CELL MEMBRANE OF Candida albicans

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THE EFFECTS OF ACTIVATED VIRGIN COCONUT OIL (AVCO) AND VIRGIN COCONUT OIL (VCO) ON

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

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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.

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ثحبلا ةصلاخ

ي دنلها زوج تيز مدختس ركبلا

( virgin coconut oil, VCO )

في تاباهتللاا جلاع يمدق نم

،نمزلا ةياقول اضيأ مدختسي امك ةيبوركيلما ىودعلا نم هتياحمو ناسنلإا مسج

و لما ةيرطفلا ببس

ة للأ رما ض

لثم ءاد ونم طرف نع ةتجان ىودع وه يومفلا تاضيبلما ءاد .يومفلا تاضيبلما يم

تاضيبلما تابورك

ءاضيبلا . ناك ذه نم فدلها وه ثحبلا ا

دنلها زوج تيز يرثأت ةسارد ركبلا

شنلما ( ط activated

virgin coconut oil, AVCO صلختسلماو )

تا مالخا ة للـ VCO لخا ءاشغلا للتح ىلع

يول

و لمحا تانوكم وتيسلا تايوت

ب ةيمزلا لما تايرطفل تاضيب

ءاضيبلا خلاا ليلتح تم . تافلات

في ىوتمح ضاحملأا

في ةينهدلا ــلا

AVCO و

ــلا VCO اغلل يلتكلا يفيطلا ليلحتلا ةقيرط مادختساب

( ز GC-MS )،

و حيضوتل نم لاك يرثأت

AVCO و

VCO

، مادختسا تم اييرتكبلا ةيويح رابتخا مقط

ا ةيلحاو ةتيلم

ققحتلل ةيويلحا نم للخا

و ةي ل لا تاضيبمل ءاضيب

في

،يلحا مسلجا جراخ تاتبنتسم هتنياعم تمو

ا رهمج تتح

يروفسف نيوتركللإا رهلمجا مادختسا تم . ذفانلا

ل يوللخا لكشتلا صحفل ل

تاضيبم ال

ءاضيب اهتلجاعم دعب

ــلاب AVCO و

ــلا VCO يولخ برست نع فشكلا تم .

لما في تاضيب لا اضيب ء م مادختساب ةعشلأا ةيفايط

ةيجسفنبلا قوفو ةيئرلما (

UV/Vis )

. دكأ زلابوتيسلا برستلا بوقث ثودح ىلع يم

في ءاشغلا يوللخا

ةطساوب AVCO .

ىدأ بارطضا لا

للخا ءاشغ و

لىإ ي ءوشن لهلاخ نم ثديح ماسم ا

برست ا ةيوللخا داولم

.)يوونلا ضملحاو ينتوبرلا(

تابثإ تم نأ

ىدل AVCO يرثأت

ا داضم ا لع تايرطفلل ءاضيبلا تاضيبلما ى

قيرط نع هيوشت

لخا ةيشغأ كيكفتو ،ايلالخا ناردج جئاتن تحترقا .ةيرطفلا ايلا

نأ ةساردلا هذه ناكمإب ه

AVCO ت

طع ي قلاطإ ببسيو يوللخا ءاشغلا ةينب ل لما

مزلابوتيسلا داو ةي

ضيبلما نم ببسب ءاضيبلا تا

ليطعت .ءاشغلا ةكبش ــلل ةبسنلاب امأ

VCO رهظأ دقف

وأ اًدودمح اًطاشن امودعم

ض تايرطفلا د ىلع

لا للخا ءاشغ ل يو

ءاضيبلا تاضيبمل دعاست .

تانوكلم يوللخا برستلا ةسارد لا

ءاشغ للخا يو ل تاضيبمل

ضيبلا ءا لمع ةيلآ مهف في ــلا

AVCO اًجلاع نوكت نأ ىلع ةردقلا اهيدل تيلا و

تايرطفلل اًداضم اًديد ج

ل يومفلا تاضيبلما ءاد

، لما تايرطفلا هذه ةحفاكلم ًلايدب نوكي نأ نكيم يذلا و تيلاو ضارملأل ةببس

تمواقم لحا اه ديازت في ةيوي ماتخ .تايرطفلل ةداضلما ةيلالحا لماوعلا دض

ا ترقت نأ ةساردلا هذه ح ىدل

ــلا AVCO طاشن

اداضم لل تايرطف دض

ضيبلا تاضيبلما ىوقأ

صلختسلما عم ةنراقم ا

مالخ

ــلل

VCO

.

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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

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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...

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COPYRIG HT PAG E

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.

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

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This thesis is dedicated to my beloved parents for laying the

foundation of what I turned out to be in life .

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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.

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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

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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

3.5Release 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

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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

Table 4.5 Significance Value for Means Comparison among 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

Table 4.7 Significance Value for Means Comparison among AVCO, VCO, Nystatin and Tween throughout 4 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

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Table 4.9 Significance Value for Means Comparison among AVCO, VCO, Nystatin and Tween throughout 4 Hours of Total Protein Concentration.

57

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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

47

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

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Figure 4.9 TEM Images of C. albicans Cells after 3 H Treatment with the Crude Extract of VCO

60

Figure 4.10 TEM Images of C. albicans Cells after 3 H Treatment with AVCO

61

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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

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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

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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).

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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).

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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

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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?

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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.

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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

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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

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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).

CELL MEMBRANE OF Candida albicans

THE EFFECTS OF ACTIVATED VIRGIN COCONUT OIL (AVCO) AND VIRGIN COCONUT OIL (VCO) ON

شنلما ( ط activated

DECLARATION

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

This thesis is dedicated to my beloved parents for laying the

TABLE OF CONTENTS

CHAPTER TWO LITERATURE REVIEW ... 6

CHAPTER THREE MATERIALS AND METHOD ... 30

CHAPTER FOUR RESULTS AND DISCUSSION ... 40

APPENDIX B ... 79

LIST OF FIGURES

LIST OF SYMBOLS

CHAPTER ONE INTRODUCTION

1.2 PROBLEM STATEMENT

1.3 GENERAL RESEARCH OBJECTIVE

1.5 RESEARCH QUESTIONS

1.6 RESEARCH HYPOTHESIS

CHAPTER TWO LITERATURE REVIEW

Clinical classification