University of Khartoum

Faculty of Medicine

Postgraduate Medical Studies Board

Antibody Response against Conserved, Polymorphic and Variant Antigens in Severe

falciparum Malaria in Gedarif State, Eastern Sudan

By

Thoraya Mohamed Elhassan A-Elgadir

BSc, MSc of Medical Biochemistry,

University of Khartoum

A Thesis Submitted for the Fulfillment of the Degree of PhD in Medical Biochemistry

January 2006

Supervisor

Professor, Mustafa Idris Elbashir, MD, PhD

List of contents

Dedication …………………………………………………………………………..

I

List of publications………………………………………………………………….

Acknowledgements ……………………………...………………………………….

Abbreviations……………………………………………………………………….

II

III

V

Abstract ……………………………………………………………………………. VIII

Arabic Abstract…………………………………………………………………….. X

List of Figures …………………………………....................................................... XII

List of Tables ………………………………………………………………………. XV

Chapter One: Introduction ………………………………………………….. 1

1. Introduction ……………………………………………………………………… 1

1.1. Malaria ………………………………………………………………………… 1

1.2. Severe malaria ………………………………………………………………… 1

1.2.1. Pathogenesis of severe malaria ……………………………………………... 2

1.2.1.1. Cytokines ………………………………………………………………….. 2

1.2.1.2. Reactive oxygen species (ROS) …………………………………………… 3

1.2.1.3. Nitric oxide (NO) ………………………………………………………….. 4

1.2.1.4. Cytoadherence, rosetting and sequestration ………………………………. 4

1.3. Malaria parasite and life cycle (Figure 1.1) …………………………………… 6

1.4. Epidemiology of malaria ……………………………………………………… 8

1.5. Malaria control ………………………………………………………………… 10

1.6. The malaria parasite genome ………………………………………………….. 10

1.7. Plasmodium falciparum Antigenic diversity …………………………………. 12

1.7.1. Antigenic polymorphism ……………………………………………………. 12

1.7.2. Antigenic variation ………………………………………………………….. 12

1.7.2.1. The var gene ………………………………………………………………. 13

1.7.2.2. Repetitive interspersed family (rif) ………………………………………... 15

1.7.2.3. Subtelomeric variant open reading frame (stevor) ……………………….... 15

1.7.2.4. Pf60 ………………………………………………………………………... 16

1.8. Immunity to malaria …………………………………………………………… 16

1.8.1. Innate natural immunity ……………………………………………………... 16

1.8.2. Acquired immunity ………………………………………………………….. 18

1.8.2.1. Arms of the acquired immunity …………………………………………… 18

1.8.2.1.1. The cell mediated immune response …………………………………….. 18

1.8.2.1.2. The humoral immune response ………………………………………….. 19

1.8.3. Immune suppression during malaria ………………………………………… 21

1.10. Malaria vaccine ………………………………………………………………. 21

1.10.1. Potential malaria vaccine candidate antigens ……………………………… 22

1.10.1.1. Sporozoite or pre-erythrocytic stage vaccine candidates ………………… 23

1.10.1.2. Transmission blocking vaccine …………………………………………... 24

1.10.1.3. Asexual blood stage vaccine candidates …………………………………. 24

1.10.1.3.1. Merozoite surface antigens …………………………………………….. 25

1.10.1.3.2. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) …. 28

1.10.1.4. Anti-toxic and anti-disease vaccine ……………………………………… 30

1.10.2. Types of vaccine formulations ……………………………………………..

1.10.3 Malaria vaccine trials……………………………………………............... ...

31

32

The objectives ………………………………………………………………… 33

Chapter Two: Materials and Methods ……............................................. 34

2.1. Study area, study design and samples ………………………………………… 34

2.1.1. Study area …………………………………………………………………… 34

2.1.2. Study design ………………………………………………………………… 34

2.1.2.1. Malaria definition and diagnosis ………………………………………….. 35

2.1.2.2. Characterization and grouping of patients with severe malaria …………… 35

2.1.3. Samples collections and storage …………………………………………….. 36

2.1.3.1. Plasma and parasitized red blood cells (PRBC) separation and storage …... 36

2.2. Measurement of antibody response directed against MSP119 and MSP2 by

ELISA ……………………………………………………………………...

36

2.3. Measurement of the antibodies directed against Plasmodium falciparum er

membrane protein 1 (PfEMP1) ………………………………………...

37

2.3.1. Parasites ……………………………………………………………………... 37

2.3.2. Plasma ……………………………………………………………………….. 38

2.3.3. Long term In vitro culture of Plasmodium falciparum from cryo-preserve

isolates ………………………………………………………………………

38

2.3.4. Magnet activated cell sorting (MACS) ……………………………………… 39

2.3.5. Analysis of plasma antibodies to variant surface antigens by fluorescent activ

sorter (FACS) ………………………………………………………...

41

2.4. Statistical analysis …………………………………………………………….. 42

Chapter Three: Results ………………………………………………………. 45

3.1. Epidemiology of severe Plasmodium falciparum malaria …………………….. 45

3.1.1. The frequency of acute uncomplicated and complicated malaria …………… 45

3.1.2. The frequency of the different types of severe complications ………………. 45

3.1.3. Age distribution of severe and mild malaria ………………………………… 46

3.1.4. Characterization of patients in the different categories of severe malaria …... 47

3.1.5. Comparison between fatal and non-fatal severe malaria ……………………. 49

3.2. The antibody response against the conserved and polymorphic antigen

merozoite surface proteins (MSP119, MSP2A and MSP2B)…………………………

49

3.2.1. Characteristics of donors ……………………………………………………. 49

3.2.2. Recognition of merozoite surface protein (MSP) …………………………… 49

3.2.3. The relation between the age and the prevalence of MSP antibodies ………. 51

3.2.4. The antibody response against MSP antigens: comparison between a

healthy malaria-free donors and malaria patients ………………………

52

3.2.5. The antibody response against MSP antigens, comparison between patie

uncomplicated and severe malaria …………………………………………….

55

3.2.6. The antibody response against MSP antigens: comparison between i

complications of severe malaria (SMA, CAM, CM and HTN) …………

56

3.2.7. The antibody response against MSP antigens: comparison between fatal

fatal cerebral malaria …………………………………………………………..

58

3.2.8. Acquisition of anti-MSP antibodies at convalescence (D28) by SM patients.. 59

3.3. Antibody response against variant surface antigens (VSA) …………………… 59

3.3.1. Spectrum and level of antibody reactivity against VSA……………………...

3.3.2. VSA antibody recognition of parasites isolated from patients with sever

uncomplicated malaria…………………………………………………………

59

60

3.3.3. Correlation between parasite donor age and parasite-VSA recognition rate… 62

3.3.4. Plasma reactivity (VSA-Ab spectrum) in malaria patient groups and asym

malaria free individuals ……………………………………………..

63

3.3.5. Correlation between plasma donor age and VSA antibody prevalence ……... 66

3.3.6. The reactivity of acute (D0) and convalescence (D28) plasma against hom

and heterologous parasite isolates …………………………………….

66

Chapter Four: Discussion …………………………………………………… 68

4.1. Conclusions …………………………………………………………………… 78

4.2. Recommendations ……………………………………………………………... 80

4.3. References ……………………………………………………………………... 81

Appendices………………………………………………………………………….A

x I: Equipment, materials and reagents

Appendix II: Questionnaire and consent

Appendix III: Publications

101

DُُُُEDICATION

To the souls of my parents and my sister Fathia

&

To my family

List of publications This thesis is based on the work presented in the following papers:

1- Giha HA, ELGhazali G, A-Elgadir TME, A- Elbasit, EM, Eltahir, Baraka OZ, Khier MM, Adam I,

Troye-Blomberg M, Theander TG and Elbashir MI. Clinical pattern of severe P. falciparum malaria in

Sudan in an area characterized by seasonal and unstable malaria transmission. Trans Roy Soc Trop Med

Hyg 2005; 99: 243-51.

2- T. A-Elgadir, T. Theandr, M. Nielsen, I.A-elbasit, G. Elghazali, I. Adam., Troye-blomberg, H. Giha,

M. Elbashir. Antigenic variation in severe Plasmodium falciparum malaria: A comparison between severe

malarial anemia and cerebral malaria. 4th MIM Pan-African Malaria conference (abstract). MIM-TA-

7406. Acta Tropica 2005; 95S: s378-9.

3- Thoraya M E A-Elgadir, Thor G Theander, Gehad ElGhazali, Morten A Nielsen, Ishraga E A-Elbasit,

Ishag Adam, Marita Troye-Blombergc, Mustafa I. Elbashir, and Hayder A. Giha. Determinants of VSA

antibody response in severe Plasmodium falciparum malaria in an area of low and unstable malaria

transmission. Scandinavian Journal of Immunology 2006; 63:232-40.

4- Thoraya M E A-Elgadir, Mustafa I. Elbashir, Klavs Berzin , Ishraga E A-Elbasit, Gehad ElGhazali

and Hayder A. Giha. IgG antibody response against merozoites surface antigens 1 and 2 associates with

protection from severe nonfatal P. falciparum malaria, in an area with unstable transmission. Accepted by

Parasite Immunology 2006.

ACKNOWLEDGEMENTS

In the name of God, the merciful and the compassionate who gave me the strength to do this work.

I am extremely grateful to my supervisor Professor Mustafa Idris Elbashir Department of Biochemistry,

Faculty of Medicine, University of Khartoum, for genuine guidance, encouragement, and supervision over

the course of this study.

I would like to express my thanks to my co-supervisor Associate Professor Hayder Ahmed Giha,

Department of Biochemistry, Faculty of Medicine, University of Khartoum for his valuable comments,

advice and unlimited support throughout the study, without his help this work would not have been

accomplished.

Professor Thor Theander, Centre for Medical Parasitology, Institute for Medical Microbiology and

Immunology, University of Copenhagen, Denmark, is thanked for his good hospitality during my stay in

Copenhagen, his help, care and support is highly appreciated.

Deepest gratitude to my friend Dr. Insaf Fadul Khalil, who gave my stay in Copenhagen nice touches.

My thanks are also extended to the Copenhagen team, Dr. Lars

Hviid for hosting me through out the course of the FACS

analysis and the access to the different facilities in his

laboratory. Thanks are also extended to Dr.Trine Staalsoe

who provided the Tanzanian plasma. Dr. Morten Nielsen, Anna

Corfitz and Maiken Christensen are thanked for their support

and assistance in FACS work.

Dr James Chipeta, University teaching hospital, Lusaka,

Zambia who, was in a training period visit to Dr. Lars

laboratory at Copenhagen and offered me a great help and

support during the FACS work. Special thanks are due to him.

Dr Gamila Ibrahim is thanked for donating the merozoite

surface antigens. Special thanks are due to Dr Yousra

Mirghani for her support and kindness.

Sincere thanks to the Associate Professor; Gehad Elghazali, who kept

supporting me even when he is abroad, by providing valuable

references and articles that are unavailable in our library

or in the internet services.

Thanks are due to all patients and their guardians for cooperation

and willingness to participate in this study. Gedarif

Teaching Hospital staff is acknowledged for the support and

facilitation of the field work, with special thanks to Dr.

Howida Elmardi, Dr. Suad Abdalla, Dr. Aisha Abdalla, Dr

Mutaz Abdalla, Dr. Usama and to the memory of Dr. Haithem.

Special thanks to Faiz Omer, Adil Amin, Ihsan Eltayeb and

Mustafa Hamid for their help during the field work in

Gedarif area. The help that received from Dr. Ahmed Fahmi

during the field work is highly appreciated.

My thanks are due to my friends Muna Attia, Ishraga Eltayeb, Maha Elhadi, Dr. Nuha

Galal, Nada Bayoumi, Hanan Babiker, Shadia Mahgoub, Fathia Mohamed Khier, Dr.

Ahmed Tamam and Adil Balal for their support and help.

I am indebted to all members of the department of Biochemistry, Faculty of Medicine,

University of Khartoum for their support and encouragement and for providing the suitable

home-like environment.

Finally my thanks are due to my brothers and sisters with special thanks to Nasr Eldein.

This study has been financially supported from the UNDP/World Bank/WHO/TDR, the

Multilateral Initiative on Malaria in Africa (MIM), project ID A00003.

Abbreviations

ADCC

ADCI

APC

APL

Antibody dependent cell mediated cytotoxicity

Antibody dependent cellular inhibition

Antigen presenting cell

Altered peptide ligand

CAM

CIDRs

CM

CR1

CSA

CSP

D0

D28

DABP

DBL

DCs

EBA-175

EDTA

EGF

EIR

ELISA

FACS

FcγRIIa

FITC

GPI

GST

HLA

HRP/IgG

ICAM-1

IL

iNOS2

LFA-1

LSA-1

MACS

MF

Convulsions associated malaria

Cysteine rich interdomain regions

Cerebral malaria

Complement receptor 1

Chondroitin sulfate A

Circumsporozoite surface protein

Day zero

Day 28

Duffy antigen binding protein

Duffy binding like proteins

Dendritic cells

Erythrocyte binding protein of Plasmodium falciparum

Ethylenediaminetetraacetic acid

Epidermal growth factors

Entomological inoculation rate

Enzyme linked immunosorbent assay

Fluorescent activated cell sorter

Fc gamma receptor IIa

Fluorescein isothiocyanate

Glycosyl phosphatidylinositol

Glutathione-S-transferase of Schistosoma japonicum

Human leukocyte antigen

Horseradish peroxidase-conjugated to rabbit anti-human IgG, sp

gamma chain

Intercellular adhesion molecules 1

Interleukin

Inducible nitric oxide synthase 2

Leukocyte function-associated molecule- 1

Liver stage-specific antigen

Magnet activated cell sorter

Malaria free donors

MSP

NK

NO

NTS

P-ABA

PAM

PBS2

PBS-Tween-20

PfEMP1

Pfs 25 & Pfs 28

PfSSP2

PRBCss

rif

ROS

SM

SMA

stevor

TGF-β

TNF

TRAP

TSP

TZ

UM

VCAM

VSA

WHO

Merozoite surface protein

Natural killer cells

Nitric oxide

Amino terminal segment

para-aminobenzoic acid

Pregnancy associated malaria

Phosphate buffered saline with 2% fetal calf serum

Phosphate buffered saline with 0.05 % Tween 20

Plasmodium falciparum erythrocyte membrane protein 1

Sexual stage antigens of Plasmodium falciparum malaria parasite S

surface protein 2 of Plasmodium falciparum

Parasitized red blood cells

Repetitive interspersed family

Reactive oxygen species

Severe malaria

Severe malarial anemia

Subtelomeric variant open reading frame

Transforming growth factor beta

Tumor Necrosis factor

Thrombospordin-related adhesive protein

Thrombospondin

Tanzanian plasma

Uncomplicated malaria

Vascular cell adhesion molecule

Variant surface antigens

World Healh Organization

ABSTRACT

The objectives of this study were to describe the clinico-epideiological feature of severe malaria in an area

of markedly unstable and seasonal transmission. Also the interest was directed to characterize the immune

response against merozoite surface proteins and variant surface antigens and to assess their impact on the

development of different manifestation of severe malaria. The study was carried out in Gedarif area,

Eastern Sudan during 2 successive malaria transmission seasons over the period 2000-2002.

This is the first report about severe malaria in the Sudan and it is important for filling the paucity in the

knowledge about the epidemiology of severe malaria.

The incidence of severe malaria was 4.4%, and the mortality rate among the severe malaria (SM) patients

was 6.4%, which accounts for 0.3% of all cases of malaria. Only four types of complications were

recognized during the study period, the commonest was the severe malarial anemia (SMA) followed by

convulsions (CAM), cerebral malaria (CM) and hypotension (HTN). Severe malaria was recognized in all

age groups. The mean age for SM peaked at the age 2-4, while uncomplicated malaria peaked at 5-19

years. The 4 types of severe malaria significantly differ from each other in clinically important clinico-

epidemiological indices, age, hemoglobin, blood glucose, parasite count and mortality rate. The antibody

response directed against merozoite surface proteins (MSP119, MSP2A and MSP2B) revealed that, the

three tested merozoite surface antigens were immunogenic. The MSP antibody response was age

dependent in this setting. The antibody response against MSP119 was more significantly

associated with protection from severe malaria but not uncomplicated malaria. More importantly, neither

the prevalence nor the levels of MSP antibodies was associated with protection from fatal cerebral

malaria.

The study of the immune response against variant surface antigens (VSA)

showed that the parasites obtained from patients with severe malaria,

namely SMA and CM, displayed predominantly recognized VSA antigens

compared to parasites obtained from patients with uncomplicated malaria

(UM). On the other hand plasma from CM patients showed high or normal

antibody response compared to the UM, while patients with SMA were

found to have poor antibody response to VSA antigens expressed by

parasites obtained from patients with different clinical grades of

malaria. In addition it was found that, the plasma/parasite interaction

is influenced by the intensity of malaria transmission in the place of

the plasma donor (Sudan or Tanzania) rather than the place of parasite

donor. On the contrary, the VSA antibody response was more affected by

the age of parasite donor than the age of plasma donor.

الخالصة

ال موسمي للمرض ة إنتق ضًا . أجريت هذه الدراسة بغرض وصف األوجه السريرية الوبائية للمالريا المعقدة في منطقة ذات طبيع أيسطحية؛ و لمتمت دراسة االستجابة ا ات ال ة ضد ميروزويت البروتين ة ناعي سطح المتباين ات ال ي تطور مختلف أنتجين ا ف يم أثره لتقي . مظاهر المالريا المعقدة

. 2002-2000تم إجراء الدراسة في منطقة القضارف بشرق السودان خالل موسمين متتابعين النتقال المرض في الفترة

ات تعتبر هذه الدراسة األولى من نوعها عن وضع المالريا المعقدة بالسودان، هذه الد راسة مهمة لملء الفراغ الناتج عن شح المعلوم . المتعلقة بوبائية المالريا المعقدة بالسودان

ي ي % 4.4أشارت الدراسة إلي إن نسبة حدوث المالريا المعقدة تصل إل ا تصل إل ات الناتجة عنه سبة الوفي سبة %46.ون ذه الن ، ه . من آل حاالت المالريا% 0.3تمثل

ة الدراسة تم التحقق من وجود أر دة في منطق ا المعق ة بالمالري واع من المضاعفات المتعلق ع أن دة المرتبطة . ب ا المعق مثلت المالريدة المرتبطة بانخفاض ا المعق رًا المالري ة وأخي ا الدماغي م المالري شنجات ث باألنيميا أآثر األنواع شيوعًا، تلتها المالريا المرتبطة بالت

دم شاف وجود المال. ضغط ال م اآت دة ت ا المعق د أن المالري ة؛ وج ي آل المجموعات العمري دة ف ا المعق ي تصل ري دالتها ف ى مع أعل . سنة15-9 سنوات، بينما آانت المالريا غير المعقدة أآثر شيوعًا في المجموعة العمرية 4-2المجموعة العمرية

سريرية أشارت النتائج إلي أن األنواع األربعة من المالريا المعقدة تختلف عن بعضها ة وال إختالفًا معنويًا في عدد من األوجه الوبائية . و معدل الوفيات آثافة الطفيل مثل عمر المريض، مستوي الهيموغلوبين، مستوي السكر في الدم، ائج االستجابة المناعي أشارت نت

ذا . ة ضد المرض يمكن إستخدامها لتوليد مناع ) MSP119, MSP2A & MSP2B(إلي أن ميروزويت البروتينات السطحية ي ه فر ) MSP119(اإلطار وجد أن ميروزويت البروتين يرتبط ارتباطًا معنويًا مع الحماية من المالريا المعقدة ولكن ليس مع المالريا غي

ة من المالري . المعقدة ا أشارت النتائج أيضًا لعدم وجود عالقة بين مستوى األجسام المضادة لمريزويت البروتينات السطحي والحماي . الدماغية القاتلة

دة ا المعق أشارت دراسة االستجابة المناعية لبروتينات السطح المتباينة إلي أن الطفيليات التي تم جمعها من مرضى مصابين بالمالريدة ر المعق ا غي ا من مرضى المالري م جمعه ي ت ات الت ة بالطفيلي ائدة مقارن ة بصورة س في . آشفت عن وجود انتجينات سطح متباين

ريض بالمالري منح ي مصل دم الم ضادة ف سام الم ستوى األج د أن م ر وج هى آخ ة بمرضى ا الدماغي ة مقارن دالت عالي ت بمع آان . المالريا غير المعقدة بينما آان مستوي األجسام المضادة منخفضًا لدى مرضي المالريا المعقدة المرتبطة باألنيميا

سودان، (المالريا تتأثر بكثافة انتقال المرض بالمنطقة التي أخذ منها مصل الدم باإلضافة إلي ذلك وجد أن عالقة مصل الدم بطفيل ال . ، على النقيض تتأثر االستجابة المناعية النتجينات السطح المتباينة بعمر الشخص الحامل للطفيل)تنزانيا

List of Figures Figure 1. Malaria parasite life cycle

8

Figure 2.1A. P.falciparum parasite (Gedarif ) from an in vitro culturedifferent developmental stages of rings and late stages.

40

Figure 2.1B. RBCs with late stage Plasmodium falciparum parasites fromarea after it had been in vitro cultured and enriched to late stage (schiztrophozoites) by MACS

40

Figure 2.2. FACS histogram results representing the antibody response sample (Bl), 11 Danish plasma samples, Pool positive plasma in 6 dilutiosevere plasma samples from Gedarif area directed against PfEMP1 of Plasmodium falciparum isolate (SG93).

43

Figure 3.1. Symptoms in the 110 patients admitted to Gederif HospitalSudan and fulfilling the WHO criteria for severe malaria, the patients wereover a period of two years

46

Figure 3.2. Age distribution of patients with severe malaria (n=110, white with uncomplicated malaria (n=1 537, black dots) in Gedarif HospitalSudan (period 2000 to 2002).

47

Figure 3.3: Comparison between four groups of patients with severe(anemia, convulsions, cerebral malaria and hypotension) admitted toHospital; in the following; a. age in years (mean ± 95% confidence interval, CI) b. parasite count (mean ± 95% CI) at diagnosis (day o) and before treatmenc. hemoglobin level (mg/dl, converted to percentage), mean ± SD, at diagbefore treatment, d. random blood glucose level, mean ± SD, at diagnosis and before tHorizontal lines with P-values span between clinical groups with sdifferences

48

Figure 3.4. The prevalence of Abs against MSP119, MSP2A and MSP(proportions ± 95% confidence interval), in the different age groups of atogether; patients with malaria (severe and uncomplicated) and malaria-fre(n=339). The age grouping was based on physiological development (infyoung children >1-5, older children >5-10, adolescents >10-15, adulthoodolder adults >45 year).

52

Figure 3.5. The prevalence of antibodies against individual MSP antigens; 54

MSP2A, MSP2B or any of the three antigens (MSPall), proportion (%) confidence interval (CI). A: Comparison between malaria-free donors (dand patients with malaria (both uncomplicated and severe malaria), (light Comparison between patients with severe malaria (dark bars) and uncommalaria (light bars), the differences were all significant although in MSMSP2B bars; there was slight overlap for CI. Figure 3.6. The MSP Ab response in SM patients, comparison between; coassociated malaria (CAM), severe malarial anemia (SMA), cerebral malaand hypotension (HTN). The proportion of patients with Abs against MSP2A or MSP2B Ags is indicated by the length of bars. Proportion ofrecognized the 3 antigens (black), two antigens (light) and one antigen (gage (mean ± SD) of the patients in each sub-group is indicated by the line wcircles.

57

Figure 3.7. The correlation between two indices used for evaof the VSA antibody (Ab) response; the Y axis shows the proof isolates recognized by individual plasma samples circle), and the X axis shows the mean level of Abs isamples. Plasma was obtained from patients with severe (n=65) at the time of diagnosis, antibody level was scoredlevels as explained before. Generally, plasma with broad sof reactivity had on average higher level of antibody individual isolates, with general positive correlation. insert, the lines represent the mean.

60

Figure 3.8. The prevalence of anti VSA antibodies in differeof plasma obtained from malaria free donors (MF), patientuncomplicated malaria (UM), severe malaria at the time of di(SM-D0), and at convalescence (SM-D28 day) and Tanzanian cwith UM (TZ). The isolates were obtained from patientuncomplicated malaria (UM), severe malarial anemia (SMcerebral malaria (CM). The insert shows the reactivity individual isolates in each group of the parasites

62

Figure 3.9. a. The prevalence of VSA antibodies in subsets of plasma ofrom patients with different entities of severe malaria;malaria anemia (SMA), malaria associated convulsions (MAcerebral malaria (CM). b. The overall prevalence of antibodies against VSA aexpressed by isolates obtained from patients with diclinical presentation of P. falciparum malaria; SMA, CM and

63

Figure 3.10. The correlation between age of parasite (children aged 1.5 to 11 years and adults aged 19 to 27 yeathe proportion of plasma from all panels able to recogniz

64

groups of parasites. Parasites obtained from cstatistically significantly more recognized Figure 3.11. The correlation between age of plasma donprevalence of antibodies against VSA expressed by all 22-pisolates. The plasma donors were Tanzanian children (A) other Sudanese plasma donors (B).

65

List of Tables Table 3.1. Clinical categorization of patients presented with malaria-like sto the malaria clinic at Gedarif Hospital, in the period, from SeptemberJanuary 2002. The patients died of severe malaria were all had cerebral mal

45

Table 3.2. Levels of variable parameters (parasitological, hematologbiochemical) in individuals who died of severe malaria, at Gedarif HospitaSudan, and comparison of the mean values of these parameters between non-fatal severe malaria groups

50

Table 3.3. Characteristics of the different groups of volunteers studiedcollected during two successive transmission seasons 2000-2002 from eastern Sudan

51

Table 3.4. The malaria-free donors (negative control) were apparently heahad negative blood smears for malaria parasite, as seen by microscope. Tshows the donors with parasitemia (detectable by PCR) and with antibodies (against any of the following MSP antigens: MSP119, MSP2MSP2B)

53

Table 3.5. The plasma level of antibodies in individuals with positive response (above the cut off point) directed against different MSP antigenthe 3 different study groups: a comparison between malaria-free donouncomplicated malaria (UM) and severe malaria (SM) patients.

55

Table 3.6. The average number of MSP antigens (MSP119, MSP2A andrecognized by individuals in different study groups: a comparison betmalaria patients and malaria-free donors, and between patients with uncommalaria and severe malaria.

56

Table 3.7. The cerebral malaria patients, characteristics and anti-MSPs response in patients died or survived following the cerebral malaria attack

58

Table 3.8. The details of the total samples used for the study of theresponse directed against variant surface antigens, plasma and parasite donmainly classified on clinical bases, date of sampling and geographical locat

61

Table 3.9. The parasite donor's characterization, levels and spectrumplasma antibody reactivity at diagnosis (D0) and convalescence (D28variant surface antigens (VSA) of parasites isolated from them (homo) adonors (hetero) respectively.

67

INTRODUCTION 1.1. Malaria

Malaria is one of the most wide spread transmissible diseases in the world, the name of malaria originated

when an Italian Lancisi, in 1917 linked malaria with poisonous vapours of swamps (mal aria-bad spoilt

air). The disease has been known to the humanity from the dawn of civilization. In the ancient Chinese

and Egyptian manuscripts and the literary sources of ancient Greece and Rome, epidemic fever,

symptomatically similar to malaria was described. Regarding the history of scientific discoveries in

malariology, in 1847, the German scientist Meckel detected brownish pigment in the leukocytes-

macrophage of malarious patients; the presence of this was associated with malaria. In 1890 Marchiafa

and Celli described Plasmodium falciparum. In 1897 Ronald Ross proved experimentally that mosquitoes

serve as vectors of human and avian malaria (1).

Malaria is considered as one of the main health problems in Sudan and the disease was known to affect

adults as well as children (2), and causes death for 1.5% of the in-patient individuals (3). Plasmodium

falciparum was found to be the causative agent for 98% of malaria infections in Eastern Sudan, with

Plasmodium vivax and Plasmodium malariae occasionally seen (4).

Globally, 300–500 million clinical cases are detected annually with an estimated mortality of 1.5–2.7

millions. Approximately one million deaths among children less than five years of age are attributed to

malaria alone or in combination with other diseases. Countries in tropical Africa are estimated to account

for more than 90% of the total malaria incidence and the great majority of malaria deaths (5).

1.2. Severe malaria

Malaria causes 3000 deaths per day, with annual total that exceeds one million deaths worldwide (6).

Severe and complicated malaria is a fatal form of human Plasmodium falciparum malaria that associated

with the failure of the host defenses to control parasite replication, excessive secretion of pro-

inflammatory cytokines and sequestration of parasitized erythrocytes in vital organs (7). In clinical

practice severe malaria can be manifested in different forms starting with the severe cerebral malaria,

prostration, impaired consciousness, severe malaria anemia, hypoglycemia, repeated multiple convulsions,

circulatory collapse, respiratory distress, hyperlactataemia, abnormal bleeding, jaundice, haemoglobinuria,

renal impairment, and pulmonary edema (radiological)(8).

1.2.1. Pathogenesis of severe malaria

The epidemiological profile and clinical pattern of severe malaria in Africa, has been shown to be

modulated by the intensity of exposure and pattern of transmission (9). Severe complications in areas

highly endemic for Plasmodium falciparum malaria such as tropical Africa predominantly affects children

in the age group of 1-5 years i.e. children that have not yet developed a sufficient anti-disease immunity.

Cerebral malaria is not so common in regions with perennial or hyper-holoendemic transmission where

Plasmodium falciparum is transmitted all around the year. The severe forms of the disease are in other

areas frequently seen both in children as well as in adults reflecting a less intense transmission pattern of

Plasmodium falciparum infections (10).

A number of substances have been proposed to be responsible for severe disease. The cytokines, reactive

oxygen species (ROS), and nitric oxide (NO) have been implicated with the possibility of a dual role for

some, in which these substances are involved in both the pathogenesis of severe disease and killing the

parasite (11). Cytoadherence is believed to be a key factor in the development of severe malaria. Although

the property of cytoadherence is universal, under some conditions parasites appear to accumulate in

particular organs in large numbers and cause damage by mechanisms that are poorly understood. These

may include impairment of the local circulation or the induction of the release of local mediators such as

cytokines or nitric oxide (12).

1.2.1.1. Cytokines

Macrophages of the spleen red pulp are apparently assumed to be involved in eradicating infected

erythrocyte under normal circumstances. Gamma delta T cells (γ/δ-T cells) also have been shown to be

involved in controlling the parasitism. These findings fit with data ascribing early production of interferon

gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) both to splenic γδ T lymphocytes and to natural

killer (NK) cells that activated by macrophages IL-12 (13). In addition it was proposed that malaria

parasite directly induce TNF-α from macrophages and this initiate the characteristic pyrogenic response to

malaria infection and up-regulate expression of endothelial adhesion molecules in the brain allowing

sequestration of Plasmodium falciparum infected erythrocytes and interference with cerebral perfusion,

leading to comma. Plasmodium vivax does not lead to cerebral malaria despite very high plasma

concentration of TNF-α (14).

Animal models of malaria have provided convincing evidence of the important role of inflammatory

processes in the development of cerebral malaria. Although none of them completely duplicates the

situation in humans. Not all pro-inflammatory cytokines are equally relevant for the development of

cerebral malaria. The best documented evidence implicates IFN-γ, TNF-α and IL-12. The elevations in

TNF may exacerbate the tendency to sequestration by the up regulation of host ligand molecules

responsible for cytoadherence of parasites, increasing mechanical obstruction of cerebral or other blood

vessels (11).

Most of the evidence supporting an immunological involvement in malarial anemia comes from data

related to the role of IL-12, a cytokine that boosts erythropoiesis. The levels of IL-12 were found to be

correlated with anemia in this pathology (15), and the administration of recombinant IL-12 was able to

ameliorate anemia in A/J mice infected with Plasmodium chabaudi (16). The implications of high levels

of immune regulatory cytokine IL-10 in Plasmodium falciparum malaria are unclear. IL-10 may down-

regulate pro-inflammatory responses and also exacerbate disease by inhibiting anti-parasitic immune

functions (17). Contradicting results were reported concerning the levels of TNF-α in serum of severe

anemia patient (18,19).

A high TNF early in malarial infection and during the acute phase of malaria predicted protective role and

rapid clinical and parasitological cure, whereas prolonged, high TNF levels may be determinal. These

finding illustrate the dual role of cytokines in the protection and pathology of malaria (20,11) and indicate

the importance of timing and amounts of different cytokines that released, particularly TNF, that might be

an important determinants of subsequent patho-physiological events and perhaps mortality. The ratio of

anti-inflammatory cytokines over pro-inflammatory cytokines in serum has been proposed to be the

accurate determinant of the severity of anemia (21,22).

1.2.1.2. Reactive oxygen species (ROS)

Clark and others (23) proposed that ROS might play a role in the pathogenesis of severe malaria. They

subsequently suggested that extravasated erythrocytes might lead to ROS generation, causing comma and

damage to local tissues, including the brain. ROS produced by leucocytes may also kill the parasites. In

humans there is some evidence that ROS play a role in pathogenesis (11). Griffiths and his colleagues (24)

demonstrated that malaria infection might be associated with oxidative damage and reduced alpha-

tocopherol reserve in erythrocyte membrane, suggesting that local antioxidant depletion may contribute to

erythrocyte loss in severe malaria. They also demonstrated that erythrocyte membrane alpha-tocopherol

appeared a better indicator of ROS exposure than plasma.

1.2.1.3. Nitric oxide (NO)

NO is constitutively produced in certain tissues, and in response to inflammatory stimuli through the

action of cytokines which up-regulate the synthesis of inducible NO synthase (iNOS2 or NOS2) (11). NO

was suggested to be produced from peripheral blood mononuclear cells (PBMCs) and other tissues and

cellular sources in response to Plasmodium falciparum malaria (25). NO has been proposed as a mediator

of malarial tolerance, where asymptomatic children have higher level of NO than children with severe

disease. The above tolerance may be mediated by one or more of the followings; inhibition of parasite

growth (26), intracellular parasite killing by reactive nitrogen metabolites (27) or having anti-adhesive

effect (28). Interestingly, NO seems not to be involved in the pathogenesis of cerebral malaria (13), and

the implication of NO in severe malaria may be due to a genetic polymorphism in the NOS2 gene or

different haplotypes (29,30). Data suggested a role of inducible nitric oxide synthase (iNOS) variants,

which affect the ability of macrophages to release reactive nitrogen metabolites in response to TNF-α and

IFN-γ, which influence the progression to cerebral malaria and to severe infection (31).

1.2.1.4. Cytoadherence, rosetting and sequestration

Rosetting is the adhesion of Plasmodium falciparum infected erythrocyte to uninfected erythrocytes,

which is suggested to be mediated by PfEMP1 as the ligand and heparin sulfate or a heparin sulfate-like

molecule as a receptor. Rosetting is regarded as a virulent parasite phenotype associated with the

occurrence of severe malaria (32).

Sequestration is the adherence of infected erythrocytes containing late developmental stages of the

parasites (mediated by PfEMP1) to the endothelium of capillaries and venules and is a characteristic of

Plasmodium falciparum infections (33) and contributes directly to acute malaria pathology (34).

Sequestration favors the development of the parasite by protecting it from the filtering action of the

spleen; it is also responsible for the severe clinical forms of cerebral malaria in which brain capillaries are

obstructed by sequestered parasites (33)(reviwed by David et al. 1983).

The mechanisms of parasite sequestration remain unclear. Ultrastructural studies have suggested that

adherence of parasitized erythrocytes to the vascular endothelium occurs by means of the knobs on the

infected erythrocyte membrane (33). These knobs are seen at the point of contact between the infected red

blood cells (iRBCs) and endothelial cells, the knobs are made up of parasite-encoded proteins that have

been exported to the surface of the infected erythrocyte. They are essential for firm cytoadherence by

facilitating the initial attachment and by concentrating the parasite ligands at a particular site. Although

knobless iRBC can adhere in static binding assays in vitro, ultrastructural studies of human tissues from

fatal malaria cases have not shown cytoadherence independent of knobs (35). Gilks and his coworker (36)

stated that there were several lines of evidence showed that variant surface antigen expression and the

sequestering phenotype are closely linked and co-expressed, or functions of the same antigen.

Individual variant surface antigens (VSA) can bind to different combinations of microvasculature

endothelial receptors, including thrombospondin, CD36, intercellular adhesion molecules 1 (ICAM-1),

vascular cell adhesion molecule, E-selectin, CD31, P-selectin, chondroitin sulfate A, and αvβ3 integrin

(37).

Until recently, Variant surface antigens (VSA) were thought to be made up exclusively of Plasmodium

falciparum erythrocyte surface protein-1, but recently rifins were described which are clonally variant

surface proteins expressed on the surface of red cells infected with Plasmodium falciparum; but their role

in the agglutination of field isolates is still unknown (37).

PfEMP1 has been reported as a parasitized erythrocyte receptor for adherence to CD36, thrombospondin

(TSP) and intercellular adhesion molecule-1 (ICAM-1) (34). It is important to note that whereas virtually

all isolates show the phenotype of adherence to CD36 and TSP, adherence to the other receptors is an

isolate- and variant-specific property (12). Isolates that bind to multiple receptors were found to be

involved in the causation of severe malaria and that several receptor-ligand interactions work

synergistically in bringing about severe disease (38).

Naturally induced antibodies binding to surface antigens of Plasmodium falciparum – infected

erythrocytes can be detected by direct agglutination or by indirect immunoflourescence, the results

obtained by both methods generally correlate well. The agglutinating antibodies predominantly recognize

variant-specific epitopes of PfEMP1 that is the adhesin mediating adhesion of Plasmodium falciparum -

infected erythrocytes to the endothelial wall (39). Using flow cytometry, Staalsoe and others (39)

demonstrated that the adhesive capacity of a parasite isolate correlates with the capacity of human

immune plasma to label the isolate and the antigen recognized are strain specific and their molecular

weights are in the range previously described for PfEMP1 antigens.

Immune serum can inhibit and reverse in vitro binding of infected erythrocytes from intact animals to

melanoma cells. Similarly, antibody can reverse in vivo sequestrations as shown by the appearance of

trophozoite/schizont-infected erythrocytes in the peripheral blood of an intact animal after inoculation

with immune serum. So the spleen was regarded as the organ that modulates the expression of parasite

alterations of the infected erythrocyte membrane responsible for sequestration and suggests that the

prevention and reversal of sequestration could be one of effectors mechanisms involved in antibody-

mediated protection against Plasmodium falciparum malaria (33).

Antibodies directed to the variant antigens on the surface of infected erythrocytes were found to be

associated with protection from malaria (40,41).

In areas of high intense Plasmodium falciparum malaria transmission, malaria among pregnant women is

both more prevalent and more severe than in non-pregnant women (42,43). Pregnancy associated malaria

(PAM) is characterized by marked accumulation of parasites in the intervillous space of the placenta, and

is the cause of maternal anemia as well as low birth weight, prematurity and increased infant mortality

(44,43). PAM is characterized by the placental accumulation of infected erythrocytes that adhere to CSA

and that the susceptibility to PAM decreases with increasing parity, apparently due to acquisition of

antibodies directed against VSACSA. There is an evidence that-specific antibodies are linked to placental

infection and that high antibody levels contribute to the control of placental infection by inhibiting

parasite adhesion to CSA. Data suggest that VSACSA is a target for vaccination against PAM (45).

1.3. Malaria parasite and life cycle (Figure 1.1)

Malaria is a protozoal infection caused by Plasmodium species. Over 70 species of malaria parasites that

infect monkeys, rodents and, birds are known (1).

The multi-stage protozoan parasites Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and

Plasmodium malariae are the etiological agents of malaria infection in human. Plasmodium falciparum is

the most widely spread and the most fatal of the human malaria. Two hosts are involved in malaria life

cycle, a vertebrate (man) and an invertebrate (female Anopheles mosquito).

Infection is initiated by the injection of sporozoites into the blood stream during feeding of infected

female Anopheles mosquitoes. The sporozoites migrate to the liver, where they invade hepatocytes (pre-

erythrocytic phase) and undergo a phase of maturation and asexual reproduction (schizogony) to release

into the blood tens of thousands of free merozoites.

The invasion of merozoites into circulating red blood cells initiates the erythrocytic phase of the life cycle.

Invasion is a complex series of events from RBC binding, to apical orientation, junction formation and

signaling the start of the invasion process. Invasion events include releasing essential molecules from the

invasive apical organelles (rhoptries, micronemes and dense granules) and initiating the actin-myosin

moving junction that bring the parasite inside the vacuole that forms in the RBC. What remains

completely unknown is which merozoite surface molecules recognize the erythrocyte surface and then

signal the start of the invasion process (46).

Schizogony is repeated within the erythrocytes, and infected red cells rupture and release more merozoites

into the circulation to begin another erythrocytic cycle. Alternatively, a proportion of invading merozoites

differentiates into male or female gametocytes which, when ingested by a mosquito, emerge from the

erythrocyte to form gametes. After fertilization, the zygote (ookinete) migrates across the mosquito

midgut wall and matures within the body cavity. The resulting

oocyst produces sporozoites that migrate to the mosquito salivary glands where they mature and become

infectious to humans. Only the asexual erythrocytic stage of the life cycle is associated with pathogenesis

of the disease.

Each phase of the life cycle is associated with the expression of a number of stage-and species-

specific antigens, many of which are located within the surface membrane of the parasite and appear to be

targets for naturally acquired immune responses (47).

It has been established that the blood forms of Plasmodium falciparum species are haploid (48). The

diploid phase is being probably restricted to the ookinete stage in mosquitoes (49). Meiosis occurs within

a few hours (50), followed by further mitotic division to produce haploid sporozoites. Each oocyst thus

contains the meiotic products of a single zygote. The mixture of clones of Plasmodium falciparum

ingested by mosquitoes undergo cross mating, meiosis of such heterozygotes leads to recombination

between genes, with the consequent production of parasites with novel genotypes (51).

Figure1. Malaria parasite life cycle

1.4. Epidemiology of malaria

Malaria continues to be a threat to hundreds of million of people living in endemic areas and to travelers

visiting these regions (52). Its transmission however is possible only when a combination of several

factors is available, including sources of infection, vectors, susceptible population and favorable natural

and climatic conditions. The source of infection is a malaria patient or asymptomatic parasite carriers in

endemic areas. Intrauterine infection, blood transfusion and the use of non-sterile syringes among drug

addicts represent another route of transmission. Under natural conditions, female mosquitoes of the genus

Anopheles usually serve as a vector or host for the sexual cycle of parasite development. Transmission of

malaria parasites occurs in the appropriate climatic conditions such as temperature higher than 16oC. and

the presence of either stagnant or slowly flowing water. The transmission intensity depending on the

number of mosquitoes and the type of species they belong to (1).

The disease is “endemic” where there is a constant measurable incidence of cases and natural transmission

over a succession of years. It is “epidemic” where the incidence of cases in an area rises rapidly and

notably above the usual levels, or where the disease suddenly occurs in an area previously non-malarious.

An epidemic becomes “pandemic” when it spread far beyond usual limits. The prevailing frequency and

intensity of malaria are referred to as “malaria endemicity”. Two systems are used to estimate the level of

endemicity, the first deals with the parasite rate among children aged 2-9 years (53). The second deal with

the spleen rate among the same age group (54). Malaria is “holoendemic”: where the parasite or the spleen

rate is constant over 75% and the adult's populations have high tolerance. Malaria is “hyperendemic”

where the parasite or the spleen rate is constantly over 50 % and also the rate is high in adults.

“Mesoendemic” where the parasite or the spleen rate is ranged 11-50 %. “Hypoendemic” where, parasite

or the spleen rate is 10 % or less. Transmission is “stable” when the prevalence is relatively steady from

year to year and season to season, and if there is a wide difference, it is called “unstable”.

Endemic and epidemic malaria results from a complex interplay of numerous factors, the most important

being, of course, man as a carrier of gametocytes, the Anopheles mosquito as vector, and man as recipient

of infection. The following factors modify this chain of transmission from man to mosquito to man: the

species and strain of Plasmodium; the immune status of host (man); bionomics or habits of man and

mosquito; environmental conditions, such as temperature, relative humidity, rainfall, topography, soil,

flora, and fauna, control measures applied to mosquitoes, and treatment of man by therapeutic and

prophylactic drugs (55). Epidemiological studies have shown that the development of resistance to

malaria in man depends on both the degree and the duration of exposure to malaria parasite. A primary

malaria infection is not followed by the development of solid immunity and most of the deaths occur in

young children in endemic areas and in infected adults with no previous history of exposure to the malaria

parasites. Immunity builds up in the course of successive infection and sterile immunity is probably never

achieved. In endemic areas, clinically significant malaria becomes infrequent in adults with the exception

of pregnant women. However, transient parasitaemia is still observed (56). Protection to malaria acquired

by individuals living in endemic areas is largely governed by the transmission pattern (57), and the

development of disease immunity is characterized by a decrease in the frequency and severity of disease

episodes over several years despite almost continuous infection (58).

The apparently slow acquisition of protective immunity in children to Plasmodium falciparum malaria

may have several reasons, including effects of the parasite on immune regulation resulting in suppression

of specific immune responses (59). Other factors include the genetic diversity or variability of the human

host or parasite antigens giving rise to potentially protective immune responses.

1.5. Malaria control

Malaria parasites have defied all attempts to eradicate, the reasons for this failure are complex, briefly

might be due to the complexity of the parasite life cycle that involves more than one host, and the

development of antigenic diversity, in addition to the wide spread of drug resistant parasites and vectors.

The low educational and socio-economical statuses of the inhabitants of malaria endemic areas also play

roles in this failure. These status lead to the failure of the control programs concerning the control of the

mosquitoes breeding sites and limit the use of the insecticide impregnated bed nets that have been

succeeded in controlling malaria vectors in different endemic areas (60,61). WHO realized that the global

eradication of malaria was impossible and the focus shifted to control.

1.6. The malaria parasite genome

The Plasmodium falciparum 3D7 clone sequence was launched in 1996 and completed in 2002 reported a

unique sequence of this eukaryotic organism. Analysis of the genome sequence provides a global view of

the metabolic potential of Plasmodium falciparum irrespective of the life cycle stage. The 22.8 mega-base

nuclear genome consists of 14 chromosomes ranging in size from approximately 0.643 to 3.29 Mb and

encodes about 5,300 genes, and it has been regarded as the most (A + T) rich genome sequenced to date.

The overall (A + T) composition is 80.6 % and rises to ~90 % in introns and intergenic regions. Excluding

introns, the mean length of Plasmodium falciparum genes was 2.3 kb, substantially larger than in the other

organisms in which the average gene lengths range from 1.3 to 1.6 kb. Genes involved in antigenic

variation are concentrated in the sub-telomeric regions of the chromosomes, so the understanding of sub-

telomere structure and functional properties is essential for the elucidation of the mechanisms underlying

the generation of antigenic diversity. Compared to the genomes of free-living eukaryotic microbes, the

genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of

genes are devoted to host-parasite interactions and immune evasion. At least 1.3 % of Plasmodium

falciparum genes are involved in cell to cell adhesion or the invasion of host cells and 3.9 % (208 genes)

known to be involved in the evasion of the host immune system (62).

The malaria genome sequencing consortium estimated that more than 60% of the 5,409 predicted open

reading frames (ORFs) lack sequence similarity to genes from any other known organism (62). The

function of these elements is still not clear. One might speculate that the ORF- sequences are transcribed

as poly-cistronic mRNA’s together with a specific var gene and encode proteins necessary for functional

var gene expression on the cell surface. Alternatively, it is tempting to speculate that some of the ORF-

elements are spliced to the large exon I of a var mRNA giving new biological properties to the var protein

(63).

The chromosome mapping data demonstrate several features common to all Plasmodium falciparum

chromosomes: house keeping genes map to the central chromosome regions whereas the genes encoding

immunodominant antigens are generally located at the polymorphic chromosomes extremities. The

compartmentalization of Plasmodium falciparum antigen genes to the highly recombinogenic

chromosome ends can lead to related variant antigen gene families scattered on several chromosome

extremities. The constant turn over of Plasmodium falciparum DNA at chromosome ends also implies that

new gene linkage groups are continually being formed. Over the long term this constant turn over is a

powerful adaptive mechanism by which Plasmodium falciparum parasites create functional diversity.

Plasmodium has in addition to the nuclear genome, two unrelated organellar genomes. One is

mitochondrial and the other probably of plastid origin of 35 kb (63). The mitochondrial genome of

Plasmodium falciparum is small about 6 kb and encodes no tRNAs (62).

Genetic diversity can be generated during the sexual or asexual stage of the parasites’ life cycle by several

mechanisms. During mitosis these include mutation, chromosome breakage, unequal sister chromatid

exchange, gene conversion or inter-chromosomal exchange. During meiosis, reassortment of

chromosomes, recombination between homologous or heterologous chromosomes, unequal sister

chromatid exchange or gene conversion can also occur (64).

1.7. Plasmodium falciparum Antigenic diversity

Mc Bride et al. (1982) has reported antigenic diversity in the human malaria parasite Plasmodium

falciparum, stage and strain specific but no clear regional differences were seen, since identical antigenic

combinations occurring in isolates from different parts of the world. There are two aspects of antigenic

diversity among malaria parasites that need to be distinguished (65).

1.7.1. Antigenic polymorphism

Polymorphisms in allelic genes give rise to the expression of structurally and antigenically distinct forms

of a particular protein in different parasites and this can be demonstrated by the variations in the merozoite

surface proteins 1 and 2 (MSP1 and MSP2). Because there are large numbers of different polymorphic

genes that are not linked, and there is recombination and re-assortment of these genes during meiosis,

there is the potential for a very large numbers of different genotypes within one species of Plasmodium

(66). A sexual blood stage of Plasmodium falciparum can be conveniently classified into those exhibiting

minor polymorphism involving point mutation and those in which there are major polymorphism often

involving change in repetitive sequences (67).

1.7.2. Antigenic variation

Antigenic variation is a phenomenon by which a clonal population of parasite can changes its antigenic

phenotype. Plasmodium falciparum contains four families of highly variable gene termed var, rif, stevor

and Pf60, which code for proteins known as Plasmodium falciparum erythrocyte membrane protein -1

(PfEMP-1), repetitive interspersed family (rifin), sub-telomeric variable open reading frame (STEVOR)

and 6.1 protein respectively (62,68). The sequencing of the 3D7 genome contains 59 var, 149 rif and 28

stevor genes, but for each family there are also a number of pseudogenes and gene truncations present

(62). These pseudogenes may code for Pf60 where the majority of the 90 copies per haploid genome code

for Pf60 appear to be pseudogenes because they contain frameshifts or internal stop codons (12).

The differential expression of these genes results in antigenic variation which is used as a mechanism for

immune evasion. Babiker & Walliker (1997) in their study of the var gene as one of these multifamily

genes assumed that if these large repertoires contain many allelic variants at each locus in the parasite

population the number of genetically distinct clones that could be generated by recombination seems

almost infinite (51).

Variant gene families have also been identified in all other malaria parasite species investigated. Some of

these genes have been shown to be located in subtelomeric regions of the chromosomes, many are

distinctly different in structure and sequence from those identified in Plasmodium falciparum, indicating

that they might have evolved independently and have different functions (69).

1.7.2.1. The var gene

When Su et al., (1995) tried to find the drug resistant gene; they fortuitously discovered a number of

adjacent genes that look like genes encoding members of the PfEMP1 family, they call these genes var

genes. Su and his coworker (70) have sequenced four genes of the var family. The deduced protein

sequences look like cellular adhesion molecules; where, they appear to consist of a large and variable

extra-cellular domain, a single trans-membrane segment, and a conserved intracellular domain. As

expected for a variant antigen, the extra-cellular domains of different members of family are very different

in sequence, although they share an overall similarity in structure. Smith et al. (1995) put an indication

that the var genes encode the variant surface antigen of Plasmodium falciparum and are the basis of

antigenic variation and binding to endothelium. Also they presented the most extensive analysis that

confirmed the differential expression of the members of the var gene family in different parasite lines.

They didn’t find evidence for duplicate copies of expressed genes, they found a deletion in var genes but

this deletion did not correlate with change in antigenic phenotypes. Multiple var expression sites exist,

since transcripts from different chromosomes were identified (71). Var genes are located predominantly at

chromosome ends (approx. 20 to 40 kb from the telomere repeats) in subtelomeric regions next to the non-

coding element rep20 as well as central chromosome regions (63). Var genes are encoded in two exons,

the first exon includes the extracellular region and a putative trans-membrane domain. The second exon

encodes the acidic terminal segment (ATS), a domain that is hypothesized to anchor PfEMP1 at knobs

(72).

A study of different clones of Plasmodium demonstrated that the differential control of var gene

expression must be tight; implying that a single infected erythrocyte may only express one or at most a

few var genes (73).

Within the 3D7 genome, there are 59 intact var genes, with 149 rif and 28 stevor genes interspersed near

the var genes (62). Whereas rif and stevor genes appear to be closely related, the var genes are unique and

are the most extensively studied variant gene family. Plasmodium falciparum populations harbour many

var forms, whose diversity and continual renewal support the success of the species against host

immunity. Var gene sequences undergo recombination at frequencies much higher than those expected

from homologous cross-over alone. These recombination events occur between sub-telomeric regions of

heterlogous chromosomes, which associate in clusters near the nuclear periphery in asexual blood stage

parasites or in bouquet-like configurations near one pole of the elongated nuclei in sexual parasite forms

(74). This clustering should bring the subtelomeric genes into alignment, thus facilitating recombination

event between genes that are positioned similarly along the chromosome (74,75).

The recombination preferentially occurs within var gene groups (76), where close examination and

analysis of the var gene sequences indicate that most of the family can be divided into three broadly

defined classes based on chromosomal location (sub-telomeric or central), direction of transcription

(towards or away from the telomeres), and type of the semi-conserved upstream flanking region (referred

to as UpsA, B or C) (69). It is speculated that the groups reflect a functional diversification evolved to

cope with the varying conditions of transmission and host immune response directed against the parasite

(76).

The work done by Rowe and his colleagues (77) was the first to describe sequence conservation in the

PfEMP1 family that binds to the host receptor chondroitin sulfate A (CSA) called var1 subfamily.

Another gene belongs to a second subfamily (var2csa) was reported to be implicated in the pathogenesis

of pregnancy associated malaria (PAM). The var2csa genes are structurally distinct from all other var

genes in the parasite genome in lacking both CIDR and DBL-γ domains. These domains have previously

been implicated in PfEMP1-mediated adhesion to CD36 and CSA (43). This conservation may be

explained by the orientation of the gene where, most sub-telomeric genes transcribed toward the

centromere but this CSA-binding gene is transcribed towards the telomere and this inverted orientation

doesn’t favor ectopic recombination (76,77).

The mechanisms of switching in the expression of the var genes have yet to be understood. The chromatin

structure could play a major role in transcriptional activation of individual var genes; mechanisms that

regulate chromatin structure such as the histone deacetylase may be key modulators in Plasmodium

falciparum gene regulation (78). For example, the deacetylation of histones is correlated with

transcriptional silencing (79). Thus Scherf and his coworker (63) speculate that chromatin structure could

be reversibly modified to activate or inactivate var gene transcription by targeting histone

acetyltransferase and deacetylases to a particular var gene. Both chromatin structure and DNA

modification might work hand in hand in the regulation of transcription control. Deitsch and his

colleagues suggested that further control elements in the intact var gene are required to control or silence

expression, when they found that silent var promoters become transcriptionally active when they removed

from their chromosomal context. They later showed that the silencing is established during the DNA

synthesis phase (S phase) of the cell cycle and that it depends on the cooperative interaction between two

elements in separate control regions of each var gene (the 5’ flanking region and the conserved intron

which separate the two exons of all var genes) (80). The var gene is found to be expressed in apparently

large and varied numbers at the sporozoite stage as well (81).

1.7.2.2. Repetitive interspersed family (rif)

Repetitive interspersed family (rif) sequences were originally identified as multi-copy Plasmodium

falciparum sequences but were not studied further because they appeared to be short open reading frames

with no obvious translation start site. The genome project revealed the presence of a spliced exon about

300 base pairs upstream of each rif that encoded a translational start and a putative signal sequence. This

suggested not only that rif genes encode proteins but that their high copy number (∼200 per genome) and

high degree of polymorphism might indicate another variant antigen family. Evidence to date suggests

that the encoded proteins (RIFINs) are expressed in trophozoites, are located on the infected red cell

surface, and undergo antigenic variation. Currently no function has been ascribed to members of this

family, although there is a suggestion that they might act as cofactors in rosetting or in binding to CD31

(12).

1.7.2.3. Subtelomeric variant open reading frame (stevor)

The subtelomeric variant open reading frame (stevor) is localized in Maurer’s clefts, unique membranous

structures located in the cytoplasm of infected erythrocytes. The timing of stevor expression that occurs

over just a few hours of the asexual parasite life cycle and the location of STEVOR are clearly distinct

from those of other parasite variant antigens suggesting that this gene family may have a novel role in

Plasmodium falciparum biology (82).

1.7.2.4. Pf60

The Pf60 family shares a high degree of homology to the second exon of PfEMP-1, is composed of

roughly 90 copies per haploid genome. From the malaria genome project, the majority of these sequences

appear to be pseudogenes because they contain frameshifts or internal stop codon (12). However the one

member of this family that has been fully characterized to date has a series of additional 5′ exons and is

expressed via a translational frameshift an interesting mechanism that has only recently been described in

Plasmodium falciparum. The Pf60.1 gene is constitutively expressed by all mature blood stages and codes

for a protein located within the nucleus (68).

1.8. Immunity to malaria

Responses to malaria infection are regulated by both the innate and adaptive acquired immune system as

well as by environmental factors. Acquired immunity is both species and stage specific. It is rarely sterile

but rather associated with low grade parasitemia and episodes of clinical disease throughout life (83). In

areas of endemic Plasmodium falciparum malaria transmission, neonates are relatively protected from

severe malaria compared with older children. It is not clear whether resistance in neonates is primarily

physiological (for example, erythrocytes containing fetal hemoglobin only poorly support parasites

growth, the breast milk is relatively deficient in para-aminobenzoic acid (P-ABA), which is an essential

nutrient for parasite growth), or immunological (mediated by maternal immunoglobulin transferred across

the placenta, or the human breast milk which contains high concentration of transforming growth factor

beta (TGF-β) which is, if absorbed intact into the infants blood stream, might exert a systemic anti-

inflammatory effect (14).

1.8.1. Innate natural immunity

The observation that host genetic factors can modify disease outcome is an importance factor when

considering global pattern of human migration. Morbidity and mortality due to malaria will be

substantially greater upon exposure of individuals who are innately susceptible to this disease. Such group

may be selectively targeted for interventions.

Several host factors play a crucial role in the survival of the parasite by determining host resistance or

susceptibility to infection by Plasmodia, (lack of the Duffy blood group antigens on erythrocytes confer

resistance to P. vivax infection). Heterozygotes for haemoglobinopathies and other disorders of the red

cell (thalassemia, sickle cell anemia and glucose-6-phosphate dehydrogenase deficiency), promote innate

resistance to Plasmodium falciparum infection; such protection is probably triggered by modifications of

parasite development within the erythrocytes (57). Recently, hemoglobin HbC has been reported to

protect against severe malaria (84) may be by reducing PfEMP-1 mediated cytoadherence (85).

Elagib and others (86) suggested that the haptoglobin phenotype (1-1) is associated with susceptibility to

falciparum malaria and the development of severe complications.

Polymorphic forms of number of host genes involved in immunity have been associated with protection or

susceptibility to malaria; Polymorphic point mutations at the human tumor necrosis factor (TNF) promoter

region have been reported to be associated with severe malaria anemia, and other variant is associated

with cerebral malaria (87).

FcγRIIa (CD32) on the surface of lymphocytes and monocytes/macrophages provides an important link

between humoral and cellular immune systems. However, the recognition of IgG by CD32 is influenced

by polymorphism within the gene. The functional activity of immunoglobulin subtypes is likely to

depend, at least in part, on the CD32 genotype of the host and an association between FcγRIIa (CD32)

polymorphism and increased susceptibility to severe malaria has been reported (88).

The genetically determined low Complement receptor type 1 (CR1; CD35) density on erythrocytes may

be a risk factor for developing a more severe form of malaria (89).

The presence of variants of genes encoding adhesion molecules, such as ICAM-1 and CD36, may also

affect the outcome of malarial infections; these molecules are also involved in the immunity regulation.

ICAM-1 is expressed on activated endothelial cells, dendritic cells and lymphocytes and is preferentially

up-regulated on memory T cells. ICAM-1 binding to infected erythrocytes exhibits parasite strain

specificity, and one host variant is associated with susceptibility to cerebral malaria in West Africa. CD36

is expressed on endothelial cells, platelets, macrophages and dendritic cells. Several polymorphic forms

have been detected, one polymorphism found to confer protection against severe anemia by reducing

parasite sequestration. There is evidence to suggest that interaction of CD36 on dendritic cells with

specific Plasmodium falciparum strains leads to defects in dendritic cell maturation. Therefore,

polymorphism in CD36 may affect the priming of immune responses during infection, as well as parasite

sequestration (31).

1.8.2. Acquired immunity

Acquired immunity to Plasmodium falciparum is species, stage, and strain-specific (90, 91) involving

interactions between several different immune mechanisms (92). Adaptive ‘specific” immune responses

are mediated by T-lymphocytes (T-cells) and B-lymphocytes (B-cells).

Defense mechanism of immunity are mediated and regulated through a sublime interplay between

different parts of the system and that all parts of the system cooperate with each other, specific as T and B

cells or non specific as the white blood cells and the natural killer cells (NK). T-cell stimulates NK-cells

and monocytes through the secretion of interferon gamma (IFN-γ). Antibodies often opsonize

microorganisms that can easily be taken up by phagocytic cells; the T-cells in turn regulate the production

of antibodies by B-cells (57).

Natural killer cells have a cytotoxic activity and can recognize potential target cells that has been coated

with antibodies through CD16 IgG Fc receptor on the NK cells which is known as antibody dependent cell

mediated cytotoxicity (ADCC) (93).

1.8.2.1. Arms of the acquired immunity

1.8.2.1.1. The cell mediated immune response

The role of T-cells in the protection against malaria was emphasized by studies in animal models in which

intact animals developed solid natural immunity after infection whereas thymectomized animals failed to

do so (94). T-cells might participate in the protection against malaria as helper cells for antibody

production by regulating the activation of B-cells. As cytotoxic cells to infected hepatocytes, T-cells also

produce IFN-γ, which is toxic to the liver stage of the parasite. Macrophage/monocytes activation is also

regulated by T-cells. Theander (57) concluded from the animal and human studies that Plasmodium

falciparum infections result in an immunological memory at the T-cell level. The functions of these cells

in the development and execution of immunity is probably to regulate macrophage activation and

antibody production. Ferreira and his coworkers (95) reported that interferon-γ released by immune T-

cells have been shown to kill intra-hepatic parasites in vivo. Both T-cells with TCRαβ+ or TCRγδ+ have

been shown to secrete IFN-gamma after malaria antigen activation. TCR γδ cells are primarily activated

by live parasites and the activation depends on secretion of interleukin 2 (IL2) by CD4+ TCRαβ+ cells.

TCRγδ cells which are the major producers of malaria-induced IFN-γ in vitro accumulate in the peripheral

blood, where they may release cytokines systematically, contributing more to pathology than to

protection. Whereas TCRαβ T-cells express high level of adhesion ligands and disappear from the

peripheral circulation, presumably migrate to liver and spleen, where they may encounter sequestered

parasites and be actively involved in parasite killing (14).

Frequencies and absolute numbers of T-cell subsets (as well as their response to antigenic stimulation) is

lower in the peripheral circulation during acute malaria, This may be explained by the sequestration of

activated T-cells expressing the adhesion molecule leukocyte function-associated molecule- 1 (LFA-1) on

their surface and this lymphopenia, the degree of which correlated with disease severity normalized

following anti-malarial drug administration (96).

Both T helper1 (Th1) and T helper 2 (Th2) responses have regulatory functions in human Plasmodium

falciparum malaria and seem to be required to control the infection, but they need to be adequately tuned

in intensity and time (13,14,97,98). Type 1 cytokines are important in controlling early parasitemia,

although they need to be counter balanced later in the infection by a type 2 response which leads to

antibody production (13).

1.8.2.1.2. The humoral immune response

In residents of endemic areas malaria infection induces strong humoral immune responses. Involving

production of immunoglobulin predominantly IgM and IgG but also of other immunoglobulin isotypes.

While a large proportion of this immunoglobulin is non-malaria-specific, reflecting polyclonal B-cell

activation, up to 5% or more represent species as well as stage specific antibodies reacting with a wide

variety of parasite antigens (83).

It has been reviewed by Edozien and his coworkers (99) that African serum known to contain more γ-

globulin than Europeans and this, to have a significant relationship with malaria parasitisation. A bulk of

evidence that antibodies are important in the control of malaria infection comes from the passively

transferred immune serum. Cohen and his colleagues (100) administered intramuscularly purified γ-

globulins that have been prepared from Gambian adults to Gambian children suffering from acute attacks

of Plasmodium falciparum malaria. It was found that gamma-globulin has marked effect on both

parasitaemia and clinical symptoms, although the temperature did not returned to normal before the 7th

day and low parasitaemia could still be detected after 9 days in about 30% of the cases. The treated

subjects were infected again 3 months later, which indicated that the protection was of limited duration. It

was not clear, how the protection was mediated, but the authors suggested that the antibodies acted either

on mature intracellular forms or on merozoites.

Edozien and others (99) confirmed the previous anti-parasitic effect of γ-globulins and the transfer of anti-

malaria immune antibody across the human placenta from mother to fetus, when used Nigerian adult and

cord-blood γ-globulins for the treatment of children with acute malaria.

Sabchareon and others (101) studied the effect of intravenous administration of African IgG antibodies

against Plasmodium falciparum malaria to Thai patients to obtain much faster effect, where the clearance

of parasites and symptoms was as fast as or faster than with drugs. The effect was stage-specific but non-

sterilizing, since recrudescence occurred after the disappearance of the transferred antibodies. They also

stated from their data that the antibodies developed after 20 years of exposure to the parasites could confer

protection on all strains tested.

It has been indicated that antibodies against sporozoite blood stage and gametocytes mediate protection.

Antibodies directed against sporozoites inhibited the uptake of Plasmodium falciparum sporozoites into

culture cells (102).

Human vaccinated with a B cell epitope of circumsporozoite surface protein (CSP) a major surface

molecule on sporozoites were particularly protected (103,104).

Antibodies directed against the blood stages of the parasite could mediate protection in several ways.

Antibodies against merozoites can block the invasion of merozoites into erythrocytes (105). Cytophilic

antibodies can opsonize parasites for phagocytosis (106,107), and specific immune serum was found to

inhibit the dispersal of the merozoites from mature schizonts (108). Antibodies also can obstruct parasites

attachment to capillary allowing these parasites to be removed by the spleen and liver (109,33,57).

Antibodies can also mediate anti-toxic immunity against malaria antigens that provoke the production of

interleukin 1 (IL-1) and tumor necrosis factor (TNF).

Antibodies against gamete antigens seem to inhibit parasite development in the mosquitoes by several

mechanisms. Antibodies from immunized animals have been shown to block the fertilization in the

mosquito mid gut (57).

In general there is a high degree of cooperation and integration between the innate and acquired immunity,

where the rate of phagocytosis of an antigen was 4000-fold higher in the presence of specific antibody to

the antigen than in its absence (93). On the other hand Bouharoun-Tayoun and her colleagues (110)

demonstrated that antibodies that protect human against Plasmodium falciparum blood stages do not on

their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes. Druilhe &

Perignon (1997) hypothesized that the relative amounts of antibodies, monocytes and merozoites can all

be expected to influence the final range within which the parasitemia is going to fluctuate (111).

1.8.3. Immune suppression during malaria

Many observations lead to the hypothesis that infection early in life lead to inflammatory immune

response effective in parasite killing but can cause immuno-pathology. With increasing exposure, immune

deviation and immuno-regulation may reduce immuno-pathology during infection; with the exception of

cerebral malaria. It is clearly in the interest of the parasite and more formally a source of selective

advantage for strains of falciparum parasites, to develop methods to inhibit immune and inflammatory

responses, potentially harmful to the parasite and the host (112). Such as impairment of antigen presenting

cell (APC) function which can be represented by the presence of altered peptide ligand (APL) antagonism

(113). Macrophages phagocytose intact infected erythrocytes and also extract parasites from recently

infected erythrocytes, leaving the erythrocytes to continue to circulate (114,115). These crucial defenses

are undermined and manipulated during malaria infection; the inhibition of macrophage function may be

in part due to haemozoin. A potential mechanism of inhibition of cellular function by haemozoin is the

generation of biologically active endo-peroxides-formed by peroxidation of arachidonic acid, where it has

been found to inhibit macrophage function in vitro (112).

Intact malaria-infected erythrocytes adhere to dendritic cells (DCs) through CD36 and CD51, inhibit the

maturation of dendritic cells and subsequently reduce their capacity to stimulate T cells (116,117). After

interaction with infected erythrocytes, DCs also acquire a capacity to suppress CD8+ T-cell response in

immunized mice (118). In vitro study demonstrates evidence that natural infection may induce both types

of antibodies, the protective inhibitory antibodies that inhibit the secondary processing of the 42 KDa

fragment of the merozoite surface protein 1 (MSP142) and antibodies that block this inhibition by

competing for binding to merozoite surface (119,120).

1.10. Malaria vaccine

An alternative hope for malaria control is the development of a vaccine that prevents the majority of naïve

recipients from developing any clinical manifestation of disease after exposure to Plasmodium falciparum,

and to prevent the development of severe disease and death in those individuals who do become ill by

limiting the effect of blood stage infection. Preventing severe disease and death will require limiting the

effects of blood stage infection either by reducing parasite replication, reducing cytoadherence and or

inhibiting the effects of toxic materials released by the parasites. The vaccine development focuses on

antigen discovery, immune mechanisms and vaccine delivery system (121).

The attempts to develop a malaria vaccine began early in the twentieth century and despite the fast

advances in science and technology that progress with high optimism no effective malaria vaccine is

available for widespread human use. This failure may be due to multiple factors where, the Plasmodium

falciparum have evolved multiple mechanisms of immune evasion at the individual and population level

including stage specific antigen expression. Where the study done by Bozdech and his colleagues (2003)

suggested that greater than 75% of the genes expressed during the intra-erythrocytic developmental stage

of this highly specialized parasitic organism are activated only once (122).

Still there is a hope for developing a malaria vaccine and this hope is supported by the fact that adults in

endemic areas develop naturally acquired immunity to disease, and the immunization of mice, non-human

primates and human volunteers with radiation attenuated sporozoites induce complete sterile immunity

(123-128). These observations demonstrated that the human host is capable of developing an effective,

protective immune response against malaria on vaccination

1.10.1. Potential malaria vaccine candidate antigens

The development of an effective malaria vaccine depends upon identification of antigens that are targets

of protective immune responses. The efforts were directed at developing a subunit vaccine, since the

acquired immunity to Plasmodium falciparum is species, stage, and strain specific (90,91).

Three approaches have been used for the selection of antigens with vaccine potential. 1) Identification of

molecules with critical functional roles in the biology of the parasite that expected to be accessible to the

immune effectors mechanisms. 2) Identification of molecules on the surface of stages in the parasite life

cycle, which might be exposed to and vulnerable to immune attack. 3) Identification of antigens, which

are responsible for inducing immune effector’s mechanisms (129). A malaria vaccine aimed at disrupting

the parasite life cycle at one or more of the 3 stages (sporozoite/pre-erythrocytic stage; asexual

erythrocytic stage and sexual/sporogonic stage) might be a long term solution (130).

The complexity of the malaria parasite life cycle implies a considerable complexity of antigens and hence

problem in selecting the optimal vaccine candidates. It is assumed, however that those molecules which

are functionally important to the parasite, for example in cell invasion, may be subject to less intra-

specific variation and may be prime targets for vaccine-induced attacks, but the intra-specific antigenic

diversity seen in malaria parasites presents a formidable challenge. Blood-stage vaccines against

Plasmodium falciparum are aimed at preventing complications of disease, such as cerebral malaria or

anemia. Both Plasmodium falciparum and Plasmodium vivax can cause severe anemia, but only

plasmodium falciparum causes the many complications of cerebral malaria, hypoglycaemia, metabolic

acidosis, and respiratory distress. Plasmodium falciparum is responsible for the great majority of deaths

and, for this reason; most effort has been devoted to Plasmodium falciparum vaccines (131).

The optimal vaccine is proposed to be multi-stage, multi-immune response i.e. containing antigens from

the different parts of the life cycle, to induce more than one type of immune response. It was reasoned that

this might ensure that mutant organisms evading immunity induced against one stage of the life cycle will

be removed by immunity induced to the next. The vaccine also must be multi-valent, which includes

multiple epitopes restricted by different MHC molecules (90,129).

The development of an effective malaria vaccine depends upon identification of antigens that are targets

of protective immune responses. A variety of promising parasite antigens from several life cycle stages

have already been discovered and are being developed as malaria vaccine component.

1.10.1.1. Sporozoite or pre-erythrocytic stage vaccine candidates

a- Circumsporozoite protein

Two proteins have been described to be expressed on the surface of sporozoite known as

circumsporozoite protein (CSP) and sporozoite surface protein 2 (PfSSP2), which is also known as

thrombospordin-related adhesive protein (TRAP). Circumsporozoite protein (CSP) has a central area of

repeated amino acids sequences that are highly immunogenic and are present in all Plasmodium

falciparum species studied to date. Many studies recommended (CSP) as an excellent vaccine candidate,

that induces both antibody and cell mediated immunity (132).

b- Liver stage-specific antigen (LSA-1)

LSA-1 is expressed in infected hepatocytes and contains 17 amino acids repeat, which is immunogenic in

man (133,134). West Africans produced HLA-restricted cytotoxic T-cells that recognized a nine amino

acid peptide from LSA-1 in association with HLA BW53. The presence of HLA BW53 correlated with

protection from severe malaria suggested that immunity to LSA-1 provides some protective immunity

against severe malaria (132,135,136).

1.10.1.2. Transmission blocking vaccine

An option for the design of a malaria vaccine is to aim at the induction of malaria immunity that protects

from death and (severe) disease but allows parasite reproduction rate just high enough to ensure natural

boosting. A transmission blocking vaccine can be an effective tool in the strategy to meet such an

objective (137). Transmission blocking vaccine aimed at disrupting the sexual development of the parasite

in the mosquito vectors which leads to the block of the transmission. Pfs 25 is a sexual stage antigen of

Plasmodium falciparum that is expressed on the surface of zygote and ookinete forms of the parasites.

Monoclonal antibodies that directed against native Pfs25 could block completely the development of

Plasmodium falciparum oocyst in the mid-gut of the mosquito vector. Thus Pfs25 has been regarded as a

potential vaccine candidate for eliciting transmission-block (138).

Pfs28 is one of the proteins that are relatively late expressed in the sexual cycle and is being present on the

parasite surface membrane of gametes and ookinetes.

Pvs 25 and Pvs28 mice anti-sera raised against Sal I strain of Plasmodium vivax using alum adjuvant

completely inhibited oocyst development for most natural human isolates of Plasmodium vivax in

Thailand, where it overcome the genetic polymorphism (139).

1.10.1.3. Asexual blood stage vaccine candidates

Plasmodium falciparum express the vast majority of its genes during this intra-erythrocytic stage of the

parasite life cycle (122). Blood-stage vaccines against Plasmodium falciparum are aimed at preventing

complications of disease, such as cerebral malaria or anemia.

The use of asexual blood-stage proteins as malaria vaccines is strongly supported by experimental data

directly implicating antibodies induced by these antigens in parasite clearance and protection from re-

challenge. These vaccines could complement other vaccines aimed at preventing infection, such as those

targeted at pre-erythrocytic or mosquito stages of the parasite. Asexual blood-stage vaccines may reduce

disease by blockade of red blood cell invasion, inhibition of parasite growth in red cells or interference

with cytoadherence of infected red cells. Clearance of blood-stage parasites is dependent primarily on

antibody-mediated mechanisms, but CD4 T cells may also play an important role in help for B cells and

probably have a direct effectors function in the clearance of blood-stage parasites (131).

1.10.1.3.1. Merozoite surface antigens

The well characterized merozoite surface proteins (MSP1 and MSP2) of Plasmodium falciparum are

considered as important vaccine candidates, which are directly accessible to immune attack. Humoral and

cellular responses to MSP1 and MSP-2 may be of great importance for the induction of protective

immunity.

a- Merozoite surface protein 1 (MSP1)

It is an integral protein known as MSA1, P195, gp185, PSA and PMMSA. This glycoprotein is processed

at the time of schizonts rupture to generate the majority of the antigens on the mature merozoite surface

(140). MSP1 has a molecular weight between 180 and 230 KDa depending on the particular parasite clone

from which it has been extracted (140-142). Each species of malaria parasite has a single MSP1 gene. The

gene controlling the expression of MSP1 is found on chromosome 9 of Plasmodium falciparum (143).

DNA sequence analysis of this gene from a number of in vitro cultured laboratory isolates suggest that the

MSP1 gene can be divided into 17 blocks that are conserved, semi-conserved or variable. Block 1 and 17

coding the N and C terminal sequence respectively are the most highly conserved regions. The variable

and semi-conserved regions exist in only two dimorphic forms. The only exception to this dimorphic rule

was observed in the polymorphic tri-peptide region at the 5’ end of the gene in block 2. The alleles K1 and

MAD20 are considered as the allelic prototypes, from which other allelic forms probably have been

generated by intra genic recombination at the 5’ end of the gene (144). The RO33 form is belonging to the

same dimorphic group as MAD20 but lacks the N-terminal tripeptide repeat (145). The sequence repeats

of MSP1 are a minor structural feature of the molecule, and much of the antigenic diversity appears to

have arisen from intra-genic recombination between two dimorphic alleles (140,144,146). Hui and others

(147) demonstrated an immunological cross-reactivity between the two gp195 protein and they referred

this to the recognition of the conserved determinants.

Post-translational proteolytic process of MSP1 generates fragments of 83, 42, 38, and 28-30 KDa, which

persist as a non-covalently linked complex on the surface of mature extra-cellular merozoites. Secondary

processing of the 42 KDa fragments produces a C-terminal 19 KDa fragment (MSP119), which is retained

on the surface of merozoites throughout invasion of erythrocytes, all other fragments being shed before or

at this event (148,149). An important role of MSP1 in the invasion process is indicated by the finding that

prevention of the secondary processing of MSP1 and the shedding of the MSP1 complex result in failure

of the merozoite to invade the erythrocytes (150).

The C-terminal of Plasmodium falciparum MSP1 consists of two epidermal growth factors EGF-like

domains, which is the first protozoan EGF example to be determined (151). MSP119 containing twelve

positionally conserved cysteine residues (6 in each domain), and this EGF are thought to be required in

interactions with the erythrocytes (152,153). The immune protection induced by this antigen in different

animal models appears to be conformation specific as reduction of the disulfide linkage abolishes this

protective immunity. This fragment is highly conserved in all Plasmodium species, and has been

characterized as a major vaccine target antigen since it will be possible to develop a vaccine that induces

protective immune responses against all Plasmodium falciparum isolates based on this conserved region

(152). PfMSP119 is highly conserved except for four amino acid residues that subject to dimorphic

substitutions. In the Wellcome sequence, these four residues are Q in the first motif and K-N-G in the

second motif, while in the MAD20 sequence they are E and T-S-R, respectively, and the major epitopes in

the different allelic forms are cross-reactive by human antibodies (154). Many studies demonstrated the

antigenicity and immunogenicity of MSP119. Immunoepidemiologic study carried out in West Africa,

demonstrated that the antibodies to PfMSP119 were shown to provide 40% protection against clinical

malaria in Sierra Leone. In Gambian children, antibodies to one of the EGF-like motifs of MSP119 were

strongly associated with resistance to both clinical malaria and high level of parasitaemia (155).

The role of MSP119 in invasion process is indicated by many experiments, where MSP119-specific

monoclonal antibody were found to inhibit the erythrocytes invasion (148), and the affinity purified

antibody to the 19 KDa, C-terminal fragment of MSP1 from malaria immune human serum was found to

compete with invasion-inhibitory monoclonal antibodies for binding to Pf MSP119 and mediate inhibition

of parasite growth in vitro (156).

Infants who were positive for MSP119 were protected against parasitaemia and febrile illness, and those

negative for IgG had a 10 times greater risk of becoming parasitaemic, suggesting a protective role for

anti-MSP119 antibodies in infants and pregnant women (157). The role of the antibody response against

MSP119 in the protection against malaria was claimed in a number of other studies (158-162). However, at

least one study argued the protective effect of MSP119 antibodies in Ghanaian children (163).

b- Merozoite surface protein 2 (MSP2)

MSP2 is a second merozoite surface antigen, integral glycoprotein of 45 KDa. MSP2 is in low abundance

in the ring stage and therefore is presumably released from the merozoite at the time of invasion, which

may indicate a role of this molecule in the merozoite attachment to the erythrocytes (164). This antigen

has been denoted as MSA2, the 45 KDa antigen, the 46-53 KDa glycoprotein, the 35-48 KDa merozoite

surface antigen, 51 KDa or GP3. It is also a polymorphic glycoprotein (165). The gene coding for this

antigen is located in chromosome 2 (66).

Sequencing of the MSP2 gene done by different workers (164,166,167) revealed a pattern of variability

that suggested the existence of at least two major family types of MSP2. All MSP2 genes characterized at

the DNA level can be divided into five blocks of sequence, MSP2 is highly conserved in its N and C

terminal regions (Block 1 and 5) but contains a more extensive domains of repeats, located centrally

(Block 3) which vary in number, length and sequence. Non-repetitive variable regions (Block 2 and 4)

flank the repeats, which define two allelic families called the IC1 or 3D7 (Group A) and FC27 (Group B)

families. All MSP2 studied were members of these two allelic families, each family being associated with

characteristic sets of repeats (168,165). When the two alleles of MSP2 group A (IC1 denoted T9/96

derived from Thai isolate) and group B (FC27, Papua New Guinean isolate) cloned and sequenced, the

comparison between the DNA and amino acid sequence revealed that the overall homologies are over

95% among variants in the same sero-group. The similarities between group A and group B variants are

only 75% and 64% for DNA and amino acids respectively. Within the central region the values are only

52% and 35% for DNA and amino acid respectively (169). Sequence repeats are a prominent feature of

the variable central region of the gene. There are more than two types of sequence repeats but one of the

dimorphic forms of MSP2 is characterized by 12 and/or 32 amino acid repeat, whereas the other

dimorphic form has much shorter repeats of four or eight amino acids. Different alleles of MSP2 of the

same dimorphic form and having identical or similar repeats may have different numbers of these repeats

in the tandem array. The recombination in MSP2 usually occurred in the repeat regions, which although

very different, have a region of homologous sequence, which presumably facilitated the recombination

(67).

Mice vaccinated with diphtheria toxoid conjugated to three octa-peptides from the C and N-terminal, C-

regions of MSP2 of Plasmodium falciparum were found to be protected against lethal inoculums of

Plasmodium berghei. This shows that the conserved region of MSA2 could form a basis of a malaria

vaccine when presented in a suitably immunogenic form, thus avoiding the problem of antigenic diversity

(170). Taylor and his coworkers (171) demonstrated that the antibodies to MSP2 are predominantly of

cytophilic types (IgG1 and IgG3). IgG3 antibodies to the serogroup A are negatively associated with the

risk of clinical malaria whereas IgG1 to the serogroup B were associated with an increased risk of clinical

infection.

c- Merozoite surface protein 3 (MSP3)

Is a 48 KDa protein, the antibody from clinically protected subject directed towards specific region of

MSP3 promotes parasite killing mediated by monocytes. Purified IgG from protected subjects are

effective in antibody dependent cellular inhibition (ADCI) and those directed against MSP3 are

predominantly cytophilic (172).

e- Other merozoite surface proteins

More merozoite surface proteins have been reported such as MSP4, MSP5 (173), MSP6, MSP7 (174),

MSP8 (175), MSP9 (176) and Apical membrane antigen 1 (AMA) (177).

1.10.1.3.2. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)

Since 1965, there have been indications that malaria parasites use the antigenic variation in the strict sense

to escape host destruction (73). In 1995 many studies established that malaria parasites contain a large

family of genes for variant antigens (70,71,178).

These variant proteins are secreted by the parasite and find their way into the erythrocyte membrane,

where they are concentrated in structure known as knobs. This suicidal action of Plasmodium falciparum

appears to be required to avoid another death trap, the host spleen, and to avoid this splenic death

Plasmodium falciparum modifies the host cell by inserting PfEMP1. These proteins make the erythrocytes

infected with mature parasites attached to vascular endothelium, resulting in their retention in vascular

bed. Although this trick allows the parasite to avoid splenic death, it now becomes vulnerable to immune

attack, so the plasmodium uses the antigenic variation of PfEMP1 to mitigate the consequences of this

attack (73).

The clonal antigenic variation exemplified by the var multigene family exerts its influence at the level of

the whole parasite population present in the host. When an effective antibody response is mounted against

one particular PfEMP1 variant, parasites expressing this variant are eliminated. A few infected RBCs with

different PfEMP1 proteins on their surface can then expand to dominate the parasitaemia (179). Snounou

and his colleagues (179) suggest that, the multigene families are a cornerstone in the survival of

Plasmodium parasites; its success relies on maintaining an infection for lengthy periods despite the

immune response of the host. While at the same time limiting their multiplication rate so as to enhance

the survival of that host. The first evidence of the in vivo switching between variant surface antigens

(VSA) in human Plasmodium falciparum infection was reported by Staalsoe and others (180).

PfEMP1 is 200-350 KDa proteins with antigenic variant and adhesive properties, which encoded by the

var gene. To date, four distinct functions have been attributed to PfEMP1 that contribute to the important

role of this protein family in malaria disease. These are antigenic variation, cytoadherence, rosetting of

uninfected erythrocytes and immunoregulatory activities on host immune cells (72).

Su and others (70) stress that the var gene products are related to the erythrocyte binding protein (EBA-

175) of Plasmodium falciparum and to the Duffy antigen binding protein (DABP) of Plasmodium vivax

and Plasmodium knowlesi. The single copy genes for EBA-175 & DABP are conserved and present in all

Plasmodium line. The common motifs in EBA-175 and var gene products may indicate a common origin

and have led Su and his colleagues (70) to group the proteins in one super family of Duffy binding like

(DBL) proteins. Every var gene encodes a high molecular weight PfEMP1 variant composed of tandemly

arranged cysteine rich domains of ~300 amino acids known as (DBL) domain, and the related cysteine

rich interdomain regions (CIDRs). Together DBL and CIDR domains account for the majority of PfEMP1

extra-cellular sequence, in addition to two conserved domains, the C2 domain and the amino terminal

segment (NTS). Each block of the four major building blocks of the PfEMP1 display regions of diversity.

Thus PfEMP1 polymorphism encompasses the whole extra-cellular domain.

The DBL domains are numbered consecutively from the N terminus and have been classified into 5 types

(α - ε) on the basis of their amino acid sequence, whereas CIDR domains grouped as three distinct

sequence classes (α, β and γ). A conserved feature of NTS domains is a central block of amino acids that

is expected to have an alpha helical fold; the second conserved domain is the C2 domain, which found in

50% of the isolates studied. Both C2 and NTS have globular structure and regions of alpha helical

structure that is far unique to PfEMP1.

The DBL and CIDR molecules that possess adhesive properties vary in number between PfEMP1

variants. PfEMP1 domain architecture is not random where certain tandem domain association was

observed (DBLαCIDRα, DBLδCIDRβ, DBLβC2). This conservation may have functional significance for

PfEMP1 folding, transport or binding activity. Parasite binding phenotype appears to be a determinant of

infected erythrocyte tissue tropism that contributes to parasite survival, transmission, and disease outcome

(72). Infected erythrocytes display multiple and diverse binding properties that vary between clones. DBL

domains of PfEMP1 have been shown to bind different molecules including intercellular adhesion

molecule-1 (ICAM-1), chondroitin sulfate A (CSA), complement receptor 1 (CR1), and undefined heparin

sulfate molecules on the erythrocyte surface, the CIDR1 has been shown to bind CD36 (72).

The antibodies capable of disrupting rosettes and those agglutinating erythrocytes infected with the late

stage of Plasmodium falciparum have been reported to correlate with protective immunity (181,182). No

significant correlation was found between parasite agglutination and response to conserved synthetic

peptides of PfEMP1 (183). Also no correlation was found between protection and antibody response to

the conserved PfEMP1 peptide epitope originating from the DBL1 domain of PfEMP1 (41).

The antibodies to variable PfEMP1 were found protective against malaria (40,41). The pre-existing anti-

PfEMP1 antibodies (variable) were found to reduce the risk of contracting clinical malaria when

challenged by novel parasite clones expressing homologous, but not heterologous variable surface

antigens. Both clinical and subclinical infections induce antibodies against variant antigens, and

antibodies against several var sero-types are induced during a malaria infection (40).

1.10.1.4. Anti-toxic and anti-disease vaccine

Playfair and his colleagues (184) suggested that complete resistance to malaria infection is probably not

feasible, and that attention should be directed not so much at vaccines designed to eliminate one or other

stage of the parasite, but rather towards the possibility of an antitoxic vaccine that prevents the serious

pathological complications of the disease.

The clinical symptoms of human malaria are mediated at least in part, by the release of tumor necrosis

factor alpha (TNF-α) by monocytes and macrophages. Monoclonal antibodies were found to inhibit TNF

inducing factors derived from Plasmodium falciparum infected erythrocytes. This monoclonal antibody

binds to liposomes containing phosphatidylinositol (GPI) but not to other phospholipids liposomes,

showing that it recognizes a common phosphatidylinositol-like epitope which has been suggested as a

suitable immunological target for the prevention or treatment of severe malaria (18). Plasmodium GPIs

have been also demonstrated to up-regulate expression of endothelial cell surface receptors implicated in

cytoadherence and to induce hypoglycemia (186). Adults who have resistance to clinical malaria contain

high levels of persistent anti-GPI antibodies, whereas susceptible children lack or have low levels of short

lived antibody response, suggesting that anti-GPI antibodies provide protection against clinical malaria

and thus are likely to be with valuable implication for the development of GPI-based therapies or vaccines

(187).

1.10.2. Types of vaccine formulations

Multiple approaches for the development of subunit vaccines have been investigated that include:

a. Recombinant proteins vaccine

Potential vaccine candidates could be produced in a reasonable amount using recombinant DNA

technology. Recombinant antigens were found immunogenic and elicit strong protection in monkeys,

indicating their potential as components of a subunit vaccine (188).

b. Gene vaccines using viral and bacterial vectors

Finding a means of introducing antigenic proteins to the cell cytoplasm to be expressed with MHC

molecules has become one of the prime challenges in vaccine development. The genes would have to be

delivered specifically to antigen presenting cells rather than to other cell types to stimulate the T- cells

(189). Harmless viruses or ones that have been rendered non-replicative in humans or attenuated bacteria

vector carrying plasmids have been used for delivery of genes derived from different pathogens (189,190).

c. DNA vaccine

Wolff and his colleagues (191) provided evidence that plasmid DNA can transfect cells in vivo and hence

can be regarded as safe alternative of viral and bacterial vectors.

d. Synthetic Vaccine

Synthetic peptide vaccines provide several practical advantages when compared to recombinant proteins

or attenuated bacterial or viral vectors expressing parasite antigens. All components contained in the

synthetic peptide vaccine are chemically defined and readily available, and provide at least in principal,

the rapid production of an unlimited supply of immunogens. It is stable and safe compared to the viral or

bacterial vectors which have the potential for eliciting pathogenic and/or reactogenic complications (192).

1.10.3. Malaria vaccine trials

A number of malaria antigens reached phase I trial; the apical membrane antigen 1 (177) and MSP3 (193).

The RTS,S/AS02A is a promising malaria vaccine that reached phase II clinical trial, composed of a

portion of the circumsporozoite surface protein (CSP) of Plasmodium falciparum linked to hepatitis B

surface antigen (HBsAg) formulated with the adjuvant system AS02A (194). RTS,S/AS02A provided

partial protection against infection in malaria-naïve adults and reduced the risk of clinical malaria, delayed

time to new infection and reduced episodes of severe malaria over 6 months in African children. The

vaccine conferred partial protection in African children for at least 18 months, and confirms the potential

of malaria vaccines to become credible control tools for public-health care (195).

The objectives

General objectives This study is designed to investigate the clinico-epidemiological pattern of severe Plasmodium falciparum

malaria in an area of low and seasonal malaria transmission. The goal also directed to study the state of

naturally acquired protection and the impact of the presence of naturally acquired antibodies directed

against merozoite surface proteins and variant surface antigens on the outcome of infection and

progression to fatal sort of the disease.

Specific objectives 1- To describe the clinico-epideiological pattern of severe malaria in Gedarif area, Eastern Sudan.

2- To measure the antibody response directed towards the conserved C-terminal merozoite surface protein 1

(MSP119) of Plasmodium falciparum by ELISA.

3- To measure the antibody response directed towards the two alleles of the polymorphic Plasmodium

falciparum merozoite surface protein 2 (MSP2) by ELISA.

4- To measure the antibody response directed towards different variants of Plasmodium falciparum

erythrocyte membrane protein-1 (PfEMP-1) using flow cytometry.

5- To identify the patterns of the immune response among severe and uncomplicated Plasmodium falciparum

malaria patients compared to malaria free volunteers.

MATERIALS AND METHODS

2.1. Study area, study design and samples

2.1.1. Study area

This study was carried out over two successive malaria seasons in the period between September

2000 and January 2002, in Gedarif Hospital. Gedarif town (350 - 400 thousand inhabitants) is

situated on the Sudanese Savannah, at an altitude of about 600 meters above sea level; 350 km

from the capital, Khartoum. The epidemiology of the febrile uncomplicated malaria episodes in

the area has been reported before (40). The area is mesoendemic for Plasmodium falciparum

malaria which is the predominant malaria species in the area (98%), Plasmodium vivax and

Plasmodium malariae occasionally seen (2). Malaria in the area is highly seasonal, and follows the

annual June–October rains; the seasonal incidence of malaria varies considerably from year to

year (196). Anopheles arabiensis is the sole malaria vector in the area. The entomological

inoculation rate (EIR) in Gedarif has not been measured, but was found to be very low and

difficult to measure by conventional methods in the surrounding villages (197).

2.1.2. Study design

Patients reporting to Gedarif hospital were seen by medical doctors at an outpatient clinic. Blood

slides were prepared from suspected malaria patients and examined for the presence of malaria

parasites. Based on clinical assessment, patients with severe falciparum malaria were admitted and

patients with uncomplicated disease were treated as outpatients. All patients who satisfied the

WHO criteria of severe malaria were enrolled in the study, and control groups (uncomplicated

malaria and healthy donors) were matched for age, gender, residence and etc. Households and

relatives were being a priority in the selection of the two control groups. Certain investigations

were routinely done for the patients with severe malaria e.g. blood glucose level (electronic

glucometer, accu trend™), blood insulin measured by radioimmunoassay (kit IMK-414, Beijing,

China; Morgan and Lazarow, 1962), urine general and complete haemogram. Other diagnostic

investigations were also done, each was selected based on the clinical presentation of the patients

e.g. chest X-ray, blood and spinal fluid culture, serum/plasma creatinine, lactate, urea, electrolytes

and etc. The results and the clinical data were entered into a data base program. Severe malaria

patients were treated with the standard dose of Quinine sulphate; patients were discharged from

hospital after clinical improvement and the ability to continue the treatment orally at home under

our supervision. Patients were followed up, clinically and parasitologically up to day 28 after

diagnosis, and were advised to visit our malaria clinic if symptoms recurred thereafter.

The consent

Informed consent was obtained from severe, uncomplicated malaria patients and malaria free

donors. In case of children or comatose patients the consent was obtained from parents or

guardians (a copy of the consent is attached in the appendix).

This study had national ethical clearance and national endorsement from the ministry of health,

Sudan. 2.1.2.1. Malaria definition and diagnosis A patient was defined as suffering from malaria if he/she complained of fever or had body

temperature measured with an oral probe of > 37.5°C plus microscopically detected asexual

parasitemia.

For diagnosis, thick and thin blood smears were prepared, stained with Giemsa and examined for

malaria parasites under a magnification of x1000. Films were considered negative after

examination of 200 thick smear fields without detection of parasites. For positive films, the

parasites were counted per 300 leukocytes and standardized per one micro liter of blood (198).

2.1.2.2. Characterization and grouping of patients with severe malaria Having the clinical information (symptoms and signs) and the laboratory results available the

patients were allocated to the different categories of severe malaria according to the WHO criteria

for severe and complicated malaria (8), one or more of the features in the presence of asexual

parasitemia. The patients with severe anemia, had normocytic normochromic anemia with

haematocrit <15% or haemoglobin <5 g/dl. Those with cerebral malaria were in unarousable coma

persisting for at least 30 minutes, while convulsions diagnosed when patients had generalized

convulsions, at least two/24 hours despite cooling. The hypotension was defined as systolic blood

pressure <50 mmHg in children (1-5 years) or < 70 mmHg in adults), with cold clammy skin.

Patients who had more than one type of complications e.g. cerebral malaria and severe anemia

were categorized separately.

Patients were treated immediately with antimalarial and other needed drugs. Patients with SMA

were further treated with cross-matched, infections-screened blood infusion.

2.1.3. Samples collections and storage

2.1.3.1. Plasma and parasitized red blood cells (PRBC) separation and storage

Five to seven ml of blood samples was collected in EDTA vacutainer tubes. The samples were

centrifuged for 10 minutes, the plasma separated and kept frozen at – 20 0C. For the PRBC, equal

amount of freezing solution (sterile mixture of 3% sorbitol, 28 % glycerol and 0.65% NaCl in

water) was added (after puffy coat removal), mixed and immediately kept in liquid nitrogen.

Blood samples were spotted on filter paper and sealed separately in plastic bags for PCR

investigations.

2.2. Measurement of antibody response directed against MSP119 and MSP2 by

indirect ELISA. Recombinant proteins representing the 19 kDa, C-terminal fragment of merozoite surface protein

(MSP119) and two antigens representing the full length of group A and group B alleles of

merozoite surface protein 2 (Group A, denoted T9/96 13/14 and group B, FC27 denoted GF/88),

and glutathione-S-transferase (GST) protein were used.

The recombinant antigens were prepared according to the method that has been described by

Ranford-Cartwright and others (199), where the gene representing these proteins were expressed

in Escherichia coli as fusions to the C-terminus of glutathione-S-transferase (GST) of

Schistosoma japonicum using the pGEX vectors, which permit the purification on a glutathione

column.

The method that has been described by Cavanagh and others (149) was used with slight

modification. The wells of the 96 well plates were coated with 100 µl of recombinant protein

fused to glutathione-S- transferase (GST). 16 ng of MSP119, 50 ng of T9/96 13 / 14 isolate of

(group A) of MSP2, 25 ng of GF 88 full length of FC27 (group B) of

MSP2, and 50 ng of GST in coating buffer (carbonate buffer pH 9.4- 9.6) as determined by

checkerboard titration. The plates incubated for three days at 4°C., the wells were washed five

times with the washing buffer that composed of phosphate buffered saline with 0.05 % Tween 20

(PBS-Tween-20). Unoccupied protein binding sites on the plate were blocked with 200 µl per well

of blocking buffer (1% skimmed milk in washing buffer) for five hours at room temperature,

again the wells washed five times. Sera diluted 1:500 in blocking buffer; (100 µl/well) were added

to duplicate antigen-coated wells and incubated over night at 4°C. After five washes, the wells

were incubated for three hours at room temperature with 100 µl (1:5000) of the conjugate

(Horseradish peroxidase-conjugated to rabbit anti-human IgG, specific for gamma chain

(HRP/IgG) DAKO A/S, Denmark).

The plates were then washed again and incubated for 10 minutes with 100 µl of the substrate

buffer. The reaction was stopped by the addition of 20 µl of 2M H2SO4. The optical densities were

measured at 492 nm, using the ELISA reader (Labsystems Multiskan MCC/340). The inter-plates

variations were controlled by the introduction of pool positive and negative sera in each plate. The

optical density value for each serum sample was calculated by subtracting the mean optical

density value of wells containing control GST protein alone from the mean optical density value

of wells containing antigen-GST.

Cut off values at which binding of antibodies was regarded as significantly above background

were calculated as the mean plus three standard deviations of optical density readings obtained

with sera from Danish donors with no history of exposure to malaria.

2.3. Measurement of the antibodies directed against Plasmodium falciparum

erythrocyte surface protein 1 (PfEMP1)

2.3.1. Parasites

Thirty five parasite isolates were thawed and cultured as described before

(200,201), 22 parasite isolates were successfully grown and used, they were

obtained from 7 cerebral malaria (CM), 4 severe malarial anemia (SMA), 1

convulsion-associated malaria (CAM) and 10 uncomplicated malaria (UM) patients.

2.3.2. Plasma

A total of 215 plasma samples used were obtained from 5 groups of donors. 139

plasma samples were collected at the time of diagnosis and before treatment; 75

from patients with severe malaria, and 64 from patients with uncomplicated

malaria. The other sets included; 17 samples from cerebral malaria patients at

convalescence (day 28), 33 samples from Sudanese malaria–free donors, and 26

samples from Tanzanian patients with malaria. The experiment was controlled by

11 Danish (not known to be exposed to malaria) plasma samples, and a pool of

African (Gomoa) hyperimmune plasma samples from African adults was used in

duplicates for 6 serial dilutions for semi quantitative scoring of the level of

immune response, where, the values above the cut off point and the 1:16

dilution gave the rank of 1, above 1:16-1:8 gave the rank of 2, above 1:8-1:4

gave the rank of 3 and so on until those above 1:1 dilution where they gave the

rank of 6. The mean of the 11 Danish plasma readings plus two standard deviations was

regarded as the cut off value above which, a sample can be regarded as positive

for PfEMP1 antibodies for each parasite isolate. 2.3.3. Long term In vitro culture of Plasmodium falciparum from cryo-

preserved patient isolates The liquid nitrogen freezed infected red blood cells were thawed in a 37 0C. water-bath, the tube

then spinned at 2000 rpm (600g) for 8 minutes, immediately after thawing the supernatant

discharged and 500 µl of thawing solution (3.5% NaCl) was added and spinned for 8 minutes,

supernatant removed and 1 ml of pre-warmed culture medium (500 ml RPMI-1640 supplemented

with 25 mg Gentamycin, 91.6 mg L-Glutamine, 2.5 g Albumax II, 10 ml normal human serum

and 10 mg hypoxanthine) was added, spinned and supernatant removed, the washing was

repeated until the supernatant became clear (maximum 3 washes). The pellet was added to a 50 ml

culture flask; 6 ml of culture medium and 200 µl of washed uninfected packed cells (type O Rh- or

Rh+) were added. The culture flask was then flushed with gas mixture of 2% O2, 5.5 % CO2, and

92.5 % N2 through 0.2 mm filter unit at 1.5 - 2 bar pressure for 30 seconds. The flask was

incubated at 37 oC. Twenty four hours later the culture medium was changed and the parasitaemia

was estimated (slides were prepared and stained by 10% Giemsa stain). The culture monitoring

was proceeded every 48 hours with medium change and parasite count and when the parasitaemia

start to increase the culture suspension was removed to a 250 ml culture flask, washed uninfected

erythrocytes and more culture medium were added and flushed with the gas mixture for 90

seconds. The monitoring was proceeded every 48 hours until a mature form of parasite with

parasitamia of 2% or more reached.

2.3.4. Magnet activated cell sorting (MACS)

Mature–form of Plasmodium falciparum cultures were enrichmed by magnetic selection, a

method that has been described by Staalsoe and her colleagues (39) that relies on the

paramagnetic properties of the parasite metabolic waste product hemozoin that firstly been

described by Paul and others in 1981 (202). The VarioMACS magnet (Miltenyi

Biotec), with size CS column was used. The three-way valve was assembled at the bottom

of the column; the tip of the outlet needle was cut by a pair of nippers and then mounted on the

downward arm of the 3-way valve. The column was mounted in the VarioMACS magnet and the

3-way valve was adjusted to allow flow through its side arm, and then the column was filled with

phosphate buffered saline with 2% fetal calf serum (PBS2) by injecting the fluid into the side arm

of the valve using a 20 ml syringe. The 3-way valve was opened to allow flow from the column

through the outlet needle and when there was no PBS2 left in the loading chamber above the

column the suspension of the infected red blood cells was added. The passing solution that

contains the uninfected red blood cells or those infected with immature form of parasites was

collected as waste. The column was washed with PBS2 until no more red blood cells were seen in

the effluent (change from red to absolutely clear). Five ml of PBS2 was added to the upper

chamber and a 20 ml syringe filled with 20 ml PBS2 was mounted on the top of the column via

the top adapter, the column was then removed from the magnet and a syringe used to flush the red

cells infected with the mature form of the parasites that have trapped inside the column through

the outlet needle.

This method was used to enrich in vitro Plasmodium falciparum cultures to > 80 % late stage

infected erythrocytes. Parasites enriched in this way are completely unaltered and fully viable

(Figure 2.1A & B).

Figure 2.1A

Figure 2.1B

Cleaning the column: for re-use, the column was rinsed by passing 3*20 ml PBS followed by

3*20 ml distilled water and finally by 3*20 ml 70% ethanol through the column by using a 20 ml

syringe through the top adapter of the column. The column was dried carefully by blowing

compressed air through the column for several minutes.

Counting of red blood cells infected with mature parasites The cells infected with late trophozoites and schizonts that have been enriched with MACS were

counted in a Neubauer chamber. The mature parasites were centrifuged for 8 minutes at 2000 rpm

and the supernatant discharged and the pellet was re-dissolved in 5 ml PBS2. Twenty five

microlitre of the cells was diluted to 500 µl with PBS2 and the number of cells was counted in

4*16 fields using the Neubauer chamber and then the number of cells per milliliter of buffer was

counted

Fig. 2.1A: P.falciparum parasite (Gedarif ) from an in vitro culture showing different

developmental stages of rings and late stages.

Fig. 2.1B: RBCs with late stage P. falciparum parasites from Gedarif area after it had

been in vitro cultured and enriched to late stage (schizonts and trophozoites) by MACS

Number of cells * dilution factor * 1000 µl/ml = Cells/ml

4 * 0.1 µl

Dilution factor = 20

4 = Number of Neubauer chamber squares counted

0.1µl = volume /square

2.3.5. Analysis of plasma antibodies to variant surface antigens by fluorescent

activated cell sorter (FACS) The FACS analysis was used for the measurement of antibodies (Abs) directed

against P. falciparum variant surface antigens (VSA), the full protocol was

described before. The antigens recognized by this method were reported strain

specific and their molecular weight are in the range previously described for

PfEMP1 antigens (39). The number of the erythrocytes infected with late stage of Plasmodium falciparum was adjusted to

2.5*106 erythrocytes/ml with PBS2, and labeled with 20 µl ethidium bromide (0.1 mg / ml in

PBS) per ml of erythrocyte suspension. Five-microlitre plasma were placed in FACS tubes then

100 µl of ethidium bromide labeled infected erythrocytes were added and incubated for 30

minutes at 5 0C. Then 3 ml of PBS2 were added to each tube, centrifuged for 8 minutes at 2000

rpm, supernatant discharged and other 3 ml PBS2 were added, centrifuged and supernatant

discharged. After the second wash the precipitate were resuspended in 100 µl of Goat anti human

IgG antibody (1:250 in PBS2) and incubated for 30 minutes at 5 0C. The tubes then washed twice

with PBS2 and the pellet was resuspended in 100 µl of Rabbit-anti goat-IgG - FITC (Green

fluorescence diluted 1:25 in PBS2) and incubated for 30 minutes at 5 0C.

The tubes then washed once and the precipitate was resuspended in 200 µl PBS2. The tubes were

kept overnight at 5 0C. and then analyzed by flow cytometer.

The flow cytometer setting was adjusted to achieve a clear separation of uninfected (ethidium

bromide negative) and infected (ethidium bromide positive) erythrocytes in an FSC/ethidium

bromide dot plot. Data acquired and stored on FSC (forward scatter), SSC (side scatter), FL1

(FITC), and FL2 (ethidium bromide). FACS data were analyzed by Win List software packages.

A gate was set around late-stage infected erythrocytes (blank tube without plasma) in an

FSC/ethidium bromide diagram plot. The FITC fluorescence of the cells within this gate was used

to quantify the antibody response of variant surface antigens, which expressed as mean/median

fluorescence (Figure 2.2). Blank tube, Danish plasma, serially diluted positive plasma, Tanzanian

plasma (hyper-endemic area) and all study samples (Sudanese patients’ and malaria free plasma)

were assayed on the same day for each parasite isolate.

Cut off values at which the samples were regarded as antibody positive were calculated as the

mean fluorescence plus two standard deviations obtained with plasma from Danish donors.

2.4. Statistical analysis The statistical package for social sciences (SPSS 10.0 for windows) software was used for the

analysis of the clinical and other characteristics of malaria patients and the control group’s data.

For comparison of groups, first the distribution of data was tested for normality by descriptive

statistics, normally distributed data analyzed by independent samples T-test, and when normal

distribution was violated, data transformed by computing, and then analyzed. Uncorrected

distributions were analyzed using non-parametric tests. Kruskal-Wallis test was used for

comparison between clinical subgroups of severe malaria. The quoted levels of significance allow

for post-hoc testing using Scheffe's method (Comparison of age, hemoglobin and blood glucose

level between individual complications) or the Least Significant Difference (LSD) method

(parasite count between individual complications).

The SigmaStat statistical software, version 2.03, was used in analysis of data of merozoite surface

proteins (MSPs) and Plasmodium falciparum erythrocyte membrane protein (PfEMP1).

For the prevalence of anti-MSP antibodies in different study groups, Chi-square test and Fisher

Exact Test were used. The t-test and Mann-Whitney Rank Sum Test were used for comparison

between malaria and malaria-free donors and between patients with UM and SM with regard to

the plasma level of antibodies (donors with positive response) and for the number of MSP

antigens recognized by the different study groups. For comparison of plasma levels of antibodies

and number of merozoite surface protein antigens recognized, between the SM, UM and MF

donors and between the subgroups of SM, One Way Analysis of Variance (ANOVA) and One

Way ANOVA on Ranks were used. For variant surface antigens (VSA) reactivity, the descriptive statistics mean

and standard deviation were calculated. For comparison between the reactivity

of plasma obtained from the different subgroups of severe malaria patients, and

Figure 2.2. FACS histogram results representing the antibody response

of blank sample (Bl), 11 Danish plasma samples, Pool positive plasma in

6 dilutions and 2 severe plasma samples from Gedarif area directed

against PfEMP1 of a severe Plasmodium falciparum isolate (SG93).

to compare between the recognition rate of isolates from CM, SMA, and UM

patients t-test, Chi-square test and One Way Analysis of Variance (One Way

Anova) were used. To test the correlation between levels and prevalence of VSA

antibodies and that between prevalence of VSA antibodies and the age of plasma

or parasite donors Pearson Product Moment Correlation

was used.

RESULTS

3.1. Epidemiology of severe Plasmodium falciparum malaria

3.1.1. The frequency of acute uncomplicated and complicated malaria

The total number of patients clinically suspected of malaria for whom blood smears were taken and

examined were 16 606 and among them, 2 488 patients (15%) were found to be parasitemic (table 3.1).

Plasmodium falciparum was the causative agent in 98.88%, while Plasmodium vivax, Plasmodium

malariae and Plasmodium ovale, accounted for 0.64%, 0.44% and 0.04% of all malaria infections,

respectively. No mixed infections were detected. Based on clinicians' decision 252 malaria patients

were hospitalized, and 110 (4.4%) fulfilled the WHO criteria for severe malaria and were included in

the study. Seven patients died, all with cerebral malaria. The mortality rate was 0.3% of all malaria

infections, 6.4% for all severe cases of malaria and 38.9% for patients with cerebral malaria.

Table 3.1. Clinical categorization of patients presented with malaria-like symptoms to the malaria clinic at Gedarif Hospital, in the period, from September 2000 to January 2002. The patients died of severe malaria were all had cerebral malaria.

A

PatiRate of malaria Uncomplicate Proportion of sever

Severe m

death r

166

15%

(2488/166

95.6%

(2378/24

4.4%

(110/2488)

6.4%

(7/11

3.1.2. The frequency of the different types of severe complications

The WHO criteria for severe malaria were used to classify malaria patients as uncomplicated and

severe. The clinical presentations of patients categorized as having severe malaria are shown in figure

3.1. Severe anemia (50 patients) was most commonly found and accounted for 45.5% of the severe

malaria cases. Cerebral malaria was found in 16.4% (18 patients) of the patients. The rest had

convulsions, hypotension or a mixture of above manifestations. There was a tendency that a higher

percentage of patients had anemia during the second malaria season, but the difference between clinical

presentations in the two years was not statistically significant. Six patients had more than one type of

complication; two patients with cerebral malaria were also anemic, while four severely anemic patients

also presented with convulsions. Other complications, like respiratory distress syndrome, renal failure,

and bleeding abnormalities (thrombocytopenia) were not recognized during the study. There was no

difference in the clinical presentations in females and males.

3.1.3. Age distribution of severe and mild malaria Uncomplicated and severe malaria affected both children and adults, but as seen in figure 3.2, small

children between two and four years constituted a much higher percentage among patients with severe

malaria compared to patients with uncomplicated disease.

Twenty seven malaria patients (14 females and 13 males) were above 12 years of age and constitute

24.5% of the severe malaria patients. The incidence of severe malaria in males and females was (1:1).

Figure 3.1. Symptoms in the 110 patients admitted to Gederif Hospital, Eastern Sudan and fulfilling the WHO criteria for severe malaria, the patients were admitted over a period of two years.

0

5

10

15

20

25

30

35

40

45

50

0 - 1 Year 2 - 4 Year 5 -19 Year 20 & above

Age groups (year)

prop

ortio

n of

pat

ient

s (%

)

Severe malaria Uncomplicated malaria

3.1.4. Characterization of patients in the different categories of severe malaria The mean age in years (mean ± SD) varied markedly between the different categories of patients with

severe malaria. Malaria patients with anemia or convulsions aged 5.6±7.7 and 5.9±5.3 years

respectively were infants and small children, patients with cerebral malaria aged 14.1±13.4 were older

children and young adults, whereas hypotensive group aged 35.2±12.1, were adults (figure 3.3a). The

mean parasite count was highest in patients with convulsions and in this group the parasitemia was

significantly higher than in patients with hypotension or anemia (figure 3.3b). The group of patients

with severe anemia by definition had low levels of haemoglobin, but the other groups did differ with

respect to this parameter (figure 3.3c). The

Figure 3.2. Age distribution of patients with severe malaria (n=110, white dots) and with uncomplicated malaria (n=1 537, black dots) in Gedarif Hospital, eastern Sudan (period 2000 to 2002).

(C)

Hem

oglo

bin

leve

l (%

)

0

20

40

60

80

100

(D)

Clinical categories of severe malaria

Blo

od G

luco

se le

vel (

gm/d

l)

0

50

100

150

200

250

Age

(Yea

rs)

-505

101520253035404550

Para

site

cou

nt (p

er m

icro

litre

)

010x10320x10330x10340x10350x10360x10370x10380x10390x103

100x103110x103120x103

(A)

(B)

P<0.001 P<0.001

P<0.001

P=0.013P=0.053

P<0.01 P<0.019

P<0.001

P<0.001

SMA CAM CM HTN

Figure 3.3: Comparison between four groups of patients with severe malaria (anemia, convulsions, cerebral malaria and hypotension) admitted to Gedarif Hospital; in the following; a. Age in years (mean ± 95% confidence interval, CI) b. Parasite count (mean ± 95% CI) at diagnosis (day o) and before treatment, c. Hemoglobin level (mg/dl, converted to percentage), mean ± SD, at diagnosis and before treatment, d. Random blood glucose level, mean ± SD, at diagnosis and before treatment. Horizontal lines with P-values span between clinical groups with significant differences

mean blood glucose levels at diagnosis and before treatment, was higher in patients with cerebral

malaria than in patients with anemia (figure 3.3d), but did not differ significantly between the other

groups. The white blood cell (WBC) count was highest in patients with cerebral malaria (6 876.5± 4

556, mean ± SD), but the differences between the groups were not statistically significant.

3.1.5. Comparison between fatal and non-fatal severe malaria

Seven patients with cerebral malaria died despite treatment. The characteristics of these patients are

shown in (table 3.2). Two of the patients were adults and the remaining were children between 5 - 11

years. The duration of symptoms before admission varied between two to seven days. The mean (±SD)

haemoglobin and the mean blood glucose levels were higher in patients who died of severe malaria

than in the survivors (p=0.035 and p<0.001, respectively, T-test). The differences in glucose levels

were not reflected in the insulin levels, which were comparable between the groups, although insulin

level in fatal malaria was above the normal physiological range (4.0 to 16.8 mIU/L).

3.2. The antibody response against the conserved and polymorphic antigens of the

merozoite surface proteins (MSP119, MSP2A and MSP2B) 3.2.1. Characteristics of donors Patients with SM (n = 109, mean age ± SD in years (yrs), 10.6 ± 13.1), and a

comparable number of patients with UM (n=114, 13.5±13.1 yrs) and malaria free

donors (MF) (n=117, 12±12.8 yrs) were included in this part of the study. The mean

age, age range and gender distribution, were comparable between the groups, see

table 3.3. Patients with SM had either severe malaria anemia (SMA) (n=49, 5.6±8

yrs), convulsions associated malaria (CAM) (n=23, 5.9±5.2 yrs), cerebral malaria

(CM) (n=18, 14.1±13.2 yrs) or with hypotension HTN (n=13, 35.2±12.1 yrs), while 6

patients had multiple complications (3.3±1 yrs). Seven patients died within the

first three days after hospital admission, they were all with CM.

3.2.2. Recognition of merozoite surface protein (MSP)

The frequency of pre-existing antibodies in plasma of all donors (n=340) at the time of malaria

diagnosis (D0) against the MSP antigens, showed a consistent differences in recognition of the three

antigens. The MSP119 was the most recognized Ag (49.8%, 166/333), followed by MSP2A (41.6%,

137/329), then MSP2B (37.2%, 123/331), in all conditions whether the data was pooled or each group

or subgroup of samples were considered separately the differences were not significant. On the other

hand there was strong statistically significant correlation in recognition of the three antigens i.e. the

plasma that recognized one antigen tends to recognize the other antigens and vice versa.

Table 3.2. Levels of variable parameters (parasitological, hematological and biochemical) in individuals who died of severe malaria, at Gedarif Hospital, eastern Sudan, and comparison of the mean values of these parameters between fatal and non-fatal severe malaria groups

Age (years Symptoms

duration#

Parasite coun

(parasite/µl)

Hemoglobi

level (%)

WBC

(cell/µl)

Blood glucos

(mg/dl)

Insulin level

(mIU/L)

11/male 3 days 4350 ND ND 219.6 32.6

35/male NK 180000 92 1900 122.0 9.0

09/female∗ 4 days 96000 60 4000 246.6 20.3

05/male 3 days 114000 65 11600 102.6 4.1

55/male 5 days 4800 46 4600 311.0 25.0

10/male 7 days 3150 ND 3500 149.4 ND

06/female 2 days 42000 40 11000 111.6 ND RI 18.7

63471±

68590

60.6±

20.3

6100±

4131

180.4±

79.7

18.2±

11.6

RII 10.5

37940±

66741

42.18±

18.3

5790±

2962

94.3±

29.9

13.8±

12.7

P-value 0.226 0.035 0.885 <0.001 0.406

WBC: white blood cell count. NK: not known. RI: Row I; mean ± SD of measured variables in patients with fatal SM. RII: Row II; mean ± SD of measured variables in patients with non-fatal SM. # before admission. ∗ The patient received a drip of 5% dextrose in saline, before blood sampling Table 3.3. Characteristics of the different groups of volunteers studied, plasma collected during two successive transmission seasons 2000-2002 from Gedarif, eastern Sudan Donors Number Gender Age in years Statistical differ

Males% mean±SD & range

Severe malaria

109 49.1% 10.6±13.1

(0.9 – 55)

Uncomplicated m

113

52.6%

13.5±13.1

(0.7 – 61)

Malaria-free don

117

49.6%

12±12.8

(1 – 70)

No s

differences

3.2.3. The relation between the age and the prevalence of MSP antibodies

The donors were divided into 6 age groups (figure 3.4.), based on the physiological development;

infants (1 year or less), young children (>1 – 5 yrs), older children (>5 – 10 yrs), adolescents (>10 – 15

yrs), adults aged (>15 – 45yrs), and older adults (>45 Y). A donor was considered as responder to MSP

antigens if his/her plasma contains antibodies to at least one of the three tested antigens, (MSP119,

MSP2A and / or MSP2B). Accordingly, the prevalence of the antibodies in each age group (proportion ±

95% CI) was; 18.2±18.2%, 52.5±8.9%, 68.3±9.1%, 80±14.3%, 89.2± 7.5% and 100%, respectively.

Thus, the prevalence of plasma anti-MSP antibodies in all study population was increased with

increasing age. When the middle age of each age group was correlated with the anti-MSP antibodies

prevalence rate, statistically significant positive correlation was found (CC=0.836, P=0.0384, Pearson

Product Moment Correlation).

Age groups (years)

prev

alen

ce o

f ant

i-MS

Ps

anitb

odie

s (%

)

0

20

40

60

80

100

120

3.2.4. The antibody response against MSP antigens: comparison between apparently healthy

malaria-free donors and malaria patients

The apparently healthy malaria-free donors (n=117) had negative blood smears for malaria

parasite by microscopy. However, using PCR, 22 (18.8%) patients were found to have had

asymptomatic sub-microscopic parasitemia, half of them had anti-MSP antibodies (50%), among the

remaining 95 donors 44 donors had anti-MSP antibodies (46%). The prevalence (proportion±95% CI)

of MSPall antibodies in this group of donors was 47±9% (table 3.4).

The prevalence of MSPall antibodies in malaria patients (uncomplicated and severe) (n=223) was

77.1±5.5%, which was significantly higher than in MF donors, (p<0.001, Chi-Square).

Figure 3.4. The prevalence of Abs against MSP119, MSP2A and MSP2B Ags (proportions ± 95% confidence interval), in the different age groups of all donors together; patients with malaria (severe and uncomplicated) and malaria-free donors (n=339). The age grouping was based on physiological development (infants 0-1, young children >1-5, older children >5-10, adolescents >10-15, adulthood >15-45, older adults >45 year).

0-1 >1-5 >5-10 >10-15 >15-45 >45.

Table 3.4. The malaria-free donors (negative control) were apparently healthy, and had negative blood smears for malaria parasite, as seen by microscope. The table shows the donors with parasitemia (detectable by PCR) and with anti-MSP antibodies (against any of the following MSP antigens: MSP119, MSP2A and/or MSP2B) Malaria-free donors

PCR positive

PCR negative

Total

MSP positive ELISA 11

50%(11/22)

44

46.3% (44/95)

55

47% (55/117)

MSP negative ELISA 11

50%(11/22)

51

53.7% (51/95)

62

53% (62/117)

Total 22 95 117

The prevalence of the individual MSP antibodies; MSP119 antibody, MSP2A antibody and MSP2B

antibody, in MF donors were; 32.5 ± 8.5, 19.7±7.2, 25.6±7.9, respectively, which were significantly

lower than in malaria patients (57.8±6.5, 44.8±6.6, 48 ± 6.5, respectively), (all p=<0.001, Chi-Square)

(figure 3.5A.). The median number of MSP Ags (n, [25% - 75%

percentile]) recognized by the malaria patients was 2, [1 – 2], which was significantly higher than in

MF donors (0, [0 – 1]), (P = <0.001, Mann-Whitney Rank Sum Test). The plasma level of MSP119

antibody and MSP2A antibody, was significantly higher in malaria patients (SM and UM) (0.739 and

0.641, respectively) than MF donors (0.518 and 0.449, respectively) (p= <0.001, t-test, and p=0.002,

Mann-Whitney Rank Sum Test, respectively). While there was no significant difference in MSP2B

antibody levels between the malaria patients (1.084) and MF donors (1.107) (p=0.858, t-test) (table

3.5).

A

M SP-A M SP-B M SP-19 M SP-all

Prev

alen

ce o

f ant

i-MSP

ant

ibod

ies

(%)

0

20

40

60

80

100

M alaria-Free donorsM alaria patients

B

The m erozoite surface antigens

M SP-A M SP-B M SP-19 M SP-all

Prev

alen

ce o

f ant

i-MSP

ant

ibod

ies

(%)

0

20

40

60

80

100

SM patientsUM patients

Figure 3.5. The prevalence of antibodies against individual MSP antigens; MSP119, MSP2A, MSP2B or any of the three antigens (MSPall), proportion (%) and 95% confidence interval (CI). A: Comparison between malaria-free donors (dark bars) and patients with malaria (both uncomplicated and severe malaria), (light bars). B: Comparison between patients with severe malaria (dark bars) and uncomplicated malaria (light bars), the differences were all significant although in MSP2A and MSP2B bars; there was slight overlap for CI.

Table 3.5. The plasma level of antibodies in individuals with positive antibody response (above the cut off point) directed against different MSP antigens among the 3 different study groups: a comparison between malaria-free donors (MF), uncomplicated malaria (UM) and severe malaria (SM) patients.

The level (mean/median) of anti-MSP antibodies The antigens

MF UM SM P-value Statistical test

MSP119

MSP2A

0.518

.449

0.728

.672

0.76

.549

<0.001

<0.001

1Tukey Test

(mean)

2Dunn,s Method

MSP2B 1.107 1.128 1.018 >0.05 ANOVA

(mean)

1 = One Way Analysis of Variance (ANOVA) 2 = Kruskal-Wallis, One Way Analysis of

Variance on Ranks

Note: values in italic were significantly lower than bolded values.

3.2.5. The antibody response against MSP antigens, comparison between patients

with uncomplicated and severe malaria The prevalence of MSP119 antibodies and MSPall antibodies, was significantly higher in patients with

UM (68.1%±8.6 and 86%±6.4, respectively) compared with SM patients (48.1±9.5 and 67.9±8.8,

respectively) (p=0.004 and p=0.002, respectively, Chi-Square). The prevalence of MSP2A antibodies

and MSP2B antibodies were significantly higher in patients with UM versus SM patients (56.1±9.1 vs

41.7±9.5 and 53.6±9.2 vs 37.4±9.2, respectively) (p=0.048 and p=0.023, respectively). For the last 2

antigens, although the differences were

statistically significant, the results should be considered with precautions, as the power of performing

both tests were below the required strength (figure 3.5B).

The average number of MSP antigens recognized by patients with UM were statistically significantly

bigger than the number recognized by SM patients, (median, [25%-75% percentile]), (2, [1 – 3] vs 1,

[0 – 2], respectively), p<0.001, Mann-Whitney Rank Sum Test (table 3.6.).

Table 3.6. The average number of MSP antigens (MSP119, MSP2A and MSP2B) recognized by individuals in different study groups: a comparison between all malaria patients and malaria-free donors, and between patients with uncomplicated malaria and severe malaria.

Donor group

Number No. of recognized MSP Ags, (Median, 25% – 75% percent

P-value

Malaria patients

223 2, [1 – 2]

Malaria-Free donors

117 0, [0 – 1]

<0.001

Uncomplicated malar

114 2, [1 – 3]

Severe malaria

109 1, [0 – 2]

<0.001

3.2.6. The antibody response against MSP antigens: comparison between individual

complications of severe malaria (SMA, CAM, CM and HTN) The prevalence of antibodies against the MSP antigens was markedly different between the individual

complications of severe malaria. While patients with HTN had MSP antibodies against, at least one of

the 3 antigens (100%), patients with CAM had MSPall antibody prevalence rate of 43.5%, which was

significantly lower than that of HTN and CM (83.3%) patients, p=<0.001, Fisher Exact Test, and

p=0.023, Chi-square, respectively. Patients with SMA had a prevalence rate of Abs against MSPall of

63.3%, which was significantly lower than that of HTN, p=0.013, Fisher Exact Test. However, patients

in the different subgroups of

SM had significantly different ages. The mean age of the subgroups correlated with the prevalence rate

of MSPall antibodies (figure 3.6.)

Clinical groups

Ant

i-MSP

s A

b pr

eval

ence

(%)

0

20

40

60

80

100

120

The

mea

n ag

e (y

ears

)

-10

0

10

20

30

40

50Response to 3 Ags Response to 2 Ags Response to 1 Ag

Mean age of clinical groups

CAM SMA CM HTN

While the plasma levels of MSP119, MSP2A and MSP2B antibodies were comparable between UM and

SM patients (0.725 vs 0.76, 0.672 vs 0.549 and 1.128 vs 1.018, respectively). The number of the MSP

antigens recognized by patients in the different SM sub-groups was different; patients with HTN

recognized statistically significant higher number of antigens than patients with CAM and SMA

(p<0.05, Kruskal-Wallis One Way Analysis of Variance on Rank, Dunn's Method). Although, the age

difference between the SM subgroups was remarkable, there was no difference in the level of

antibodies against the three MSP antigens, the mean levels varied between 0.66 and 0.83 (p=0.593).

Figure 3.6. The MSP Ab response in SM patients, comparison between; convulsion associated malaria (CAM), severe malarial anemia (SMA), cerebral malaria (CM) and hypotension (HTN). The proportion of patients with Abs against MSP119 , MSP2A or MSP2B Ags is indicated by the length of bars. Proportion of patients recognized the 3 antigens (black), two antigens (light) and one antigen (grey). The age (mean ± SD) of the patients in each sub-group is indicated by the line with open circles.

3.2.7. The antibody response against MSP antigens: comparison between fatal and

non-fatal cerebral malaria Seven patients were died due to CM (38.9%, 7/18). The MSP antibody response was comparable

between fatal and non-fatal CM. The prevalence of MSPall antibodies (100% vs 72.7%), and the

average number of recognized MSP antigens (1.9 vs 1.3), in fatal and non-fatal CM, respectively, were

not significantly different. Similarly, were the differences between the levels of antibodies against the

MSP antigens between the two groups of CM (table 3.7).

Table 3.7. The cerebral malaria patients, characteristics and anti-MSPs antibody response in patients

died or survived following the cerebral malaria attack

Ab level Cerebral patients

Number Age Mean± SD

Anti-MSPAbs

No. of Agsrecognize

MSP19 MSPA MSPB

Died 7 18.7±

19

100% 1.9 0.74 0.69 1.19

Survived

11

11.1± 7.4

72.7%

1.3

0.89

0.5

0.77

No significant differences

No. = number, Ags = antigens Ab = antibody

3.2.8. Acquisition of anti-MSP antibodies at convalescence (D28) by SM patients. The plasma antibodies against MSP antigens were measured in 84 plasma samples obtained from

patients with SM at convalescence, 28 days after malaria diagnosis, but not all samples were tested for

antibodies against all MSP antigens. There was no significant change in the prevalence of antibodies

against MSP antigens 28 days after infection. The prevalence of anti-MSP Abs at D0 to MSP119,

MSP2A and MSP2B was 45.7%, (32/70), 43.6% (34/78) and 33.3% (25/75), respectively, which was

almost similar to that at D28, 55.7% (39/70), 50% (39/78) and 33.3% (25/75), respectively. However,

even if we considered the change of response for the individuals in each group, the changes were not

significant. For MSP119, 14.3% (10/70) acquired and 4.3% (3/70) lost antibodies against the antigens,

for MSP2A and MSP2B, 16.7% (13/78), 8% (6/75) acquired and 10.3% (8/78), 8% (6/75) lost antibody

to the respective antigens. However, upon breakdown of data of SM, the most recognizable finding was

that, 24.2% (8/33) of patients with SMA, and 00% (0/11) of patients with CM, acquired anti-MSP119

antibody response at convalescence, but the difference was not significant. In general, the acquisition

of anti-MSP antibodies at convulscence was neither age nor complication-dependent.

3.3. Antibody response against variant surface antigens (VSA)

3.3.1. Spectrum and level of antibody reactivity against

VSA

The spectrums of antibody reactivity account for how many isolates were recognized

by the test plasma samples, irrespective of the antibody level. To correlate

between the 2 indices, we used plasma (n=65) obtained from patients with severe

malaria at day 0 and the 22 isolates (not all parasites tested with all plasma).

There was strong correlation between the number of isolates recognized by each

plasma sample, and the average level of VSA antibody in plasma against each of the

recognized isolates (CC, 0.727 P < 0.001, Pearson Product Moment Correlation)

although a number of outliers were recognized (figure 3.7).

E stim a tio n o f m e a n A b le ve l (se m i-q u a n titive sco rin g )

0 1 1 .1 1 .2 1 .3 1 .5 1 .6 1 .7 1 .8 1 .9 2 2 .1 2 .6 2 .9

Spe

ctru

m o

f rea

ctiv

ity

(% o

f par

asite

s re

cogn

ized

)

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

p la sm a sa m p le

0 1 1.1 1.2 1.3 1 .5 1 .6 1 .7 1 .8 1 .9 2 2.1 2.6 2 .9

0

10

20

30

40

50

60

70

80

90

100

Thus, when all plasma were considered together and data re-analyzed, the rate of

recognition

3.3.2. VSA antibody recognition of parasites isolated from

patient wseverand uncomplicated malaria

3.3.2. VSA antibody recognition of parasites isolated from

patients with severe and uncomplicated malaria

Parasites and plasma were collected before initiation of the treatment from

patients with clinically defined malaria syndromes. Plasma also collected from

asymptomatic malaria free donors and convalescence samples (D28) from some patients

who have had severe malaria. Plasma also obtained from children with uncomplicated

malaria living in an area of high malaria transmission in Tanzania (table 3.8).

Parasites causing severe malaria have previously been shown to express variable

surface antigens (VSA) that are more often recognized by plasma IgG than VSA

expressed by parasites from patients with uncomplicated malaria. Thus, we compared

the plasma reactivity to parasites isolated from the different patients groups. The

reactivity to parasites obtained from patients with severe malarial anemia tended

to be consistently higher than the reactivity to parasites causing cerebral

malaria, whether or not all panels of plasma were considered together, but the

Figure 3.7. The correlation between two indices used for evaluation of the VSA antibody (Ab) response; the Y axis shows the proportion of isolates recognized by individual plasma samples (closed circle), and the X axis shows the mean level of Abs in these samples. Plasma was obtained from patients with severe malaria (n=65) at the time of diagnosis, antibody level was scored into 6 levels as explained before. Generally, plasma with broad spectrum of reactivity had on average higher level of antibody against individual isolates, with general positive correlation. In the insert, the lines represent the mean.

differences were not significant (figure 3.8 and figure 3.9B).

Table 3.8. The details of the total samples used for the study of the immune response directed against variant surface antigens, plasma and parasite donors were mainly classified on clinical bases, date of sampling and geographical location

plasma donors

Parasite donors

MF

(n=33)

UM

(n=64)

SM-D0

(n=75)

SM-D28

(n=17)

TZ

(n=26)

Total

(215)

UM (n= 10 )

264 568 636 133 260 1861

CM (n=7 )

165 364 421 102 182 1234

SMA (n= 4 )

132 249 276 55 104 816

CAM (n=1)

33 62 73 15 26 209

Total

(22)

594 1243 1406 305 572 4120

Abbreviations: MF= malaria-free donors, UM= uncomplicated malaria, SM= severe, TZ: Tanzanian

donors.

However, when we compared between parasites causing SM and UM there were

significant differences of parasite causing SMA (50.5%, CI of 47.1 and 53.9

[412/816]) and CM (48.9%, CI of 46.2 and 51.7 [604/1234]) were significantly higher

than the rate of recognition of isolates causing UM (42.5, CI of 40.3 and 44.8

[792/1861]), P < 0.001, Chi-square test (figure 3.9B.)

There was a considerable difference between the recognition of the individual

isolates, the best recognized isolate was recognized by 85.6% of the plasma tested

(119/139). On the other hand only 7% (15/214) of the plasma donors had VSA

antibodies to the least-recognized parasite. Both of these isolates were obtained

from patients with UM.

3.3.3. Correlation between parasite donor age and parasite-

VSA recognition rate

For each parasite the recognition rate counts for how many samples of the tested

plasma recognized that specific parasite. When we correlated age of parasite donors

with the frequency of the parasite VSA recognition rate by different panels of

plasma (antibody), there was an overall trend of an inverse relationship between

the parasite donor age and parasite recognition rate. But no significant

correlation was recognized, whether all plasma sets taken together (CC, -0.273, P =

0.288, Pearson Product Moment Correlation) or each set considered separately.

However, as seen in figure 3.10, parasites obtained from children (all were aged

between 1.5 and 11 years), were significantly more recognized than isolates

obtained from adult (all adults aged between 19 to 27 years), P = 0.021.

Plasma donors

MF SM D-0 UM D-0 SM D-28 TZ

Pro

porti

on o

f res

pond

ers

(bar

s ar

e 95

% C

I)

0

20

40

60

80

100

120

Clinical groups of parasite donors

Individual parasites recognition

rate (%)(plasma from S

M-D0)

0

20

40

60

80

100

SMA CM UM

SMA (4 isolates)CM (6-7 isolates)UM (9-10 isolates)

Figure 3.8. The prevalence of VSA Abs in different sets of plasma obtained from malaria free donors (MF), patients with; uncomplicated malaria (UM), severe malaria at the time of diagnosis (SM-D0), and at convalescence (SM-D28) and Tanzanian children with UM (TZ). The isolates were obtained from patients with SMA, CM and UM, The insert shows the reactivity to the individual isolates in each group of the

it

Plasma donors (D0)

SMA MAC CM

Pro

porti

on o

f res

pond

ers

(%)

-10

0

10

20

30

40

50

60

70

80

90

100

B

Clinical groups of parasite donors

SMA CM UM

Ove

rall

para

site

s re

cogn

ition

rate

s

-10

0

10

20

30

40

50

60

70

80

90

100

A

3.3.4. Plasma reactivity (VSA-Ab spectrum) in malaria

patient groups and asymptomatic malaria free individuals

Children (3-5 years of age) living under conditions of high transmission in

Tanzania had much higher levels and a broader spectrum of anti-VSA antibodies to

the parasites collected from Sudan than Sudanese children and adults living under

low and seasonal transmission (figure 3.11).

Figure 3.9.a. The prevalence of VSA antibodies in subsets of plasma obtained from patients with different entities of severe malaria; severe malaria anemia (SMA), malaria associated convulsions (MAC), and cerebral malaria (CM). Figure 3.9.b. The overall prevalence of antibodies against VSA antigens expressed by isolates obtained from patients with different clinical presentation of P. falciparum malaria; SMA, CM and UM.

2D Graph 4

Parasite donors (age)

Para

site

reco

gniti

on ra

te (%

)

0

20

40

60

80

100

120

Children (1.5 - 11 years) Adults (19 - 27 years)

Among the Sudanese donors the VSA-levels were higher in the patients than in the

asymptomatic malaria-free individuals. There was a tendency that the levels were

higher in patients with mild disease compared to patients with severe disease, but

this difference was not statistically significant (figure 3.8).

Plasma obtained at diagnosis from patients with severe malaria was divided into

three sub-sets; SMA (n=37), CAM (n=9) and CM (n=17), and data were re-analyzed to

compare between the three groups. Results showed that plasma from CM patients

recognized the different isolates in 190 of 370 occasions in which CM plasma mixed

with parasites (51.4%, 95% confidence interval, CI of 46.3 and 56.4). In SMA

plasma, the overall parasite

Figure 3.10. The correlation between age of parasite donors

(children aged 1.5 to 11 years and adults aged 19 to 27

years) and the proportion of plasma from all panels able to

recognize the 2 groups of parasites. Parasites obtained

from children statistically significantly more recognized

than isolates obtained from adult.

(A)

TZ plasma donors age (years)

1 2 3 4 5 6

(A)%

of i

sola

tes

reco

gniz

ed

0102030405060708090

100110

(B)

Age of Sudanese plasma donors (year)0 10 20 30 40 50 60 70

% o

f iso

late

s re

cogn

ized

0

20

40

60

80

100

120

Figure 3.11. The correlation between age of plasma donors and

prevalence of antibodies against VSA expressed by all 22-parasite

isolates. The plasma donors were Tanzanian children (A) and all

other Sudanese plasma donors (B).

recognition rate was 30.6%, CI95% of 27.2 and 34.1 (212/692), which was

significantly lower than in CM. (P = 0.001, Chi-square), while CAM plasma had an

intermediate capacity for parasite recognition of 39.9%, CI of 32.8 and 47 (73/183)

(figure 3.9A). Furthermore, analysis was done for testing the reactivity of the 3 sub-sets of

plasma from SM patients with the 3 categories of isolates used in the experiments,

the SMA, CM and UM parasites. Results showed no change in the order of parasite

recognition seen in figure 3.8 by plasma sub-groups, irrespective of parasite

source i.e. no unique pattern of reactivity between CM parasite with CM plasma or

SMA parasite and SMA plasma.

3.3.5. Correlation between plasma donor age and VSA antibody prevalence

Whether taken together or considered separately as different sets of plasma donors,

there was no correlation between age and prevalence of VSA antibodies (CC= 0.279 to

– 0.089 and P=0.51 to 0.76), except for plasma obtained from patients with UM where

there was an inverse relationship (CC=0.012, P = 0.93, Pearson Product Moment

Correlation). However, there was a general trend of insignificant decline in

prevalence of VSA antibody with increasing age for the plasma from Sudanese donors.

On the other hand there was a positive correlation between age of Tanzanian plasma

donors and the frequency of parasites VSA recognition, but still not statistically

significant (CC=0.279, P=0.168) (figure 3.11).

3.3.6. The reactivity of acute (D0) and convalescence (D28) plasma

against homologous and heterologous parasite isolates

At the time of diagnosis, 73% (8/11) of the patients with SM and 43% (3/7) of

patients with UM had detectable VSA antibodies against their own parasites (table

3.9). Convalescence samples obtained 28 days after initiation of the treatment were

available from 8 patients with severe disease. In 5 of the patients there was a

marked increase in the Anti -VSA titer to the homologous parasite and in three an

overall better recognition of heterologous parasites.

Table 3.9. The parasite donor's characterization, levels and spectrum of their plasma antibody

reactivity at diagnosis (D0) and convalescence (D28) against variant surface antigens (VSA) of

parasites isolated from them (homo) and other donors (hetero) respectively.

Ab response to isolates

at Day 0

Ab response to isolates

at Day 28

Plasma

Donors

Type of

Donor

Homo isolate

(Ab level)

Hetero isolates

(% responders) Homo isolates

Hetero isolates

(%)

MG29 UM 1.5 0 04% (01/22)

MG16 UM 3.6 0 04% (01/22)

MG95 UM 5 0 09% (02/22)

MG35 UM 8 0 68% (15/22)

MG50 UM 11 2 26% (04/15)

MG51 UM 6 2 50% (10/20)

MG31 UM 27 2 68% (15/22)

Plasma samples were not taken at D

UM patients

SG60 SMA 1.5 0 10% (02/19) 5 ND

SG90 SMA 2 1 16% (03/18) 0 26% (04/15)

SG65 SMA 9 1 31% (06/19) ND ND

SG112 SMA 22 4 94% (18/19) ND ND

SG45 CM 9 0 63% (14/22) 0 52% (10/18)

SG93 CM 11 0 00% (00/22) 1 30% (06/20)

SG21 CM 5 1 68% (15/22) 4 68% (13/19)

SG9 CM 8 1 81% (18/22) 4 95% (19/20)

SG35 CM 25 2 23% (05/21) 1 50% (10/20)

SG43 CM 5 3 63% (14/22) ND ND

SG42 CAM 9 1 50% (11/22) 3 57% (11/19)

DISCUSSION

The first objective of this study was to characterize the clinico-epideiological feature of severe malaria

from a unique setting in an area of markedly unstable transmission. The assessment of the impact of the

development of immune response against the Plasmodium falciparum merozoite surface protein and

variant surface antigens was the second objective of this study.

The malaria transmission in Gedarif area, as in most of central Sudan (where ∼ 70% of Sudan population

live), is confined to a short window of two or three months each year, and sometimes is shorter or absent.

Under such pattern of transmission, a partially protective immunity is acquired and the inhabitants are best

described as semi-immune population (40,196).

The overall frequency of severe malaria (SM) among the confirmed cases of malaria was 4.4%, and the

mortality rate among the severe malaria patients was 6.4%, which accounts for 0.3% of all cases of

malaria, this finding is coherent with a previously reported rate from other settings (203). Only four types

of complications were recognized during the study period, the severe malarial anemia (SMA) was the

commonest (45.4%), followed by convulsions (CAM), cerebral malaria (CM) and hypotension (HTN),

and 5.4% of the patients had multiple complications. In general, the difference in the rate of severe

malaria morbidity and mortality between the two seasons was not statistically significant, but the

frequency of the individual complications changed considerably. While the SMA represented 26.4% of all

complications in the first season it was increased by 2.4 fold (63.2%) in the second season, the high

frequency of SMA was not due to multiple visits of individual patients. But, whether or not the unstable

transmission of malaria accelerates the change of parasite population and consequently the pattern of

clinical complications is not known and worth prolonged observation. Some complications were not

recognized during the two malaria seasons e.g. respiratory distress syndrome and renal disorders. These

complications are commonly recognized in regions like South East Asia (131,204). The absence of the

respiratory distress syndrome cases among the study population may be due to the fact that most of

patients with this syndrome die before reaching the hospital.

Previously, it was reported that uncomplicated malaria in the same area was recognized in all age groups,

and individuals aged between 5 and 19 years had the highest risk (40). In this study, the highest proportion

of patients with mild malaria was recognized in the same age group (5 – 19 years), while that for severe

malaria was in the age group of two to four years. The earlier protection from severe malaria compared

with uncomplicated malaria is believed to be due to an earlier and faster acquisition of immunity against

parasite strains causing non-cerebral severe malaria (205). However, due to the inherent limitations in

urban hospital-based studies in general (not all malaria patients seen in hospital) it was difficult to

calculate with accuracy the incidence and the age risk for both mild and severe malaria.

There is some evidence that malaria infection in the older age groups results in a different form of

complications as compared to that seen in children, in areas of stable transmission (206). In these areas,

distinct age-specific patterns of infection and disease are seen (207,208). In this study, we showed that the

different clinical groups of severe malaria (anemia, convulsions, cerebral malaria, and hypotension) differ

from each other in critically important clinico-epidemiological indices. We compared five parameters; the

age, parasite count, hemoglobin level, blood glucose level, and mortality rate, among the groups. Taken

together, the four clinical entities of severe malaria were found to be strongly statistically significantly

different from each other in all the five tested parameters (p=0.022 – 0.000, Kruskal-Wallis test).

However, the comparison between these complications showed that the greatest difference was between

CM and SMA, and the least difference was between CM and convulsions. The similarity between CM and

convulsions (except for the age), and the dissimilarity between CM and SMA on clinical and

epidemiological aspects, looks interesting, and possibly denote resemblance and disparity in pathogenesis

of these complications, respectively.

The difference in the age distribution of the different complications in this setting, and between this and

other settings, was marked and of special importance. It is well known that, there are differences in the

peak age for CM compared to SMA and also in the distribution of CM and SMA depending on the level of

Plasmodium falciparum transmission in the area (203,207). The SMA peaks at the age of 6 months to one

year and CM at the age of two to three years in hyperendemic areas (209-211). In this study, the mean age

for SMA was around five years, while that for CM was approximately 14 years. The mean age for both

complications, was three times higher in our study area, while the ratio of the peak age for the two

complications in each site is constant, (a ratio of approximately 1:3). Hypotension was only recognized

during adulthood with a mean age of 35 years; however, the outcome of treatment was good and fast, with

the shortest duration of hospital admission. On the contrary, the mean age for malarial convulsions was

similar to SMA, five years. In general, more than 60% of patients with severe malaria were above five

years of age, while all patients who died of severe malaria were above this age (with a mean age of 18

years).

The lowest parasite count was recognized in patients with SMA, the youngest group of patients, a low

parasite count was also recognized in individuals with hypotension, the eldest group. The low parasite

count in patients with hypotension could be due to the acquired anti-parasitic immunity which increases

with age (47). However, the role of age-associated acquisition of immunity can not explain the low

parasite count in patients with SMA.

Generally, in other studies hypoglycemia is considered as one of the complications of Plasmodium

falciparum malaria with a prevalence of 10%, it is ascribed to inhibition of gluconeogensis (212), and is

considered as independent risk of mortality in severe malaria (213). In this study the blood glucose level

was highest in patients with CM and lowest in patients with SMA, the difference was significant; also,

hyperglycemia was very remarkable in the fatal cases of malaria.

In other studies the glucose production, glucose concentration and gluconeogenesis in UM patients were

significantly increased, whereas glycogenolysis is decreased and the mechanism by which these changes

occur is uncertain (214,215). The counterregulatory hormone concentrations were found higher compared

to healthy control subjects (215,216). In CM patients a doubling in glucose production was reported and

the authors conclude that CM is characterized by massive stimulation of gluconeogenesis, where the blood

glucose was found to be 100% derived from gluconeogenesis. The plasma insulin and the hormones that

antagonize its action were found comparable to those in healthy control except the elevation of plasma

cortisol (5 times) which is higher than the

elevation observed among UM patients (2 fold) (212). Hyperglycemia also has been reported by Chadee

and others to be associated with cerebral malaria (217).

In this study, hyperglycemia in fatal severe malaria was not due to quantitative deficiency of insulin, on

the contrary insulin levels were extraordinary higher than in nonfatal severe malaria and above the upper

physiological limits. Using animal models infected with P. coatneyi, Davis and his coworker (218)

reported the occurrence of hyperglycemia and insulin resistant in severe malaria and found biochemical

and histological evidence of severe hepatic pathology. Another study reported reduced tissue insulin

sensitivity during acute malaria compared with convalescence and also they reported a reduction in liver

blood flows that associated with hyperlactatemia in fatal malaria cases (219).

The strong association between hyperglycaemia, hyper-insulinaemia and cerebral malaria fatality, need

further biochemical explanation. It is of interest to know, how hyperglycemia and hyperinsulinemia

coexist, and which preceded the other. The other observation is that the youngest patient who died of

severe malaria had a relatively lower blood glucose level and insulin level, although the sample size is not

large enough to make such a judgment.

In this setting, the CM had an average mortality rate of 39% (45% and 29% in the first and second season,

respectively), while there was no mortality from other complications. We compared levels of hemoglobin

and blood glucose between severe fatal and severe non-fatal malaria. To our surprise, patients died of

severe malaria had higher hemoglobin and glucose levels when compared with patients survived the

severe attack of malaria. Interestingly, none of the CM patients who also had SMA died (2 patients).

In order to link the clinical patterns with the immune response to known malaria antigens, the antibodies

directed toward specific Plasmodium falciparum malaria antigens (Merozoite surface proteins and variant

surface antigens) were measured. The importance

of the antibody in the modulation of the disease was reported in previous studies, where; hyperimmune

sera from African adults were used successfully for management of children or non-immune patients with

Plasmodium falciparum malaria (99-101). That was an evidence for the importance of antibodies as part

of the protective, naturally acquired immunity in adults living under intense malaria transmission, since

the passive transfer of antibodies relief the malaria symptoms. Thousands of parasite antigens were

identified so far, but antigens eliciting and boosting sufficiently protective immunity were not well-

defined.

The merozoite surface proteins (MSP) were among the major vaccine candidate proteins (220), and in

natural infections they were found to modulate infection outcome in uncomplicated malaria

(158,159,162). However, little is known about the immune response of patients with SM against MSP

antigens (221). Even more uncommon knowledge is the comparison between the individual complications

of SM with regard to the MSP response.

The IgG immune responses against the individual MSP antigens (MSP119, MSP2A and MSP2B), and all

three Ags taken together (MSPall), were measured. The same data was analyzed from three different

aspects, to know if any could be related to protection; a. the prevalence of antibodies b. the level of

antibodies against the recognized antigens c. the number of recognized antigens. Comparisons were made

between different study groups and sub-groups as explained hereunder. The comparison was made in the

following order: 1- Between malaria patients and healthy malaria-free donors. 2- Between patients with

SM and UM. 3- Within the SM group, between patients with SMA, CAM, CM, HTN. 4 - In the CM sub-

group, between fatal CM and non-fatal CM.

In this study the prevalence of antibodies against MSP antigens, was significantly associated with partial

protection from SM. Although, the prevalence of antibodies to MSP2A &B was higher in patients with UM

compared with SM, the most pronounced effect was that of MSP119 antibodies. Furthermore, the number

of the MSP antigens (maximum of three) recognized was also significantly associated with protection

from SM. However, we found no significant difference in the level (concentration) of anti-MSP antibodies

between patients with SM and UM.

The protective effect of anti-MSP antibodies against malaria was thought to be mediated through

inhibition of erythrocytes invasion by merozoite (150) and that was confirmed in in-vitro studies (222).

The above-mentioned mechanism implies that, anti-MSP antibodies reduces the parasite multiplication

(one of the risk factors in SM) rather than prevents the establishment of the infection. That is very much

coherent with our findings in which the high prevalence of MSP antibodies didn’t prevent the occurrence

of UM, while the lower prevalence of these antibodies (in the SM group) could be incriminated in the

progression of the disease to the severe form. A worth noting point about our epidemiological setting is

that the population is semi-immune to malaria, accordingly, infection mostly presents clinically as

symptomatic malaria. This study was designed in such a way that it aborts most of the bias possibilities

referred to by Dodoo and others (163) regarding the difference in exposure of the study groups, by

including SM and UM patients in the study. Both groups of patients were similarly exposed and sampled

at the same time. Thus, our findings regarding the protective effect of anti-MSP antibodies against SM but

not UM, can explain the contradicting results of other studies, that support (155, 158) or not (163) the

protective role of MSP antibodies.

The antibody response to MSP antigens was consistently and significantly age–dependent, half of the

donors between 1 and 5 years of age were found to have anti-MSP antibodies, and all donors above 45

years were responders to one or another MSP antigen. The findings of high prevalence and age-

dependence of MSP antibody response were opposite to the findings from West Africa (163), even if we

correct for age differences of the study populations. The age dependency of the acquisition of anti-MSP is

in agreement with (158,160,223).

Further analysis of the SM data, revealed significant differences in prevalence of anti-MSPall antibodies in

patients with HTN (high) compared with CAM and SMA (low). The prevalence of MSP Abs was higher

in CM than SMA patients, the difference was not significant, but the prevalence in CAM was significantly

lower than in CM, although the ages of SMA and CAM were comparable. The differences in MSP

antibody prevalence between the SM subgroups was largely but not absolutely due to age factor, that

means there was other complication-specific pattern of response to MSP antigens. The subgroups of this

same cohort of SM were found to differ significantly in a number of clinico-epidemiological parameter as

reported above.

Around 40% (7/18) of the CM patient died, unexpectedly, the MSP antibody response (prevalence and

concentration) was generally high in CM, and it was slightly but not significantly higher in fatal CM than

non-fatal CM. The protective effect of anti-MSP antibodies against malaria was thought to be mediated

through inhibition of erythrocytes invasion by merozoite as mentioned before (150). If this is true these

antibodies are not fine specified where the fine specificity (blocking antibodies with overlapping epitopes

with the inhibitory antibodies that have minor differences in the binding specificity) of natural acquired

human antibodies were reported to have marked variation between individuals and were found crucial in

determining their functional efficacy (224). These detected antibodies might not be of the cytophilic

isotype where the IgG1 and IgG3 were found to predominate in protected subject (225). Or there is a sort

of polymorphism such as that of FcγRIIa (CD32) among the study population (88)(Cooke et al. 2003).

This finding may indicate that, the pathogenesis of CM involves factors more important than the

erythrocytes invasion by merozoite and the parasite multiplication.

The apparently healthy, MF donors, had significantly low prevalence and concentration of anti-MSP

antibodies and the number of recognized MSP antigens (MSP119, MSP2A, MSP2B) compared with malaria

patients. About 19% of the MF donors had PCR-detectable parasitemia. But, there was no difference in

the MSP antibody response in MF donors with or without PCR-detectable parasitemia. It could be that, in

asymptomatic donors, submicroscopic parasitemia is not sufficient to elicit MSP antibody response as in

symptomatic submicroscopic parasitemia (226). The low anti-MSP antibodies response in MF donors was

an indicator for low exposure to malaria rather than increased susceptibility to SM.

Considering the different aspects of the antibody responses with respect to clinical outcome of malaria

infection, it seems that the prevalence of MSP antibodies were more important than the level of MSP

antibodies, in this setting. However, significantly lower levels of MSP antibodies were recognized in

apparently healthy MF donors, where it was considered as a sign of exposure. In other studies the

protective role of anti-MSP antibody response was mainly attributed to the level of antibodies

(155,160,227,228).

The most immunogenic of the three MSP antigens, was MSP119, followed by MSP2A and the least

recognized was MSP2B. This result is in agreement with Riley and her colleagues (158) where the highest

prevalence of antibodies being detected against regions which are highly conserved between parasites

isolates. There was a consistency in this pattern of antibody response in all groups and sub-groups of

plasma analyzed except for the plasma obtained from patients with SMA, in which the most recognized

antigen was MSP2B. The prevalence of antibodies against MSP antigens in patients with SM was not

significantly changed one month after malaria diagnosis. This antibody response inertia over the

convalescence period in SM patients was in line with previous study carried out in Gabon (221).

Interestingly, while the prevalence of the MSP antibodies at diagnosis (D0) was strongly age-dependent,

the acquisition of this Abs over the convalescence period (28 days) was not affected by age or type of SM.

The explanation for the above findings is that, in SM, either the parasites have fast boosting effect on the

MSP antibody response, or the patients with SM were more sensitive to MSP antigens. Thus, patients with

SM have quick response revealed in a form of a high prevalence (63.3 – 100%) of Abs within few days

after infection (D0), and no significant change thereafter. For further simplification, the high prevalence of

anti-MSP antibodies at diagnosis (D0) might not be due to a pre-existing antibody, but was likely to be

due to the fast triggering of response (boosting) following infection. The finding supported that; the MF

donors had significantly low prevalence of antibodies compared with the malaria patients (at Do), in this

setting. It is interesting if we can confirm that, the prevalence of anti-MSP Abs was mostly boosting of

antibody response by current infections, in semi-immune population.

Plasmodium falciparum erythrocyte surface protein 1 (PfEMP1) is a variant antigen expressed on the

surface of infected erythrocyte and serve as the parasite- sequestration ligand to endothelial cells. This

cytoadherence in the deep circulation or the cytoadherence of PRBC with two or more uninfected RBC

(rosetting) is regarded as an important virulence factor. PfEMP1 antibodies were believed to protect from

disease by inhibiting sequestration, thus facilitating the destruction of infected erythrocytes in the spleen.

In addition antibodies disrupting erythrocyte rosettes have been reported to be associated with protection

from cerebral malaria.

A total of 4120 parasite/plasma samples were evaluated by flow cytometry to estimate the prevalence and

level of VSA antibodies against VSA antigens expressed by parasite isolates obtained from patients with

different clinical grades of malaria. In general, it is believed that the host immune competence with regard

to VSA antibody response indicates the number of variants recognized and the level of antibody acquired.

In this line, the plasma that is able to recognize variants expressed by large number of parasites, on

average contains higher level of antibodies than the plasma that recognized small number of isolates. This

suggests a link between the two immunological indices; the ability to recognize large number of parasites

and the ability to mount high level of antibodies against the recognized isolates.

The overall pattern of interaction between three clinically distinct groups of parasites with the five panels

of plasma revealed an association between VSA expression/response and malaria infection outcome.

However, in figure 3.8, the three lines (parasite groups) did not cross, indicating differences between the

different clinical entities of malaria with regard to VSA expression/responses. This whole pattern of

interaction, suggest that neither VSA expression nor VSA Ab response are random events, but are

relatively structured by the degree of virulence. It was stated before that, following Plasmodium

falciparum infection (convalescence), there is an induction of VSA Ab response to

homologous and heterologous isolates (2). In addition to that, we here report that, infections with parasites

which caused SM induced VSA Ab response to parasites caused the UM. Importantly, the convalescence

plasma from patients with SM recognized the broadest range of isolates compared to the other panels of

Sudanese plasma. Another interesting finding was the strikingly high proportion of Tanzanian responders

(aged 3 – 5 years) to VSA of the Sudanese isolates compared to Sudanese plasma donors (aged 1.5 to 55

years). Thus, the major determinant of VSA antibody response in this situation was the endemicity of

malaria in the place of the plasma donor as shown before (229) (hyper-endemic in Tanzania and meso-

endemic in Sudan) rather than the age of the donors.

The VSA expression of parasite causing SM and UM were different. The best recognized parasites

isolates were obtained from patients with SMA followed by CM and in all instances they were better

recognized than UM parasites, and the differences were statistically significant. The trend was maintained

even when each set of plasma was considered separately, but the differences were below the statistical

significance. This finding is in line with previous observations (37,230-232). The wide recognition of

VSA expressed by SMA parasites either indicate; a. a restriction in the number of expressed variants

(231), b. a dominance of the causative parasite due to chronicity of infection or c. a higher

immunogenecity of variants expressed by severe malaria parasites, specifically SMA.

An inverse correlation between the ages of the parasite donors and recognition was found i.e. isolates

obtained from younger patients were better recognized, irrespective of the type of malaria the patient had.

This general trend was consistent whether the different sets of plasma were analyzed separately or not, but

in both situations the correlation was not statistically significant. However, when the parasite donors were

divided into children (1.5 - 11years) and adults (19 - 27 years), isolates obtained from children were

statistically significantly better recognized. This observation confirms previous reports when only

uncomplicated malaria samples were used (2), and when both SM and UM samples were used (229,231).

This consistent finding indicates that, VSA is unique among other malaria antigens, since the cassette of

variants expressed by the parasite is somehow determined by the host age. That is independent of VSA Ab

immune pressure, as the VSA Ab prevalence was not age-dependent in our setting.

Among the Sudanese donors the VSA-levels were higher in the patients than in the asymptomatic malaria-

free individuals. There was a tendency that the levels were higher in patients with mild disease compared

to patients with severe disease, but this difference was not statistically significant. This is in agreement

with the results obtained from Gabon where the VSA antibody level was found significantly lower among

severe malaria children than their match counterpart mild malaria patients (232). Where this low VSA

antibody was proposed to be associated with severe malaria and increased susceptibility to malaria attacks.

An important and unexpected finding was the significantly broader spectrum of reactivity of pre-existing

VSA Ab in plasma of patients with CM compared with SMA and CAM at the time of diagnosis. However,

the broadness of plasma reactivity of CM and UM patients were comparable, so, it is more tempting to

describe SMA patients as having a significantly limited (lower)VSA Ab response. The younger age of

SMA patients cannot explain the above observation as we mentioned before. These observations argue the

immune competence of patients with SMA. This assumption is supported by the failure of patients with

SMA to clear their chronic parasitemia for months. Equally, it can be said that, patients with CM had a

normal VSA Ab response at least to the heterogonous parasites i.e. the patients with CM were not

immunologically jeopardized.

The VSA Ab response was age independent in this setting. Thus there was no positive correlation between

plasma donor age and prevalence of antibodies, rather in the other panels of plasma there was decline in

prevalence of antibodies with increasing age. The lack or the slightly inverse correlation between age of

plasma donor and parasite recognition rate can explain the susceptibility to malaria in all age groups, in

this setting. An inverse relation between the age of the plasma donors and parasite recognition rate has

been reported earlier in a study of a cohort of Gabonese children, in an area of low transmission (232).

However, other studies conducted in malaria hyperendemic areas, proved the opposite (37,231). The age-

independent acquisition of VSA Abs is more indicative for protection than exposure.

Although our sample size was not large, the presence of low level of VSA Ab against the homologous

isolates at the time of diagnosis was more common in SM than UM. This is in agreement with the study

done by Kinyanjui and others (233) where they found small proportion (19%) of patients had antibodies to

the infecting parasites. On the other hand this result is in contrast to a study of Kenyan children where

homologous antibodies were absent at the time of diagnosis (58). The reasons for these discrepancies

could be that in their study the materials were obtained from patients with UM, that they exclusively used

children, or the method used was the less sensitive technique, the fluorescent microscopy. The presence of

homologous antibody at the time of diagnosis might reflect the duration of infection before presentation as

acquisition of this Abs, as reported before (233) take about two weeks and their decay up to few months

(234). If that is the case, then SM (including CM) took longer time before presentation than previously

expected or at least not less than UM do.

4.1 Conclusions

• Severe malaria (SM) accounts for 4.4% among the confirmed cases of malaria

• The mortality rate among the severe malaria patients was 6.4%, which accounts for 0.3% of all

cases of malaria.

• Only four types of severe malaria complications were reported, the severe malaria anemia (SMA)

was the commonest (45.4%), followed by convulsions (CAM), cerebral malaria (CM) and hypotension

(HTN), and 5.4% of the patients had multiple complications. • A distinct clinical and immuno-epidemiological pattern of severe malaria was found, which is

markedly different from that described in areas of high malaria transmission.

• The three merozoite surface proteins (MSP119, MSP2A and MSP2B) studied were

immunogenic and the prevalence of antibodies against MSP antigens was significantly associated with

partial protection from SM.

• The pronounced effect was that of the conserved fragment of MSP1 (MSP119) which is the most

immunogenic MSP antigens studied.

• On the other hand VSA immunogenicity was found to differ from parasite isolate to another.

• No difference was observed between the acquisition of Ab against conserved and polymorphic MSP

Ag, where the responsiveness were age dependent. On the other hand the acquisition of Abs against the

variant antigens (PfEMP1) was age independent.

• VSA is unique among other malaria Ags, since the cassette of variants expressed by the parasite is

somehow determined by the host age.

• There was an association between VSA expression/response and malaria infection

outcome, suggesting that neither VSA expression nor VSA Ab response are random

events, but are relatively structured by the degree of virulence.

• The best recognized parasite isolates were obtained from patients with SMA

followed by CM and in all instances they were better recognized than UM parasites.

• On the other hand, CM plasma had the broadest spectrum of VSA Ab reactivity

compared with SMA patients who can be described as having a significantly limited

(lower) VSA Ab response.

• The patients with CM were not immunologically jeopardized because they had

normal Ab response compared to UM patients, at least to the heterologous parasites,

which may indicate the involvement of other factors.

• MSP (conserved and polymorphic Ags) responses among SM patients were not significantly

changed one month after malaria diagnosis, whereas convalescent plasma from patients with severe

malaria developed VSA Ab that recognized the broader range of isolates VSA compared to D0 response.

4.2. Recommendations

• The strong association between hyperglycaemia, hyper-insulinaemia and cerebral malaria fatality,

need further biochemical explanation. • Study of the antibody subclasses and if possible their fine specificity (minor differences in the

binding specificity).

• Study of the different type of polymorphism that might occur in this population and lead to the

modulation of the immune response such as FcγRII.

• Finally, the findings of this study in general, suggest that the immunity against severe malaria might

not be similar to that against uncomplicated malaria, and we support the idea of developing malaria

vaccines of selective proficiency.

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APPENDIX I: Equipment, material and reagents

Giemsa stain:

The Giemsa stock solution was prepared by dissolving 3.8 grams of Giemsa (Avondale Laboratories

(supplies and services) limited, Banbury, Oxon., England ) powder in 250 ml methanol and 250 ml

glycerol with vigorous shaking over 3 days. The working solution was prepared by diluting the Giemsa

stock 1in10 with phosphate buffer pH 7.2 and filtered.

Parasite freezing Solution: 180 ml of 4.2 % sorbitol in Normal saline and 70 ml glycerol were mixed and sterile through 0.22 µm

Millipore filter and stored frozen.

ELISA equipment material and reagents

ELISA reader (Labsystems Multiskan MCC/340)

The antigens

Recombinant proteins representing:

1- The 19 kDa C-terminal fragment of merozoite surface protein (MSP119)

2- Group A of MSP2, denoted T9/96 13/14

3- Group B of MSP2, FC27 denoted GF/88)

4 Glutathione-S-transferase (GST) protein

The conjugate

Horseradish peroxidase-conjugated to rabbit anti-human IgG, specific for gamma chain (HRP/IgG)

DAKO A/S, Denmark.

Coating buffer:

1.5 grams sodium carbonate (Na2CO3) and 2.93 grams sodium hydrogen carbonate (NaHCO3) were

dissolved in distilled water and the pH was adjusted to 9.4 - 9.6, the volume completed to one liter with

distilled water.

Washing buffer:

Phosphate buffered saline with 0.05% Tween-20:

8 grams sodium chloride (NaCl)

0.2 grams potassium chloride (KCl)

1.14 grams disodium hydrogen phosphate (Na2HPO4)

0.2 grams potassium dihydrogen phosphate (KH2PO4)

Dissolved in distilled water and the pH adjusted to 7.4 and then 0.5 ml of Tween-20 (0.05%) was added

and the volume completed to one liter with distilled water.

Substrate buffer:

Solution A: 0.1 M citric acid solution (9.6 grams of citric acid / 500ml distilled water).

Solution B: 0.2 M phosphate solution (14.2 gram of disodium hydrogen phosphate / 500 ml distilled

water).

The substrate buffer was then prepared by mixing

6 ml of solution A

6.4 ml of solution B

12.5 ml of distilled water

11 µl of 27.5% hydrogen peroxide (H2O2). From Aldrich

10 mg O-Phenylenediamine Dihydrochloride tablets. (OPD). From Sigma Chemical Company.

Stop solution: two molar sulphuric acid (2M H2SO4):

55.6 ml of concentrated sulphuric acid (BDH) was added carefully to 400 ml of distilled water and finally

the volume was completed to 500 ml with distilled water.

In vitro parasite culture and FACS (reagents and buffers)

Thawing solution (sterile 3.5 % NaCl solution in distilled water)

Washing medium (500 ml RPMI-1640 supplemented with 25 mg Gentamycin (Gibco))

Parasite culture medium (500 ml RPMI-1640 (Gibco) supplemented with 25mg Gentamycin (Gibco),

91.6 mg L-Glutamine (Sigma), 2.5 gram Albumax II (Life Technologies), 10 ml normal human serum and

10 mg Hypoxanthine (Sigma))

a. The AlbumaxII and Hypoxanthine were prepared as mixture where, 100 grams Albumax II and 400 mg

Hypoxanthine were dissolved in 2 liter of RPMI-1640 and then the solution was filtered through 0.8 mm

and 0.2 mm filters, then the sterile solution was kept frozen in 50 ml portions till used.

b. 2.5 ml of Gentamycin stock (10 mg / ml) per 500 ml RPMI-1640 medium.

Uninfected blood Uninfected blood type O Rh- or Rh+ collected in sterile filtered Citrate-Phosphate-Dextrose solution

(CPD) washed 3 times in washing medium and the puffy coat removed the packed cells were suspended in

washing media and stored in the fridge at 4 OC.

MACS equipment, material and reagents

VarioMACS magnet (Miltenyi Biotec)

Size CS column (Miltenyi Biotec)

3-way valve for the column

Top-adapter for column (Miltenyi Biotec)

Outlet needle for the column (0.9 mm*40 mm 20G needles). The dimensions of the outlet needle regulate

the flow rate through the column.

Nippers for securing the needle, for safety the tip of the outlet needle was cut by a pair of nippers before

passing infected material through the column.

Phosphate buffered saline with 2% fetal calf serum (PBS2).

FACS equipment, material and reagents

-2-colour flow cytometer

Phosphate buffered saline plus 2 % fetal calf serum (PBS2), Plasmodium falciparum late stage infected

erythrocytes purified by magnetic separation at 2.5 * 106 erythrocytes/ml in PBS2.

Ethidium bromide (0.1 mg / ml in PBS).

Human plasma.

Goat anti-human IgG antibody (DAKO A473).

Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-goat IgG antibody (DAKO F250).

APPENDIX II Severe Malaria Protocol, Clinical questionnaire

Gedarif teaching hospital & New Helfa Hospital Sample I D:………. Dates: admission: …/…../2000; discharge …/…../2001 Name: ……………………………………………………….. …….Gender: M ( ), F ( ) Date of birth: …………………….. …………… age: ……………………………………………. Residence (detailed): …………………………………………………………….…………….…… Contact address:…………………………………………………………………….…………..…… Occupation: …………………………………………………………………………………………… Tribe: ………………………………………………………………………………………………….. For females: pregnant ( ), lactating ( ). Last menstrual cycle ………………….

C/O: 1.History obtained from: patient ( ), relative ( ) 2.Fever : A/ Duration: …………….. Days. B/ Grade: mild ( ), moderate ( ), severe ( ) C/ continuous ( ), intermittent ( ). D/ regular ( ) irregular ( ). E/ rigors (Yes, No ). F/ sweating (Yes, No ). 3.General ill health, headache ( ), joint pain ( ), back pain ( ), fatigue ( ), bitter taste ( ) 4.Bleeding: no bleeding ( ), epistaxis ( ), haematemsis ( ), haemoptysis ( ), bleeding from other site (……………………………….). 5.CNS: confusion ( ), convulsions ( ), loss of consciousness ( ), Hallucination ( ), Coma (Y, N), duration ……… Seizures : (partial, general), (L side, R side), duration…… 6.Abdominal symptoms: pain ( ), nausea ( ), anorexia ( ), vomiting ( ), diarrhoea ( ). 7.Respiratory symptoms: Cough ( ) , breathlessness ( ). 8.Urinary system: anuria, polyurea, haematurea Medical History: 1. Malaria history • Number of attacks ≥ 1 a year ( ), 1 every two years ( ), 1 ≤ every three years ( ), not at all (). 2. History of severe malaria (Yes, No), what type……………………… • Last clinical attack:…………………… Method of diagnosis : laboratory ( ), clinical ( ) • Treatment received: ………………………………………………………………… 3. History of: Diabetes mellitus ( ), hypertension ( ), renal diseases ( ), asthma ( ), other diseases : ………………………………………. 4. History of prolonged drug use (more than 1 week): ……………………………………… Clinical examination: General condition: A/ ill ( ) well ( ); B/ Normal ( ), pale ( ), Jaundiced ( ), cyanosed ( ) C/ drowsy ( ), confused ( ), comatose ( ), convulsions ( ), stiff neck ( ). Oral Temperature ……..°C Weight….kg Nails: normal ( ) clubbing ( ), spooning ( ). Blood pressure: ……………, Pulse ……………….., RR ……………… Speech: normal ( ), slurred speech ( ), dysphasia ( ). Tongue: normal ( ), dry ( ), coated ( ), angular stomatitis ( ). Skin: dry ( ), rash ( ).

Systemic examination 1. Central Nervous System: ………………………………………………………………. Position: normal ( ), decerebrate ( ), decorticate ( ), opisthotonus ( ). ……………………………………………………………………… 2. Respiratory system: rhythm (regular, irregular), hyperventilation ( ), deep slow breathing ( ), shallow fast breathing ( ) intercostals recession ( ). 3. Gastrointestinal system : Liver size, ……cm , spleen,………cm Others……………………………………………………………………………………… 4. Genitourinary system: ………………………………………………………………………. 5. Muscle-skeletal system: …………………………………………………………………….. Investigations: Blood film for malaria: BF…………. Species: falciparum ( ), vivax ( ), malariae ( ), ovale ( ). Parasitxe count: ………/µl Complete haemogram : Hb ……% (……gm/dl), Haematocrit ……….. Reticulocytes………….TWBC: …………, lymphocytes …….%, Neutrohpils ……%, …….Basophils ……%, Esinophils ……% Thrombocyte count …………………. ESR (1 hour) …….. Biochemical analysis: Blood glucose: …………………………Urine general: …………………………………. - Renal Function Test : Blood : pH …….. Urea . …………., serum creatinine…………plasma bicarbonate. ……… Widal test for typhoid : ………………………Widal test for brucelosis : ………………………… Chest X-ray (when needed) : …………………………………………………………. Current treatment 1- Anti-malarial……………………………………………2.Fluid:……………………………… 3. Other drugs ……………………………………………4. Blood transfusion:………………. Annex (I) For Cerebral malaria (Adults)

Eye: Ptosis ( ), Nystagmus ( ), squint ( ).

Pupils: normal ( ), dilated ( ), constricted ( ), fixed ( ).

Fundi exam: …………………………………………………………………..…………………….

Cranial nerves: ………………………………………………………………………………………

Upper limbs: ………………………………………………………………………………………….

Lower limbs: ………………………………………………………………………………………….

Plantar reflex (down going, equivocal, up going)

Modified Glasgow coma scale ( ) a. Best motor response b. Verbal response c. Eye movement 5 - Obeys commands ( ) - Oriented ( ) - ………………… 4 - Localized pain ( ) - Confused ( ) - Spontaneously ( )

3 - Flexion to pain ( ) - Inappropriate words ( ) - To speech ( ) 2 - Extension to pain ( ) - Incomprehensible sounds ( ) - To pain ( ) 1 - None ( ) - None ( ) - Never ( )

Annex (II) For Children

Fontanelle; normal ( ), sunken ( ), bulging ( ), absent ( ).

Can the child sit without support (Y,N), drink/breastfeed (Y,N).

Nasal flaring (Y,N)

Blantyre coma scale a. Motor response b. Verbal response c. Eye movement 2. - Localized painful stimulus - Appropriate cry - 1. - Withdraw limb from pain - moan / inappropriate cry - Directed 0. - Non-specific or absent response - None - Not directed

Annex (III) For pregnant ladies Gravidity: ……………………………………………………………………………………………... Abortions: No…………… causes: fever ( ) others ( ). Current pregnancy: …….weeks. 1st trimester ( ), 2nd trimester ( ), 3rd trimester ( ).

Follow up Day 3 ………………………………………………………………………………………………… Day 7 ………………………………………………………………………………………………… Day 28 ………………………………………………………………………………………………… ………………………………………………………………………………………………………

THE CONSENT

h

الموافقة عن علم

جامعة الخرطوم بالتعاون مع بعض المؤسسات – آلية الطب –مجموعة أبحاث المالريا التابعة إلي مرآز أبحاث المالريا -اط وخصا ة بدراسة أنم دها وظهور السودانية واألوربية مهتم ساهم في تعقي دة والعوامل التي ت ة والمعق ا المزمن ئص المالري

.في السودان) الخ ... ، األنيميا، المالريا الدماغية( الحاالت المستعصية سابقة - ة ال سنين القليل لقد تم إختيار مستشفي القضارف التعليمي لهذه الدراسة نسبًة لتفشي هذا النوع من المالريا في ال

طقة، وسيكون موضوع البحث هو دراسة وفهم طبيعة المرض وهذا الفهم لخصائص المالريا المزمنة سوف يساعد في في المن .تقليل حاالت اإلصابة وتخفيف حدتها أو علي أقل تقدير في سرعة معالجتها

ي آشف طبي متك - ا سوف يخضعوا إل وع من المالري ذا الن شفي به ي المست الهم إل تم إدخ ذين ي تم آل المرضي ال ل وت ام .متابعتهم طبيًا لفترة أقلها أسبوعين للتأآد من شفائهم التام

أيام وبعد 7 أيام، 3من المريض فور دخوله المستشفي، ثم بعد ) سي سي 5(بعد الفحص الطبي الشامل يتم أخد عينة دم -سبة (،)Complete Haemogram(شهر، هذه العينات سوف يتم أستخدامها إلجراء بعض الفحوصات مثل فحص آامل للدم ن

ا و ) المالريا(، )السكر في الدم ل المالري ول (للتأآد من خلو الدم من طفي ن يتحمل المريض أو ذووه أي ). فحص عمومي للب ول .أعباء مادية نتيجة إلشتراآه في هذا البحث

.الدراسةالحق في الموافقة أو الرفض في الدخول في هذه ) حاالت اإلغماء واألطفال( للمريض أو مرافقه - .المرضي الذين ال يرغبون في الدخول في هذه الدراسة سوف تتم متابعتهم بالطريقة المعتادة في المستشفي -ة طوال - تستفيد من إشتراآك في هذه الدراسة ألنك أو قريبك المريض سوف تتم متابعتكم متابعة لصيقة وبكثيٍر من العناي

.فترة الدراسة

. *نحن علي أتم إستعداد لإلجابة علي أي سؤال أو إستفسار •