Comparison of Minimal Inhibitory Concentration Level in Vitro of

Itraconazole and Fluconazole against Malassezia furfur in Patients

with Pityriasis Vesicolor in Makasar

Dian Anggraeni, Olivia Wibisono, Safruddin Amin, Andi Muhammad Adam, Khairuddin Djawad

Department of Dermatology and Venereology, Hasanuddin University Faculty of Medicine, Makassar, Indonesia.

Keywords: Fluconazole, Itraconazole, Minimal Inhibitory Concentration, MIC, Malassezia furfur, Pityriasis vesicolor

Abstract: Pityriasis Versicolor (PV), the second most common dermatomycosis in Indonesia, is a superficial fungal

infection that is frequently reported in the tropics with warm temperatures and high humidity, as in Makassar.

Recurrence and long course of disease in PV is most often caused by M. furfur species. Hence, oral antifungal

is commonly used in extensive, recalcitrant and recurrent infections. The aim of this study is to assess the

MIC of Itraconazole (ITC) and Fluconazole (FLC) against M. furfur of PV patients in Makassar. This is a

multi-center cross sectional observational study with consecutive sampling of 21 isolates from PV patients in

Makassar, with the identification of M. furfur from morphological (culture using modified Dixon agar) and

biochemical criteria (catalase test and lipid assimilation test using Tween-20, 40, 60, 80, Cremophor El). Our

study shows MIC for ITC and FLC against M. furfur ranged from <0.03-0.25 μg/mL and <0.03-2 μg/mL,

respectively with MIC of ITC is lower than FLC. The regimen of ITC as systemic antifungal therapy for PV

patients in Makassar, particularly caused by M. furfur might be a more effective option.

1 INTRODUCTION

Pityriasis Versicolor or tinea versicolor is a

superficial fungal infection characterized by changes

in skin pigment caused by colonization of lipophilic

dimorphic fungi from the normal skin flora of the

stratum corneum (Moniri et al, 2009). This disease is

spread throughout the world but more often in the

tropics with warm temperatures and high humidity, as

in Makassar(Muhammad et al, 2009). In the tropics

the prevalence is 30-40% and the frequency becomes

higher in summer. Pityriasis Versicolor is the second

most common dermatomycosis after dermatophytosis

in Indonesia(Krisanty et al, 2009) Malassezia is a

lipophilic dimorphic fungus belonging to normal

flora and can be isolated from skin scrapings that

originate from almost all areas of the body, especially

in areas rich in sebaceous glands such as the chest,

back and head area (Krisanty et al, 2009). Identifies

Malassezia species in 98 PV patients based on

morphological observation and biochemical

evaluation in which M. furfur (42.9%) was the most

prevalent species.

Pityriasis Versicolor does not affect health

significantly but has psychological and social

implications. The optimal treatment of PV should

consider the effectiveness of the drug, safety, cost,

and patient complaints. Topical antifungal is the first

line of therapy, but in some patients complain of

unsatisfactory response, short-term success, and

regular application or longer treatment periods

especially for large lesions, prompt the consideration

of systemic antifungal (Silva et al, 1998). Antifungal

has its breakthrough since the discovery of azole

group, which has been shown to be safer than

previous antifungal agents. The azole agents exert its

antifungal property by inhibiting the cytochrome

P450, 14-alpha-demethylase enzyme. In addition

topical therapy has a high recurrence rate up to 60%

in the first year and 80% increase in the second year

(Hu and Bigby, 2010). Oral antifungal is commonly

used in extensive, recalcitrant and recurrent

infections, as it can penetrate keratin, while M. furfur

thrive at the base of the keratin layer (Pantazidou and

Tebruegge, 2007). Several studies have suggested

that ketoconazole (KTC), itraconazole (ITC), and

fluconazole (FLC) have been shown to produce high

clinical and mycological cure rates in patients with

PV.

ITC is a triazole group antifungal which is a

powerful keratophilic and lipophilic agent, having a

Anggraeni, D., Wibisono, O., Amin, S., Adam, A. and Djawad, K.

Comparison of Minimal Inhibitory Concentration Level in Vitro of Itraconazole and Fluconazole against Malassezia furfur in Patients with Pityriasis vesicolor in Makasar.

DOI: 10.5220/0008153101630166

In Proceedings of the 23rd Regional Conference of Dermatology (RCD 2018), pages 163-166

ISBN: 978-989-758-494-7

163

similar mechanism by inhibiting 14-alpha-

demethylase resulting in disruption of sterol synthesis

in the cell wall of the fungus. In vitro ITC is not only

active against Malassezia species and Candida

species but is also active against dermatophyte and

nondermatophyte fungi. The dose of ITC used for PV

is 200 mg/day for 7 days, with a minimum

accumulative dose of 1000 mg for effective therapy.

Four weeks after initial therapy, a cure rate of 80-90%

has been reported (Faergemann et al, 2002). FLC is

another antifungal class of azole with a high

absorption rate, in which optimum concentrations can

be found in the skin several hours after being

consumed in small doses. Benefits of FLC includes

rare side effects, mostly available, and preferred as it

requires only two or three weekly doses, compared

with a 7-day regimen for ITC (200 mg/day) (Silva et

al, 1998). In vitro susceptibility tests of the

Malassezia species to KTC, Voriconazole (VRC),

ITC and FLC were performed by Miranda et al. which

reported that the Malassezia species are highly

susceptible to the four azole preparations, but the

susceptibility to KTC and ITC appears higher

(Miranda et al, 2007).

This study was conducted to assess the minimal

inhibitory concentration (MIC) antifungal ITC and

FLC against M. furfur as the causative agent of PV in

Makassar in vitro. Previous in vitro research in

Makassar has not been done even though ITC and

FLC are one of the most effective modes of PV

therapy for recurrent cases after treatment with

topical antifungal, safer than other antifungal, and

readily available.

2 METHODS

This study is a multi-center cross sectional

observational study with consecutive sampling that

was performed in microbiology laboratory of

Hasanuddin University Faculty of Medicine in 2013.

Specimen of 21 samples were collected by skin

scraping from the back or shoulder, upper arm, chest,

face and neck of PV patients from Dr. Wahidin

Sudirohusodo Hospital and Hasanuddin University

Dermatovenereology Department’s networking

hospital in Makassar, of whom the diagnosis of PV

was confirmed by Wood’ s lamp, direct microscopic

KOH preparation, culture and signed the informed

consent. Then the specimen was planted on the

modified Dixon agar plate, incubated at 32-34°C and

was regularly assessed to confirm the growth of yeast

until the 3

week. Furthermore, the yeast was

identified by their morphology, catalase test and lipid

assimilation test (growth of yeast in the presence of

Tween-20, 40, 60, 80 and Cremophor El).

The in vitro susceptibility test by determining

MIC value of both antifungals was conducted using

broth microdilution that was performed in accordance

with the NCCLS guidelines in document M27-A2.

The inoculum suspension was prepared by the

spectrophotometric method obtaining a final

inoculum of (0.5-2.5)x10

cells/mL. The final

concentrations of the antifungal agents (ITC and

FLC) were 128µg/mL which then diluted half in

series and was inoculated to suspension hence

obtaining concentrations of 128 µg/mL, 64 µg/mL, 32

µg/mL, 16 µg/mL, 8 µg/mL, 4 µg/mL, 2 µg/mL, 1

µg/mL, 0.5 µg/mL, 0.25 µg/mL, 0.125 µg/mL, 0.06

µg/mL, and 0.03 µg/mL with false positive and false

negative control prepared. Growth of each various

concentrations of all two drugs was recorded every 24

h for 5 days of incubation at 32 ◦C. Cell growth was

compared with growth in a drug-free control. The

MIC was defined as the lowest concentration of agent

that produced none or 90% growth in comparison

with the control. Data analysis was performed using

SPSS. The Fisher Exact test was used to analyze the

mean and distribution frequency of each drug with P

value <0.05 is considered significant.

3 RESULT

Based on the morphological and biochemical

characteristics, the 21 isolates were identified as M.

furfur. MIC showed apparent differences in

antifungal susceptibility against M. furfur. For all 21

isolates, the MIC for ITC ranged from <0.03–

0.25µg/mL and <0.03–2µg/mL for FLC. The MIC

ranged, MIC90 values and MIC percentage for M.

furfur are presented in Table 1 and Figure 1.

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Table 1. MIC comparison of ITC and FLC against M. furfur isolates

Antifun

als

MIC cumulative

ercenta

e (%) of 21 isolates M. furfur

128 64 32 16 8 4 2 1 0.5 0.25 0.125 0.06 0.03 <0.03 MIC90

ITC 0 0 0 0 0 0 0 0 0 0 14.3 19 38.1 61.9 0.25

FLC 0 0 0 0 0 0 4.8 14.3 19 28.6 28.6 42.9 61.9 33.3 2

Figure 1. MIC comparison of ITC and FLC against M. furfur isolates.

4 CONCLUSION

In summary, our study shows MIC for ITC and FLC against

M. furfur ranged from <0.03-0.25 μg/mL with MIC90 0.25

μg/mL and <0.03-2 μg/mL with MIC90 2 μg/mL,

respectively. M. furfur isolates of our PV patients in

Makassar are still sensitive to both antifungals, with MIC

of ITC is lower than FLC. Thus, the regimen of ITC as

systemic antifungal therapy for PV patients in Makassar,

particularly caused by M. furfur might be a more effective

option although further studies based on clinical trials are

needed to confirm this

ACKNOWLEDGEMENT

We would like to express our gratitude to our

supervisors in Hasanuddin University Department of

Dermatology and Venereology for their guidance,

encouragement and useful critiques of this research.

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