MULTIPLEX REAL-TIME PCR FOR THE

DETECTION OF PATHOGENIC INTESTINAL

PARASITES AND COMPARISON WITH

PARASITOLOGICAL TECHNIQUES

MADIHAH BINTI BASUNI

UNIVERSITI SAINS MALAYSIA

2012

brought to you by COREView metadata, citation and similar papers at core.ac.uk

provided by Repository@USM

MULTIPLEX REAL-TIME PCR FOR THE DETECTION OF PATHOGENIC

INTESTINAL PARASITES AND COMPARISON WITH PARASITOLOGICAL

TECHNIQUES

by

MADIHAH BINTI BASUNI

Thesis submitted in fulfillment of the

requirements for the degree of

Master of Science

January 2012

ii

ACKNOWLEDGEMENTS

All praises and gratitude is due to Allah - alhamdulillah, may salawat and salam be granted

to His beloved Prophet Muhammad s.a.w., to his family and companions r.a.

My sincere thanks and appreciation is extended to my supervisor, Professor Rahmah

Noordin who is generous for sharing her wide knowledge, guidance and encouragement

throughout the duration of this work. Her comments, help, advices, suggestions and criticism

have been a great value for me. My warmest thanks also goes to my co-supervisor Dr

Zeehaida Mohamed, who has contributed significantly in my project.

My sincere appreciation is extended to all lecturers, laboratory and administration staffs of

INFORMM who have helped me from time to time during the study. Besides, I would also

like to acknowledge Dr Jamail Muhi, the Deputy Director of the State Health of Sarawak,

and his staffs; En Jamadi, Pn Lila, Pn Norkiah and Pn Rosa from Kuching Health

Department, Hospital Serian and Hospital Lundu. A lot of appreciation also goes to the

contributions from Pn Maimunah Ahmad, En Hafiz Mohamad, Cik Jannah Iman and

laboratory staffs at HUSM. My sincere thanks to Dr Hasnah from IPPT, Dr Lin Naing, Dr

Low and Dr Noorani Ahmad from School of Mathematical Sciences and Dr Sasidharan from

INFORMM for statistical consultation.

Last but not least I would like to extend my warm and sincere thanks to all my friends and

colleagues whose gave me great support, help, advices and companionship; especially Kak

Munah, Kak Anizah, Aishah Azman, Kalpu, Thanes, Izan, Kak Emi, Wani, Shue, Kak

Nurul, Tihah, Kak Syida, Zul, Farhanah, Nik Syaza, Dyana, Kak Linda, Shaza, Chun Wei,

Teng Kew, Akbar, Geita, Sin Yee, Kak Hawani, Ann, Teh, Efa, Syahira, Husna, Ju, Zubed,

Bahiyah and all friends in INFORMM and any other places during my postgraduate study,

whom I fail to mention their names due to space constraint.

iii

On a personal note, I am deeply grateful to extend my warm gratitude to my ummi Chanisah

Hj Ahmad and my abi Basuni Hj Abdul Ghani for their prayers, encouragement and love.

Special thanks to all my family members especially my beloved brothers, sisters, nephews

and nieces whom have inspired, encouraged, supported and helped me indirectly in

completing this study. Ultimately, I am deeply thankful to Allah and all of those who

supported me in any aspect during this study.

The financial supports from EU grant (COINFECT), no. INCO-C7-2006-031714 and USM

Fellowship are gratefully acknowledged.

iv

TABLE OF CONTENTS

Page

Acknowledgements ii

Table of Contents iv

List of Tables ix

List of Figures xi

List of Abbreviations xiv

Abstrak xvi

Abstract xx

CHAPTER ONE

INTRODUCTION Page

1.1 Overview of parasitic infection 1

1.2 Intestinal parasitic infection 2

1.2.1 Distribution and prevalence of intestinal parasitic infection 2

1.3 Intestinal helminth

1.3.1 Overview 3

1.3.2 General life cycle of soil-transmitted intestinal helminth 4

1.3.3 Ascaris lumbricoides 5

1.3.4 Hookworm 9

1.3.5 Strongyloides stercoralis 13

1.4 Intestinal protozoa

1.4.1 Overview 18

1.4.2 General life cycle of intestinal protozoa 19

1.4.3 Entamoeba histolytica 19

1.4.4 Giardia lamblia 26

1.4.5 Cryptosporidium parvum 31

1.5 Multiple intestinal parasitic infections cases in individuals 35

1.6 Screening methods

1.6.1 Routine laboratory practice for intestinal parasites 35

1.6.2 Other diagnostic approaches and current problems 37

1.7 Nucleic acid based diagnostic methods

1.7.1 Overview 39

1.7.2 Polymerase chain reaction (PCR) 40

v

1.7.3 Real-time PCR 41

1.7.3.1 Methods of monitoring in real-time PCR amplification 43

1.7.3.2 Advantages of real-time PCR 45

1.7.3.3 Studies on intestinal helminths and intestinal protozoa in

Malaysia

45

1.8 Statement of the problem & rationale of the study 49

1.9 Objectives of the study 51

1.10 Flow chart for research approach 52

CHAPTER TWO

MATERIALS AND METHODS

2.1 Materials

2.1.1 Stool sample 53

2.1.2 Oligonucleotide primers and probes

2.1.2.1 Primers 54

2.1.2.2 Probes 54

2.1.3 Bacterial strain 58

2.1.4 Chemicals and reagents 58

2.1.5 Sterilization 58

2.1.6 Kits 59

2.1.7 Cloning vector 59

2.1.8 Laboratory equipments 59

2.1.9 Preparation of media

2.1.9.1 Luria-Bertani (LB) broth 59

2.1.9.2 SOC medium 60

2.1.9.3 LB agar 60

2.1.9.4 LB agar with ampicillin 60

2.1.10 Preparation of buffers and reagents

2.1.10.1 Phosphate buffered saline (PBS), pH 7.2 61

2.1.10.2 TE buffer, pH 7.0 61

2.1.10.3 Ampicillin (stock solution 100 mg/ml) 61

2.1.10.4 Ethanol (70%) 62

2.1.11 Preparation of reagent for preservation of sample

2.1.11.1 Sodium acetate-acetic acid-formalin (SAF) preservative 62

2.1.12 Preparation of reagents for parasitological techniques

vi

2.1.12.1 Normal saline [sodium chloride, 0.85% (w/v)] 62

2.1.12.2 Iodine solution 62

2.1.12.3 Acid ethanol (90%) 63

2.1.12.4 Zinc sulphate (ZnSO4) solution 63

2.1.12.5 HCl-ethanol (1%) 63

2.1.12.6 Malachite green (3%) 63

2.1.12.7 Glycerol-malachite green solution 63

2.1.13 Preparation of reagents for agarose gel electrophoresis

2.1.13.1 Tris-Borate-EDTA (TBE), buffer (10X) 64

2.1.13.2 Ethidium bromide (EtBr), solution 10 mg/ml (w/v) 64

2.1.14 Preparation of reagents for DNA extraction

2.1.14.1 Polyvinylpolypyrolidone (PVPP) 2% 64

2.1.15 Preparation of reagents for cloning

2.1.15.1 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside

solution (X-gal), 40 mg/ml

65

2.1.15.2 Calcium chloride (CaCl2), 100 mM 65

2.1.15.3 Magnesium chloride (MgCl2), 100 mM 65

2.1.15.4 Glycerol (100%) 65

2.2 Methods

2.2.1 Parasitological detection 66

2.2.1.1 Direct smear 66

2.2.1.2 Zinc sulphate concentration/floatation method 67

2.2.1.3 Trichrome staining 68

2.2.1.4 Modified Ziehl-Neelsen technique (acid-fast stain) 68

2.2.1.5 Kato-Katz technique 69

2.2.2 DNA extraction 74

2.2.3 Phocine herpesvirus 1 (PhHV-1) optimization 75

2.2.4 Polymerase chain reaction (PCR) 76

2.2.4.1 Preparation of PCR master mix 76

2.2.4.2 Conventional PCR 76

2.2.4.2.1 Optimization of primer concentration for

conventional PCR

77

2.2.4.2.2 Selection of PCR thermal profile 77

2.2.4.3 Real-time PCR assay 78

2.2.4.3.1 Optimization of primer concentrations for

real-time PCR

79

vii

2.2.4.3.2 Optimization of multiplex real-time PCR

assay

80

2.2.4.3.3 Determination of detection limit 83

2.2.5 Agarose gel electrophoresis 83

2.2.6 Cloning

2.2.6.1 Preparation of competent cells 84

2.2.6.2 Cloning PCR product into TOPO® cloning vector 85

2.2.6.3 Transformation of vector plasmid into bacterial host 85

2.2.6.4 Screening of positive clones 86

2.2.6.5 Plasmid extraction 87

2.2.6.6 Determination of purity and concentration of DNA 88

2.2.6.7 DNA sequencing 89

2.2.7 Statistical analysis 89

CHAPTER THREE

RESULTS

3.1 Cloning of specific target regions of A. lumbricoides, Ancylostoma, N.

americanus, S. stercoralis, E. histolytica, G. lamblia and C. parvum

3.1.1 Optimization of primer concentrations for conventional PCR and

selection of PCR thermal profile

90

3.1.2 Screening of positive clones 96

3.1.3 Plasmid extraction 101

3.1.4 DNA sequencing 104

3.2 Parasitological techniques detection 107

3.3 Optimization of real-time PCR assay

3.3.1 Phocine herpesvirus 1 (PhHV-1) optimization 112

3.3.2 Optimization of primer concentrations for real-time PCR assay 115

3.3.3 Optimization of multiplex real-time PCR assay 117

3.3.4 Determination of detection limit 120

3.4 Evaluation of multiplex real-time PCR assay 123

3.4.1 Analysis of real-time PCR assay and the quantitation of DNA

(Median Ct-value of positive samples and the approximate DNA

copy number)

135

3.4.2 Distribution of parasite species 138

viii

3.4.3 Statistical comparison of the techniques, sensitivity and specificity

tests

141

CHAPTER FOUR

DISCUSSION

4.1 Intestinal parasitic infections 143

4.2 Parasitological techniques

4.2.1 Microscopic examination 144

4.2.2 Concentration techniques 146

4.2.3 Other diagnostic methods 148

4.3 DNA-based diagnostic method

4.3.1 PCR assay 150

4.3.2 PCR limitations 153

4.3.3 PCR assay optimization 154

4.3.4 Real-time PCR 159

4.3.5 Multiplex real-time PCR 163

4.4 Prevalence of intestinal parasitic infections 163

4.5 Research related to the present study

4.5.1 Intestinal parasitic studies 164

4.5.2 Intestinal helminths studies 165

4.5.3 Intestinal protozoa studies 166

4.6 Conclusion and further studies 170

REFERENCES 173

LIST OF PUBLICATIONS 189

APPENDICES

A - List of chemicals and reagents

B - List of equipments

C - Table of Corbett Rotorgene

ix

LIST OF TABLES

Table Page

2.1.a Sequences of primers for intestinal helminths.

55

2.1.b Sequences of primers for intestinal protozoa.

56

2.1.c Sequences of primers for internal control.

56

2.2 Sequences of probes.

57

2.3.a DNA template added in multiplexing and background test of

intestinal helminths with and without the presence of internal

control.

81

2.3.b DNA template added in multiplexing and background test of

intestinal protozoa with and without the presence of internal

control.

82

3.1 BLAST NCBI results of the DNA sequences from

recombinant plasmids.

106

3.2.a Detection of intestinal helminths by parasitological techniques.

108

3.2.b Detection of intestinal protozoa by parasitological techniques.

109

3.3.a Detection of intestinal helminths by specific parasitological

techniques.

110

3.3.b Detection of intestinal protozoa by specific parasitological

techniques.

111

3.4 Mean and standard deviation (SD) of the Ct-values obtained

for optimization of primer concentrations in real-time PCR

assay.

116

3.5.a Ct-values of multiplex real-time PCR assay for the

multiplexing and background test of intestinal helminths.

118

3.5.b Ct-values of multiplex real-time PCR assay for the

multiplexing and background test of intestinal protozoa.

119

3.6.a Mean Ct-values and standard deviation for the determination

of detection limit for intestinal helminths.

121

3.6.b Mean Ct-values and standard deviation for the determination

of detection limit for intestinal protozoa.

122

3.7.a Detection of intestinal helminths by multiplex real-time PCR.

124

x

3.7.b Detection of intestinal protozoa by multiplex real-time PCR.

125

3.8 Median Ct-value of positive samples by real-time PCR

(positive or negative by microscopy), and approximate DNA

copies.

137

3.9 Distribution of intestinal parasites according to the species

(N=302).

140

3.10 Statistical analysis on the comparison of detection by

microscopy and real-time multiplex PCR for detection of (i)

intestinal helminths and (ii) intestinal protozoa.

142

xi

LIST OF FIGURES

Figure Page

1.1.a A. lumbricoides unfertilized egg.

7

1.1.b A. lumbricoides fertilized egg.

7

1.1.c A. lumbricoides life cycle.

8

1.2.a Hookworm larva.

11

1.2.b Hookworm egg.

11

1.2.c Hookworm life cycle.

12

1.3.a S. stercoralis rhabditiform larva (L1) in iodine stained

smear.

16

1.3.b S. stercoralis short buccal cavity in unstained wet mount.

16

1.3.c S. stercoralis filariform larva (L3) characteristic; notched

tail.

16

1.3.d S. stercoralis egg.

16

1.3.e S. stercoralis life cycle.

17

1.4.a Distribution of E. histolytica in human body.

23

1.4.b E. histolytica trophozoites in trichrome stained smear.

24

1.4.c E. histolytica cyst with four nuclei.

24

1.4.d E. histolytica life cycle.

25

1.5.a G. lamblia iodine stained cyst.

29

1.5.b G. lamblia trophozoites.

29

1.5.c G. lamblia life cycle.

30

1.6.a C. parvum oocyst in modified acid-fast stained smear.

33

1.6.b C. parvum oocyst in wet mount.

33

1.6.c C. parvum life cycle.

34

1.7 Amplification plot of real time PCR.

42

2.1.a - 2.1.g Standard procedure of Kato-Katz technique. 70

xii

3.1.a - 3.1.b Optimization of primer concentrations and selection of PCR

thermal profiles for Ancylostoma.

92

3.2.a - 3.2.b Optimization of primer concentrations and selection of PCR

thermal profiles for A. lumbrocoides.

92

3.3.a - 3.3.b Optimization of primer concentrations and selection of PCR

thermal profiles for N. americanus.

93

3.4.a - 3.4.b Optimization of primer concentrations and selection of PCR

thermal profiles for S. stercoralis.

93

3.5.a - 3.5.b Optimization of primer concentrations and selection of PCR

thermal profiles for E. histolytica.

94

3.6.a - 3.6.b Optimization of primer concentrations and selection of PCR

thermal profiles for G. lamblia.

94

3.7.a - 3.7.b Optimization of primer concentrations and selection of PCR

thermal profiles for C. parvum.

95

3.8 Screening of positive clones that has inserted target gene for

Ancylostoma.

97

3.9 Screening of positive clones that has inserted target gene for

A. lumbricoides.

97

3.10 Screening of positive clones that has inserted target gene for

N. americanus.

98

3.11 Screening of positive clones that has inserted target gene for

S. stercoralis.

98

3.12 Screening of positive clones that has inserted target gene for

G. lamblia.

99

3.13 Screening of positive clones that has inserted target gene for

C. parvum.

99

3.14 Screening of positive clones that has inserted target gene for

E. histolytica.

100

3.15 Agarose gel electrophoresis analysis of recombinant

plasmids for Ancylostoma.

102

3.16 Agarose gel electrophoresis analysis of recombinant

plasmids for N. americanus.

102

3.17 Agarose gel electrophoresis analysis of recombinant

plasmids for A. lumbrocoides.

102

xiii

3.18 Agarose gel electrophoresis analysis of recombinant

plasmids for S. stercoralis.

102

3.19 Agarose gel electrophoresis analysis of recombinant

plasmids for E. histolytica.

103

3.20 Agarose gel electrophoresis analysis of recombinant

plasmids for G. lamblia.

103

3.21 Agarose gel electrophoresis analysis of recombinant

plasmids for C. parvum.

103

3.22.a Positive amplification curves and the Ct-values of real-time

PCR for determination of optimized PhHV-1 dilution prior

to DNA extraction.

113

3.22.b Analysis of PCR product for determination of optimized

PhHV-1 dilution prior to DNA extraction based on agarose

gel electrophoresis.

114

3.23.a Quantitation data for Cycling B red channel (for detection of

PhHV-1).

126

3.23.b Quantitation data for Cycling B orange channel (for

detection of A. lumbricoides).

127

3.23.c Quantitation data for Cycling B green channel (for detection

of N. americanus).

128

3.23.d Quantitation data for Cycling B yellow channel (for

detection of Ancylostoma).

129

3.23.e Quantitation data for Cycling B crimson channel (for

detection of S. stercoralis).

130

3.24.a Quantitation data for Cycling B red channel (for detection of

PhHV-1).

131

3.24.b Quantitation data for Cycling B yellow channel (for

detection of E. histolytica).

132

3.24.c Quantitation data for Cycling B green channel (for detection

of G. lamblia).

133

3.24.d Quantitation data for Cycling B orange channel (for

detection of C. parvum).

134

xiv

LIST OF ABBREVIATIONS

Description Abbreviations

1 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside X-gal

2 Acquired Immune Deficiency Syndrome AIDS

3 Base pair bp

4 Basic Local Alignment Search Tool BLAST

5 Black hole quencher BHQ

6 Bovine serum albumin BSA

7 Calcium chloride CaCl2

8 Calcium chloride dehydrate CaCl2.2H2O

9 Celcius C

10 Centers for Disease Control and Prevention CDC

11 Centimeter cm

12 Complementary DNA cDNA

13 Cycle threshold Ct

14 Deoxyribonucleic acid DNA

15 Direct immunofluorescence assay DFA

16 Disodium hydrogen phosphate Na2HPO4

17 Double-stranded DNA dsDNA

18 Eggs per gram EPG

19 Enzyme-linked immunosorbent assay ELISA

20 Ethidium bromide EtBr

21 Ethylenediaminetetraacetic acid EDTA

22 Fluorescence resonance energy transfer FRET

23 Full examination microscopic examination FEME

24 High-performance liquid chromatography HPLC

25 Hydrochloric acid HCl

26 Immunofluorescence assay IFA

27 Indirect hemagglutination assay IHA

28 Internal transcribed spacer ITS

29 Iodine I2

30 Larvae stage 1 L1

31 Larvae stage 2 L2

32 Larvae stage 3 L3

33 Larvae stage 4 L4

34 Loop-mediated isothermal DNA amplification LAMP

35 Magnesium chloride MgCl2

36 Microliter µl

37 Micrometer µm

38 Micromolar µM

39 Mililiter ml/mL

40 Milligram mg

41 Millimeter mm

42 Milimolar mM

43 Minor groove binder MGB

xv

44 Multiplex tandem PCR MT-PCR

45 Nanometer nm

46 Nanomolar nM

47 National Center for Biotechnology Information NCBI

48 Non fluorescent quencher NFQ

49 Non template control NTC

50 Number No.

51 Optical density OD

52 Phosphate buffered saline PBS

53 Plaque forming unit PFU

54 Polyvinylpyrrolidone PVPP

55 Polymerase chain reaction PCR

56 Potassium dihydrogen phosphate KH2PO4

57 Potassium iodide KI

58 Quantitative polymerase chain reaction qPCR

59 Random amplification of polymorphic DNA RAPD

60 Restriction fragment length polymorphism RFLP

61 Revolutions per minute rpm

62 Ribonucleic acid RNA

63 Ribosomal DNA rDNA

64 Single-stranded DNA ssDNA

65 Small subunit ribosomal RNA SSUrRNA

66 Sodium acetate NaOAc

67 Sodium chloride NaCl

68 Soil-transmitted helminth STH

69 Specific gravity SG

70 Synergy Brands, Inc. SYBR

71 Tetramethylrhodamine TAMRA

72 Tris-borate-EDTA TBE

73 Ultra violet UV

74 Unit U

75 Weight per volume w/v

76 World Health Organization WHO

77 Zinc sulphate ZnSO4

78 Zinc sulphate heptahydrate ZnSO4.7H2O

xvi

PENGESANAN PARASIT PATOGENIK USUS MELALUI KAEDAH ‘REAL

-TIME MULTIPLEKS PCR’ DAN PERBANDINGAN DENGAN TEKNIK-

TEKNIK PARASITOLOGI

ABSTRAK

Jangkitan parasit dalam usus oleh helmin (cacing) dan protozoa adalah antara

jangkitan yang paling biasa berlaku dan kekal sebagai masalah besar di kalangan

masyarakat awam terutamanya di negara membangun. Jangkitan parasit usus yang

berkaitan dengan helmin biasanya menyebabkan kesakitan di bahagian abdomen dan

disertai oleh anereksia, loya dan cirit-birit, manakala protozoa usus lazimnya

menyebabkan cirit-birit.

Kaedah pengesanan rutin yang dijalankan untuk mengesan jangkitan parasit usus

biasanya bergantung kepada pengesanan dengan mikroskop. Bagaimanapun, kaedah

ini tidak sensitif dan memerlukan kepakaran mikroskopi untuk mengelakkan

‘misdiagnosis’ mahupun ‘overdiagnosis’. Kekurangan ini telah mencetuskan

pembangunan kaedah yang lebih sensitif menggunakan DNA. PCR adalah satu

kaedah yang terbukti sensitif dan spesifik untuk mengesan kehadiran organism yang

patogenik di dalam usus. Memandangkan PCR secara konvensional mengambil masa

panjang serta mudah terdedah kepada pencemaran, maka pembangunan kaedah

pengesanan ‘real-time PCR’ yang pantas dan mampu memberikan data secara

langsung dan kuantitatif ke atas produk PCR adalah bermanfaat.

Dalam kajian ini, satu kaedah ‘real-time multipleks PCR’ untuk mengesan spesies

helmin usus yang berkaitan rapat iaitu Ascaris lumbricoides, Strongyloides

xvii

stercoralis, Necator americanus dan Ancylostoma duodenale dan satu kaedah ‘real-

time multipleks PCR’ untuk mengesan protozoa usus yang juga berkaitan rapat iaitu

Entamoeba histolytica, Cryptosporidium parvum dan Giardia lamblia telah

dioptimumkan dan dinilai. Keputusan yang diperolehi telah dibandingkan dengan

keputusan yang didapati menggunakan teknik parasitologi.

Sebanyak 302 sampel tinja telah dipungut daripada pesakit yang mengalami masalah

gastrousus daripada Hospital Serian, Hospital Lundu dan Hospital Universiti Sains

Malaysia. Teknik parasitologi yang dilakukan ke atas sampel tersebut adalah kaedah

smir langsung, pengapungan / pemendapan dengan larutan zink sulfat, pewarnaan

trikrom untuk E. histolytica dan G. lamblia, pewarnaan ‘acid fast’ untuk C. parvum

dan kaedah Kato-Katz untuk mengesan ova helmin.

Primer yang digunakan dalam kajian ini telah diuji menggunakan lima tahap

kepekatan primer dan dua profil suhu PCR. Produk PCR diklon ke dalam vektor

pengklonan TOPO® dan plasmid DNA yang diperolehi disimpan sebagai kawalan

positif untuk pembinaan lengkung piawai (‘standard curve’). Daripada lengkung

piawai yang diperolehi, had pengesanan bagi setiap organisma adalah seperti berikut:

satu salinan DNA untuk G. lamblia, 10 salinan DNA untuk Ancylostoma, A.

lumbrocoides dan S. stercoralis, dan 102 salinan DNA untuk N. americanus, E.

histolytica dan C. parvum. Untuk kaedah ‘real-time multipleks PCR’,

pengoptimuman had kepekatan primer juga dilakukan untuk mengelakkan

perencatan kaedah PCR yang mungkin berlaku disebabkan oleh templat DNA yang

mempunyai ketumpatan tinggi. Phocine herpesvirus 1(PhHV-1) dengan kepekatan

yang optimum iaitu 10-2

PFU/ml telah dimasukkan bersama dalam setiap

xviii

pengekstrakan templat, ini bertujuan untuk mengesan sebarang keputusan negatif

palsu yang mungkin disebabkan oleh kehadiran bahan perencat dalam sampel tinja

ataupun disebabkan kegagalan proses PCR.

Setiap tindak balas yang dijalankan diuji dengan satu atau beberapa templat DNA

dalam satu tiub, samaada dengan kehadiran DNA daripada PhHV-1 ataupun tanpa

kehadiran DNA tersebut. Dua pengesanan menggunakan kaedah ‘real-time

multipleks PCR’ dilakukan ke atas semua DNA, iaitu satu pengesanan untuk helmin

dan pengesanan untuk protozoa. Keputusan yang diperolehi dibandingkan dengan

keputusan teknik parasitologi berdasarkan ujian sensitiviti, spesifisiti dan Chi-square.

Berdasarkan keputusan daripada 302 sampel yang diuji, untuk kaedah pengesanan

cacing, 13 (4.3%) sampel positif dikesan melalui kaedah mikroskopi manakala

94 (31.1%) sampel positif dikesan menggunakan kaedah ‘real-time PCR’. Untuk

pengesanan protozoa pula, lima (1.7%) sampel positif dikesan melalui kaedah

mikroskopi manakala 23 (7.6%) sampel positif dikesan menggunakan kaedah ‘real-

time PCR’ (p <0.05). Jangkitan oleh dua atau lebih parasit usus (samaada helmin

atau protozoa) dicatatkan dalam 29 daripada 110 sampel yang positif (26%)

menggunakan kaedah ‘real-time PCR’ manakala kaedah mikroskopi tidak dapat

mengesan kehadiran jangkitan berganda.

Kedua-dua kaedah ‘real-time multipleks PCR’ yang telah dioptimumkan dalam

kajian ini telah berjaya mengesan semua organisma yang disasarkan. Kaedah-kaedah

ini juga telah dinilai ke atas sampel pesakit dan berjaya mengesan empat jenis cacing

usus dan tiga jenis protozoa usus yang penting. Untuk pengesanan cacing, kaedah

xix

PCR berjaya mengesan 7.2 kali lebih banyak sampel positif manakala untuk

pengesanan protozoa, kaedah PCR mengesan 4.6 kali lebih banyak sampel positif

berbanding teknik parasitologi.

Kesimpulannya, kaedah ‘real-time PCR’ dalam kajian ini menyediakan satu kaedah

alternatif untuk diagnosis parasit usus. Kaedah ini boleh diaplikasikan untuk

diagnosis rutin pesakit, pemantauan rawatan dan kajian epidemiologi berkaitan

jangkitan parasit usus.

xx

MULTIPLEX REAL-TIME PCR FOR THE DETECTION OF PATHOGENIC

INTESTINAL PARASITES AND COMPARISON WITH

PARASITOLOGICAL TECHNIQUES

ABSTRACT

Intestinal parasitic infections by helminths and protozoa are among the most

prevalent infections and remain a major public health burden in underdeveloped

countries. Most intestinal helminth infections cause abdominal pain accompanied by

aneroxia, nausea and diarrhea while intestinal protozoa cause diarrheal diseases.

Routine diagnostic methods for intestinal parasitic infections which rely heavily on

microscopic detection are insensitive, and require well-trained microscopists to avoid

misdiagnosis or overdiagnosis of the infection. These limitations have led to the

development of highly sensitive DNA-based assays. PCR has been proven to be

sensitive and specific for detection of enteric pathogens. Since conventional PCR is

time consuming and prone to cross-contamination, it is desirable to develop a real-

time PCR assay which is rapid and can provide quantitative and real-time

information on the amplified products.

In this study, a real-time multiplex PCR assay for the detection of closely related

intestinal helminths namely Ascaris lumbricoides, Strongyloides stercoralis, Necator

americanus and Ancylostoma duodenale and an assay for the detection of three

closely related intestinal protozoa which comprised Entamoeba histolytica,

Cryptosporidium parvum and Giardia lamblia were optimized and evaluated.

xxi

A total of 302 stool samples were collected from patients with gastrointestinal

problems from Hospital Serian, Hospital Lundu and Hospital Universiti Sains

Malaysia. Parasitological techniques were performed by direct wet smear, zinc

sulphate floatation/sedimentation, trichrome staining for E. histolytica and G.

lamblia, acid-fast staining for C. parvum and Kato Katz technique for helminth ova.

The primers were tested at five concentrations and two PCR thermal profiles. The

PCR products were cloned into TOPO® cloning vector and the DNA plasmids were

used as positive control for standard curve construction. The detection limit for each

organism was as follows: one DNA copy for G. lamblia; 10 copies for each

Ancylostoma, A. lumbrocoides and S. stercoralis; and 102 copies for each N.

americanus, E. histolytica and C. parvum. For real-time multiplex PCR assay

optimizations, primer limitation steps were performed in order to avoid any

inhibition due to high abundant template. Phocine herpesvirus 1 (PhHV-1) with an

optimum dilution of 10-2

PFU/ml was included at the template preparation stage in

order to detect false negative results due to the presence of any inhibitor compounds

or PCR failure. Each assay was tested with one or multiple DNA template with or

without the addition of PhHV-1 DNA in each reaction. Two real-time multiplex PCR

assays i.e. for detection of intestinal helminths and intestinal protozoa were

performed on all DNA samples. The real-time PCR results were compared with those

obtained by parasitological techniques based on sensitivity, specificity and Chi-

square tests.

For the detection of intestinal helminths, out of 302 samples, microscopic

examination detected 13 (4.3%) positive samples while real-time PCR assay detected

xxii

94 (31.1%). For the detection of intestinal protozoa, microscopic examination

detected five (1.7%) positive cases and real-time PCR detected 23 (7.6%) with

p<0.05. Multiple infections by two and more organisms (either helminths or

protozoa) were recorded in 29 out of 110 positive cases (26%) by real-time multiplex

PCR, while no cases of multiple infections were reported by microscopic

examination.

The real-time multiplex PCR assays optimized in this study successfully detected all

the target organisms. They were also successfully evaluated on patients’ samples for

the detection of four important intestinal helminths and three common intestinal

protozoa. The PCR assay detected 7.2 times more positive samples for intestinal

helminths and 4.6 times for intestinal protozoa as compared to parasitological

techniques.

In conclusion, the real-time PCR assays described in this study provide an alternative

laboratory diagnostic method for intestinal parasitic infections and would be useful

for treatment monitoring and epidemiological studies.

1

CHAPTER ONE

INTRODUCTION

1.1 Overview of parasitic infection

Parasitic infections are often caused by two most common type of organisms which

are protozoa and helminths. Protozoa is single-celled organism meanwhile helminth

is multicellular organism. Parasitic infection occurs when organisms live on or

within a living host (usually mammals e.g. human). In general, parasite harms its

host or survives by utilizing the host nutrients (http://www.cdc.gov/parasites).

Parasitic infections not only cause morbidity but also mortality in humans (Haque,

2007). Children and the elderly are highly susceptible to parasitic infections

compared to other age groups due to their weakened immune systems and less

capability to respond effectively to infections (Nagamani et al., 2007).

Immunocompromised or immunosuppressed people in cases of injury, surgery or

chronic illness are also more vulnerable to acquire serious infections (Graczyk and

Fried, 2007). In addition, children who are more exposed to soil when playing, have

higher chances to be infected with soil-transmitted helminths. Parasitic infections are

also responsible for many ill health conditions. The symptoms usually depend on the

type of the parasite and the affected organ of the host. Moreover, prolonged or

undetected infection may cause systemic problems which can affect the whole

system of the human body. Most of the parasites undergo a complicated life cycle

which includes the sexual and asexual types of reproduction within mammalian or

invertebrate hosts.

2

1.2 Intestinal parasitic infections

Parasitic infections caused by intestinal helminths and protozoan parasites are among

the most prevalent infections among humans in underdeveloped and developing

countries (Haque, 2007). It is also among the leading causes of death and disease in

tropical and subtropical regions of the world. Intestinal parasites refer to organisms

that live in the intestine and consume the nutrition of the host. Common

complications due to intestinal parasitic infections are abdominal discomfort,

dysentery or mechanical irritation of intestinal mucosa and other general symptoms

such as bloating, diarrhea and fever. Furthermore, when the burden of the infection is

prominent, intestinal parasites may also cause serious health conditions and problems

such as malnutrition, mental retardation and death especially in children (Amuta et

al., 2009).

1.2.1 Distribution and prevalence of intestinal parasitic infections

Intestinal parasites are distributed worldwide and many countries have high

prevalence rates of the infection. It is estimated that there are more than three billion

people infected with intestinal parasites throughout the world (Balcioglu et al., 2007;

Kurt et al., 2007). Several factors affect the distribution and the prevalence of

intestinal parasites. Personal hygiene, dietary habits, education level of the

community, socio-economic status and climates are among the most common factors

that influence the prevalence of intestinal parasitic infections (Balcioglu et al., 2007;

Mahsol et al., 2008). Furthermore, overcrowded areas with inadequate hygiene and

sanitation may increase transmission of parasitic infections. Therefore, the

prevalence of intestinal parasitic infection is usually high in urban slum communities

3

where the socio-economic status is low, environmental sanitation and living

condition is poor and water supply is unsafe with unhygienic personal habits (Noor

Azian et al., 2007).

1.3 Intestinal helminths

1.3.1 Overview

Intestinal helminths are macroparasites or multicellular organisms. Intestinal

helminths include nematodes (roundworms), cestodes (tapeworms) and trematodes

(flatworms) which live in the human gut. The common nematodes are the large

roundworm Ascaris lumbricoides, the whipworm Trichuris trichiura, two species of

blood-feeding hookworm Necator americanus and Ancylostoma duodenale, and the

threadworm Strongyloides stercoralis (Chigozie et al., 2007; Haque, 2007). They are

known as geohelminths or soil-transmitted helminths (STHs).

STHs infect more than a billion people and among the most common cause of

chronic infection to human (de Gruijter et al., 2005; Wiria et al., 2010). Recent

estimates suggest that A. lumbricoides infects 1221 million people, T. trichiura

infects 795 million, and hookworms infect 740 million people (de Silva et al., 2003).

Furthermore, infections with S. stercoralis, which affect 30 to 100 million people

worldwide, are probably even more underestimated (Wiria et al., 2010).

Commonly, infection with STH does not typically result in clinical disease.

However, the effect of infections depends on several factors namely the helminth

4

species, the intensity of infection and the host immunological status. Majority of the

infected individuals exhibit asymptomatic infections. This is because the pathology

of STH is strongly related to the intensity of infection which is the number of worms,

and usually, only a few worms are found in human body especially in early infection.

The most common symptoms of intestinal helminth infection include diarrhea, foul

breath, headache, nausea, abdominal pain and itching. Constipation and bloating can

also arise, due to the intestinal organs obstruction (Baba et al., 2009). Other

symptoms associated with intestinal helminth infections are anaemia and asthma (da

Silva et al., 2008; Dori et al., 2011).

1.3.2 General life cycle of soil-transmitted intestinal helminths

Intestinal helminth infection can be transmitted by fecal-oral route in the course of

contact with parasite eggs or larvae in contaminated soil. Usually, infection occurs

through accidental ingestion of A. lumbricoides or T. trichiura eggs or penetration of

the skin by hookworm or S. stercoralis larvae. Intestinal helminths do not divide in

the host (Haque, 2007) but they reproduce sexually. Male and female adult worms

mate in the intestinal of the host and produce eggs. Adult worms inhabit in specific

part of the host intestine; A. lumbricoides, hookworm and S. stercoralis live in the

small intestine while T. trichiura inhabits the colon. Adult worms can produce large

number of eggs and are able to survive for several years in the host, depending on the

species. The female worms then discharge their eggs or larvae in human feces (Garg

et al., 2005).

5

Depending on the environmental conditions, the eggs (of A. lumbricoides and T.

trichiura) can remain viable in the soil for several months while larvae (of

hookworms and S. stercoralis) can remain viable for several weeks. Higher humidity

of external environment is associated with faster development of the eggs to hatch

into larvae. Some of the helminth larvae (namely A. duodenale and S. stercoralis)

can undergo hypobiosis in the human body for several months. Hypobiosis is an

arrested development at a specific point in the nematode life cycle. In this stage, the

larvae do not grow and stop moving. They can survive for weeks or months before

resuming development and may be resistant to some antihelminthics.

1.3.3 Ascaris lumbricoides

Ascaris lumbricoides is one of the most common and most widespread human

infections, although Ascaris suum (the pig nematode) has also produced human

infection. It is estimated that A. lumbricoides causes 1.2 million cases of acute illness

and 10000 deaths annually. A. lumbricoides infection is rarely found in developed

countries. However the infection rate is increased with travelling and migration in

developing countries. The infection by A. lumbricoides is known as ascariasis. It

occurs by ingestion of the infective Ascaris eggs which hatch into larvae in the small

intestine. The larvae migrate to the caecum and proximal colon where they penetrate

the mucosa. The larvae then move via the portal blood to reach the liver. After

migration in the liver, the larvae advance to the lungs, penetrate the alveolar space

and move to the pharynx where they are swallowed and return to the small intestine.

Upon reaching the small intestine, they develop into adult worms, mate and produce

eggs (Dold and Holland, 2011). The morphology of A. lumbricoides eggs in

6

diagnosis stage are shown in Figures 1.1.a - 1.1.b while the complete life cycle of

Ascaris is further explained in Figure 1.1.c.

Infection of A. lumbricoides may be asymptomatic. Though, it may become life

threatening as seen with hepatobiliary and intestinal obstructions due to heavy worm

burdens (de Silva et al., 1997; Crompton et al., 1999). Hepatobiliary and intestinal

obstruction are the main causes of morbidity and mortality due to ascariasis.

Otherwise the infection may contribute to impaired nutrition, development, and

educational progress especially in children (Hlaing, 1993).

In the stage when the larvae migrate to the pulmonary system, eosinophilic

pneumonitis or Loeffler syndrome may develop. It happens when the eosinophils

accumulate in the lung due to tissue destruction and releasing of the larval antigens.

During this period, the patient may also develop asthma with hypersecretion of

mucus, bronchiolar inflammation or has sputum containing eosinophils. In allergy-

prone people, urticaria or itchiness of the skin may also occur. Infrequently, the

larvae emerge through fistulae or fallopian tubes, urinary bladder, lungs or heart and

pancreatic or bile duct (Noor Azian et al., 2007; Varkey et al., 2007). Women are

believed to be more affected since the progesterone hormones play a role in inducing

Oddi’s sphincter relaxation which allows the nematode to access the biliary duct

(Galzerano et al., 2010). However, the most intense infections usually occur in

children aged five to fifteen years due to higher exposure to contaminated

environment and low immune system of the children. Treatment with albendazole

was reported to give 93% reduction in egg count and a better cure rate (85% to

100%) than ivermectin treatment (78.4%) (Belizario et al., 2003).

7

Figure 1.1.a

Ascaris lumbricoides unfertilized egg. Elongated and covered by a

visible mammillated layer.

Figure 1.1.b

Ascaris lumbricoides fertilized egg. Rounded, has a thick shell and

smaller than unfertilized egg.

Source: http://www.phsource.us/PH/PARA/Diagnosing_Medical_Parasites.pdf

8

Figure 1.1.c

Ascaris lumbricoides life cycle 1. The life cycle begins with the

production of 40 to 60 eggs by adult females living in the small

intestine and deposited into external environment (soil) 2. Eggs

become infectious within several weeks and transmitted to the human

host by ingestion or by inhalation of contaminated soil or dust 3.

Within the host, Ascaris larvae hatch in the jejunum, penetrate the

intestinal wall, and migrate by hepatic venules to the right heart and

pulmonary circulation 4. They subsequently break through into

alveolar spaces, ascend the trachea, and are swallowed back into the

intestine 5. They undergo a final moult and develop into adult worms

(15 cm to 40 cm), which mate and generate a new generation of eggs.

Notes Under normal condition, the time from ingestion of eggs to development of

new eggs is 10 to 12 weeks. Adult worms live for approximately one year

before expelled from human body. Occasionally, male-only adult worms

infection occur with yielding no eggs in stool while female-only adult worms

infection produce infertile eggs, which never become infectious.

Source: https://online.epocrates.com/u/2924908/Ascariasis/Basics/Etiology

9

1.3.4 Hookworm

Primarily two species of hookworms infect humans; Ancylostoma duodenale and

Necator americanus. The World Health Organization has catogerized A. duodenale

and N. americanus as very destructive parasites (WHO, 2005). N. americanus tends

to be prevalent in tropical climates, while A. duodenale is commonly found in a

cooler and drier environment. However, their geographical distributions overlap

considerably, and both species are endemic to many areas (Albonico et al., 1998;

Brooker et al., 2004; Hotez et al., 2005). Currently, it is estimated that 740 million

people are infected worldwide, and more than 80 million of them are severely

affected clinically (Bethony et al., 2006).

Both species of the hookworm are transmitted through percutaneous route. The eggs

are excreted in human feces. However, as opposed to Ascaris, hookworm eggs hatch

into infective larvae in the feces or appropriate soil conditions. The infective larvae

then infect humans by active invasion of the skin. They find and recognize their hosts

by the behavioural phases of activation, directed crawling and penetration (Haas et

al., 2005). The infective, third-stage filariform larvae (L3) penetrate human skin and

migrate via the circulatory system and the lung to finally reside as the adult stage (8

mm to 20 mm in length) usually in the duodenum. The adult worms can persist in the

host within one to two years before being eliminated from the intestine (Gasser et al.,

2008).

Iron-deficiency anaemia caused by intestinal blood loss is the most outstanding

feature of hookworm infection. Blood loss occurs when the adults worms attach via

their buccal capsule to the small intestines, rupture capillaries and suck the blood

10

from the intestinal mucosa (Albonico et al., 1998). The level of blood loss is

proportionally related to the number of adult worms that inhabit in the host intestine.

The risk and severity of iron-deficiency anaemia is described by the balance between

iron intake and iron loss from intestinal bleeding. In heavy infections, hookworm can

cause physical and mental retardation and deaths in children as well as adverse

maternal-fetal outcomes in pregnant women (Hotez et al., 2005).

A. duodenale is capable of oral infection through accidental ingestion of the eggs

(Hoagland and Schad, 1978; Schad, 1991). Furthermore, A. duodenale larvae may

undergo a dormant state (arrested stage of development in the musculature and/or

intestine) after penetrating into the skin (Albonico et al., 1998). Infection by A.

duodenale may occur via transmammary routes; whereas N. americanus requires the

transpulmonary migration routes (Setasuban et al., 1980).

Currently, the control of hookworms has relied predominantly on antihelminthics

drugs such as albendazole, mebendazole, pyrantel pamoate or levamisole (Bethony et

al., 2006). The morphology and the life cycle of hookworm are summarised in

Figures 1.2.a, 1.2.b and 1.2.c.

11

Figure 1.2.a

Hookworm larva. General morphology of both hookworm larvae is

similar. Approximate length; 10 to 13µm for female and 8 to 11 µm

for male worm.

Figure 1.2.b Hookworm egg. General morphology of both hookworm eggs is

similar. Oval shaped, 56 to 75 µm by 36 to 40 µm in size, with

transparent and smooth thin shell.

Source: http://www.phsource.us/PH/PARA/Diagnosing_Medical_Parasites.pdf

12

Figure 1.2.c Hookworm life cycle 1. Hookworm eggs are passed in the stool. Under

favourable condition; warmth, humidity and shade, the eggs develop

into rhabditiform (L1) larvae 2. Rhabditiform larvae hatch within one

to two days 3. The larvae grow and develop in the feces and/or the

soil. After five to ten days (including two moults) they become

filariform (L3) larvae which are infective to the human host. These

ensheathed larvae can survive for three to four weeks under favourable

environmental conditions 4. Upon contact with the suitable host, the

larvae penetrate through the skin of the host 5. The larvae transported

via the blood stream to the heart and then to the lungs. They migrate

into the pulmonary alveoli, ascend the airways to the pharynx 6. They

are then swallowed and reach the small intestine and develop into

mature adults. The adult worms attach to the small intestinal wall and

feed on blood 7. The worms copulate, and the females produce

fertilized eggs, which are released in the feces.

Source: http://sprojects.mmi.mcgill.ca/tropmed/disease/intest-hookworm/life.htm

13

1.3.5 Strongyloides stercoralis

Strongyloides sp. can infect mammals, birds, reptiles and amphibians. To date, only

two species of Strongyloides have been known to be able to infect humans;

Strongyloides stercoralis and Strongyloides fuelleborni. However the most common

and clinically important pathogenic species in humans is S. stercoralis whereas S.

fuelleborni is found periodically in Africa and Papua New Guinea (Ashford et al.,

1992). S. stercoralis inhabits the small intestine of the host. Abdominal pain, nausea,

vomiting, wheezing, and asthma are the common symptoms of S. stercoralis

infection. During the infection, immunocompetent individuals may produce a

moderate clinical symptom of diarrhea whereas it can be fatal in

immunocompromised patients (Adenusi et al., 2003; Olsen et al., 2009; Feely et al.,

2010).

Strongyloides can undergo two types of development which are known as

heterogenic development and homogenic development. In heterogonic development,

Strongyloides eggs hatch in the feces and develop into L1 (first-stage larvae). L1

undergo L2, L3 and L4 stages and develop into rhabditiform male and female worms

which are the free-living nematodes. These rhabditiform or the free-living nematodes

will mate and the female lays eggs which hatch to release L1 which moult to an L2,

then into infective filariform L3. These infective L3 are long lived and can persist in

the environment until they come across a suitable host. This type of development is

also known as indirect or sexual development. On the other way, homogenic

development occurred when the L1 larvae is directly moult via L2 into infective L3

and persist in the environment until they encounter a suitable host. This type of

development is also known as direct or asexual development (Viney and Lok., 2007).

14

Only the female worms of Strongyloides will be infective to the host. The female can

either turn into L3 (infective) or free-living adult (non pathogenic) while the male

worm can only develop into a free-living nematode. The infective larvae (L3) can

penetrate into the skin host and migrate through the lungs until they are swallowed to

reach the small intestine or else, they migrate in the host and moult via an L4 stage to

develop adult parasitic female worms (Olsen et al., 2009).

S. stercoralis also undergoes a dormant state as the same as A. duodenale. This

dormancy of infective L3 larvae in human can cause chronic infection. At this stage,

the infective L3 larvae can also cause transmammary transmission by re-activation of

the larvae at lactation after their migration and arrestment in the mammary glands

(Miller, 1981). Autoinfection can also occur in Strongyloides life cycle.

Autoinfection is repeated generations of development in the same host individual.

During autoinfection, the fast development of female larvae into infective L3 occurs

within the gut and penetrates directly into the tissues of the primary host. Therefore,

the whole life cycle is occurred within the host. During this time, infective L3 larvae

may distribute through many organs and tissues of the host as well as the

development of new female larvae in the gut (Vadlamudi et al., 2006; Viney and

Lok, 2007).

Strongyloides infections can be detected by the presence of Strongyloides eggs or

larvae in the feces. Direct examination of thick smear on fresh fecal sample will

reveal L1 stages and, or eggs. However, eggs are rarely found as they hatch in the

intestine. High intensity of Strongyloides L1 can be detected by direct smear.

Meanwhile, for lower intensity infections, larvae can be collected from feces using

15

concentration techniques. Fecal samples can also be grown to obtain the infective

L3s. In severe and disseminated infection, sputum testing is also done to detect

Strongyloides larvae (Smith et al., 1985; Maayan et al., 1987).

The drug of choice to treat Strongyloides infection is thiabendazole. However,

ivermectin is also effective especially for disseminated infection (Datry et al., 1994;

Viney and Lok, 2007). The morphology and the life cycle of S. stercoralis are

summarised in Figures 1.3.a, 1.3.b, 1.3.c, 1.3.d and 1.3.e.

16

Figure 1.3.a Strongyloides stercoralis rhabditiform larva (L1) in iodine stained

smear. Large genital primordium (arrow) with short buccal cavity, and

sharp tail.

Figure 1.3.b Strongyloides stercoralis short buccal cavity in unstained wet mount of

stool.

Figure 1.3.c Strongyloides stercoralis filariform larva (L3) characteristic; notched

tail.

Figure 1.3.d Strongyloides egg. Oval and thin shelled, similar morphology with

hookworm egg but smaller in size (50 to 58 by 30 to 34 µm). It is

rarely found in stool as they hatch in the intestine.

Source: http://www.med.cmu.ac.th/dept/parasite/nematode/ssrlarva.htm

http://www.tropicalmed.eu/Page/WebObjects/PageTropE.woa/wa/displayPage?name=Readi

ngCultureAgarMicro

17

Figure 1.3.e Strongyloides stercoralis life cycle. The shaded box indicates the host.

The figure shows three possible development of first stage larvae (L1)

1. The autoinfective cycle, unique to S. stercoralis, in which

development from the L1 to the autoinfective L3 (aL3) occurs within

the gut of the primary host. Autoinfection can lead to explosive cycles

of development and a highly pathogenic disseminated infection. All

post-parasitic male L1 develop to free-living adult males. Post-

parasitic female L1 passed in the feces may undergo development by

either of two alternative pathways 2. The homogonic cycle involves

direct development to the infective L3 (iL3) 3. Heterogonic

development involves development to the free-living female and,

following mating, production of a generation of free-living progeny.

All progeny of the free-living adults develop to the iL3.

Source: http://www.ncbi.nlm.nih.gov/books/NBK19663/figure/A14969/?report=objectonly

18

1.4 Intestinal protozoa

1.4.1 Overview

Protozoa are unicellular microparasites. Majority of the protozoa are non-pathogenic

commensals. However, some of them can cause severe disease under certain

circumstances. For example, Giardia (waterborne protozoa) produces mild diarrhea

in immunocompetent individuals though in immunocompromised people, the

infection can result in a severe disease. Intestinal protozoa are commonly found in

tropical countries or areas with poor sanitary conditions. They are the more common

causes of gastrointestinal infections in the developed countries as compared to

helminths (Haque, 2007). Most intestinal protozoa has complex life cycle which

enables the protozoa to adapt and replicate at high rates within the host (Garg et al.,

2005; Haque, 2007).

Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum have been

recognized as the three most important intestinal parasitic protozoa that cause

diarrhea in human and other mammals (Marshall et al., 1997; Garcia et al., 2000;

Guy et al., 2004; Verweij et al., 2004; Wang et al., 2004; Coklin et al., 2007; Haque,

2007). These protozoan parasites are acquired orally through contaminated food and

water. In severe cases, they can produce chronic diarrhea which may lead to

malabsorption, weight loss, and dehydration. The diseases they cause are known as

amebiasis, giardiasis, and cryptosporidiosis respectively.

19

1.4.2 General life cycle of intestinal protozoa

A typical protozoan life cycle consists of cysts and trophozoites. Many intestinal

protozoa exhibit a similar life cycle. After ingestion by the host, the cysts transform

into trophozoites. Trophozoites is an active phase, in which protozoa are motile,

acquires nutrient and undergoes asexual replication. If desiccation or low nutrients

occurs, the trophozoites will secrete a thick wall that functions to protect them from

dehydration and enter into a dormant period. The trophozoites develop into cysts

instead of undergoing replication and is excreted with the feces, then ingested by the

next host. The cysts will convert back into trophozoites when conditions are

favourable.

Source: http://www.tulane.edu/~wiser/protozoology/notes/intes.html

1.4.3 Entamoeba histolytica

Amebiasis is a major cause of morbidity and mortality in the developing world

(Furrows et al., 2004). Currently, it is seen more frequently in developed country

through the increase in travel (Tanyuksel et al., 2005) and resulting in 100000 deaths

per year (Verweij et al., 2004). Several members of the genus Entamoeba infect

humans. E. histolytica and E. dispar is morphologically similar and previously

considered to be the same species but, genetic and biochemical data indicate that this

20

two species are distinct. However, E. histolytica or E. dispar can be distinguished

from another species of Entamoeba by having peripheral chromatin in the nuclear

and a smaller karyosome than Entamoeba coli and Entamoeba hartmanni.

Among various Entamoeba sp., only E. histolytica is considered pathogenic. E.

histolytica can cause a severe intestinal disease characterized by amebic dysentery.

Humans are the only host of E. histolytica. E. histolytica exhibits a typical fecal-oral

life cycle which consists of infectious cysts that are passed in the feces and

trophozoites that replicate within the large intestine. Infection occurs through the

ingestion of cysts primarily by consuming contaminated food and water. Once the

cysts ingested, excystation occurs to release the trophozoites. The trophozoites then

secrete enzymes that digest the intestinal lining and leads to a perforated colon and

peritonitis; an inflammation of the lining of the abdominal wall. Adherence of the

trophozoites, cytotoxicity, and disruption of the tissues are important factors in the

pathogenesis of E. histolytica (Ravdin, 1986; Petri et al., 2002).

In severe cases, dehydration and anaemia may result from the loss of fluids and

blood especially in children. Instead of infection in the intestinal tract of the host, the

amebas can also metastasize to other organs such as liver and lungs and produce

extraintestinal amebiasis. The liver is the most commonly affected organ and this is

probably due to the direct transport of trophozoites from the large intestine to the

liver via the hepatic portal vein and causing amebic liver abscesses. Haematogenous

spread of trophozoites to other sites, such as the lungs, brain, spleen or pericardium

21

and cutaneous lesions can also occur, although it is extremely rare (Pelton et al.,

2006; Shenoy et al., 2010; Maldonado et al., 2011).

Only about 10% of the infected individuals will develop symptomatic invasive

amebiasis and the remaining will undergo a non-invasive infection. The non-invasive

infection is often asymptomatic but can cause diarrhea or other gastrointestinal

symptoms (Tanyuksel et al., 2005). This non-invasive infection can persist to an

invasive amebiasis when the trophozoites invade the intestinal mucosa and kill the

epithelial cells to produce ulcers and dysentery.

Previous studies show anti-ameba humoral responses in both asymptomatic and

symptomatic E. histolytica infections (Sánchez et al., 2002). Thus, this suggests that

even in asymptomatic cases, there is a limited amount of invasion of the

trophozoites. The symptom of infection depends on the organ infected. Hepatic

infections are characterized by hepatomegaly, liver tenderness, and pain in the upper

right quadrant, fever and anorexia while the symptoms of pulmonary amebiasis

include cough, chest pain, dyspnea (difficult breathing) and fever. The sputum of the

infected patient may be purulent or blood-stained and contain trophozoites (Wiser,

2011).

The best diagnosis of amebiasis requires the demonstration of E. histolytica cysts or

trophozoites in feces or tissues (Fotedar et al., 2007). Fresh stools can be

immediately examined for motile trophozoites or preserved, stained and

microscopically examined for cysts or trophozoites. Cysts will tend to predominate

22

in formed stools and trophozoites in diarrheic stools. Trophozoites with ingested

erythrocytes are commonly found in the dysenteric feces (Wiser, 2011).

The extraintestinal infection is diagnosed by sigmoidoscopy for ulcers, especially in

more severe disease (Madanagopalan et al., 1968; Harries, 1982). Besides, the

aspirates or biopsies can be examined microscopically for trophozoites that are most

likely to be found at the abscess wall. Other methods such as antigen and antibody

detection tests using enzyme-linked immunosorbent assay (ELISA), direct

fluorescent antibody (DFA) or indirect haemagglutination (IHA) are available for

detection of E. histolytica (Fotedar et al., 2007).

The choice of drug for amebiasis depends on the clinical stage of the infection and

the location of infection (lumen or tissue). The treatment is using antibiotics which

kill the organism in the body, and followed or combined with an agent (lumenal anti-

amebic drugs) which kills the parasite throughout the intestine. Several antibiotics

are recommended for all symptomatic infections including metronidazole, tinidazole,

tetracycline, and chloroquine (Kimura et al., 2007). Iodoquinol, paromomycin or

diloxanidefuroate are the luminal agents to treat asymptomatic cases (Petri and

Singh, 1999; Wiser, 2011). In endemic areas where the rates of re-infection are high

and treatments are expensive, only symptomatic cases would be treated. However,

lumenal anti-amebic drugs are given to asymptomatic cyst passers to prevent the

progression to severe disease and to control the spread of the disease (Wiser, 2011).

The distribution of E. histolytica in human body, the morphology and the life cycle

of E. histolytica are summarised in Figures 1.4.a, 1.4.b, 1.4.c and 1.4.d.

23

Figure 1.4.a Distribution of Entamoeba histolytica in human body A. E. histolytica

is found primarily in the colon where it can live as a non-pathogenic

commensal or invade the intestinal mucosa B. The ameba can

metastasize to other organs via a blood stream; primarily involving the

portal vein and liver C. The ameba can also spread via a direct

expansion causing a pulmonary infection, cutaneous lesions or

perianal ulcers.

Source: http://www.tulane.edu/~wiser/protozoology/notes/intes.html

A

A A

A

B

B

B

C

C

24

Figure 1.4.b Entamoeba histolytica trophozoites in trichrome stained smear. Two

diagnostic characteristics are observed. Two trophozoites have

ingested erythrocytes, and all three have nuclei with small, centrally

located karyosomes.

Figure 1.4.c Entamoeba histolytica cyst with four nuclei.

Source: http://umanitoba.ca/science/zoology/faculty/dick/z346/entahome.html

25

Figure 1.4.d Entamoeba histolytica life cycle 1. Upon ingestion, the cyst passes

into the small intestine and excystation occurs with transformation to

the trophozoite stage 2. The trophozoites migrate to the large intestine

and colonized the host by asexual multiplication via binary fission.

They can remain in the lumen or invade the wall of the intestine and

multiply (for the pathogenic species). From here they can be

transported via the circulation to other organs such as liver and lungs

3. The cysts and trophozoites are passed in the feces of the infected

host 4. Infective stage of E. histolytica is the mature cyst 5. The

diagnostic stages are the trophozoite or cyst in stool or tissue

specimens.

Source: http://commons.wikimedia.org/wiki/File:Entamoeba_histolytica_life_cycle-en.sgv