vulvovaginitis cases and study of their potential virulence factors, from a tertiary care center, North India


1 Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
2 Department of Gynaecology and Obstetrics, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

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Date of Web Publication23-Sep-2013
 

   Abstract 

Purpose: The purpose of this study was to determine the prevalence of various Candida species and study some of their virulence factors among thevulvovaginal candidiasis(VVC)patients. Study Design and Settings: The study was conducted in a Tertiary Care University Hospital in North India. Materials and Methods: This study was carried out prospectively for a period of 1 year. High vaginal swabs (HVSs) were collected from women in childbearing age group attending the gynecology and obstetrics out-patient departments with the complaints suggestive of vulvovaginitis. Samples were plated on Sabouraud's dextrose agar slope. Candida spp. isolated was further speciated based on microscopy, biochemical tests and culture characteristics on special media. Virulence factors of these strains were determined by biofilm formation and phospholipase activity. Result: A total of 464 HVS from 232 patients with the complaints of vulvovaginitis were included in this study. Following laboratory workup, 71 specimens were positive for genus Candida (30.6%). Further speciation showed 32.4% as Candida albicans, 45.07% Candida parapsilosis and 22.53% of Candida glabrata. Biofilm production was shown by 50 candidal strains (70.4%) and phospholipase activity was given by 41 candidal strains (57.74%). Conclusion: Our study suggests increasing prevalence of non-albicans Candida among the VVC cases along with their virulence factors. Therefore, we recommend that microbiological investigation upto species level should be mandatory to determine the emergence of non-albicans Candida as a major cause of VVC.

Keywords: Biofilm, non-albicans, phospholipase, vulvovaginal candidiasis

How to cite this article:
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

How to cite this URL:
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 [serial online] 2013 [cited 2019 Dec 16];56:144-7. Available from: http://www.ijpmonline.org/text.asp?2013/56/2/144/118703



   Introduction Top


Fungal infections have become a prominent problem over the last decade mainly due to world-wide increase in the number of immunocompromised patients, who are highly susceptible to opportunistic infections, including mycoses. [1] Vulvovaginal candidiasis (VVC) is defined as signs and symptoms of inflammation of the vulva and vagina in the presence of Candida spp. And in the absence of other infectious etiology. [2] The most common clinical manifestations of VVC are pruritus, hyperemia, vaginal discomfort and leucorrhea, burning, soreness, dyspareunia and vaginal or vulvar erythema, which may cause a problem in marital and sexual relations. [3] Candidal vulvovaginitis is a common infection in females, primarily during the fecund period. Prevalence of these infections increase in particular groups, such as pregnant or diabetic women, those using oral contraceptives or after antibiotic treatment. The predisposing factors can be associated primarily, with the host and also with some virulence attributes of the pathogens such as increased adherence ability. [4] Although VVC is the most common fungal disease in the world, little information is known about the distribution and etiology of candidiasis because microbiology tests are not routinely performed in most of the laboratories. [5]

The present study was undertaken to determine the prevalence of various Candida species among the VVC patients and study of the important virulence factors.


   Materials and Methods Top


A total of 232 women within the child-bearing age group of 19-45 years with the complaints of vaginal discharge, itching, dyspareunia, lowback-ache and pain in the lower abdomen, attending the obstetrics and gynecology out-patients department and clinically diagnosed as VV cases were included in this study. Prior consent was taken from every patient before sample collection and the study was approved by the ethical committee. A pair of high vaginal swab (HVS) from every patient was collected aseptically with the help of speculum and posterior vaginal wall retractor. One swab was used for preparation of smear for Gram's staining and the other was inoculated on Sabouraud's dextrose agar (SDA) slope containing chloramphenicol and incubated at 37°Cup to 7 days with daily inspection. Colonies suggestive of Candida spp. in SDA tube were further identified and speciated by Gram's staining, germ-tube test, chlamydospores formation, sugar fermentation test, sugar assimilation test and color production on CHROM agar (HiMedia, Mumbai). [6] The strains were maintained in stock media (prepared by mixing 85 ml of distilled water and 15 ml of glycerol) at –70°C with regular subculture for future reference.

For detection of biofilm production, a loopful of yeast from SDA was inoculated in 15ml SD broth in polypropylene 'falcon tube' and incubated for 48h at 35°C. After 48h, the tubes were aspirated gently and washed with distilled water twice and then stained with 1% safranin for 10 min, after which they were examined on walls for the presence of an adherent layer. Each isolate was tested twice and read independently by two different observers. Biofilm production was scored as negative(-), weak (+), moderate (++), or strong (+++) positive based on the protocol given elsewhere. [7],[8] Biofilm producer Staphylococcus epidermidis ATCC 35984 was used as a positive control. For phospholipase activity, about 10μl of yeast suspension in sterile saline were placed on the surface of agar medium containing egg yolk (pH = 4.3). The plates were incubated at 37°C for 4 days. The presence of phospholipase was determined by the formation of an opaque zone around the yeast colonies. Enzyme activity precipitation zone (PZ) was measured by dividing the diameter of the colony by the diameter of the colony plus PZ. A PZ of 1.0 was evaluated as negative (-), 0.99-0.9 as weak (+), 0.89-0.8 as mild (++), 0.79-0.7 as relatively strong (+++) and <0.69 (++++) as very strong positive. [9] The Candida albicans ATCC 90028 strain was used as a positive control.


   Results Top


Out of 232 vulvovaginitis patients, 71 were positive for Candida species (30.60%). The Gram's staining findings and culture characteristics on CHROM agar media have been shown in [Figure 1] and [Figure 2] respectively. All the isolates were speciated comprising of three species, namely C.albicans (32.39%), Candida parapsilosis (45.07%) and Candida glabrata (22.53%). Themorphological and biochemical features of the isolates have been summarized in [Table 1].
Figure 1: Gram's staining of different Candida spp

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Figure 2: Color production of different Candida spp on CHROM agar

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Table 1: Morphological features and biochemical properties of the candidal isolates

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Biofilm production was detected in 50 strains (70.4%) of which as shown in [Figure 3], majority of which were among the non-albicans Candida (81.25%). Different scores of biofilm production by different species of Candida have been tabulated in [Table 2].
Figure 3: Biofilm production by various candidal strains

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Table 2: Different scores of biofilm production by various Candida spp

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Phospholipase activity was seen in 41 strains (57.74%) as shown in [Figure 4], of which 30.43% of C.albicans, 81.25% of C. parapsilosis and 18.75% of C. glabrata were strongly positive as shown in [Table 3]. None of the phospholipase positive Candida spp. showed weak phospholipase activity.
Figure 4: Phospholipase activity on egg-yolk media

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Table 3: Different PZ values of different Candida spp

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   Discussion Top


Candidal vulvovaginitis is a global problem influencing millions of women annually. At the same time, it is important to emphasize that in the past three decades, there has been an increasing percentage of infections caused by non-albicans Candida species. We found a prevalence of 30.6% candidal vulvovaginitis in our setup, which was higher than the previous reports of 20.47% and 26.6%. [10],[11] This study clearly documented the increasing prevalence of non-albicans Candida spp. among the vulvovaginitis cases in the child bearing age group.

In this study, Gram's staining from HVS material showed budding yeast cells with pseudohyphae in only 21 cases, thus showing positivity in only 9.05% of the cases. Similarly, there are reports of 27.84% positivity of yeast from microscopic examination of direct specimen. [10] Another study from India [11] showed 167 positive microscopic finding out of 1050 patients (15.90%). The difference insensitivity of direct microscopy may be due to the difference in the concentration of yeast in different vaginal secretions. [12],[13] With the issue like insufficient clinical diagnosis and failure of simple microscopy in screening cases other than severe cases, vaginal swab cultures are important for diagnosing VVC.

Different laboratory tests revealed the presence of three Candida species in the present study, with overall percentage of non-albicans Candida species much higher (67.6%). According to a comparable study on 1050 women, [13] it was indicated that 215 women were associated with candidal vulvovaginitis and the highest frequency was related to C.albicans (46.9%) out of six Candida species. The percentage of non-albicans species was 53.04%, composing of C.glabrata (36.7%), C.parapsilosis (10.2%), Candida tropicalis (2.8%), Candida kefyr (1.9%) and Candida krusei (1.4%). Another study from India [14] revealed the overall percentage of non-albicans vaginitis as 64.8%. These studies showed the increasing trends of non-albicans Candida. Though the number of isolation of Candida species in the present study were less (three) than the previous studie who isolated six and seven species respectively. [11],[15] However, in all these studies predominance of non-albicans Candida was notably seen. The difference might be due to fewer patients enrolled in the study or the level of social activities, drug abuse,sexual promiscuity and environmental variation.

As has been hypothesized previously, more frequent isolation of non-albicans species from vulvovaginitis patients might be due to widespread and inappropriate use of antimycotic treatment in the form of self-medication, long-term maintenance treatments and repeated treatments for candidiasis episodes, use of a single dose oral and topical azoles. C.albicans eradication by these means result in the selection of species such as C. glabrata that are resistant to commonly used agents. [16]

Candida species have the ability to produce virulence factors that enhances their capacity to colonize mucosal surfaces and to invade host tissues by disrupting host-cell membranes. Phospholipases act as a virulence factor by degrading cell membranes. In this study, the phospholipase activity was more associated with non-albicans Candida species. Phospholipase positivity among non-albicans Candida strains was reported in some studies. [17],[18],[19] It should be mentioned that the role of phospholipases in C.parapsilosis pathogenesis is less clear as there have been contradictory findings, with some investigators reporting phospholipase activity in as many as 51% of C.parapsilosis strains [20] and others finding no activity. [17],[21],[22] Such in consistencies in data could be the result of relatively small sample sizes as well as the biological differences between the tested strains.

Although bacterial biofilms and their role in diseases have been investigated in details over a number of years, much less is known about fungal biofilms. Studies, [7],[23] investigating biofilm production among Candida species detected higher levels of positivity among non-albicans Candida strains than that of C.albicans strains (51-61% among non-albicansvs.8-12% among albicans spp.). Similar findings were reflected in the present study too.

This study documents the scenario in north India in accordance to the changing trends of causative role of candidal vulvovaginitis in different studies from different parts of the world and from India. The emergence of nonalbicans Candida spp. from VVC cases and their association with virulence factors cannot be overlooked. We suggest increased isolation and complete identification of Candida spp. In all microbiology laboratories so that the epidemiology, emergence and spread of non-albicans Candida could be revealed. As treatment of these cases are quite different from the C.albicans VVC cases, [24] their complete identification prior to therapy is very much required to contain their emergence.

 
   References Top

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Correspondence Address:
Ragini Tilak
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.118703

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    Figures

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    Tables

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