Changing Pattern of Candida Species in Vulvovaginal Candidiasis

using Vitek 2

Linda Astari, Dewi Puspitorini, Cita Rosita Sigit Prakoeswa, Sunarso Suyoso

Dermatovenereology Departement Airlangga University/ Dr. Soetomo Hospital Surabaya, Indonesia

Keywords: Candida sp, vulvovaginal candidiasis, Vitek 2

Abstract: Vulvovaginal candidiasis (VVC) is a common fungal infection. VVC causes inflammation of vulva and

vagina, characterized with erythematous vulva and vagina, with symptoms of pruritus and vaginal discharge.

Candida albicans remains the cause of VVC in almost 80% cases. Candida non-albicans now are emerging

threat as a cause of VVC, due to extensive usage of antifungal drugs. The importance of identifying Candida

species within clinical samples is in order to provide information concerning the proper treatment for patients.

Rapid identification of Candida species are essential in clinical laboratories. Vitek 2 is an automated

microbiology identification system that evaluates an optical signal generated by biochemical reactions

contained within a variety of identification cards. Vitek 2 could be used to identify Candida sp to the species

level. This microbiological system gives some advantages, it is faster than other diagnostic tools for VVC, it

works automatically and accurately, and it practically do not need manual work so it also minimizes error.

But for teaching hospital, it still need conventional method to study fungal morphology and to do Vitek 2

examination (from cornmeal-Tween 80 agar or CHROMagar Candida).

1 INTRODUCTION

Candida species are microorganisms which live

normally in our body, the skin, mouth,

gastrointestinal tract, and genitourinary tract,

including vulva and vagina. It usually lives as benign

commensals and produce no diseases. However, in

women with some predisposition factors, such as

vaginal douching usage, pregnancy, and

immunosuppressed condition, the colony of Candida

sp will grow higher and causing VVC. This disease is

usually a common problem on child-bearing age

women and 5% will have recurrent infectious

episodes (Vermitsky J, Self MJ, Chadwich SG,

Trama JP, Adelson ME, Mordechai E, et al, 2008;

Zeng J, Zong LL, Mao T, Huang YX, Xu ZM, 2011).

Candida albicans is responsible for infection in

80 to 90% of VVC cases, but now VVC due to

Candida non-albicans have increased steadily over

the latest decades (Srihartati E, Hoetomo MM,

Ervianti E, 2006; Ervianti E, Sawitri, Murtiastutik D,

Agusni RI., 2011; Jimoh O, Inabo HI, Yakubo SE,

Ankuma SJ, Olayunka AT., 2016; Cassone A, 2016).

The rise in VVC infections that more specifically

caused by Candida non-albicans species, could be

due to an increase in over-the-counter antifungal use.

Nowadays rapid identification of microorganisms

that cause diseases are important in clinical

laboratories. Better and faster diagnostic system for

VVC would let physicians could be able to make

therapeutic decisions based on species causative

agents of VVC so enhance proper treatment for their

patients. Phycisians necessitate the rapid and accurate

identification of yeasts to the species level by the

clinical microbiology laboratory. Vitek 2 is an

automated microbiology identification system that

evaluates an optical signal generated by individual

biochemical reactions contained within a variety of

microbe identification cards. After inoculation with a

standardized suspension of the unknown organism,

each self-contained card is incubated and read by the

instrument’s internal optics. Vitek 2 provides a highly

automated, objective yeast identification method with

excellent performance. This system is useful for

timely and accurate identification of significant yeast

species in the clinical microbiology laboratory. Vitek

2 showed faster and better species identification result

than other diagnostic systems such as conventional

methods and nonculture methods because they can be

time-consuming and manual-labour (Meurman O,

Koskensalo A, Jalava-Rantakoko K, 2006;

Rajkumari N, Mathur P, Xess I, Misra MC., 2014)

328

Astari, L., Puspitorini, D., Prakoeswa, C. and Suyoso, S.

Changing Pattern of Candida Species in Vulvovaginal Candidiasis using Vitek 2.

DOI: 10.5220/0008156803280332

In Proceedings of the 23rd Regional Conference of Dermatology (RCD 2018), pages 328-332

ISBN: 978-989-758-494-7

2 METHODS

This study was a cross-sectional descriptive study that

identifying causes of VVC to the species level by

using Vitek 2. Patients attending the Sexually

Transmitted Infection (STI) Division

Dermatovenereology Clinic of Dr Soetomo General

Hospital Surabaya that suspected VVC were

examined by the physician. The samples of this study

were all VVC patients that fulfilled the inclusion

criteria. The inclusion criteria were VVC patient,

women age 15 years or more than 15 years, married

or unmarried and willing to follow the research and

signed the informed consent. The exclusion criteria

were patients with negative culture result.

Patients were examined for VVC signs and

symptoms. Samples were taken from vaginal swab,

then underwent the direct microscopic examination

(wet preparation, and Gram stain). Samples were

tested for conventional methods and Vitek 2.

Conventional methods consisted of Sabouraud

dextrose agar (SDA) then Cornmeal-Tween 80 agar,

carbohydrate fermentation test, and CHROMagar

Candida (CAC). The first method is using SDA then

Cormeal-Tween 80 agar. This method showed

structures of Candida species on microscope in 3

days (72 hours). The other method was carbohydrate

fermentation test that consisted of 6 carbohydrates.

The result of this test positive if there was changing

color of broth to yellow and the tube inside the broth

(Durham tube) filled with gas in 2-3 days (48– 72

hours). The third one was CHROMagar Candida that

showed colonies in 1,5-2 days (36-48 hours). Color

of colony showed the Candida sp. Overall all of this

conventional method need 1,5-3 days (36-72 hours)

to identify the species of Candida.

Colony from Cornmeal-Tween 80 agar or CAC

were also checked by using Vitek 2. Four until six

fresh colonies (16-24 hours age of colonies) and were

taken and suspension with NaCl 0,85% in order to get

standardized suspension (1.8-2 McF) were made.

After inoculation with a standardized suspension of

the unknown organism, each self-contained card is

incubated and read by the instrument’s internal

optics. The reading result were compared to the

baseline data of Vitek 2.

3 RESULTS

The most common age of patients is on young adult

15-24 age group. History of high frequency or

recurrency of VVC is on 72% of patients. Clinical

examination showed edematous and erythematous

vulva and vagina on all patients. There were variation

result on direct microscopic examination from wet

preparation and Gram stain. There were positive and

negative result. There were no negative culture result.

Sabouraud dextrose agar then Cornmeal-Tween

80 agar examination showed specific characteristic of

each species of Candida. Terminal vesicles

(chlamydoconidia) with pseudohifa, blastoconidia

looked like a flower, could be concluded as Candida

albicans. Other samples with divaricated pseudohifa

and oval blastoconidia, could be concluded as

Candida tropicalis. Few samples with budding yeast

like cell and no pseudohypha may reveal the species

of Candida glabrata. The carbohydrate fermentation

test showed positive result based on the changing

color to yellow on 6 carbohydrates tested and the gas

filled in the Durham tube. Positive result on dextrose

and trehalose means that the sample grown was

Candida glabrata. Positive result on dextrose,

maltose, galactose and trehalose means that the

sample was Candida albicans. Other positive result

showed on dextrose, sucrose, maltose, galactose and

trehalose means that the sample was Candida

tropicalis, and one positive result showed on dextrose

means Candida parapsilosis or could be other

Candida sp, so it will need other conventional

method, to get the final result. The last positive result

on dextrose and sucrose only, this means could be

other Candida sp, so that it will need other

conventional method to support the definite result.

CHROMagar Candida revealed the growth of

Candida sp by its colony color. All samples revealed

1 type of colony color or 1 Candida sp in every

sample, but only 4 samples showed 2 colony color (2

Candida sp). Colony color light green means the

fungi was Candida albicans (if dark green means C

dubliniensis). Purple colony color means the fungi

was Candida glabrata. White colony color means

Candida parapsilosis. Blue color means Candida

tropicalis. This method should be completed with 2

other conventional methods (SDA then Cornmeal

Tween 80 agar and carbohydrate test). The

conventional methods from 25 sample showed 14

samples of Candida albicans, and others were

Candida non-albicans. Five samples were Candida

glabrata, 1 sample was Candida parapsilosis and 4

samples were grown with 2 Candida sp or infected by

2 Candida sp. Result of samples: 1 sample were

combination Candida albicans with Candida

glabrata, 1 sample were combination of Candida

albicans with

Candida famata, and 2 samples: each

sample contained Candida albicans with Candida

tropicalis.

Changing Pattern of Candida Species in Vulvovaginal Candidiasis using Vitek 2

329

Table 1: Comparation between Conventional methods and Vitek 2.

Conventional Methods Vitek 2

All samples revealed growth of Candida sp to

the species level: C albicans (the highest), C

glabrata, C tropicalis, C parapsilosis, C famata

Time-consuming (36-72 hours)

Laborious

All samples revealed growth of Candida sp to

the species level: C albicans (the highest), C

glabrata, C tropicalis, C parapsilosis, C

famata

Faster (18 hours)

Automatic machine, minimizing error

Figure 1: Conventional methods (SDA then Cornmeal agar, Carbohydrate fermentation test, CHROMagar Candida).

Figure 2: Result sheet of Vitek 2.

In Vitek 2, 25 of samples showed result of Candida

sp 100% same as conventional methods. Fourteen

samples were Candida albicans, and others were

Candida non-albicans. Five samples were Candida

glabrata, 1 sample was Candida parapsilosis and 4

samples were grown with 2 Candida sp or infected by

2 Candida sp. Result of 4 samples: 1 sample were

combination Candida albicans with Candida

glabrata, 1 sample were combination of Candida

albicans with Candida famata, and 2 samples: each

sample contained Candida albicans with Candida

tropicalis. From table 1 showed that the result of

species from 25 samples of Vitek 2 was 100% the

same as in conventional methods. Vitek 2 has some

advantages that it is faster and works automatically.

4 DISCUSSION

The most common age of patients was an age of

young adult 15-24 group from total of 25 patients. It

could be explained that the 15-24 age group is child-

bearing age, which is high of estrogen hormone,

allowing increase growth of Candida sp. History of

high frequency or recurrency of VVC was on 72% of

subjects. It means that a lot of women with VVC were

often not going to the doctor seeking for treatment,

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330

because maybe they think this disease is not life-

threatening and would heal itself, and it could be

because of the predisposition factors that still exist on

them (Khairnar R, Khairnar A.,

2017; Fidel PL,

Cutright J. Steele C., 2000; Kalia N, Singh J, Sharma

S, Kamboj SS, Arora H, Kaur M., 2015). Clinical

examination showed edematous and erythematous

vulva and vagina on all patients. This is concordance

that clinical signs of VVC are edematous and

erythematous vulva and vagina. Direct microscopic

result from wet preparation and Gram stain showed

positive and also negative result, but there were no

negative colony culture result. This showed that

direct microscopic examination is only an additional

tool supporting the examination. The negative result

of direct microscopic examination did not exclude the

diagnosis of VVC, but signs of clinical examination

establish the diagnosis (Kundu RV, Garg A., 2013;

Sobel JD, 2008).

There are 3 conventional methods that support

each other. SDA then Cornmeal Tween 80 agar

showed specific characteristic of each species of

Candida. The carbohydrate fermentation test showed

positive result based on the changing color to yellow

on 6 carbohydrate tested and the gas filled in the

Durham tube. CAC revealed the growth of Candida

sp by its colony color. The conventional methods

from 25 samples showed 14 samples of Candida

albicans, and others were Candida non-albicans, 5

samples were Candida glabrata. One sample were

Candida parapsilosis and 4 samples were grown with

2 Candida sp or infected by 2 Candida sp. Result of

samples: 1 sample were combination Candida

albicans with Candida glabrata, 1 sample were

combination of Candida albicans with Candida

famata, and 2 samples: each sample contained

Candida albicans with Candida tropicalis (Suyoso S.

Mucosal candidiasis. In: Bramono K, Suyoso S,

Indriatmi W, Ramali LM, Widaty S, Ervianti E,

editors., 2013; Larone DH, 2011)

In Vitek 2 from 25 samples showed result of

species identification Candida sp 100%, the same as

conventional methods. The highest number species

were on 14 samples (Candida albicans). The

conventional methods need about 36-72 hours to

identify the species , but Vitek 2 only needs 18 hours.

Vitek 2 has more advantages than conventional

methods. Vitek 2 immediately yielded the result of

species by the machine itself, Vitek 2 has faster time

than conventional methods to identify to the species

level and can work automatically so it does not need

manual labour, and it minimizes error (Mona et al.,

2015; Esmat MM, Mohamed T, Abdelrahman AH,

2005).

5 CONCLUSION

The best method of identification of the species is

using the combination of this 3 conventional

methods. Conventional identification methods are

still considered to be the reference standard for the

identification of yeast isolates and also for education

purposes, but are laborious and time-consuming.

Beside that, conventional methods can show

structures of Candida sp clearly that this structures

could not be seen using Vitek 2. Conventional

methods are important and useful for learning fungi

especially in teaching hospital, Dr Soetomo Surabaya

Hospital. A fast and accurate technique for yeast

identification is very important for microbiological

laboratories. According to the results found in the

present study, the Vitek 2 system (from cornmeal-

Tween 80 agar or CAC) identified most clinically

important Candida sp reliably within 18 hours, and

appears to be an excellent alternative identification

method for performing fungal diagnostics.

The result showed the most common yeast

causing VVC was Candida albicans (56%), but there

were increasing of Candida non-albicans that cause

VVC. Most Candida non-albicans are usually

causing antifungal resistance. It is therefore important

that there should be increased awareness among

physicians on the rising prevalence of Candida non-

albicans, due to the reduced susceptibility to azoles.

Prior identification to the species level on VVC is

essential to ensure early diagnosis of Candida non-

albicans infection and in order to give proper

treatment.

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