the effects of activated virgin coconut oil (avco) and

iii

THE EFFECTS OF ACTIVATED VIRGIN COCONUT

OIL (AVCO) AND VIRGIN COCONUT OIL (VCO) ON

CELL MEMBRANE OF Candida albicans

BY

NOR IZZAH BINTI MUKHTAR

A thesis submitted in fulfillment of the requirement for the

Master of Science (Biosciences)

Kulliyyah of Science

International Islamic University Malaysia

MAY 2019

ii

ABSTRACT

Virgin coconut oil (VCO) has been used in treating infections for ages. VCO can be

used in preventing and protecting human body from microbial and fungal infections

which can cause disease such as oral candidiasis. Oral candidiasis is an infection which

is caused by the overgrowth of Candida albicans. However, the knowledge on the

mechanism of action of AVCO and VCO on the cell membrane of C. albicans is limited.

The aim of this study is to investigate the effects of activated virgin coconut oil (AVCO)

and the crude extract of VCO on the cell membrane lysis and the components of

cytoplasmic contents of Candida albicans. The differences of fatty acids composition

in AVCO and VCO was analysed by using gas chromatography-mass spectrometry

(GC-MS) method. To elucidate the effects of AVCO and VCO, live/dead bacterial

viability kit was used to investigate the cell viability of Candida albicans in vitro

cultures and was observed under fluorescence microscopy. Transmission electron

microscopy was used to examine the cellular morphology of Candida albicans after

being treated with AVCO and VCO. The cellular leakage of Candida albicans was

detected by using UV/Vis spectrophotometer. Cytoplasmic leakage confirms creation

of holes in the membrane by AVCO. Disturbance of cell membrane causes pore through

which cellular materials (protein and DNA) leakage takes place. AVCO has been

proven to have an antifungal effect on Candida albicans by altering the cell walls,

disintegrating fungal cell membranes. The results of this study suggested that AVCO

may disrupt the structure of the cell membrane and causes the release of cytoplasmic

materials of Candida albicans due to the destruction of membrane network.

Nevertheless, VCO has shown limited or no antifungal activity on the cell membrane

of C. albicans. The study of cellular leakage of cell membrane components of Candida

albicans may help to understand the mechanism of action of AVCO which has the

potential to be a new antifungal treatment in treating oral candidiasis that can be an

alternative to combat this pathogenic fungus which increases its resistance against

current antifungal agents. In conclusion, this study proposes that AVCO has higher

antifungal activity against Candida albicans compared to the crude extract of VCO.

iii

خلاصة البحث

من قديم علاج الالتهاباتفي ( virgin coconut oil, VCO) البكرستخدم زيت جوز الهند يض امر لأل ةسببالفطرية المو جسم الإنسان وحمايته من العدوى الميكروبية كما يستخدم أيضا لوقاية الزمن،كروبات المبيضات مي المبيضات الفموي. داء المبيضات الفموي هو عدوى ناتجة عن فرط نمو داءمثل

activatedط )المنشالبكر دراسة تأثير زيت جوز الهند ا البحث هو الهدف من هذكان . البيضاءvirgin coconut oil, AVCOـلل ةالخام ات( والمستخلصVCO لوي على تحلل الغشاء الخ

الأحماض محتوىفي تلافات. تم تحليل الاخالبيضاء بيضاتلفطريات الم لازميةبتويات السيتو مكونات المحو ،(GC-MSز )باستخدام طريقة التحليل الطيفي الكتلي للغا VCOالــو AVCOالــ الدهنية في

لميتة والحيةاطقم اختبار حيوية البكتيريا تم استخدام ،VCOو AVCO تأثير كلا منلتوضيح و تحت مجهر اوتم معاينته مستنبتات خارج الجسم الحي،في بيضاءلمبيضات اللية و الخل من الحيويةللتحقق بعد معالجتها بيضاءلامبيضات للفحص التشكل الخلوي ل النافذ . تم استخدام المجهر الإلكترونيفسفوري

طيافية الأشعة باستخدام م ءبيضاالبيضات في الم. تم الكشف عن تسرب خلوي VCOالــو AVCOبالــ الخلوي الغشاءفيمي على حدوث ثقوب التسرب السيتوبلاز أكد. (UV/Vis) المرئية وفوق البنفسجية

لمواد الخلوية اتسرب امسام يحدث من خلاله نشوءي إلى و غشاء الخلالاضطراب أدى. AVCOبواسطة ى المبيضات البيضاء للفطريات عل امضاد اتأثير AVCO لدى أن تم إثبات)البروتين والحمض النووي(.

ه بإمكان هذه الدراسة أن لايا الفطرية. اقترحت نتائججدران الخلايا، وتفكيك أغشية الخ تشويهعن طريق AVCO ات البيضاء بسبب من المبيض يةواد السيتوبلازمالمل بنية الغشاء الخلوي ويسبب إطلاق يعطت

على د الفطرياتضمعدوما نشاطاً محدودًا أو فقد أظهر VCOأما بالنسبة للــشبكة الغشاء. تعطيللمبيضات ل ويالخل غشاءالدراسة التسرب الخلوي لمكونات . تساعدلمبيضات البيضاءوي لغشاء الخلال

ديدًا مضادًا للفطريات جالتي لديها القدرة على أن تكون علاجًا و AVCOالــ في فهم آلية عمل اءالبيضسببة للأمراض والتي الذي يمكن أن يكون بديلًا لمكافحة هذه الفطريات المو ،داء المبيضات الفمويل

لدى ح هذه الدراسة أنتقتر ا ضد العوامل الحالية المضادة للفطريات. ختام يوية في تزايدها الحمقاومت.VCOللــلخام امقارنة مع المستخلص أقوى المبيضات البيضضد فطريات لل مضادا نشاط AVCOالــ

iv

APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion, it conforms

to acceptable standards of scholarly presentation and is fully adequate, in scope and

quality, as a thesis for the degree of Master of Science (Biosciences)

…………………………………..

Zurainie Abllah

Supervisor

…………………………………..

Mohd. Azrul Naim Mohamad

Co-Supervisor

…………………………………..

Intan Azura Shahdan

Co-supervisor

I certify that I have read this study and that in my opinion it conforms to acceptable

standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis

for the degree of Master of Science (Biosciences)

…………………………………..

Hanani Ahmad Yusof @ Hanafi

Internal examiner

…………………………………..

Wan Himratul Aznita Wan Harun

External examiner

This thesis was submitted to the Department of Biotechnology and is accepted as a

fulfilment of the requirement for the degree of Master of Science (Biosciences)

………..……………………….......

Mardiana Mohd Ashaari

Head, Department of Biotechnology

This thesis was submitted to the Kulliyyah of Science and is accepted as a fulfilment of

the requirement for the degree of Master of Science (Biosciences)

…………………………

Shahbudin Saad

Dean, Kulliyyah of Science

v

DECLARATION

I hereby declare that this thesis is the result of my own investigations, except where

otherwise stated. I also declare that it has not been previously or concurrently submitted

for any other degrees at IIUM or other institutions.

Nor Izzah binti Mukhtar

Signature ........................................... Date........................................

vi

COPYRIGHT PAGE

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION

OF FAIR USE OF UNPUBLISHED RESEARCH

THE EFFECTS OF ACTIVATED VIRGIN COCONUT OIL

(AVCO) AND VIRGIN COCONUT OIL (VCO) ON CELL

MEMBRANE OF Candida albicans

I declare that the copyright holders of this thesis are jointly owned by the student and

IIUM.

Copyright © 2018 (Nor Izzah binti Mukhtar) and International Islamic University Malaysia. All rights

reserved.

No part of this unpublished research may be reproduced, stored in a retrieval system,

or transmitted, in any form or by any means, electronic, mechanical, photocopying,

recording or otherwise without prior written permission of the copyright holder except

as provided below

1. Any material contained in or derived from this unpublished research may

be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print or

electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieved system

and supply copies of this unpublished research if requested by other

universities and research libraries.

By signing this form, I acknowledged that I have read and understand the IIUM

Intellectual Property Right and Commercialization policy.

Affirmed by Nor Izzah binti Mukhtar

……..……..…………… …………………..

Signature Date

vii

This thesis is dedicated to my beloved parents for laying the

foundation of what I turned out to be in life.

viii

ACKNOWLEDGEMENT

In the name of Allah, The Most Gracious and The Merciful. Alhamdulillah, all praise

to Allah for the strength and His blessing on me throughout the sweat and tears in

preparing this thesis. It will never be possible without all the sincere prayers from the

amazing servants of Allah around me.

I would like to express my deepest gratitude to my beloved parents; Mukhtar

bin Ahmad and Rohani binti Ahmad and to my siblings for their endless love and

support throughout my study. They have been with me through thick and thin till the

end of this journey.

Special appreciation goes to my supervisor and co-supervisor, Asst. Prof. Dr.

Zurainie Abllah, Asst. Prof. Dr. Intan Azura Shahadan and Dr. Mohd. Azrul Naim for

their supervision and constant support. Their invaluable help through their constructive

comments and suggestions throughout the experimental and thesis-writing works have

led to the success of this research.

Sincere thanks to all my friends Ummi Aqilah, Amilin, Fatin, Umi Kalthum,

Ainatul, Fatimah, Fatihah, Faiqah, Kasmadiana, Maisarah, Syazila and Ruhan for their

kindness during my study. Thanks for the friendship and memories. Not to forget, all

Science Officers and the laboratory staffs who have been assisting me with the

laboratory works and acquiring the most valuable knowledge and experiences.

To all the above specifically mentioned, and to others not mentioned in this

piece of writing, I would like to say jazakumullahu khairan kathira and thank you so

much.

ix

TABLE OF CONTENTS

Abstract ....................................................................................................................... ii Abstract in Arabic ....................................................................................................... iii Approval Page ............................................................................................................. iv Declaration .................................................................................................................. v

Copyright Page ............................................................................................................ vi Acknowledgement ...................................................................................................... viii Table of Contents ........................................................................................................ ix List of Tables .............................................................................................................. xi List of Figures ............................................................................................................. xiii

List of Symbols ........................................................................................................... xv List of Abbreviations .................................................................................................. xvi

CHAPTER ONE INTRODUCTION ...................................................................... 1 1.1 Research Background ................................................................................ 1

1.2 Problem Statement ..................................................................................... 3 1.3 General Research Objective ....................................................................... 4 1.4 Research Objectives ................................................................................... 4

1.5 Research Questions .................................................................................... 4 1.6 Research Hypothesis .................................................................................. 5

1.7 Significance of Study ................................................................................. 5

CHAPTER TWO LITERATURE REVIEW ......................................................... 6 2.1 Oral Candidiasis ......................................................................................... 6

2.2 Candida albicans ....................................................................................... 9 2.2.1 Structure of C. albicans................................................................... 10

2.2.2 Pathogenicity of C. albicans ........................................................... 14

2.3 Current Treatment of Oral Candidiasis ...................................................... 18 2.3.1 Mechanism of Action of Antifungal Agents ................................... 20

2.3.2 Resistance against Antifungal Agents ............................................. 21 2.4 Virgin Coconut Oil (Cocos nucifera L.) .................................................... 23

2.4.1 Hydrolysis of Coconut Oil .............................................................. 24

2.4.2 Medium Chain Fatty Acids (MCFA) .............................................. 25 2.4.3 VCO as A Potential Treatment against Oral Candidiasis ............... 26 2.4.4 AVCO with Broad Antimicrobial/Antifungal Spectrum ................ 28

2.5 Mechanism of Action of Plant Oils against C. albicans ............................ 28 2.5.1 Antifungal Action of Plant Oils on C. albicans .............................. 28

CHAPTER THREE MATERIALS AND METHOD ............................................ 30 3.1 Preparation of Virgin Coconut Oil ....................................................................... 30

3.1.1 AVCO and VCO Stock Preparations .............................................. 31 3.2 Preparation of Fatty Acids Methyl Esters (Fame) ..................................... 31

3.2.1 Gas Chromatography Mass Spectrometry ...................................... 31 3.3 Growth and Maintenance of Candida albicans ......................................... 32 3.4 Cell Viability .............................................................................................. 32

x

3.4.1 Culture Conditions and Preparation of Fungal Suspensions ........... 32

3.4.2 Staining Fungal Suspension with Kit L7012 .................................. 33 3.4.3 Microscopic Observations Using Fluorescence Microscopy .......... 34

3.5 Release of Cytoplasmic Material ............................................................... 34

3.5.1 Release of Cytoplasmic Material Absorbing at 260 Nm and

280 Nm Assay .......................................................................................... 35 3.5.2 Concentration of Protein Content Measurement ............................. 35

3.6 Transmission Electron Microscopy (TEM) ............................................... 36 3.6.1 Uranyl Acetate Replacement Stain (UAR) ..................................... 37

3.7 Statistical Analysis ..................................................................................... 38 3.8 Flow Chart of Methodology ....................................................................... 39

CHAPTER FOUR RESULTS AND DISCUSSION .............................................. 40 4.1 Analysis of the Composition of Fatty Acids In AVCO and VCO ............. 40 4.2 Determining the Viability of C. albicans Using Fluorescence

Microscopy ...................................................................................................... 42

4.3 Quantification of Cytoplasmic Leakage of C. albicans ............................. 49 4.4 Transmission Electron Microscopy ........................................................... 58

CHAPTER FIVE CONCLUSION AND OUTLOOK ........................................... 62

CHAPTER SIX REFERENCES ............................................................................. 63

APPENDIX A ............................................................................................................ 76

APPENDIX B ............................................................................................................ 79

APPENDIX C ............................................................................................................ 80

xi

LIST OF TABLES

Table 2.1 Clinical Classification of Oral Candidiasis 6

Table 2.2 Types of Secreted Proteins of C. albicans 15

Table 2.3 Virulence factors of C. albicans 16

Table 2.4 Summary of Drugs for the Treatment of Oral

Candidiasis

19

Table 2.5 Summary of Previous Studies on Virgin Coconut Oil 27

Table 3.1 Category of Sample Used for Cell Viability Test 33

Table 3.2 Category of Sample Used for Release of Cytoplasmic

Material

34

Table 4.1 Fatty Acid Composition (%) of VCO and AVCO

Compared with APCC Standard Range

40

Table 4.2 Percentage Distribution of Dead Fungi after Staining 43

Table 4.3 Significance Value for Means Comparison among

AVCO, VCO, PC and NC throughout 3 Hours.

43

Table 4.4 Cytoplasmic Release of DNA at 260 Nm from C.

albicans after Treatment with AVCO, VCO, Nystatin

and Tween.

52


AVCO, VCO, Nystatin and Tween throughout 4

Hours of Cytoplasmic Release of DNA.

52

Table 4.6 Cytoplasmic Release of Protein at 280 Nm from C.

albicans after Treatment with AVCO, VCO, Nystatin

and Tween.

53



Hours of Cytoplasmic Release of Protein.

53

Table 4.8 Concentration of Protein Leakage of C. albicans after

Treatment with AVCO, VCO, Nystatin and Tween.

57

xii



Hours of Total Protein Concentration.

57

xiii

LIST OF FIGURES

Figure 2.1 The Image of Different Lesions of Oral Candidiasis. 9

Figure 2.2 Different Growth Morphologies of C. albicans. 11

Figure 2.3 Cellular Organization of Candida albicans 11

Figure 2.4 Candida albicans Cell Wall Structure 13

Figure 2.5 Candida albicans Plasma Membrane Microdomains 14

Figure 2.6 Chemical Structure of Capric Acid and Lauric Acid 26

Figure 3.1 The Crude Extract of VCO on the Second Layer of

Fermented Coconut Oil

30

Figure 4.1 The Viability of C. albicans Cells in the Presence of

70% Ethanol, Saline, AVCO and VCO after 1-H

Incubation

46



Incubation

47



Incubation

48

Figure 4.4 Cytoplasmic Leakage of C. albicans Cells (A) DNA

and (B) Protein due to Nystatin, VCO and AVCO

Treatments Respectively at Different Time Points (1,

2, 3 and 4 H) at Room Temperature

51

Figure 4.5

Standard Curve of Bovine Serum Albumin (BSA)

55

Figure 4.6 Protein Leakage Analysis Using Bradford Assay:

Absorbance at 595 Nm of Supernatant Mixed with

Bradford Reagent

56

Figure 4.7 TEM Images of C. albicans in the Presence of Tween

as A Control.

59

Figure 4.8 TEM Images of C. albicans Cells after 3 H Treatment

with Nystatin

60

xiv


with the Crude Extract of VCO

60


with AVCO

61

xv

LIST OF SYMBOLS

g Gram

h Hour

µg/ml Microgram per Millilitre

m Metre

min Minute

mm Micrometre

ml Millilitre

m/z Mass-to-Charge Ratio

mol Mole

nm Nanometre

M Molar

℃ Degree Celsius

% Percentage

xvi

LIST OF ABBREVIATIONS

AVCO Activated Virgin Coconut Oil

BSA Bovine Serum Albumin

C. albicans Candida albicans

et al. (et alia) and others

FAME Fatty Acids Methyl Esters

FFA Free Fatty Acids

GC-MS Gas Chromatography- Mass Spectrometry

VCO Virgin Coconut Oil

MARDI Malaysia Agriculture Research and Development Institute

MCFA Medium Chain Fatty Acids

MFC Minimal Fungicidal Concentration

OD Optical Density

PI Propidium Iodide

SD Standard Deviation

TEM Transmission Electron Microscope

UAR Uranyl Acetate Replacement Stain

YPD Yeast Peptone Dextrose

BF3 Boron Trifluoride

MeOH Methyl Hydroxide

NaCl Sodium Chloride

NaOH Sodium Hydroxide

OsO4 Osmium Tetraoxide

1

CHAPTER ONE

INTRODUCTION

1.1 RESEARCH BACKGROUND

Naturally, humans are continuously susceptible and harbouring to different normal

microflora. Colonisation of fungi may be reliant on several factors like host’s immune

system, fungal virulence factors and antifungal therapy utilisation. Candida is the

foremost genus causing fungal disease like oropharyngeal candidiasis. Oral candidiasis

has come into existence since the era of Hipocrates (Anderson & Odds, 1985). The

commonest fungus that has highest prevalence in candidiasis is Candida albicans

(Jacobsen et al., 2008). Normally, some Candida species are non-pathogenic and

inhabit the mucosal surfaces and the skin surface of humans. Mostly, these yeasts reside

in the oral cavity, gastrointestinal tract and vagina (Ganguly & Mitchell, 2011; Nobile

& Johnson, 2015).

Candidal infections can be fatal if it is circulated through the bloodstream and

upper gastrointestinal tract (Akpan & Morgan, 2002). The colonization of Candida can

bring to local discomfort, a modified taste sensation, dysphagia, slow recovery and

prolonged hospital admission. Candida infection is possibly occurring in healthy

persons in the range of 10%-100% asymptomatic (Akpan & Morgan, 2002). Isolation

of C. albicans from oral cavity was reported to be 45% in neonates, 45%-65% in healthy

children, 30%-45% in healthy adults, 50%-65% in dental wearers, 65%-88% from

patient in long term care facilities and 90% from leukaemia patient which undergone

chemotherapy treatment (Akpan & Morgan, 2002).

2

Previous studies stated that biofilm formation of fungi might contribute to most

of diseases caused by C. albicans (Martinez, & Fries, 2010; Ramage, Saville, Thomas,

& Lopez-Ribot, 2005). Planktonic cells of C. albicans are grouped together forming

multicellular community embedded in self-produced matrix of host cells or extracellular

polymeric substances such as prostheses, catheters and other surfaces (Soll, 2008). C.

albicans can form biofilm through cellular recognition such as specific interaction, for

instance, adhesion-ligand bonds and non-specific interactions, like hydrophobic and

electrostatic forces (Ramage et al., 2005; Chaffin, 2008; De Groot, Bader, de Boer,

Weig, & Chauhan, 2013; Epstein, & Nicholson, 2016; Lipke, 2018).

In clinical use, three types of major antifungal agents have been tested against

C. albicans. They are amphotericin B, echinocandins (caspofungin, anidulafangin and

micafungin) and two triazole agents (posaconazole and voriconazole) have been used

against C. albicans (Hacioglu, Tan, Dosler, Inan, & Otuk, 2018). Nevertheless, the

resistance of C. albicans biofilms towards these agents may be up to 1000 times

compared to planktonic cells (Shuford, Piper, Steckelberg, & Patel, 2007; Tobudic,

Lassnigg, Graninger, & Presterl, 2012; Tobudic, Lassnigg, Kratzer, & Presterl, 2010;

Sardi, Scorzoni, Bernardi, Fusco-Almeida, & Mendes Giannini, 2013). Previous studies

showed that Candida has prodigious adaptability to the different physiological

environment such as immunocompromised situations. For example, biofilms of C.

albicans are less vulnerable to antifungal agents compared to planktonic cells (Ramage

et al, 2005; Taff, Mitchell, Edward, & Andes, 2013). Recently natural resource like

virgin coconut oil have been studied as an alternative treatment against C. albicans

(Tjin, Setiawan, & Rachmawati, 2016).

3

Coconut, Cocos nucifera L from the family of Arecaceae (Palmae) is the source

of virgin coconut oil (VCO). VCO has been used in this study to investigate its

antifungal effect on C. albicans. VCO has high content of lauric acid which is one of

the components of medium chain triglycerides. Lauric acid is known to have

antibacterial, antiviral and antimicrobial properties (Kamariah, et al., 2008). Due to the

high content of high saturated fatty acids which mostly composed of lauric acid, VCO

seems to have high resistance against oxidation and prevents rancidity due to the

functionality and stability of the fatty acids (Ahmad et al., 2011; O’Brien, 2008). In

this study, AVCO and VCO was used to evaluate the mechanism of action of on the cell

membrane of C. albicans.

1.2 PROBLEM STATEMENT

Virgin coconut oil is obtained from the kernel of the coconut, a tropical plant

which has many health benefits in cooking, confectionary, cosmetics and health

supplement area. AVCO that has been modified from the catalytic activity of specific

lipase composed of medium chain fatty acids and their corresponding monoglycerides

have been claimed to have broad spectrum of antimicrobial/antifungal activity against

bacteria and fungus (Long, 2010). AVCO is well known for its lauric and capric acids

that can kill C. albicans effectively and may be used to treat infections caused by other

pathogens which is one of the complication from broad-spectrum

antibacterial/antifungal treatment (Bergsson, Arnfinnsson, Steingrimsson & Thormar,

2001).

The resistance of C. albicans to the current antifungal agents has increased by

time and becoming a major concern worldwide (De Castro et al., 2015; Tanwar, Das,

Fatima, & Hameed, 2014). The advancement of antifungal resistance of C. albicans

4

has supported the maturation process of biofilm of the fungus (Sardi, Almeida, &

Giannini, 2011). Thus, another option for therapeutic treatments are necessary and new

antifungal agents are needed to come up with a better solution facing the problems of

antifungal resistance in fungi. Although there were several studies about the antifungal

activity of AVCO on C. albicans, yet the mechanism of action of AVCO on C. albicans

is still unknown. Thus, this study is to investigate the mechanism of action of AVCO in

killing C. albicans which can become pathogenic fungi in oral cavity that can lead to

oral candidiasis.

1.3 GENERAL RESEARCH OBJECTIVE

This study aims at evaluating the effects of AVCO and the crude extract of VCO on cell

membranes of Candida albicans.

1.4 RESEARCH OBJECTIVES

The specific objectives of this study are:

1. To compare the composition of fatty acids in AVCO and the crude extract

of VCO.

2. To evaluate the effects of AVCO and the crude extract of VCO on the cell

membrane of Candida albicans.

3. To analyse the release of cytoplasmic contents of Candida albicans after

exposure to AVCO and the crude extract of VCO.

1.5 RESEARCH QUESTIONS

1. What is the highest composition of fatty acids in AVCO and the crude

extract of VCO?

5

2. What are the effects of AVCO and VCO on cell membrane lysis of Candida

albicans?

3. What is the release of fungal components of Candida albicans after

exposure to AVCO and VCO?

1.6 RESEARCH HYPOTHESIS

1. Medium chain fatty acids are the highest composition in AVCO and the

crude extract of VCO.

2. AVCO lyses the cell membrane of Candida albicans.

3. AVCO releases protein and DNA of Candida albicans after exposure.

1.7 SIGNIFICANCE OF STUDY

1. This study is carried out to find out the mechanism of action of AVCO

which needs further insight in killing C. albicans that may help to reduce

the cases of oral candidiasis in patients.

2. This study is done to investigate the antifungal activity of AVCO in order

to find the new antifungal treatment based on natural product.

6

CHAPTER TWO

LITERATURE REVIEW

2.1 ORAL CANDIDIASIS

Oral candidiasis also known as oral thrush or oropharyngeal candidiasis. The

overgrowth of Candida species, the commonest being is Candida albicans, in the oral

cavity may lead to the oral lesions and this is related to oral candidiasis which can be

classified as acute, chronic and other lesions. The recognition of oral lesions is crucial

because the treatment given is depends on the type of oral lesions as shown in Table

2.1. Numerous predisposing factors have been reviewed by previous study such as

nutritional deficiency, extremes of age, oral cancer, malignancy, chemotherapy,

phagocyte dysfunction, and individuals with AIDS. Oral candidiasis also can infect

immunocompetent or healthy people who are using broad-spectrum of antibiotics,

wearing dentures and smoking (Krishnan, 2012; Pankhurst, 2012).

Table 2.1: Clinical Classification of Oral Candidiasis (Garcia-Cuesta, Sarrion-Pérez,

& Bagán, 2014).

Clinical

classification

Acute Chronic Other lesions

Type of oral

candidiasis

Pseudomembranous Pseudomembranous Angular cheilitis

Erythematous Erythematous Denture-associated

erythematous

Hyperplastic Median rhomboid

glossitis

7

A high prevalence of oral candidiasis which is the most common muco-

cutaneous disease was also reported amongst HIV patients and this has led to the initial

manifestation of HIV infection (Akpan & Morgan, 2002). In Malaysia, the incidence of

oral candidiasis associated with HIV patient is 43.4% (Nissapatorn, Lee, Rohela &

Khairul Anuar, 2004). Immunosuppressed patients are prone to invasive oral

candidiasis by several factors such as undergoing cytotoxic chemotherapies,

transplantation, multiplying the use of broad-spectrum antibiotics and invasive

procedures such as the use of intravenous catheters and total parenteral nutrition (Ortega

et al., 2011). Oral infections can also occur in neonatal intensive care, in people with

head and neck cancers who have undergone radiotherapy and in people who inhaled

steroids for asthma treatment (Brown et al., 2012).

To prevent complications of oral candidiasis, practicing a good oral hygiene can

be a worthy management. Starting points towards better oral health can begin with

cleansing the teeth, buccal cavity, tongue and dentures daily. A thorough examination

of the mouth is essential to make a right diagnosis especially to those wearing dentures

in response to antifungal therapy (Akpan & Morgan, 2002). The first-line treatment

advocated for less complicated oral candidiasis is topical antifungal therapy whereas

the systemic antifungal therapy is given under certain occasions such as those who have

high chance of developing systemic infection (Akpan, & Morgan, 2002).

In a study reported by Nissapatorn et al., (2004), significant associations were

found between oral candidiasis and main opportunistic systemic diseases such as

tuberculosis, pneumocystis pneumonia, penicillosis, toxoplasmic encephalitis and

cytomegalovirus retinitis. In addition, the most regular oral manifestation of HIV

infection is related to oral candidiasis (Shiboski et al., 2014). The diagnosis of oral

8

candidiasis can be performed by visual examination of the mouth by the health care

providers with proper training (Chidzonga, et al., 2008).

Having a dry mouth, loss of taste sensitivity and a burning sensation are some

of the clinical features of the patients with oral candidiasis. Furthermore, the

exacerbation of candidal infection can threaten the patients by disseminating through

blood circulation and affecting major internal organs. Thus, an early identification of

the disease and applicable intervention of the underlying diseases are necessary to avoid

the complications of oral candidiasis (Zhou, Hua, & Liu, 2017). Early diagnosis of

candidiasis is significant because it can lead to the manifestation of systemic disorder

including HIV infection (Warrier & Sathasivasubramanian, 2015).

The diagnosis of oral candidiasis majorly is based on the clinical features of the

patients. However, the interference of other mucosal diseases accompanied by oral

candidiasis in clinical differentiation is quite a problem. Thus, oral candidiasis can be

recognized from the lesions in the mouth as shown in Figure 2.1 which can be identified

by microscopic identification of Candida from oral samples or isolation in culture

(Zhou et al., 2017). To differentiate Candida spp. between oral candidiasis patient and

healthy people, it is compulsory to ascertain the threshold amount of Candida spp. In

healthy population, 20% to 40% is the normal range of Candida found in their saliva

(Jenkinson & Douglas, 2002; McCullough et al., 2002).

the effects of activated virgin coconut oil (avco) and

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