Profile of Candida Species in Vulvovaginal Candidiasis using

Conventional Methods

Cita Rosita Sigit Prakoeswa, Dewi Puspitorini, Yuri Widya, Sylvia Anggraeni, Linda Astari, Evy

Ervianti, Sunarso Suyoso

Dermatology and Venereology Departement Universitas Airlangga/ Dr.Soetomo Hospital Surabaya

Keywords: Candida sp, vulvovaginal candidiasis, cornmeal Tween 80 agar, carbohydrate fermentation test, CHROM

agar Candida.

Abstract: Vulvovaginal candidiasis (VVC) is an inflammatory disease of vulva and vagina that caused by Candida

sp.Candida albicans was the predominant cause of candidiasis. However, a shift toward non-albicans

Candida species has been recently observed. These non-albicans Candida species demonstrated reduced

susceptibility to commonly used antifungal drugs. Identification of the infecting Candida to the species

level is of utmost importance for clinical microbiological services for prediction of likely drug susceptibility

and to guide treatment. Conventional methods for the diagnosis of candidiasis are sensitive, low-cost, and

although time consuming, are still considered the references standard for identification of yeast isolates.

There were three conventional methods that is still the standard tests to identify the Candida sp. The tests

are Sabouraud dextrose agar (SDA) thencornmeal-Tween 80 agar, carbohydrate fermentation test, and

CHROM agar Candida (CAC). These tests usually give some specific colony, morphology andcolor for

every Candida sp.

1 INTRODUCTION

Vulvovaginal candidiasis (VVC) refers to a disorder

characterized by signs and symptoms of

vulvovaginal inflamation in the presence of Candida

species. It is the second most common cause of

vaginitis symptom after bacterial vaginosis. It is

estimated that at least 75% of healthy adult women

will experience one episode of vulvovaginal

candidiasis during their reproductive phase. The

signs and symptoms of VVC include a thick cheese–

like discharge associated with intense vaginal and

vulvar pruritus, pain, burning, erythema, and/or

edema (Kunduk and Garg, 2012; Moyes and Naglik,

2011; Sobel, 2008).

Candida sp are among the most common fungal

pathogens. They are capable of initiating infections

in both immunocompetent individuals and

immunocompromised hosts. Candida sp,

arecommensal organisms that normally colonize

mucosal surfaces in an asymptomatic manner, but it

also can become one of the most significant causes

of disabling and lethal infection. Candida sp are

responsible for various clinical manifestations

ranging from mucocutaneous overgrowth to life

threatening disseminated infections like candidemia.

Candida albicans remains the most common

causative agent for VVC in approximately 85%-95%

of the cases. However,there is an alteration of

species that cause VVC in the last decade as

incidence of VVC due to non-albicans Candidasp is

increasing. This rise could be due to an increase of

over-the-counter antifungal use. The clinical

manifestations of infections caused by non-albicans

Candidassp are usually indistinguishable with VVC

caused by Candida albicans except in its poor

response to common anti-fungal drugs due to either

inherent or acquired. Due to the changing

epidemiology of Candida, physicians may no longer

be able to make therapeutic decisions based on

species levels study to enhance proper treatment for

their patients (Hedayati et al., 2015; Deorukhkar et

al., 2014; Farooqi et al., 2013; Richter et al., 2005;

Fidel et al., 1999).

Species identification requires isolation and

biochemical or physiological characterization.

Candida sp, being non-fastidious organism, readily

grows on laboratory media used for the isolation of

fungus. Conventional methods, including Sabouraud

Prakoeswa, C., Puspitorini, D., Widya, Y., Anggraeni, S., Astari, L., Ervianti, E. and Suyoso, S.

Proﬁle of Candida Species in Vulvovaginal Candidiasis using Conventional Methods.

DOI: 10.5220/0008155702810285

In Proceedings of the 23rd Regional Conference of Dermatology (RCD 2018), pages 281-285

ISBN: 978-989-758-494-7

281

dextrose agar(SDA), cornmeal-Tween 80 agar,

carbohydrate fermentation test, and CHROM Agar

Candida (CAC), are recommended to identify

Candida spto the species level. Each method has its

own eminence at identifying Candida species. The

formation of blastospore, pseudohyphae, hyphae and

chlamydospores which aids identification of

Candida sp requires the use of nutritionally deficient

media like cornmeal agar Tween 80 as these

nutritionally deficient media suppress the vegetative

growth and promote sporulation. Carbohydrate

fermentation test classifies Candida based on the

color transformation on carbohydrate broth and gas

formation on the tube. Candida sp metabolizes

carbohydrates both aerobically (assimilation) and

anaerobically (fermentation) (Mutiawati, 2016;

Suyoso, 2013; Larone 2011). Lastly, enzymatic

reaction methods using chromogenic substrates in

Chromogenic agar (CHROM Agar Candida)

medium has high sensitivity and specificity in

differentiating Candida species in clinical samples.

This media contains chromogenic substrates that

react with enzymes secreted by yeast cells, resulting

in various pigmentations. Using this medium, it is

possible to identify Candida species based on color

characteristics. CAC can differ one Candida sp to

another Candida sp by its colony color. The

presence of two colonies with different color

indicates there were two species that grew on CAC.

2 METHODS

This was a cross-sectional descriptive study that

identifies causes of VVC down to the species level

by using conventional methods of fungal

examination. The sample of this study were all VVC

patients that fulfilled the inclusion criteria, and

underwent examination at Sexually Transmited

Infection Division, Dermatoveneorology Clinic of

Dr.Soetomo Hospital, Surabaya. There were a total

of 25 enlisted patients. The patients' data

information in data collection sheet.

The Inclusion criteria were VVC patients, aged

15 years or older, married or unmarried, willing to

follow the research and sign the informed

consent.The exclusion criteria were patients with

negative culture result.

All women who attended Sexually Transmitted

Infection (STI) Division, Dermatoveneorology

Clinic of Dr.Soetomo Hospital, Surabaya were

interviewed for medical history and clinically

examined. Samples were taken from vaginal swab

and checked for Candida and Gram stain

examination. Patients diagnosed with VVC were

included in inclusion criteria.

Consecutively, the samples were first grown in

SDA, followed by cornmeal-Tween 80 agar and then

performed on carbohydrate fermentation test. The

results were available in 2-3 days. Therewere 6

carbohydratesused in this study: urea, dextrose,

lactose, sucrose, maltose, galactose and trehalose.

Positive result was marked bythe changing color of

broth to yellow and the gas formation in the Durham

tube. The other test was CHROM agar Candida

(CAC), showed colony color in18 hours-3 days.

Each color represents one specific species of

Candida.

3 RESULTS

This study involved 25 female patients with VVC.

The patients were predominated by 15-24 age group.

Most patients were private employees and most of

the patient's education were bachelor.

Most common duration of complaint was 1

month until 9 months with vaginal douching usage

being the highest predisposition factor. Most patients

admitted have not consumed any therapy for VVC

yet. Clinical examination revealed edematous and

erythematous vulva and vagina on all 25 patients.

Direct examination from wet specimen and Gram

stain were: wet specimen contained positive

blastospore with negative pseudohifa 20%, positive

blastospora with positive pseudohifa 48%, negative

blastospora with negative pseudohifa 32%, no

negative blastospore with positive pseudohifa(0%)

and Gram positive blastospore with negative

pseudohifa 16%, negative blastospore with positive

pseudohifa 4%, positive blastospore with positive

pseudohifa 52%, and negative blastospore with

negative pseudohifa 28%.

There were various results for each conventional

method. Cornmeal agar showed the specific

formation of every Candida. The presence of

terminal vesicles (chlamydoconidia) with pseudohifa

and flower-like blastoconidia in cornmeal agar

indicates that the fungi was Candida albicans. Other

sample showing divaricated pseudohifa with oval

blastoconidia indicates the presence of Candida

tropicalis.

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282

Table 1: Comparation between direct examination and

conventional methods

As for the carbohydrate fermentation test, for

example, positive result on dextrose and trehalose

means that the sample was Candida glabrata.

CAC revealed color of colony. All samples

showed one type of colony color, meaning one

Candida sp in every sample, and only 4 samples

shown two colony color (2 Candida sp). Light

green-colored colony is specific for Candida

albicans, while dark green colony is specific for

Candida dubliniensis. Purple-colored colony

characterized the colony of Candida glabrata.

These three conventional methods from 25

samples revealed that 14 sample were positive

forCandida albicans, and others were non-albicans

Candida sp. Five samples were positive for Candida

glabrata, 1 sample for Candida parapsilosis and 4

samples were positive for2 Candida sp (double

infections). From those 4 samples,1 sample were

positive for Candida albicans and Candida glabrata,

1 sample for Candida albicans and Candida famata,

and 2 samples were positive for Candida albicans

and Candida tropicalis. From the table 1 we can see

the comparation between microscopic and

conventional methods. Conventional methods

revealed species of the fungi while direct

microscopic examination can only show

pseudohypha and blastospore.

4 DISCUSSION

This was a descriptive cross-sectional study that

aimed to identify the causative agents of VVC. After

3 months sampling, 25 participants fulfilled the

inclusion criteria. The result showed that most

participants were in 15-24 age group. This is a

reproductive age group, with high level of estrogen.

Estrogen has been found to reduce the ability of

vaginal epithelial cells to inhibit the growth of

Candida albicans and also decreases immunoglobins

in vaginal secretions resulting in increased

vulnerability of women to vaginal Candidiasis.

Most patients were private employees. This group of

people tend to use vaginal hygiene products or

vaginal douche that contained antiseptic, and

prolonged misuse of antiseptic can cause VVC.

Most patient education were bachelor; they might

have better senses of personal vaginal hygiene than

patients with lower education group So it made them

went to hospital and checked their complaint and to

get a proper treatment from the hospital (Fidel et al.,

2000).

Most common duration of complaint of the

patients of VVC is 1 month until 9 months. From

this, we can deduct that there might be a recurrent

VVC that was ignored by the patientand causing

delayed proper treatment.

Clinical examination revealed edematous and

erythematous vulva and vagina on all 25 patients.

This means clinical sign of VVC is the first and

definite diagnosis of VVC withthe direct

microscopic examination was not the only parameter

to diagnose VVC (Sobel, 2008).

Figure 1: Conventional methods (SDA then Cornmeal Tween 80 agar, carbohydrate fermentation test, and CHROM agar

Candida).

Wet specimen & Gram

stain

Conventional methods

Microscopic

examination (wet

specimen and Gram

stain) can show fungal

morphology

(blastospore,

pseudohyphae, and

hae

)

Conventional methods

can reveal species of the

fungi (Candida sp itself).

Proﬁle of Candida Species in Vulvovaginal Candidiasis using Conventional Methods

283

Cornmeal Tween 80 agar showed the specific

morphology of every Candida. One sample with

budding yeast-like cell with no pseudohyfa indicates

the presence ofCandida glabrata. However, there

was also a sample that showed yeast cell only and,

no pseudohyfa.This could not conclude the presence

of Candida glabrata and additional examination

using other conventional method was needed to

identify the species (Golia et al., 2013; Suyoso,

2013).

The carbohydrate fermentation test shown

positive result based on the changing color of broth

to yellow and the gas formationin the Durham tube.

This test, however, is not reliable when used without

other additional method. For example, positive result

of dextrose and trehalose indicates presence of

Candida glabrata, while positive result of dextrose

and maltose, galactose and trehalose indicates

presence of Candida albicans. Therefore, a dubious

positive result of only dextrose and maltose could

not accurately indicate the presence of Candida

albicans.CAC will be useful for the additional

examination (Devi et al., 2014; Larone, 2011).

CAC revealed specific color for

eachcolony.There was a sample which color was

pink to purple, thus almost similar to Candida

glabrata. The carbohydrate fermentation test could

be used to differentiate dubious results from this

examination in order to get the final diagnosis (Faraz

et al., 2016; Suyoso, 2013; Vijaya et al., 2011).

5 CONCLUSION

There were increasing of non-albicans Candida sp

in the agents causing VVC. Non-albicans Candida

sp is an emerging threat fungi due to its antifungal

resistance. Identification of causative agent of VVC

down to the species level is important to give proper

treatment to the VVC patients. There are 3

conventional methods that can be used to identify

Candida sp to the species level. Cornmeal agar

showed the morphology of Candida sp.

Carbohydrate fermentation test revealed positive

result if there is brothcolor transformation to yellow

and Durham tube inside filled with gas. CHROM

agar Candida showed Candida sp by its colony

color. These 3 methods are still recommended for

identification of Candida sp to the species level and

should be performed simultaneously to support each

other's data to get the final definite species

identification result.

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