|Year : 2015 | Volume
| Issue : 1 | Page : 92-95
Detection of phospholipase activity of Candida albicans and non albicans isolated from women of reproductive age with vulvovaginal candidiasis in rural area
SR Fule, D Das, RP Fule
Department of Microbiology , Jawaharlal Nehru Medical College, Sawangi, Meghe, Wardha, Maharashtra, India
|Date of Submission||19-Sep-2013|
|Date of Acceptance||27-Mar-2014|
|Date of Web Publication||5-Jan-2015|
R P Fule
Department of Microbiology , Jawaharlal Nehru Medical College, Sawangi, Meghe, Wardha, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Vulvovaginal candidiasis (VVC) is most common accounting for 17 to 39% of symptomatic women. Both Candida albicans and non albicans Candida species are involved in VVC. Amongst various virulence factors proposed for Candida, extracellular phospholipases is one of the virulence factor implicated in its pathogenicity. With this background the present study was carried out to find the prevalence of different Candida species and to detect phospholipase producing strains isolated from symptomatic women with VVC. Materials and Methods: At least two vaginal swabs from 156 women of reproductive age with abnormal vaginal discharge were collected. Direct microscopy and Gram's stained smear examined for presence of budding yeast and pseudo mycelia followed by isolation and identification of Candida species. Extracellular phospholipase activity was studied by inoculating all isolates on Sabouraud's dextrose egg yolk agar (SDA) medium. Results: Of the 156 women with curdy white discharge alone or in combination with other signs, 59 (37.82%) women showed laboratory evidence of VVC. A total of 31 (52.54%) women had curdy white discharge followed by 12 (20.33%) with other signs and symptoms. C. albicans (62.59%) and non albicans Candida (37.28%) in a ratio of 1.68:1 were isolated. Of the 37 strains of C. albians 30 (81.08%) showed the enzyme activity. Seventeen (56.66%) strains showed higher Pz value of < 0.70 (++++). Conclusion: Although there may be typical clinical presentation of Candidiasis. all the patients did not show laboratory evidence of infection. Pregnancy was found to be major risk factor for development of VVC. C. albicans was prevalent species but non albicans species were also frequently isolated. Extracellular phospholipase activity was seen in C. albicans and not in non albicans Candida isolates.
Keywords: Candida albicans, non albicans Candida, phospholipase, vulvovaginal Candidiasis
|How to cite this article:|
Fule S R, Das D, Fule R P. Detection of phospholipase activity of Candida albicans and non albicans isolated from women of reproductive age with vulvovaginal candidiasis in rural area. Indian J Med Microbiol 2015;33:92-5
|How to cite this URL:|
Fule S R, Das D, Fule R P. Detection of phospholipase activity of Candida albicans and non albicans isolated from women of reproductive age with vulvovaginal candidiasis in rural area. Indian J Med Microbiol [serial online] 2015 [cited 2019 Sep 19];33:92-5. Available from: http://www.ijmm.org/text.asp?2015/33/1/92/148392
| ~ Introduction|| |
Of the many causes of vaginal infections, bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) are believed to be the two most common causes accounting for an estimated 22% to 39% and 17% to 39% of symptomatic women, respectively.  Although vulvovaginal complaints, such as itching, burning, abnormal discharge and odour, frequently may be trivialised or ignored, vaginitis is significantly associated with direct or indirect healthcare costs. Despite the importance of VVC and BV as a common infection with significant costs and morbidities they are frequently overlooked and as a result often misdiagnosed. Ferris et al., reported in a prospective study of symptomatic women that the purchase of antifungal products over-the-counter (OTC) only 34% had pure VVC.
Women having VVC present with spectrum of manifestations ranging from asymptomatic colonisation to severe acute symptomatic infection.  Some symptomatic women with VVC may have an occasional sporadic episode, or others may have frequent severe symptoms. Certain patients may develop primarily vulvar symptoms instead of vaginal manifestations of VVC. As large number of women are affected, VVC is categorised into uncomplicated and complicated disease.  Host or microbial factors distinguish the infection and these factors have profound impact on therapeutic outcome. 
C. albicans is responsible for the vast majority of symptomatic episodes of VVC.  Of the non-albicans yeast species, C. glabrata is considered the most common. ,, Relative contribution of other non-albicans Candida to the burden of VVC is difficult to measure. Trama et al.,  reported in their study that C. albicans accounted for 80.2% of VVC followed by C. glabrata 14.3%, C. parapsilosis 5.9% and C. tropicalis 8%. Other less frequent causes of VVC include C. krusei,  C. lusitaniae and Saccharomyces cervevisiae.
Strains of C.albicans differ in their virulence. Both virulent and non-virulent strains may be isolated from clinical specimens. Multiple characteristics of C. albicans have been proposed to be virulence factors that enable the organism to cause symptomatic and complicated VVC. These putative virulence factors include germination, adherence to host cells, and secretion of proteinase and phospholipases.  However, not a single putative virulence factor has been linked unequivocally with pathogenicity.  Phospholipases contribute to pathogenicity of bacteria,  rickettsiae  and protozoa,  the role of these membrane damaging enzymes  is proposed in the pathogenicity of C. albicans. Ibrahim et al.,  have strongly implicated phospholipases as a virulence factor in the pathogenesis of C. albicans.
Therefore, the present study was carried out to determine the relative contribution of C. abicans and non albicans species and to find the prevalent phospholipase producing Candida species causing symptomatic VVC.
| ~ Materials and Methods|| |
Two high vaginal swabs were collected from 156 women of reproductive age group attending Obstetrics and Gynaecology (OBGY) department of tertiary care rural hospital of central India from October 2010 to September 2011 at random for the present study. The study was granted approval by institutional ethics committee. All the patients had vaginal discharge as a main complaint. A detailed clinical examination was done and documented. All vaginal swabs were subjected to KOH wet mount microscopy and Gram's stain for presence of budding yeast and pseudohyphae. Subsequently, second swab was inoculated on SDA for yeast isolation. Growth of Candida was confirmed by colony morphology, Gram's stain, and distinctive colour production by different Candida species on CHROME agar (Hi- media Mumbai) and sugar assimilation test for speciation of different candida isolates using KB006 HiCandida Identification kit (Hi media Mumbai).
Extracellular phospholipase activity was determined by the egg yolk agar plate method. All the Candida species isolated were screened for production of phospholipase activity by growing them on the egg yolk agar and measuring the size of the zone of precipitation by method of Price et al.  Briefly, the egg yolk agar medium consisted of 65 g SDA, 58.4 g NaCl, and 5.5 g CaCl 2 dissolved in 980 ml distilled water and sterilised at 121 ° C for 15 minutes. Egg yolk was centrifuged at 5000 g for 30 minutes, 2 ml of the supernatant was added to medium cooled at 45-50 ° C. An aliquot of 10 μl yeast suspension was spot inoculated on egg yolk agar medium and incubated at 37 ° C for 4 days, after which the diameter of precipitation zone around the colony was determined [Figure 1]. Each isolate was tested in replicate of two and experiment was carried out on two occasions. Phospholipase activity i.e. Pz value was calculated by applying following equation:
|Figure 1: Phospholipase activity of Candida albicans on egg yolk agar medium|
Click here to view
On the basis of Pz value, phospholipase activity was classified in 5 types i.e., Pz value = 1 means, test strain is negative for phospholipase, Pz value < 0.90-0.99 = weak phospholipase activity (+), 0.80-0.89 = poor phospholipase activity (++), 0.70-0.79 = moderate phospholipase activity (+++) and <0.70 = intense phospholipase activity (++++).
| ~ Results|| |
Of the 156 women with abnormal vaginal discharge 134 (85.89%) were pregnant and 22 (14.10%) non pregnant. Fifty nine (37.82%) women who showed evidence of VVC 47 (79.66%) were pregnant and remaining 12 (20.33%) were non-pregnant. Out of the 59 women with VVC, 31 showed curdy white discharge while other showed curdy white discharge plus other signs [Table 1]. Both C. albicans 37 (62.59%) and non albicans species 22 (37.28%) in the ratio of 1.68:1 were isolated. Among non albicans, C. tropicalis 7 (37.81%), C. parapsilosis 6 (27.27%), C. glabrata 5 (22.72%) and C. dubiliensis and C. krusei (2) strains each were isolated [Table 1].
|Table 1: Prevalence of Candida albicans and non albicans species in relation to clinical presentations|
Click here to view
All the strains of C. albicans and non albicans Candida species studied for phospholipase activity. Of the 37 strains of C. albicans 30 (81.08%) showed the enzyme activity of which 17 (56.66%) strains showed higher Pz value of < 0.70 (++++) [Table 2] and [Figure 1].
|Table 2: Phospholipase activity exhibited by C. albicans isolated from VVC|
Click here to view
| ~ Discussion|| |
The state of host is of primary importance in determining Candida pathogenicity. However, there are factors associated with the organism that contribute to the ability of the organisms to cause disease and explain the differences in the pathogenicity among species.  Risk factors for VVC are frequently absent but severe forms may be associated with use of oral contraceptives, corticosteroids or antibiotics  and pregnancy.  In the present study 59 women with VVC, 47 (78.66%) were pregnant indicating a major risk factor for development of VVC.
Clinical prevalence of different Candida species is reported; however, C. albicans is most commonly implicated species.  It is responsible for the vast majority of symptomatic episodes of VVC accounting for over 80% of cases as reported earlier, , but its prevalence is declining and non abicans species rapidly supervening. Similar declining trend in prevalence of C. albicans was observed in the present study. Because vaginal yeast culture are not done routinely in the management of uncomplicated VVC, the relative contribution of non albicans yeast species to the burden of VVC is difficult to measure. But in the present study yeast culture revealed that non albicans yeast species supervened. Changes in species distribution can occur not only overtime but also in different locations. Although, exposure to antifungal agents has long been considered the main factor for this change, not a single woman gave history of exposure to antifungal agents in the present study. In the present study C. albicans (62.59%) was found to be predominant yeast species to cause VVC followed by non albicans species (37.28%). The most frequent non albicans species isolated was C. tropicalis followed by C. parapsilosis, C. glabrata, C. krusie and, C. dubiliensis [Table 1], hence non albicans species are regarded as pathogens and are frequently isolated.  Changes in species distribution can occur from time to time as well as at different geographical locations.
Several features of C. albicans have been described and proposed to be virulence factors that enable the organism to cause local and disseminated infections in susceptible hosts. Potential role of phospholipase in virulence and fungal pathogenesis has been studied extensively.  Evidence implicating phospholipase as a virulence factor of C. abicans is mounting.  Ashraf et al.,  had strongly suggested that phospholipases are important factors in the pathogenesis of C. albicans experimentally in new borne mouse model. Therefore, in the present study all C. albicans and non albicans species studied for in-vitro phospholipase activity, 30 (81.08%) of C. albicans showed the enzyme activity. Seventeen (56.66%) strains showed higher Pz value of < 0.70 (++++). None of the non albicans Candida species showed this activity. Mahmoudabadi et al.,  reported that all clinical isolates of C. albicans from VVC showed phospholipase activity while Basu et al.,  reported 66.6% of vaginal isolates exhibited the enzyme activity. [Table 1] shows the clinical correlation of phospholipase producing C. albicans. The C. albicans strains isolated from women with only curdy white discharge were positive for phospholipase activity, while variable number showed this activity when isolated from women with other associated symptoms.
Considering the observations of the present study it may be concluded that non albicans Candida species is slowly replacing C. albicans in VVC. Though not all the strains of C. albicans showed phospholipase activity the potential role of phospholipase in virulence and pathogenesis is mounting.
| ~ References|| |
Anderson MR, Klink K, Cohrssen A. Evaluation of vaginal complaints. JAMA 2004;291:1368-79.
Ferris DG, Nyirjesy P, Sobel JD, Soper D, Pavletic A, Litaker MS. Over-the-counter antifungal drug misuse associated with patient- diagnosed vulvovaginal candidiasis. Obstet Gynecol 2002;99:419-25.
Sobel JD, Faro S, Frocs RW, Foxman B, Ledger WJ, Nyirjesy PR. Vulvovaginal candidiasis: Epidemiological, diagnostic and therapeutic considerations. Am J Obstet Gynecol 1998;178:203-11.
Nyirjesy P. Vulvovaginal candidiasis and bacterial vaginosis. Infect Dis Clin North Am 2008;22:637-52.
Sobel JD, Kapernick PS, Zervos M, Reed BD, Hooton T, Soper D, et al.
Treatment of complicated Candida vaginitis: Comparison of single and sequential doses of fluconazole. Am J Obstet Gynecol 2001;185:363-9.
Nyirjesy P, Seeney SM, Grody MH, Jordan CA, Buckley HR. Chronic fungal vaginitis: The value of cultures. Am J Obstet Gynecol 1995;173:820-3.
Sobel JD, Wiessenfeld HC, Martens M, Danna P, Hooton TM, Rompalo A, et al
. Maintenance fluconazole therapy for recurrent vulvovaginal candidiasis. N Engl J Med 2004;351:876-83.
Trama JP, Adelson ME, Raphaelli I, Stemmer SM, Mordechai E. Detection of Candida species in vaginal samples in a clinical laboratory setting. Infect Dis Obstet Gynecol 2005;13:63-7.
Singh S, Sobel JD, Bhargava P, Boikov D, Vazquez JA. Vaginitis due to Candida krusie: Epidemiology, clinical aspects, and therapy. Clin Infect Dis 2002;35:1066-70.
Richter SS, Galask RP, Messer SA, Hollis RJ, Diekema DJ, Pfaller MA. Antifungal susceptibilities of Candida species causing vulvovaginitis and epidemiology of recurrent cases. J Clin Microbiol 2005;43:2155-62.
Cutler JE. Putative virulence factors of Candida albicans. Annu Rev Microbiol 1991;45:187-218.
Wright GC, Weiss J, Kim KS, Verheij H, Elsbach P. Bacterial phospholipid hydrolysis enhances the destruction of Escherichia coli
ingested by rabbit neutrophils. Role of cellular and extracellular phospholipases. J Clin Invest 1990;85:1925-35.
Silverman DJ, Santucci LA, Meyers N, Sekeyova Z. Penetration of host cells by Rickettsia rickettsii appears to be mediated by a phospholipase of rickettsial origin. Infect Immun 1992;60:2733-40.
Saffer LD, Long Krug SA, Schwartzman JD. The role of phospholipase in host cell penetration by Toxoplasma gondii. Am J Trop Med Hyg 1989;40:145-9.
Salyers AA, Whitt DD. Salmonella
species. In: Salyers AA, Whitt DD, editors. Bacterial Pathogenesis: A Molecular Approach. Washington: ASM Press; 2002. p. 381-97.
Ibrahim AS, Mirbod F, Filler SG, Banno Y, Cole GT, Kitajima Y, et al
. Evidence implicating phospholipase as a virulence factor of Candida albicans. Infect Immun 1995;63:1993-8.
Price MF, Wilkinson ID, Gentry LO. Plate method for detection of phospholipase activity in Candida albicans. Sabouraudia 1982;20:7-14.
Dignani MC, Solomkin JS, Anaissie EJ. Candida. In: Clinical Mycology. In: Anaissie EJ, McGinnis MR, Pfaller MA, editors. 2 nd
ed., Ch. 8. New York: Churchill Livingstone; 2009. p. 197-229.
Oriel JD, Waterworth PM. Effects of minocyclin and tetracyclin on the vaginal yeast flora. J Clin Pathol 1975;28:403-6.
Morton RS, Rashid S. Candidal vaginitis: Natural history, predisposing factors and prevention. Proc R Soc Med 1977;70:3-6.
Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: A persistent public health problem. Clin Microbiol Rev 2007;20:133-63.
Ghannoum MA. Potential role of phospholipase in virulence and fungal pathogenesis. Clin Microbiol Rev 2000;13:122-43.
Zarei Mahmoudabadi A, Zarrin M, Miry S. Phospholipase activity of Candida albicans isolated from vagina and urine samples. Jandishapur J Microbiol 2010;3:169-73.
Basu S, Gugnani HC, Joshi S, Gupta N. Distribution of Candida species in different clinical sources in Delhi, India, and proteinase and phospholipase activity of Candida albicans isolates. Rev Iberoam Micol 2003;20:137-40.
[Table 1], [Table 2]