Genetic Diversity of Plasmodium falciparum Isolated

from Yemen Based on the Genes of Merozoite Surface

Proteins (MSP-1 and MSP-2)

By

Nazeh Mohammed Al-Abd Ali

A DISSERTATIONSubmitted as partial fulfillment of the requirements for the degree of Master

of Biotechnology

University Malaya

Faculty of science

2011

I

ABSTRACT

Malaria is a major health problem causing substantial morbidity and mortality with

Plasmodium falciparum being the causative agent of the most severe and deadly form of

malaria in humans. In Yemen Malaria is one of the most serious health problems. About

60% of the populations live in areas with malaria transmission. P. falciparum is the

predominant species and accounts for more than 90% of malaria cases. Information on the

nature and extent of genetic diversity within P. falciparum is essential for understanding

the mechanism underlying the pathology of malaria, the acquisition of immunity, the

spread of drug resistance and the condition of the transmission. This study was conducted

in the high transmission area in Yemen (Taizz, Dhamar, and AL-Hudiydah) and was

guided by the objective that were to establish molecular characterizations of field isolate P.

falciparum in Yemen measured with polymorphic genetic markers of merozoite surface

protein msp-1 and msp-2.

Blood samples were screened for malaria parasites using Giemsa-stained thick and thin

blood films. A total of 74 blood samples had P. falciparum, for which their MSP-1 and

MSP-2 genes were studied using nested PCR. All the three families (K1, MAD20 and

RO33) of MSP-1 and the two families (FC27 and 3D7) of MSP-2 were detected in this

study. 3D7 allelic family was the most frequent (68%), followed by K1 (45%), RO33

(42%), FC27 (42%) and MAD20 (22%). The four allelic families, (MAD20, RO33, FC27

and 3D7), were significantly more prevalent in the hinterland areas as compared to

II

coastland and highland areas (p < 0.05) of Yemen. The K1 allele type was most frequent in

the highland (p < 0.02). The complexity of the infection was significantly (p < 0.05) the

highest in the hinterland followed by coastland and highland. Urban areas had higher

complexity of infection as compared to rural areas (p < 0.05). No significant difference was

shown in the complexity of falciparum infection between the age groups (p > 0.05) nor the

different levels of parasitaemia (p > 0.05). MSP-2 had higher number of alleles than MSP-1

(42 vs 18). The highest number of alleles of MSP-1 and MSP-2 was observed in the

coastland and the rural areas.

In conclusion, significant differences in complexity and the distribution of the family

alleles of MSP-1 and MSP-2 genes between hinterland, coastland and highland areas were

observed, reflecting the intensity of malaria transmission between areas. This observation

should be taken into consideration in implementing malaria control strategies in Yemen.

III

ABSTRAK

Malaria merupakan masalah kesihatan utama yang menyebabkan kadar kematian dan

morbiditi yang tinggi. Plasmodium falciparum adalah spesies dominan yang merupakan

agen penyebab malaria yang paling parah dan mengakibatkan kematian yang menyumbang

kepada penderitaan manusia yang signifikan. Parasit intraseluler ini disebarkan di kalangan

hos vertebrata oleh gigitan nyamuk Anopheles yang telah dijangkiti.Di Yemen, malaria

merupakan satu masalah kesihatan yang amat serius. Sekitar 60% populasi penduduk hidup

di kawasan transmisi malaria. P. falciparum adalah spesies yang dominan yang

bertanggungjawab terhadap lebih dari 90% kes malaria.

Maklumat berkenaan kepelbagaian genetik dalam P. falciparum adalah penting bagi

memahami mekanisma yang mendasari patologi malaria, kebolehan memperoleh

immunisasi, penyebaran ubat ketahanan dan bagaimana transmisi berlaku. Kajian ini

dijalankan di daerah yang mempunyai kadar transmisi malaria yang tinggi di Yemen

(Taizz, Dhamar, dan AL-Hudiydah). Kajian ini adalah berdasarkan objektif bagi

mengenalpasti sifat dan pencinan molekul isolat lapangan pencilan P. falciparum di

Yemen yang diukur dengan penanda genetik polimorfik protein permukaan protein

merozoit msp-1 dan msp-2.

Sampel darah diperiksa untuk mengenapasti kehadiran parasit malaria dengan

menggunakan teknik pewarnaan Giemsa dengan filem darah tebal dan nipis. Dalam kajian

ini sebanyak 74 sampel darah mempunyai P. falciparum, di mana gen MSP-1 dan MSP-2

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dikaji menggunakan PCR tersarng. Kesemua tiga keluarga (K1, MAD20 dan RO33) dari

MSP-1 dan dua keluarga (FC27 dan 3D7) dari MSP-2 berjaya dikesan dalam kajian ini.

Keluarga alelik 3D7 adalah yang paling kerap dapat dikesan (68%), diikuti oleh K1 (45%),

RO33 (42%), FC27 (42%) dan MAD20 (22%). Empat keluarga alelik (MAD20, RO33,

FC27 dan 3D7), secara signifikan lebih banyak ditemui di daerah pendalaman berbanding

dengan daerah pesisiran dan dataran tinggi (p <0.05) di Yemen. Alel jenis K1 paling kerap

ditemui di dataran tinggi (p <0.02). Kerumitan jangkitan malaria adalah signifikan (p

<0.05) dan paling tinggi di daerah pendalaman diikuti oleh daerah penisiran dan dataran

tinggi. Kawasan bandar mempunyai kerumitan jangkitan yang lebih tinggi jangkitan

berbanding dengan kawasan luar bandar (p <0.05). Tiada perbezaan yang signifikan

ditunjukkan dalam kerumitan jangkitan falciparum antara kumpulan umur (p> 0.05) dan

pelbagai peringkat parasitemia (p> 0.05). MSP-2 mempunyai jumlah alel yang lebih tinggi

dari MSP1 (42 vs 18). Kadar tertinggi alel dari MSP-1 dan MSP-2 didapati di daerah

penisiran dan kawasan luar bandar.

Kesimpulannya, perbezaan signifikan dalam kerumitan dan taburan keluarga alel gen MSP-

1 dan MSP-2 antara kawasan pendalaman, daerah penisiran dan dataran tinggi yang dikaji,

mengambarkan intensiti transmisi malaria di antara ketiga-tiga kawasan tersebut.

Pemerhatian ini harus dipertimbangkan dalam pelaksanaan strategi kawalan malaria di

Yemen.

V

ACKNOWLEDGEMENTS

First and foremost, I would to thank Allah for the blessing and the guidance, strength and

courage to preserve throughout the duration of whole my life.

I would like to thank and extend my sincere gratitude and appreciation to my supervisor,

Dr. Mohammed Mahday AL-Sharabi for his excellent support and guidance throughout the

study. His enthusiasm, boundless ideas, expertise, experience, criticism, encouragement

and challenges were very much appreciated.

I would also like to express my gratitude to my co-supervisor Prof. Dr. Fong Mun Yik for

his assistance, guidance , and the opportunity to carry out my work in his laboratory . I

would like to express great thanks to my co-supervisor Dr. Nazia Abdul-Majed for her

assistance ,advice, guidance and comments throughout the study.

Special thanks to the head and staff of Department of Parasitology, Faculty of Medicine,

for their support. I thank University of Malaya for supporting the research under the

postgraduate grant (PPP) no.P0083/2010B. I would like to thank all my lecturers in the

faculty of science, colleagues, and friends for their unending encouragements. I am grateful

for Mr Abdulsalam Mohammed Qasem Al-mekhlafi for sharing with me the field samples

and his kind help. I would like to thank all members of molecular laboratory of

Parasitology Department and my close friend Mustafaa Kassim,Ahmed Gumel, Fahad

Farid and Farid Amir for their encouragement

Most importantly, I would like to thank my parents, my brothers, my sisters, and my wife

for supporting me. It is through their encouragement, love, support and prayers that I have

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made it through all the steps to reach this point in life, and I could not have done it without

them.

Special thank to my sister Amal she didn’t skimp on her time and she sacrifice her time in

order to provide advice and assistance me in different situation during my studies.

I am also thankful to the Higher Education Ministry of my country for the support,given to

me the scholarship to study in Malaysia.

Finally, I would like to thank everyone for putting up with me for the last several years. I

hope that this dissertation has made some contribution to the field of malaria diversity and I

hope that everyone who reads this dissertation finds it useful in their work.

VII

Table of Contents

Page

IABSTRACT

IIIABSTRAK

VACKNOWLEDGEMENT

VIITable of Contents

XLIST OF TABLES

XILIST OF FIGURSE

XIILIST OF ABBREVIATION

XIIIDEFINITIONS

XIVDEDICATION

CHAPTER I INTRODUCTION

1.1 INTRODUCTION 1

1.2 Justification of the study 2

1.3 General Objective 3

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1.4 Specific Objectives 3

CHAPTER II LITERATURE REVIEW

2.1 Plasmodium Species and Life Cycle 4

2.2 The Mosquito Vector 7

2.3 Distribution of malaria worldwide 8

2.4 Transmission and Epidemiology of Malaria 10

2.5 Genetic Diversity of P.Falciparum Infections Based

on Genes Encoding MSP Proteins 11

2.6 Status of Malaria in Yemen 17

2.7 General Glance on previous studies 19

CHAPTER III METHODOLOGY

3.1 STUDY AREA AND POPULATION 22

3.2 SAMPLES AND MICROSCOPY 25

3.3 GENOTYPING OF P.FALCIPARUM BASED ON 25

MSP -1 AND MSP-2 GENES

3.3.1 GENETIC DNA EXTRACTION 25

3.3.1 NESTED POLYMERASE REACTION 26

3.4 PCR PRODUCT ANALYSIS 30

3.4.1 GEL PREPARATION 30

En

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3.4.2 GEL ELECTROPHORESIS 30

3.5 STATISTCAL ANALYSIS 31

3.6 Ethical clearance 31

CHAPTER IV RESULT

4.1 Characterization of study population and the distribution of P.Falciparum 32

4.2 Nested PCP 35

4.3 Prevalence and distribution of all elic families of MSP-1 and MSP-2 41

4.3 Complexity of families of MSP-1 and MSP-2 in YEMEN P. falciparum isolates 43

. .

CHAPTER V DISCUSSION

5.1 Discussion 45

5.2 Conclusion 49

5.3 Recommendation 50

References 51

APPENDIX 70

X

LIST OF TABLESTable Page

2.1 Review of some previous study on the genetic diversity of merozoite

Surface protein (MSP-1 and MSP-2) gene 20

2.2 Distribution of alleles of MSP-1 and MSP-2 21

3.1 The primer sequence MSP-1 and MSP-2 alleles 28

4.1 Distribution of the a Alleles of MSP-1 in Yemen P. falciparum isolates 42

4.2 Distribution of families of MSP-2 in Yemen P. falciparum isolates 43

4.3 complexity of P.falciparum based on MSP-1 and MSP-2 in Yemen isolates 44

XI

LIST OF FIGURESFigure Page

2.1 Giemsa-stained thin smears depicting the life cycle of 3D7 P. falciparum 5

2.2 Distribution of Malaria worldwide 9

2.3 Yemen location map 18

3.1 Geographical location of study area in Yemen 23

4.1 The distribution of participants according to gender in the three locations 33

4.2 Percentage of samples collected from study areas (Taizz, Damar, and Al-Hudaydah)

in Yemen 34

4.3 Parasite density among patients from the three study locations 35

4.4 Different allelic forms (100 and 250 bp alleles) of K1 36

4.5 Different allelic forms of RO33 and MAD20 detected 37

4.6 FC27 allelic forms of MSP-2 37

4.7 Size and frequency of K1 allele detected in the study areas 38

4.8 Size and frequency of MAD20 observed in the study areas 39

4.9 Size and frequency of RO33 observed in the study areas 39

4.10 Size and frequency of 3D7 detected in the study areas 40

4.11 Size and frequency of FC27 observed in the study areas 40

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LIST OF ABBREVIATIONS

ACT Artemisinin-based combination therapybp Base pair

CDC Centre for Disease ControldNTP Deoxynucleotide triphosphateEDTA Ethylene diamine tetra acetic acidEIR Entomological inoculation rateIPT Intermittent preventive treatmentMSP-1 Merozoite surface protein 1PCR Polymerase chain reactionRBC Red blood cellU UnitUV UltravioletWHO World Health OrganizationRDT Rapid diagnostic testHRP-2 Histidine-rich protein 2GLURP Glutamate-rich proteinRT Reverse transcriptaseMS Microsatellite analysisMALDI-TOF Matrix- assisted laser desorption-ionization time

of flight mass spectrometryMVR Minisatellite variant repeatLDR-FMA Ligase detection reaction-fluorescent

microsphere assayGPI glycosylphosphatidylinositolTEA Tris-EDTASPSS Statistical Package for the Social SciencesMOI Multiplicity of infectionUSA United States of AmericaNMCP National Malaria Control ProgrammeEGF Epidermal growth factor

XIII

DEFINITIONS

Definitions of terms used through this thesis:-

Allele: one of the several alternative forms of genes that occupy the same locus.

Allelic type: alleles of a gene that can be grouped based on similar characteristics e.g.

sequence similarity of the allelic types of MSP-1 and MSP-2, also referred to as allelic

families.

Genotype: combination of alleles that determine a particular genetic characteristic.

Infection diversity: the number of clones detected within one sample. This number

represents the minimum number of circulating clones; also referred as genetic diversity of

infections or multiplicity of infection.

Parasitaemia: is the quantitative content of parasites in the blood. It is used as a

measurement of parasite load in the organism and an indication of the degree of an active

parasitic infection.

Rural areas: are large and isolated areas of an open country with low population density.

An urban area: is characterized by higher population density and vast human features in

comparison to areas surrounding it.

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DEDICATION

To all of you who made this possible, thank you