Genetic Mutation of Malaria Drug Resistance Genes after Adoption of Artemisinin Combination

136 Pages·2017·2.14 MB·English

Sudan University of Science and Technology
College of Graduate Studies

Genetic Mutation of Malaria Drug Resistance Genes after Adoption
of Artemisinin Combination Therapy in Sudan

يف نينسي(cid:996)ت(cid:941)لال ع(cid:996)جت(cid:996)لا جلاعلا (cid:940)يفنت دعب جلاعلل (cid:916)مواق(cid:996)لا اي(cid:941)لا(cid:996)لا (cid:917)انيجل (cid:916)ينيجلا (cid:917)ارفطلا
(cid:997)ادوسلا
A thesis submitted in fulfilment for the requirements of degree of Ph.D. in Medical Laboratory
Science (Parasitology and Medical Entomology)
By
Ahmed Bakheet Abd Alla Ahmed
- B. Sc. in Medical Laboratory Science (Parasitology and Medical Entomology), Sudan
University of Science and Technology, 2005
- M. Sc. in Medical Laboratory Science (Parasitology and Medical Entomology), Sudan
University of Science and Technology, 2013

Supervisor
Prof. Mohammed Bhaa El-din

Co- supervisor
Dr. Tayseer Elamin Mohamed Elfaki

2018
ةيلآا

ميحرلا نمحرلا الله مسب

:ىلاعت لاق

ُ ه نهأه نومُ هلعْ هيهف اوُنمه آه نيذِ لا ام هأهف اهه هقوْ هف امه فه ً ةضه وُعبه امه لً هثمه به رِ ضْ يه نْ هأ ييِ حْ هتسْ هيلَه هللّ ا ن إ﴿
هِ ِب يدِ هْ هيوه ارً يثِ كه هِ ِب لضِ ُي لً هثمه اهذههَٰ ِب ُللّ اه داره هأ اهذامه ه نوُلوُقهيهف اورُ هفكه ه نيذِ لا ام هأوه ْ مهِ ِبره ْ نمِ ُ ق حه لْا
﴾ نه يقِ سِ اهفلْا لَ ِإ هِ ِب ُ ل ضِ ُي امه وه ارً يثِ كه
ميظعلا الله قدص

)26 ةيلاا ،ةرقبلا ةروس(

I
Dedication

To my parents

To my wife and daughters

To my sister and brothers

To my colleagues

To my friends

I dedicate this work.

II
Acknowledgement

All great thanks are firstly to Allah. I would like to express my gratitude and thanks to my
supervisors Prof. Mohammed Bhaa El-din and Dr. Tayseer Elamin for their guidance, helpful
suggestions, solving problems and their precious advices as well as continuous assistance
through the whole process of the research.
Thanks also extend to the members of Parasitology and Medical Entomology department and
staff of research laboratory for provided me with good materials and equipments and their
helpings.
Special thanks for patients participated in study and their guardian’s.
Finally, thanks to everyone helped me and was not mentioned.

III
Abstract
This study was carried out in three states in Sudan (Sennar, Khartoum and River Nile) to detect
the prevalence of antimalrial genetic markers; Plasmodium falciparum chloroquine resistance
transporter (Pfcrt), P.f multidrug resistance gene-1 (Pfmdr-1), P.f dihydorofolate reductase
(Pfdhfr) and P.f dihydropotrate synthesize (Pfdhps). A cross-sectional study was carried out
during the period from July 2015 to December 2017.
300 patients were included in this study their age ranged between 1- 90 years old of mean age
was 31 ± 23 years, from them 133 were males and 167 were females.
Blood samples were taken from all patients to detect malaria parasite by using Giemsa stained
blood smears.
Out of 300 positive malaria samples, the prevalence rates of these genes were 32%, 36%, 36.7%
and 33% respectively by using of restriction fragments length polymorphisms - polymerase chain
reaction (RFLP-PCR) method.
The highest prevalence mutant allele rate (47%) was reported in Khartoum state with Pfmdr,
while the lowest prevalence rate (28%) was reported in two genes (Pfcrt in Sennar and Pfdhfr in
Khartoum). Among the gender, the rates were 22% and 10% in females and males respectively.
The highest rate (15.7%) was reported in both Pfmdr-1 and Pfdhfr among age group 20-50 years
old, while the lowest prevalence rate (3.7%) was reported in Pfdhps among age group 11-19
years old. Also, the highest prevalence rate (12.7%) was reported in Pfdhfr among parasite count
++, while the lowest prevalence rate 3% was reported among parasite count ++++.
The highest prevalence rate (37%) of single point mutation of Pfcrt K76T was reported in River
Nile, while the lowest prevalence rate (28%) was reported in Sennar. In females reported 22%
and males 10%. Among age groups the highest prevalence rate (10.7%) of Pfcrt K76T was
reported in age group 20- 50 years, while the lowest prevalence rate (7.7%) was reported among
both age groups (<10 and >50). The highest prevalence rate (12%) was reported among parasite
count ++, while the lowest prevalence rate (3%) was reported among parasite count ++++.
The results revealed that, the highest prevalence rate (15.7%) of single point mutation of Pfmdr-1
N86Y was reported in Khartoum state, while the lowest prevalence rate (9.7%) was reported in
Sennar state. The result showed equal rate of 18% among the gender with presence of mixed
allele with a rate of 0.7%. Also, the results revealed that, the highest Pfmdr-1 N86Y rate of
15.7% was reported among age group 20-50 years, while the lowest prevalence rate of 4.7% was
IV
reported among age group 11 -19 years . The prevalence rate among parasite count was 10.7%
among the + and ++ followed by 10.3% and 4.3% among +++ and ++++ respectively.
From the results, the highest prevalence rate (14.3%) of single point mutation Pfdhfr C59Rwas
reported in Khartoum, while the lowest prevalence rate (9.3%) was reported in River Nile with
the presence of mixed allele (1.7% and 4%) in River Nile and Khartoum respectively. Among
the gender, the prevalence rates of 20.7% and 16%were among females and males respectively
with the presence of mixed allele 1.3% and 4.3% respectively. The highest rate (15.7%) was
reported among age group 20 -50 years, while the lowest rate (5.7%) was reported among age
group 11 -19 years with the presence of mixed allele among all age groups. The highest
prevalence rate (12.7%) of Pfdhfr C59R was reported among parasite count ++, while the lowest
prevalence rate (3%) was reported among parasite count ++++ with the presence of mixed allele
among all parasite counts.
From the results, the prevalence rate of single point mutation of Pfdhps A436G (13%) was
reported in Khartoum and (10%) in both Sennar and River Nile with the presence of mixed allele
in Khartoum and River Nile with rates of 1.7% and 3% respectively. Among gender 19.3% rates
was reported among females and 13.7% among males with mixed allele of 3% and 1.7%
respectively. However, the highest prevalence rate (15.7%) was reported among age group 20-
50 years, while the lowest rate (3.7%) was reported among age group 11-19 years with mixed
allele among all age groups except the age group 11-19 years. The highest prevalence rate (11%)
of Pfdhps A436G was reported among parasite count ++, while the lowest prevalence rate
(3.7%) was reported among parasite count ++++ with the presence of mixed allele among all
parasite counts.
The study indicated that, the genetic mutation of malaria drug resistance markers after adoption
of artemisinin combination therapy were still found in the study areas.

V
صلختسملا

ةداضملا ةينيجلا تامساولا راشتنا نع فشكلل )لينلا رهنو موطرخلاو رانس( ةينادوس تايلاو ثلاث يف ةسرا دلا هذه تيرجأ
تلاوفورو ديهيد فب ،)Pfmdr-1( تانيجلا ةمواقم لرا تنيج فب ،)Pfcrt( نيكورولكلا ةمواقم مورابيسلاف مويدومسلاب ،ةيودلأل
ىلإ 2015 ويلوي نيب ةرتفلا يف تيرجأ ةضرعتسملا ةسرا دلا . )Pfdhps( فيلوت ترتوبورديهيد فب و )Pfdhfr( ةلزتخم
.2017 ربمسيد
"راوكذ 133 مهنم ناك ,ةنس 31±23 يرمع طسوتمب ةنس 90 -1 نيب مهرامعأ تحورات اصخش 300 يلع ةسرادلا تلمش
."اثانإ 167و
ةغوبصملا مدلا تاحسم مادختسإ ةطساوب ايرلاملا ليفط ىلع فرعتلل ةسرا دلا عوضوم صاخشلاا عيمج نم مدلا تانيع تذخأ
.اسميج ةغبصب
نم يلاوتلا ىلع ٪33 و ٪36.7 و ٪36 و ٪32تانيجلا هذه راشتنا تلادعم تناك ايرلاملل ةيباجيإ ةنيع 300 نيب نم
.ةلسلستملا ةرملبلا لعافت ةقيرط -لاكشلأا ةددعتم تادييقتلا اياظش مادختسإ للاخ
)28٪( راشتنا لدعم ىندأ نأ نيح يف ،)Pfmdr-1( عم موطرخلا ةيلاو يف تلجس )47٪( ةلوحتم ليلأ راشتنا ةبسن ىلعأ
% 10 و % 22 تلادعملا تناك ،نيسنجلا نيب نمو .)موطرخلا يف Pfdhfr و رانس يف Pfcrt( تانيجلا نم نينثا يف لجس
-20 ةيرمعلا ةئفلا يف )Pfdhfr( و )Pfmdr-1( نم لك يف لجس )15.7%( لدعم ىلعأ .يلاوتلا ىلع روكذلاو ثانلإا يف
لدعم ىلعأ ليجست مت امك .ةنس 19-11 ةيرمعلا ةئفلا نيب Pfdhps يف ٪3.7 راشتنا لدعم ىندأ لجس نيح يف ،ةنس 50
.++++ تايليفطلا ددع نيب ٪3 راشتنا لدعم ىندأ تغلب نيح يف ،++ تايليفطلا ددع نيب Pfdhfr يف ٪12.7 راشتنا
لدعم ىندأ نأ نيح يف ،لينلا رهن يف تلجس Pfcrt K76T نم ةدحاو ةطقن ةرفط نم )37٪( راشتنا لدعم ىلعأ
(٪10.7 )راشتنا لدعم ىلعأ ةيرمعلا تائفلا نيب . % 10 روكذلاو % 22 ثانلإا يف تلجس .رانس يف لجس )28٪(راشتنا
نم لك نيب لجس )7.7٪ ( راشتنا لدعم ىندأ نأ نيح يف ،ةنس 50-20 ةيرمعلا ةئفلا نيب لجس Pfcrt K76T نم
نأ نيح يف ،++ تايليفطلا دادعأ نيب لجس )12٪ (راشتنا لدعم ىلعأ ,"را يخا . )ةنس 50 نم ربكأو لقأ10( ةيرمعلا تائفلا
. ++++تايليفطلا دادعأ نيب لجس )3٪ ( راشتنا لدعم ىندأ
VI
ةيلاو يف اهليجست مت Pfmdr-1 N86Y نم ةدحاو ةطقن ترا فط نم ٪15.7 راشتنا لدعم ىلعأ نأ جئاتنلا ترهظأ
عم نيسنجلا طسو ٪18 ةيواستم ةبسن جئاتنلا ترهظأ .رانس ةيلاو يف لجس ٪9.7 راشتنا لدعم ىندأ نأ نيح يف ،موطرخلا
ةئفلا نيب لجس ٪ 15.7 Pfmdr-1 N86Y ل لدعم ىلعأ نأ جئاتنلا تنيب "اضيأ .٪0.7 لدعمب طلتخم ليلأ دوجو
لدعم "رايخأ .ةنس19- 11 نيب ةيرمعلا ةئفلا يف لجس ٪4.7 راشتنا لدعم ىندأ نأ نيح يف ،ةنس 50- 20 ةيرمعلا
.يلاوتلا ىلع )++++ و +++( نيب ٪4.3 و ٪10.3 اهيلت )++ و +( نيب ٪10.7 تناك ليفطلا دادعأ طسو راشتنلاا
ىندأ نأ نيح يف ،موطرخلا يف تلجس Pfdhfr C59R ةدحاو ةطقن ةرفط نم ٪14.3 راشتنا لدعم ىلعأ نع ،جئاتنلا نم
نيب نم .يلاوتلا ىلع موطرخلاو لينلا رهن يف ٪4 و ٪1.7 طلتخم ليلأ دوجو عم لينلا رهن يف لجس ٪9.3 راشتنا لدعم
ىلع ٪4.3 و ٪1.3 طلتخم ليلأ دوجو عم يلاوتلا ىلع روكذلاو ثانلإا نيب ٪16 و ٪20.7 راشتنلاا تلادعم ,نيسنجلا
ةيرمعلا ةئفلا نيب لجس ٪5.7 لدعم ىندأ نأ نيح يف ، ةنس 50-20 ةيرمعلا ةئفلا نيب لجس ٪15.7 لدعم ىلعأ .يلاوتلا
لجس Pfdhfr C59R نم ٪12.7 راشتنا لدعم ىلعأ ,"را يخأ .ةيرمعلا تائفلا عيمج نيب طلتخم ليلأ دوجو عم ةنس 19 -11
عيمج نيب ةطلتخم ليلأ دوجو عم )++++( ليفطلا ددع نيب لجس ٪3 راشتنا لدعم ىندأ نأ نيح يف ،)++( ليفطلا ددع نيب
.ليفطلا دادعا
رانس نم لك يف ٪10 و موطرخلا يف لجس Pfdhps A436G نم ةدحاو ةطقن ةر فط نم )13٪(راشتنا لدعم جئاتنلا نم
لجس ٪19.3 نيسنجلا نيب .يلاوتلا ىلع ٪3 و ٪1.7 تلادعمب لينلا رهنو موطرخلا يف طلتخم ليلأ دوجو عم لينلا رهنو
لجس ٪15.7 راشتنا ةبسن ىلعأ ،كلذ عمو .يلاوتلا يلع ٪1.7 و ٪3 طلتخملا ليللأا عم روكذلا نيب ٪13.7 و ثانلإا نيب
نيب طلتخم ليلأ عم ةنس 19-11 ةيرمعلا ةئفلا نيب لجس ٪3.7 لدعم ىندأ نأ نيح يف ،ةنس 50-20 ةيرمعلا ةئفلا نيب
لجس Pfdhps A436G نم ٪11 راشتنا لدعم ىلعأ ,"را يخأ .ةنس19 -11 ةيرمعلا ةئفلا ءانثتساب ةيرمعلا تائفلا عيمج
نيب طلتخم ليلأ دوجو عم )++++( ليفطلا ددع نيب لجس ٪3.7 راشتنا لدعم ىندأ نأ نيح يف ،)++( ليفطلا ددع نيب
.ليفطلا دادعا عيمج
قطانملا يف ةدوجوم لزا ت ام جودزملا جلاعلا ذيفننت دعب ةمو اقملا ايرلاملا تامساول ةينيجلا ترافطلا نا يلع ةسرا دلا تلد
.ةسرادلا عوضوم
VII
List of contents

Contents
ةيلآا ................................................................................................................................................ I
Dedication ................................................................................................................................... II
Acknowledgement ..................................................................................................................... III
Abstract ..................................................................................................................................... IV
صلختسملا ..................................................................................................................................... VI
List of contents ........................................................................................................................ VIII
List of tables ............................................................................................................................ XIII
List of figures ......................................................................................................................... XIV
Abbreviations ........................................................................................................................... XV
Chapter 1
Introduction
1.1 Introduction ........................................................................................................................... 1
1.2 Rationale ............................................................................................................................... 4
1.3 Objectives .............................................................................................................................. 5
1.3.1 General objective .............................................................................................................................. 5
1.3.2 Specific objectives ............................................................................................................. 5
Chapter 2
Literature review
2.1 Background ........................................................................................................................... 6
2.2 Transmission of malaria ........................................................................................................ 6
2.3 Epidemiology of malaria ....................................................................................................... 7
2.4 Life cycle ............................................................................................................................... 7
2.4.1 Human phase ...................................................................................................................... 7
2.4.2 The mosquito phase ............................................................................................................ 9
2.5 Pathology of malaria ........................................................................................................... 10
2.5.1 Severe malaria .................................................................................................................. 12
2.5.2 Cerebral malaria ............................................................................................................... 13
VIII
2.5.3 Respiratory distress .......................................................................................................... 14
2.5.4 Haemoglobinuria and blackwater fever ........................................................................... 14
2.5.5 Pulmonary oedema ........................................................................................................... 15
2.5.6 Hypoglycemia .................................................................................................................. 15
2.5.7 Anemia ............................................................................................................................. 16
2.5.8 Algid malaria .................................................................................................................... 17
2.6 Immunity to malaria ............................................................................................................ 17
2.6.1 Natural or innate immunity .............................................................................................. 18
2.6.2 Acquired or adaptive immunity ....................................................................................... 19
2.7 Diagnosis of malaria ........................................................................................................... 22
2.7.1 Microscopic tests .............................................................................................................. 22
2.7.1.1 Peripheral smear study for malarial parasites ............................................................... 22
2.7.1.2 Thick smear ................................................................................................................... 23
2.7.1.3 Thin smear ..................................................................................................................... 23
2.7.1.4 Staining ......................................................................................................................... 24
2.7.2 Quantitative buffy coat (QBC) ......................................................................................... 24
2.7.3 Rapid diagnostic test (RDTs) ........................................................................................... 25
2.7.4 Polymerase chain reaction (PCR) .................................................................................... 26
2.7.5 Detection of antimalarial antibodies ................................................................................ 26
2.7.6 Intraleukocytic malaria pigment ...................................................................................... 26
2.7.7 Flow cytometry ................................................................................................................ 26
2-7-8 Mass spectrometry ........................................................................................................... 27
2-7-9 Other investigations ......................................................................................................... 27
2.8 Treatment of malaria ........................................................................................................... 27
2.8.1 Classification .................................................................................................................... 28
2.8.1.1 According to anti-malarial activity ............................................................................... 28
2.8.1.2 Tissue schizonticides for causal prophylaxis ................................................................ 28
2.8.1.3 Tissue schizonticides for preventing relapse ................................................................. 28
2.8.1.4 Blood schizonticides ..................................................................................................... 28
2.8.1.5 Gametocytocides ........................................................................................................... 28
2.8.1.6 Sporontocides ................................................................................................................ 29
IX