Indian Journal of Medical Microbiology, (2015) 33(1): 92-95 1

Original Article

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

Abstract
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 Grams 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
Sabourauds 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.

Key words: Candida albicans, non albicans Candida, phospholipase, vulvovaginal Candidiasis

Introduction they are frequently overlooked and as a result often

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.[1] 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
*Corresponding author (email: <rameshfule2010@gmail.com>)
Department of Microbiology (SRF, DD, RPF),
Jawaharlal Nehru Medical College, Sawangi,
Meghe, Wardha, Maharashtra, India
Received: 19-09-2013
Accepted: 27-03-2014
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DOI:
10.4103/0255-0857.148392
misdiagnosed. Ferris et al.,[2] 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.[3] 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.[4] Host or microbial factors distinguish
the infection and these factors have profound impact on
therapeutic outcome.[5]
C. albicans is responsible for the vast majority of
symptomatic episodes of VVC.[3] Of the non-albicans
yeast species, C. glabrata is considered the most
common.[6-8] Relative contribution of other non-albicans
Candida to the burden of VVC is difficult to measure. Trama
et al.,[9] 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,[10] C. lusitaniae
and Saccharomyces cervevisiae.[11]
January - March 2015 Fule, et al.: Phospholipase enzyme activity of Candida 93

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.[12] However, not a single putative virulence
factor has been linked unequivocally with pathogenicity.[12]
Phospholipases contribute to pathogenicity of bacteria,[13]
rickettsiae[14] and protozoa,[15] the role of these membrane
damaging enzymes[16] is proposed in the pathogenicity of
C. albicans. Ibrahim et al.,[17] have strongly implicated
phospholipases as a virulence factor in the pathogenesis of
C. albicans.
Figure 1: Phospholipase activity of Candida albicans on egg yolk agar
Therefore, the present study was carried out to determine medium
the relative contribution of C. abicans and non albicans
species and to find the prevalent phospholipase producing two occasions. Phospholipase activity i.e. Pz value was
Candida species causing symptomatic VVC. calculated by applying following equation:
Materials and Methods Colony diameter
Pz =
Colony diameter + Zone of precipitation
Two high vaginal swabs were collected from
156 women of reproductive age group attending On the basis of Pz value, phospholipase activity was
Obstetrics and Gynaecology (OBGY) department classified in 5 types i.e., Pz value = 1 means, test strain is
of tertiary care rural hospital of central India from negative for phospholipase, Pz value < 0.90-0.99 = weak
October 2010 to September 2011 at random for the present phospholipase activity (+), 0.80-0.89 = poor phospholipase
study. The study was granted approval by institutional activity (++), 0.70-0.79 = moderate phospholipase
ethics committee. All the patients had vaginal discharge activity (+++) and <0.70 = intense phospholipase
as a main complaint. A detailed clinical examination was activity (++++).
done and documented. All vaginal swabs were subjected to
KOH wet mount microscopy and Grams stain for presence Results
of budding yeast and pseudohyphae. Subsequently, second
Of the 156 women with abnormal vaginal discharge
swab was inoculated on SDA for yeast isolation. Growth
134 (85.89%) were pregnant and 22 (14.10%) non pregnant.
of Candida was confirmed by colony morphology, Grams
Fifty nine (37.82%) women who showed evidence of VVC
stain, and distinctive colour production by different Candida
47 (79.66%) were pregnant and remaining 12 (20.33%)
species on CHROME agar (Hi- media Mumbai) and sugar
were non-pregnant. Out of the 59 women with VVC, 31
assimilation test for speciation of different candida isolates
showed curdy white discharge while other showed curdy
using KB006 HiCandida Identification kit (Hi media
white discharge plus other signs [Table 1]. Both C. albicans
Mumbai).
37 (62.59%) and non albicans species 22 (37.28%) in
Extracellular phospholipase activity was determined the ratio of 1.68:1 were isolated. Among non albicans,
by the egg yolk agar plate method. All the Candida species C. tropicalis 7 (37.81%), C. parapsilosis 6 (27.27%),
isolated were screened for production of phospholipase C. glabrata 5 (22.72%) and C. dubiliensis and C. krusei (2)
activity by growing them on the egg yolk agar and strains each were isolated [Table 1].
measuring the size of the zone of precipitation by method of
All the strains of C. albicans and non albicans Candida
Price et al.[18] Briefly, the egg yolk agar medium consisted of
species studied for phospholipase activity. Of the 37 strains
65 g SDA, 58.4 g NaCl, and 5.5 g CaCl2 dissolved in 980 ml
of C. albicans 30 (81.08%) showed the enzyme activity
distilled water and sterilised at 121oC for 15 minutes. Egg
of which 17 (56.66%) strains showed higher Pz value
yolk was centrifuged at 5000 g for 30 minutes, 2 ml of the
of < 0.70 (++++) [Table 2 and Figure 1].
supernatant was added to medium cooled at 45-50oC. An
aliquot of 10 l yeast suspension was spot inoculated on Discussion
egg yolk agar medium and incubated at 37oC for 4 days,
after which the diameter of precipitation zone around the The state of host is of primary importance in
colony was determined [Figure 1]. Each isolate was tested determining Candida pathogenicity. However, there are
in replicate of two and experiment was carried out on factors associated with the organism that contribute to the

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January - March 2015 Fule, et al.: Phospholipase enzyme activity of Candida 93

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.[12] However, not a single putative virulence
factor has been linked unequivocally with pathogenicity.[12]
Phospholipases contribute to pathogenicity of bacteria,[13]
rickettsiae[14] and protozoa,[15] the role of these membrane
damaging enzymes[16] is proposed in the pathogenicity of
C. albicans. Ibrahim et al.,[17] have strongly implicated
phospholipases as a virulence factor in the pathogenesis of
C. albicans.
Figure 1: Phospholipase activity of Candida albicans on egg yolk agar
Therefore, the present study was carried out to determine medium
the relative contribution of C. abicans and non albicans
species and to find the prevalent phospholipase producing two occasions. Phospholipase activity i.e. Pz value was
Candida species causing symptomatic VVC. calculated by applying following equation:
Materials and Methods Colony diameter
Pz =
Colony diameter + Zone of precipitation
Two high vaginal swabs were collected from
156 women of reproductive age group attending On the basis of Pz value, phospholipase activity was
Obstetrics and Gynaecology (OBGY) department classified in 5 types i.e., Pz value = 1 means, test strain is
of tertiary care rural hospital of central India from negative for phospholipase, Pz value < 0.90-0.99 = weak
October 2010 to September 2011 at random for the present phospholipase activity (+), 0.80-0.89 = poor phospholipase
study. The study was granted approval by institutional activity (++), 0.70-0.79 = moderate phospholipase
ethics committee. All the patients had vaginal discharge activity (+++) and <0.70 = intense phospholipase
as a main complaint. A detailed clinical examination was activity (++++).
done and documented. All vaginal swabs were subjected to
KOH wet mount microscopy and Grams stain for presence Results
of budding yeast and pseudohyphae. Subsequently, second
Of the 156 women with abnormal vaginal discharge
swab was inoculated on SDA for yeast isolation. Growth
134 (85.89%) were pregnant and 22 (14.10%) non pregnant.
of Candida was confirmed by colony morphology, Grams
Fifty nine (37.82%) women who showed evidence of VVC
stain, and distinctive colour production by different Candida
47 (79.66%) were pregnant and remaining 12 (20.33%)
species on CHROME agar (Hi- media Mumbai) and sugar
were non-pregnant. Out of the 59 women with VVC, 31
assimilation test for speciation of different candida isolates
showed curdy white discharge while other showed curdy
using KB006 HiCandida Identification kit (Hi media
white discharge plus other signs [Table 1]. Both C. albicans
Mumbai).
37 (62.59%) and non albicans species 22 (37.28%) in
Extracellular phospholipase activity was determined the ratio of 1.68:1 were isolated. Among non albicans,
by the egg yolk agar plate method. All the Candida species C. tropicalis 7 (37.81%), C. parapsilosis 6 (27.27%),
isolated were screened for production of phospholipase C. glabrata 5 (22.72%) and C. dubiliensis and C. krusei (2)
activity by growing them on the egg yolk agar and strains each were isolated [Table 1].
measuring the size of the zone of precipitation by method of
All the strains of C. albicans and non albicans Candida
Price et al.[18] Briefly, the egg yolk agar medium consisted of
species studied for phospholipase activity. Of the 37 strains
65 g SDA, 58.4 g NaCl, and 5.5 g CaCl2 dissolved in 980 ml
of C. albicans 30 (81.08%) showed the enzyme activity
distilled water and sterilised at 121oC for 15 minutes. Egg
of which 17 (56.66%) strains showed higher Pz value
yolk was centrifuged at 5000 g for 30 minutes, 2 ml of the
of < 0.70 (++++) [Table 2 and Figure 1].
supernatant was added to medium cooled at 45-50oC. An
aliquot of 10 l yeast suspension was spot inoculated on Discussion
egg yolk agar medium and incubated at 37oC for 4 days,
after which the diameter of precipitation zone around the The state of host is of primary importance in
colony was determined [Figure 1]. Each isolate was tested determining Candida pathogenicity. However, there are
in replicate of two and experiment was carried out on factors associated with the organism that contribute to the

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94 Indian Journal of Medical Microbiology vol. 33, No. 1

Table 1: Prevalence of Candida albicans and non albicans species in relation to clinical presentations
Clinical presentation No. isolated C. albicans C. parapsilosis C. dubliniensis C. krusei C. glabrata C. tropicalis
(n=59) (n=37) (n=6) (n=2) (n=2) (n=5) (n=7)
Curdy white discharge 31 23 (23) 3 1 - 2 2
Curdy white discharge with 5 3 (2) 2 - - - -
pruritus
Curdy white discharge with 12 6 (3) 1 - 1 2 2
lower abodominal pain
Curdy white discharge 6 1 (0) - - 1 1 3
with pruritus, dysuria and
abdominal pain
Curdy white discharge 5 4 (2) - 1 - - -
with pruritus and lower
abdominal pain
Number in parenthesis shows positive phospholipase activity of C. albicans

Table 2: Phospholipase activity exhibited by C. albicans
isolated from VVC
Phospholipase No. of strain showing phospholipase
activity-Pz value activity. (no. of strains tested n=37) (%)
1(negative) 7 (18.91)
<0.90-0.99 (+) 2 (5.40)
0.80-0.89 (++) 4 (10.81)
0.70-0.79 (+++) 7 (18.91)
<0.70 (++++) 17 (45.94)
All non albicans candida species were negative for phospholipase
activity, VVC: Vulvovaginal candidiasis
ability of the organisms to cause disease and explain the
differences in the pathogenicity among species.[19] Risk
factors for VVC are frequently absent but severe forms may
be associated with use of oral contraceptives, corticosteroids
or antibiotics[20] and pregnancy.[21] 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.[22] It is responsible for the vast
majority of symptomatic episodes of VVC accounting for
over 80% of cases as reported earlier,[3,9] 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.[18] 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.[23] Evidence implicating phospholipase as
a virulence factor of C. abicans is mounting.[17] Ashraf
et al.,[17] 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.,[24] reported
that all clinical isolates of C. albicans from VVC showed
phospholipase activity while Basu et al.,[25] 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

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January - March 2015 Fule, et al.: Phospholipase enzyme activity of Candida
C. albicans showed phospholipase activity the potential role
of phospholipase in virulence and pathogenesis is mounting.
References
1. Anderson MR, Klink K, Cohrssen A. Evaluation of vaginal
complaints. JAMA 2004;291:1368-79.
2. 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.
3. 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.
4. Workowski KA, Berman SM. Sexually transmitted diseases
treatment guidelines, 2006 MMWR Recomm Rep 2006;55:54.
Available from: http://www.cdc.gov/std/treatment/2006/
rr5511.pdf. [Last accessed on 2013 Jun 27].
5. Nyirjesy P. Vulvovaginal candidiasis and bacterial vaginosis.
Infect Dis Clin North Am 2008;22:637-52.
6. 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.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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.
12. Cutler JE. Putative virulence factors of Candida albicans.
Annu Rev Microbiol 1991;45:187-218.
13. Wright GC, Weiss J, Kim KS, Verheij H, Elsbach P.
Bacterial phospholipid hydrolysis enhances the destruction
of Escherichia coli ingested by rabbit neutrophils. Role
www.ijmm.org
95
of cellular and extracellular phospholipases. J Clin Invest
1990;85:1925-35.
14. 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.
15. 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.
16. Salyers AA, Whitt DD. Salmonella species. In: Salyers AA,
Whitt DD, editors. Bacterial Pathogenesis: A Molecular
Approach. Washington: ASM Press; 2002. p. 381-97.
17. 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.
18. Price MF, Wilkinson ID, Gentry LO. Plate method for
detection of phospholipase activity in Candida albicans.
Sabouraudia 1982;20:7-14.
19. Dignani MC, Solomkin JS, Anaissie EJ. Candida. In: Clinical
Mycology. In: Anaissie EJ, McGinnis MR, Pfaller MA, editors.
2nd ed., Ch. 8. New York: Churchill Livingstone; 2009. p. 197-229.
20. Oriel JD, Waterworth PM. Effects of minocyclin and
tetracyclin on the vaginal yeast flora. J Clin Pathol
1975;28:403-6.
21. Morton RS, Rashid S. Candidal vaginitis: Natural history,
predisposing factors and prevention. Proc R Soc Med
1977;70:3-6.
22. Pfaller MA, Diekema DJ. Epidemiology of invasive
candidiasis: A persistent public health problem. Clin
Microbiol Rev 2007;20:133-63.
23. Ghannoum MA. Potential role of phospholipase in virulence
and fungal pathogenesis. Clin Microbiol Rev 2000;13:122-43.
24. 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.
25. 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.
How to cite this article: Fule SR, Das D, Fule RP. 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.
Source of Support: Nil, Conflict of Interest: None declared.