JOS.
BY
JOS UNIVERSITY TEACHING HOSPITAL, JOS.
NPMCN/AF/008/09/008/643
MEDICAL COLLEGE OF NIGERIA, IN PARTIAL FULFILMENT OF THE
REQUIREMENTS FOR THE AWARD OF THE FELLOWSHIP OF THE
COLLEGE IN PATHOLOGY
CERTIFICATION
2
This is to certify that the research work and the subsequent preparation of this
dissertation by DR ITA, OKOKON ITA of the Department of Medical Microbiology,
-------------------------------------------------------------------------
Consultant Medical Microbiologist, Jos University Teaching Hospital,
P.M.B 2076, Jos, Nigeria.
Consultant Medical Microbiologist, Jos University Teaching Hospital
P.M.B 2076, Jos, Nigeria.
DECLARATION
3
I declare that this dissertation for the award of fellowship of the National
Postgraduate Medical College of Nigeria was written by me and that it is a result of
my research work. To the best of my knowledge it has not been presented anywhere
---------------------------------
Jos University Teaching Hospital
Nigeria.
CANDIDATE
DEDICATION
I dedicate this work to my family and above all our heavenly father, Jehovah.
4
ACKNOWLEDGEMENT
I am very grateful to our heavenly father Jehovah for the undeserved kindness
he has shown to me. I remain much obliged to my parents, Mr./Mrs. Francis/Faith O.
Ita for their love and support. I remain eternally indebted to my trainers, teachers,
mentors and supervisors Professor Daniel Z. Egah and Professor Edmund B. Banwat
5
for their patience in guiding, proof reading and correcting my work, which has
enabled me to have this work ready in time. I thank Dr. Steve Oguche who pointed
me in the direction of this research and Professor Dyann Wirth of the Havard Malaria
Initiative who responded to my desperate request for help in the molecular diagnosis
of malaria. I am grateful to Professor Christian Happi of the Malaria Research
Laboratories, Ibadan. I also thank Dr. Onikepe Folarin for teaching me the diagnosis
of P. falciparum malaria by microscopy, and by the polymerase chain reaction.
I here acknowledge the material and moral support given to me by my in-laws
Mr./Mrs. John/Gloria Enyiegor Urom. I thank my fellow residents in the department
of Medical Microbiology and Parasitology in Jos University Teaching Hospital and
University of Calabar Teaching Hospital for their encouragement. To the patients and
the guardians of the minors who participated in this study, I say a big thank you. I
appreciate the assistance given to me by Dr. and Mrs. Jombo G. T who provided
accommodation for me when I first came to Jos for training.
TABLE OF CONTENTS
CHAPTER TWO
2.3 Epidemiology of Malaria - - - - - - 11
2.4 Pathogenesis of Malaria - - - - - - - 16
2.5 Pathology of Malaria - - - - - - - 25
2.6 Clinical features of Malaria - - - - - -- 26
2.7 laboratory diagnosis of Malaria - - - - - 26
2.8 Treatment of Malaria - - - - - - 36
2.9 Complications of Malaria - - - - - - 36
2.10 Prevention of Malaria - - - - - - 37
2.11 Control of Malaria - - - - - - 37
CHAPTER THREE
3.1 Study Design - - - - - - - 39
3.2 Study Area - - - - - - - 40
3.3 Study Population - - - - - - 41
3.4 Inclusion Criteria - - - - - - - 41
3.5 Exclusion Criteria - - - - - - 41
3.7 Laboratory Methods - - - - - - 43
3.8 Ethical Considerations - - - - - 56
3.9 Data Analysis - - - - - - - 56
CHAPTER FOUR
103
VII. Plates - - - - - - - 112
LIST OF TABLES
8
4.1: Age and sex distribution of participants in the study - - 66
4.2: Accuracy of RDT in detecting P. falciparum in patients clinically - 67
diagnosed with malaria in Jos.
4.3: Accuracy of microscopy in detecting P. falciparum in patients - 68
clinically diagnosed with malaria in Jos.
4.4: Sensitivity, Specificity and Predictive values of RDT and - - 69
Microscopy when compared against PCR
4.5: Sensitivity and Specificity of RDT when compared against - - 71
Microscopy
FIGURE PAGE
3.1 Principle of histidine rich protein II P. falciparum malaria RDT - 48
4.1 Bar chart showing parasite counts and the corresponding RDT result- 70
9
II. Oscillator, Microscope and Manual Cell Counter - - 113
III. MJ PTC-200 Peltier Thermal Cycler PCR Machine - - 114
IV. Electrophoresis Setup and Ultra Violet Transilluminator Equipment -
`115
10
VI. Thick Film Showing Malaria Parasites - - - - 117
VII. Thin Film Showing Ring Forms of P. falciparum - - 118
VIII. Gel Picture of DNA After Electrophoresis - - - 119
SUMMARY
In Nigeria, malaria is responsible for 60% outpatient visits to health facilities, 30%
childhood deaths and 11% maternal death. Misdiagnosis leads to wastage of resources,
unnecessary exposure to anti-malarials’ toxicity and the creation of selective pressure for drug
resistance. This makes fast and accurate diagnosis of malaria a life saving necessity.
However, microscopy, which is the gold standard for diagnosis is labourious and time
consuming. Therefore, this research set out to compare the performance of SD Bioline rapid
diagnostic test (RDT) against microscopy using polymerase chain reaction (PCR) as the
reference standard. The RDT was also compared with microscopy.
This study was a prospective, cross sectional, hospital based study. Specimens were
collected from children and adult patients of both sexes who presented with at least fever and
concerning whom a clinical diagnosis of acute uncomplicated malaria had been made.
Demographic and clinical data were collected from the patients by means of a structured
questionnaire. Specimens were processed for the diagnosis of malaria by RDT, microscopy
and PCR.
11
The prevalence of malaria in this study by PCR was 17%. The sensitivity, specificity,
positive and negative predictive values of RDT when compared to PCR was 76.5%, 98.8%,
92.3% and 95.4% respectively. The sensitivity, specificity, positive and negative predictive
values of microscopy when compared against PCR was 88.2%, 100%, 100% and 97.7%
respectively.
When compared against microscopy, RDT showed an overall sensitivity and
specificity of 80% and 96% respectively, while corresponding figures at parasite densities of
≥200 parasites/µL were 95.8% and 95.9% respectively.
Genotyping the P. falciparum isolates in this study using the GLURP-2 gene revealed
that 29.4% of patients whose samples were positive by PCR were infected by one clone of P.
falciparum, while 70.6% were infected by two clones of P. falciparum.
The performance of nested PCR in this study was better than conventional giemsa
stain microscopy which in turn was better than RDT in the diagnosis of uncomplicated P.
falciparum malaria in Jos. A patient with clinical features of malaria whose RDT result is
positive for P. falciparum should be treated for malaria, while a patient with clinical features
of malaria whose RDT result is negative should be re-tested by microscopy.
CHAPTER ONE
Malaria is the most important parasitic disease of man1. Approximately
5% of the world’s population is infected and it causes over one million deaths each
year1. The disease is a protozoan infection of the red blood cells transmitted by the
bite of a blood feeding female anopheline mosquito. Four species of the genus
Plasmodium have been known to infect humans viz: Plasmodium malariae,
Plasmodium vivax, Plasmodium ovale and Plasmodium falciparum. However, a fifth
species Plasmodium knowlesi, which causes malaria in long-tailed and pig-tailed
macaque monkeys is an important cause of human malaria on the island of Borneo
and peninsular Malaysia.1
12
A widely utilized clinical algorithm for malaria diagnosis, showed very low
specificity (0-9%) but 100% sensitivity in African settings2. This lack of specificity
reveals the perils of distinguishing malaria from other causes of fever on clinical
grounds alone. Thus, the diagnosis of malaria is based on laboratory methods and
microscopy of giemsa-stained thick and/or thin smears, is traditionally the “gold
standard” for diagnosis1. However, it is labour intensive, requires significant skills
and time (about one hour) which can cause therapeutic delays.
Several alternative laboratory methods have been developed, including
the quantitative buffy-coat centrifugal haematology system, immunofluorescence, and
the polymerase chain reaction (PCR). None of these tests are used routinely because
they are either too complicated or too expensive1. Rapid blood tests have lately
become commercially available. These use dipstick or test strip with monoclonal
antibodies directed against the target parasite antigen: Plasmodium falciparum
Histidine-Rich Protein 2 (Pf HRP-II), parasite-specific lactate dehydrogenase or
aldolase. The tests can be done in less than 15 minutes, require little training, and are
subject to less investigator-related variation compared to microscopy3.
1.1 STATEMENT OF THE PROBLEM
According to the World Malaria Report, 2011, it has been estimated
that 91% of deaths in 2010 were in the African Region, followed by the South-East
Asia (6%) and Eastern Mediterranean Regions (3%) 4. About 86% of deaths globally
were in children under 5 years of age. Of the 35 countries that accounted globally for
approximately 98% of malaria deaths, 30 were located in sub-Saharan Africa, with
four countries - Nigeria, Democratic Republic of Congo, Uganda and Ethiopia - alone
accounting for approximately 50% of deaths on the continent4.
13
In Nigeria, malaria is responsible for 60% outpatient visits to health facilities,
30% childhood deaths of children under one year and 11% maternal death5, 6. This
makes early and accurate diagnosis of malaria absolutely imperative.
The burden on the economy is also enormous. A study in Nigeria found out
households are prepared to pay an average of about 1,112 naira per month for the
treatment of malaria, and an average of 7,324 naira per month for the control of
malaria7. This amount represents about 611.7 naira per head per month and 7,340
naira per year. For a country with a population of more than 120 million this translates
to about 880,801 million naira per annum, representing about 12.0 % of the Gross
Domestic Product (GDP). Hence, the malaria burden in Nigeria has a devastating
impact on economic growth. Studies have shown that diagnosing malaria based on
clinical findings alone leads to over-diagnosis and overtreatment with the
consequences of delay in seeking an alternative diagnosis in parasite negative
patients, drug wastage, wastage of financial resources and exposing parasite negative
patients to anti-malarials’ toxicity as well as creating selective pressure for drug
resistance2, parasite based diagnosis is considered the standard of care.
The “gold standard” for malaria diagnosis is microscopy of properly prepared
thick and/or thin films that have been stained using Romanowsky stains1. However,
apart from its requirements of trained microscopists and time, there are the problems
of lack of high quality equipment, erratic power supply, the use of low quality stains
and other reagents and lack of supervision in our health centers8. The World Health
Organization has recognized rapid diagnostic tests as potential solution to improve
malaria diagnosis9. RDTs require minimal training, are easy to use, provide rapid
results and do not require electric power or the use of stains 9.
14
1.2 GENERAL OBJECTIVE
The objective of this study is to evaluate how rapid diagnostic test compares
with microscopy in the diagnosis of uncomplicated P. falciparum malaria in Jos,
Plateau State, using the polymerase chain reaction as the reference standard.
1.3 SPECIFIC OBJECTIVES
1. To determine the prevalence of malaria due to P. falciparum infection in the
Jos environment using the polymerase chain reaction.
2. To assess the sensitivity, specificity and predictive value of rapid diagnostic
tests in malaria diagnosis in Jos, Plateau State.
3. To determine the sensitivity, specificity and predictive values of light
microscopy in malaria diagnosis in Jos, Plateau State.
4. To compare the rapid diagnostic test, conventional light microscopy and PCR
in the diagnosis of uncomplicated P. falciparum malaria in Jos.
15
2.1 HISTORICAL BACKGROUND
The first recorded diagnostic feature of malaria was made in 1716 when the
Italian Physician Giovanni Maria Lancisi described a characteristic black
pigmentation of the brain and spleen in the victims of malaria10. In 1847, a German
physician, Heinrich Meckel, identified round, ovoid, or spindle-shaped structures
containing black pigment granules in protoplasmic masses in the blood of a patient
with fever and in the spleen during the autopsy of an insane person10. Thus Meckel
probably saw the malaria parasites for the first time, but could not recognize the true
importance of his finding.
On November 6, 1880, while examining a fresh blood specimen taken from a
new hospital arrival, a moving object on the slide caught the eye of Charles Louis
16
Alphonse Laveran. Under high power, this proved to be a tiny malarial body
wriggling vigorously. Laveran named the parasite Oscillairia malariae10. In his work,
his descriptions included crescents (gametocytes), pigmented trophozoites, and the
process of exflagellation thus recognizing for the first time the different diagnostic
forms of the malaria parasite10. Four years later in 1884, Marchiafava and Celli, while
studying wet blood smears from malarious patients with the new oil-immersion lens,
looked at unstained blood and saw an active amoeboid ring (trophozoite) in the red
blood cells. They published this finding and named it Plasmodium10.
A significant technical advancement in the diagnosis of malaria was made by
Ernst Malachowski and Dmitri Leonidovich Romanowsky who in 1891 independently
developed techniques using a mixture of Eosin Y and modified Methylene Blue that
produced a surprising hue attributable to neither staining component: a beautiful,
distinctive shade of purple. While Malachowski used alkali treated methylene blue
solutions, Romanowsky used methylene blue solution which were moulded or aged.
This discovery advanced to the development of the Giemsa stain for malaria
microscopy which is today considered the gold standard for malaria diagnosis11.
The first successful continuous malaria culture was established in 1976 by
William Trager and James B. Jensen, which facilitated research into the molecular
biology of the parasite and the development of new drugs subsequently11. Newer
diagnostic tests have been developed for malaria. The use of antigen-based malaria
rapid diagnostic tests (RDTs) was pioneered in the 1980s and in 1991 Becton and
Dickenson developed a fluorescence staining technique using a capillary tube called
Quantitative Buffy Coat test11. In 2002, the genomes of Anopheles gambiae and
Plasmodium falciparum were sequenced paving the way for molecular methods to be
applied in the diagnosis of malaria11.
17
The malaria parasite Plasmodium is a mosquito-transmitted protozoan.
Plasmodia are sporozoan parasites of red blood cells transmitted to animals by the
bites of mosquitoes. They belong to the family Plasmodiidae, the order
Haemosporidia and the phylum Apicomplexa. There are currently 450 recognized
species in this order. They contain three genetic elements: the nuclear and
mitochondrial genomes characteristic of virtually all eukaryotic cells and a 35-
kilobase circular extra chromosomal DNA. This encodes a vestigial plastid, the
epicoplast. All Plasmodium species examined to date have 14 chromosomes, with one
mitochondrion and one plastid genome. The chromosomes which have been
sequenced vary in length from 500 kilobases to 3.5megabases. It is presumed that this
is the pattern throughout the genus. The genomes of four Plasmodium species have
been sequenced. These species are Plasmodium falciparum, Plasmodium vivax,
Plasmodium knowlesi and Plasmodium yoelli. All these species contain genomes of
about 25 megabases. The remarkable Adenine-Thymine (AT) rich P. falciparum is
approximately 23 Mb in size and encodes about 5300 genes. Some 60% of the
encoded proteins are of unknown function1, 12.
2.2.1 Life cycle of the parasite
The life cycle of the parasite is divided into sexual and asexual stages. The
asexual stage is further divided into a pre-erythrocytic phase and an erythrocytic
phase.
Infection with human malaria begins when the feeding female anopheline
mosquito inoculates plasmodial sporozoites at the time of feeding. The small
18
sporozoites are injected during the phase of probing as the mosquito searches for a
vascular space before aspirating blood. After infection, the sporozoites enter the
circulation; either directly or via lymph channels and rapidly target the hepatic
parenchymal cells. Within 45 minutes of the bite, all sporozoites have either entered
the hepatocytes or have been cleared. Each sporozoite bores into the hepatocyte and
there begins a phase of asexual reproduction. This stage lasts on the average between
5.5 (P. falciparum) and 15 days (P. malariae) before the hepatic schizont ruptures to
release merozoites into the blood stream. In P. vivax and P.ovale infections, a
proportion of the intrahepatic parasites do not develop, but instead rest inert as
sleeping forms or hypnozoites to awaken weeks or months later, and cause the
relapses which characterize infections with these species. During the pre-erythrocytic
or hepatic phase of development considerable asexual multiplication takes place and
many thousands of merozoites are released from each ruptured infected hepatocyte.
However, as only a few liver cells are infected, this phase is asymptomatic for the
human host1.
The merozoites liberated into the blood stream closely resemble sporozoites.
They are motile ovoid forms which invade red cells rapidly. The process of invasion
involves attachment to the erythrocyte surface, orientation so that the apical complex
abuts the red cell, and then interiorization takes place by wriggling or boring motion
inside a vacuole composed of the invaginated erythrocyte membrane. Once inside the
erythrocyte, the parasite lies within the erythrocyte cytosol enveloped by its own
plasma membrane and a surrounding parasitophorus vacuolar membrane. The
attachment of the merozoite to the red cell is mediated by the attachment of one or
more of a family of erythrocyte binding proteins, localized to the micronemes of the
19
merozoite apical complex, to a specific erythrocyte receptor. In P. vivax this is related
to the Duffy blood group antigen Fya or Fyb. The absence of these phenotypes in West
Africa, or people who originate from this region, explains their resistance to infection
with P. vivax, and the absence of vivax malaria in West Africa1. For P. falciparum the
merozoite protein EBA 175, a product of the “Duffy-binding like” (DBL) super
family of genes encoding ligands for host cell receptors is clearly important. This
binds to sialic acid and the peptide backbone and the red cell membrane
sialoglycoprotein, glycophorin A. But other sialic dependent and independent
pathways of invasion also occur indicating considerable reserve in the invasion
system. Binding is linked to activation of a parasite actin motor which provides the
mechanical energy for the invasion process. The red cell surface receptors for P.
malariae and P. ovale are not known.
During the early stage of intra-erythrocytic development (<12 hours), the
small ring forms of the four parasite species appear similar under light microscopy.
The young developing parasite looks like a signet ring or, in the case of P. falciparum,
like a pair of stereo-headphones, with darkly staining chromatin in the nucleus,
circular rim of cytoplasm, and a pale central food vacuole. Parasites are freely motile
within the erythrocyte. As they grow, they increase in size logarithmically and
consume the erythrocyte’s contents most of which is haemoglobin. With this increase
in size, P. falciparum-infected erythrocytes become spherical and less deformable13-16,
whereas P. vivax enlarges the infected red cell which becomes more deformable.
Proteolysis of haemoglobin within the digestive vacuole releases amino acids which
are taken up and utilized by the growing parasite for protein synthesis, but the
liberated haem poses a problem. When haem is liberated from its protein scaffold, it
20
oxidizes to its toxic ferric form. Intra-parasitic toxicity is avoided by spontaneous
dimerization to an inert crystalline substance, haemozoin1.
As P. vivax grows it enlarges the infected red cell. Red granules known as
Schuffner’s dots appear throughout the erythrocyte. Similar dots are also prominent in
P. ovale infection. P. malariae produces characteristic “band forms” as the parasite
grows. It is usually present at low parasitaemias. High parasitaemias (over 20%) are
usually caused by P. falciparum. Approximately 36 hours after merozoite invasion (or
fifty four hours in P. malariae), repeated nuclear division takes place to form a
“segmenter” or schizont (the term “meront” is etymologically more correct).
Eventually the growing parasite occupies the entire red cell which has become
spherical, depleted in haemoglobin, and full of merozoites. It then ruptures, so that
between six and thirty six merozoites are released, destroying the remnants of the red
cell. The released merozoites rapidly re-invade other cells and start a new asexual
cycle. Thus, the infection expands logarithmically at approximately 10-fold per
cycle1.
2.2.1.3 Sexual stages and Development in the Mosquito:
After a series of asexual cycles in P. falciparum, a subpopulation of parasites
develops into sexual forms (gametocytes) which are long lived and motile. These are
the stages that transmit the infection. The process of gametocytogony takes about 7-
10 days in P. falciparum. Thus there is an interval of approximately one week
between peaks of asexual and sexual stage parasitaemia in acute falciparum malaria.
In contrast P. vivax begins gametocytogenesis immediately, and the process of
gametocytogony in the bloodstage infection takes only four days1. Symptomatic P.
vivax infections are more likely to present with patient gametocytaemia and to
transmit before treatment than acute P. falciparum infections.
21
The male-to-female gametocyte sex ratio for P. falciparum is approximately
1:4. One male (containing eight microgametes) and one female (macrogamete) are
required per mosquito blood meal (approximately two micro liters) for infection to
occur. Thus gametocyte densities of one per micro liter are theoretically sufficient to
infect mosquitoes, a density beneath the limit of detection for most routine
microscopy1.
Following ingestion in the blood meal of a biting female anopheline mosquito,
the male and female gametes become activated in the mosquito’s gut. The male
gametocytes undergo rapid nuclear division and each of the eight nuclei formed
associates with a flagellum (20-25 micro meters long). The motile male microgametes
separate and seek the female macrogametes. Fusion and meiosis then takes place to
form a zygote. For a brief period, the malaria parasite genome is diploid. Within 24
hours the enlarging zygote becomes motile and this form (the ookinete) penetrates the
wall of the mosquito mid-gut (stomach) where it encysts (as an oocyst). This spherical
bag of parasites expands by asexual division to reach approximately 500 micro
meters, i.e. it becomes visible to the naked eye. During the early stage of oocyst
development there is a characteristic pigment pattern and colour that allows speciation
but these patterns become obscure by the time the oocyst has matured to contain
thousands of fusiform motile sporozoites. The oocyst finally burst to liberate myriads
of sporozoites into the coelomic cavity of the mosquito.
The sporozoites then migrate to the salivary glands to await inoculation into
the next human host during feeding. The development of the parasite in the mosquito
is termed sporogony, and it takes between eight and thirty five days depending on the
ambient temperature and species of parasite and mosquito.1
22
2.3.1 Geographical Distribution
Malaria is endemic in 109 countries and is found throughout the tropics1. In
Africa P. falciparum predominates as it does in Papua New Guinea and Haiti,
whereas P. vivax is more common in central and parts of South America, North
Africa, the Middle East and the Indian Subcontinent. The prevalence of both species
is approximately equal in other parts of South America, South East Asia and Oceania.
P. vivax is rare in Sub-Saharan Africa (except for the horn of Africa), whereas P.
ovale is common only in West Africa. P. malariae is found in most areas but is
relatively uncommon outside Africa1. Ninety five percent of malaria infections in
Nigeria are caused by Plasmodium falciparum and five percent by Plasmodium
malariae17.
2.3.2 Morbidity and Mortality
There are an estimated 100 million malaria cases with over 300,000 deaths per
year in Nigeria. This compares with 215,000 deaths per year in Nigeria from
HIV/AIDS. Malaria accounts for 60% of outpatient visits, 30% childhood death, 25%
of death in children under one year and 11% maternal death in Nigeria5.
2.3.3 Age and Sex Distribution
Babies develop severe malaria infrequently (although, if they do mortality is
high). The factors responsible for this include passive transfer of maternal immunity,
and the high immunoglobin F content of the infants’ erythrocytes which retards
parasite development.
23
In holoendemic areas the baby is inoculated repeatedly with sporozoites in the
first year of life, but the blood stage infection is seldom severe. In this
epidemiological context, the main clinical impact of falciparum malaria is to cause
severe anaemia in the 1-3 year age group18, 19. With less intense or more variable or
unstable transmission the age range affected by severe malaria extends to older
children, and cerebral malaria becomes a more prominent manifestation of severe
disease.
Although mortality falls with decreasing transmission intensity, it remains
substantial until the entomological inoculation rate falls well below one. In
hyperendemic and holoendemic areas indigenous adults never develop severe malaria,
unless they leave the transmission area and return years later and even then malaria is
seldom life threatening. Immunity is constantly boosted and effective premonition
prevents parasite burdens reaching dangerous levels. Most infections in adults are
asymptomatic1.
There is no known sex predilection among children. However among adults,
malaria is commoner among pregnant females than in non- pregnant females and
adult males 20-23.
Though Malaria is holoendemic in Nigeria, malaria transmission is however
geographically specific. As a focal disease, malaria will therefore differ in its
characteristics from place to place, since all malaria vectors do not exhibit identical
behavior and ability to transmit parasites. Thus, there are considerable variations in
the intensity of malaria transmission in Nigeria 17.
2.3.4.1 The vector:
Malaria is transmitted by some species of anopheline mosquitoes1. Malaria
transmission does not occur at temperatures below 160C or above 330C, and at
altitudes >2000m because development in the mosquito cannot take place. The
optimum conditions for transmission are high humidity and an ambient temperature
between 200C and 300C1. Although rainfall provides breeding sites for mosquitoes,
excessive rainfall may wash away mosquito larvae and pupae. The epidemiology of
malaria is complex and may vary considerably even within relatively small
geographical areas. Intensities of malaria transmission vary from very low (on
average one infectious bite per person every 10 years) to extremely high (three
infectious bites per person per day).
Though situated in the tropical zone, a higher altitude of 1238m means that the
Jos plateau has a near temperate climate with an average temperature of between 18
and 22°C. Harmattan winds cause the coldest weather between December and
February. The warmest temperatures usually occur in the dry season months of March
and April. The mean annual rainfall is 146 cm (57 inches) on the Plateau. The highest
rainfall is recorded during the wet season months of July and August. The average
lower temperature on the Jos plateau has led to a reduced incidence of some tropical
diseases such as malaria24.
Malaria transmission to man depends on several interrelated factors. The most
important is the anopheline mosquito vector and in particular its longevity. Generally,
the transmission of malaria is described by the model put forward by MacDonald
where malaria transmission is directly proportional to the density of the vector, the
square of the number of times each day that the mosquito bites man, and tenth power
of the probability of the mosquito surviving for one day1.
to transmit malaria (vectorial capacity). There are nearly 400 species of anopheline
mosquitoes and many are species complexes1. Approximately 80 anopheline species
can transmit malaria, sixty-six are considered natural vectors and about 45 are
considered important vectors1. Each vector has its own behaviour pattern and even
among species this can vary between geographical areas. In the tropics, malaria
occurs all year round with higher incidences coinciding with the rainy season which
provides water for mosquito breeding, and increased humidity which favour mosquito
survival. Other factors which are not well understood also influence mosquito
populations and lead to fluctuations in the prevalence of malaria17.
2.3.4.2 Reservoir of infection:
There must be a human reservoir of viable gametocytes to transmit the
infection. In areas where there is a long dry season, the reservoir for malaria
transmission is in people who asymptomatically harbour parasites for long periods
until when conditions for transmission are optimal 1, 25.
2.3.4.3 Route of transmission:
This is mainly through blood inoculation of plasmodial sporozoites by an
infected female anopheline mosquito during feeding1. However, cases of malaria
transmitted through blood transfusion, congenital malaria through trans-placental
transfer, transmission through sharing needles and syringes among drug addicts,
accidental transmission among health care workers through needle and instrument
puncture and transmission through plasmapherisis and organ transplantation have
been reported26, 27.
26
The endemicity of malaria is best defined by the entomological inoculation
rate (EIR), or number of infectious mosquito bites received per person per year (ib/p).
Overall for the whole of Nigeria, mean EIR has been calculated from human bait
catches alone to be 13.6ib/p for the rainy season. The highest EIR is in the southern
rainforest (24.7ib/p) and the lowest in the drier northern guinea savannah (7.7ib/p).
The mean EIR indicates that in Nigeria about 13 infective Anopheles could transmit
malaria parasite successfully in the rainy season17.
Traditionally, the endemicity is defined in terms of the spleen or parasite rates
in children aged between two and nine years1.
i. Hypoendemic: spleen rate or parasite rate is <10%.
ii. Mesoendemic: spleen rate or parasite rate 10-50%.
iii. Hyperendemic: spleen rate or parasite rate 51-75% and adult spleen is also
high.
iv. Holoendemic: spleen rate or parasite rate >75% and low adult spleen rate.
Parasite rates in the first year of life are high.
2.3.4.5 Seasonal variation:
Malaria is usually a “rainy season disease” coinciding with increased mosquito
abundance. In some areas, parasite rates (i.e. the proportion of people with positive
blood smears) are relatively constant throughout the year, but the majority of cases
still do occur during the wet season1. Rainfall is the real climatic variable in Nigeria
with June to September being the months with the highest rainfall throughout the
country. With a short rainy season in Northern Nigeria, the region has the shortest
transmission period17. On the Jos plateau, the number of months with suitable climate
27
for malaria transmission is two to three months and the distribution of endemic
malaria in this area has been described as marginal28.
2.3.5 Occupational risk
Those involved in occupations that bring them into close contact with the
vector for extended periods of time e.g. farmers, lumberjacks, fishermen etc. are more
at risk of having malaria attacks than those whose jobs bring them into limited
contact with the vectors e.g. office workers1.
2.4 PATHOGENESIS OF MALARIA INFECTION
The pathogenesis of malaria infection is discussed in terms of parasite factors
and host factors.
2.4.1 Parasite factors
2.4.1.1 Cytoadherence and Sequestration:
The species of Plasmodium to which high morbidity and mortality of malaria
infection has been attributed is P. falciparum. At approximately 12-14 hours of
development, P. falciparum begin to exhibit a high molecular weight strain-specific
variant antigen, Plasmodium falciparum erythrocyte membrane protein1 (PfEMP1) on
the exterior surface of the infected red cell which mediates attachment of infected
erythrocyte to vascular endothelium. This is associated with knoblike projections
from the erythrocyte membrane29-31. These ‘knobby’ or K+ red cells progressively
disappear from the circulation by attachment or ‘cytoadherence’ to the walls of
venules and capillaries in the vital organs- a process referred to as ‘sequestration’.
Sequestration occurs predominantly in the venules of vital organs. It is not distributed
uniformly throughout the body, being greatest in the brain, particularly in the white
matter, prominent in the heart, eyes, liver, kidneys, intestines and adipose tissue, and
28
least in the skin. Even within the brain the distribution of sequestered erythrocytes
varies markedly from vessel to vessel, possibly reflecting differences in the
expression of endothelial receptors1. Ring forms of the parasites are also concentrated
in the spleen and placenta32-37, raising the possibility that the entire asexual cycle
could take place away from the peripheral circulation.
Once infected cells adhere, they do not enter the circulation again, remaining
stuck until they rupture at merogony (schizogony). Under febrile conditions
cytoadherence begins at approximately 12 hours after merozoite invasion and reaches
50% of maximum after 14-16 hours. Adherence is essentially complete in the second
half of the parasites’ 48 hours asexual cycle1. The other three benign human malarias
do not cytoadhere in systemic blood vessels and all stages of development circulate in
the bloodstream1. As a consequence, whereas in the other malarias of man mature
parasites are commonly seen on blood smears, these forms are rare in falciparum
malaria, and often indicate serious infection.
2.4.1.2 Rosetting:
erythrocytes38-41. This process leads to the formation of “rosettes” when suspensions
of parasitized erythrocytes are viewed under the microscope. Resetting shares
characteristics with cytoadherence. It starts at around 16 hours of asexual life cycle
development (slightly after cytoadherence begins) and it is trypsin sensitive. But
parasite species which do not sequester do rosette and unlike cytoadherence, rosetting
is inhibited by certain heparin sub-fractions and calcium chelators1.
2.4.1.3 Aggregation:
29
Recently, a new adherence property of parasitized red cells has been
characterized which is associated with disease severity. This is platelet mediated
aggregation of parasitized erythrocytes and is mediated via platelet CD36. This
clumping together is seen in ex vivo cultures42-46.
Cytoadherence and the related phenomena of rosetting and aggregation lead to
micro-circulatory obstruction in falciparum malaria. The consequences of
microcirculation obstruction are activation of the vascular endothelium, endothelium
dysfunction, together with reduced oxygen and substrate supply, which leads to
anaerobic glycolysis, lactic acidosis and cellular dysfunction1, 47-51.
2.4.1.4 Toxicity and Cytokines:
No toxin in the strict sense of the word has ever been identified1, but malaria
parasites do induce the release of cytokines in much the same way as bacterial
endotoxins. A glycolipid material with many of the properties of bacterial endotoxin
is released on meront rupture. This material is associated with the glycosyl-
phosphatidyl-inositol anchor which covalently links proteins including the malaria
parasite surface antigens to the cell membrane lipid bilayer1. These products of
malaria parasites, and the crude malaria pigment which is released at schizont rupture,
induce the activation of the cytokine cascade in a similar manner to the endotoxin of
bacteria via signaling through toll-like receptor 2 and to a lesser extent toll-like
receptor 1.1. Cells of the macrophage-monocyte series, gamma/delta T cells,
alpha/beta T cells, CD14+ cells and endothelium are stimulated to release cytokines in
a mutually amplifying chain reaction. Initially tumor necrosis factor (TNF) which
play a pivotal role, interleukin (IL)-1, and gamma interferon are produced and these in
turn induce release of a cascade of other ‘pro-inflammatory’ cytokines including IL-6,
30
IL-8, IL-12, IL-18. These are balanced by production of the ‘anti-inflammatory’
cytokines, notable IL-101, 52. Cytokines are responsible for many of the symptoms and
signs of the infection, particularly fever and malaise1. Plasma concentrations of
cytokines are elevated in both acute vivax and falciparum malaria.
Cytokines are probably involved in placental dysfunction, suppression of
erythropoiesis, inhibition of gluconeogenesis, coma, coagulopathy,
thrombocytopaenia, and fever in malaria53, 54. Tolerance to malaria or premonition
reflects both immune regulation of infection but also reduced production of cytokines
in response to malaria (“antitoxic immunity”). Cytokines up-regulate the endothelial
expression of vascular ligands for P. falciparum-infected erythrocytes, notably
ICAM-1, and thus promote cytoadherence1. They may also be important mediators of
parasite killing by activating leucocytes, and possible other cells, to release toxic
oxygen species, nitric oxide, and by generating parasiticidal peroxides, and causing
fever. Thus, whereas high concentrations of cytokines appear to be harmful, lower
levels probably benefit the host.
In a study carried out by researchers in Ibadan, Nigeria, IFN-γ and IL-10 were
significantly higher in the symptomatic children than in the asymptomatic controls but
no differences were seen for IL-1255. Estimated higher ratios of IFN-γ/IL-10 and IFN-
γ/IL-12 were also observed in the symptomatic children in the same study55.
2.4.1.5 Immune Paresis:
While innate immune responses are very important in controlling malaria,
acute infections are associated with malaria antigen unresponsiveness. This selective
paresis is one of the factors contributing to the slow development of an effective and
specific immune response in malaria. Acute malaria is characterized by non-specific
31
polyclonal B-cell activation. There is reduction in circulating T cells with an increase
in the gamma/delta T-cell subset, but other T-cell populations are usually normal.
Although residents of hyperendemic or holoendemic malarious areas have
hypergammaglobulinaemia, most of this antibody is not directed against malaria
antigens. In non-immune individuals, the acute antibody responses of infection often
comprise mostly IgM or IgG2, isotypes which are unable to arm cytotoxic cells and
thus kill asexual malaria parasites. These observations have led to the suggestion that
Malaria induces an ‘immunological smokescreen’ with broad spectrum and non-
specific activation that interferes with the orderly development of specific cellular
immune responses and immune memory. In severe malaria there is evidence of a
broader immune suppression, with defects in monocyte and neutrophil chemotaxis,
reduced monocyte phagocytic function, and a tendency to bacterial super-infection. In
the nephrotic syndrome associated with chronic P. malariae infection, malaria antigen
and immune complexes can be eluted from kidney, indicating an immunopathological
process in this condition1.
Also, the levels of micronutrients and antioxidants have been found to be
lowered in P. falciparum infection. The antioxidant status of 148 children with
Plasmodium falciparum malaria was evaluated in Edo State, Nigeria. The study
revealed that the levels of vitamin A, vitamin C, 5 carotenes and vitamin E were all
found to be depressed56. The depressed levels of plasma antioxidants in the P.
falciparum-infected children suggested lowered immunity of the children, which may
contribute to the morbidity and mortality of malaria in our locality. In Ekiti state, the
serum levels of copper, calcium, magnesium, zinc, iron, vitamin A, folic acid,
cobalamine and antioxidants such as vitamins C and E, carotenes, glutathione and
superoxide dismutase were found to be low in pregnant women with malaria57.
32
2.4.1.6 Antigenic Variation
The persistence of P. falciparum during blood stage proliferation in man
depends on the successive expression of variant molecules at the surface of infected
erythrocytes. This variation is mediated by the differential control of a family of
surface molecules termed PfEMP1 encoded by approximately 60 var genes. Each
individual parasite expresses a single var gene at a time, maintaining all other
members of the family in a transcriptionally silent state58-61.
2.4.2. Host Factors
2.4.2.1 Genetics
There is good evidence from detailed epidemiological studies that show that
people who are heterozygous for red cell abnormalities such as thalassaemia or sickle
cell disease have some protection against malaria, although the mechanisms of
protection vary considerably among the different erythrocyte abnormalities62-68. The
greatest protection is conferred by sickle cell trait and Melanesian ovalocytosis1.
These patients’ red cells resist parasite invasion (in the case of sickle cell trait under
low oxygen tensions), and once invaded the AS cells sickle readily, facilitating their
clearance by the reticuloendothelial system. The protective effect conferred by the
thalassaemia or glucose-6-phosphate dehydrogenase deficiency (which shares a
geographical distribution with malaria) is generally weaker1. Blood group O is also
protective against severe P. falciparum malaria69, 70.
The findings above are also supported by research carried out in Ogbomosho
and Ibadan Nigeria. The researchers found out that H b A S was associated with a
r e d u c e d r i s k o f severe m a l a r i a 7 1 , 7 2 . Am on g s ev er e malaria
subjects, HbAS was associated with significantly lower parasite densities71.
33
The protective effect of blood g r o u p O was demonstrated with a d ec r eas ed
r i sk o f severe malaria72. B l oo d g r ou p B was associated with increased
risk of severe malaria72.
2.4.2.2 Immunity:
The precise mechanisms controlling malaria infection in the host are still
incompletely understood1. Effective immunity, as distinct from premonition, may be
reached when there has been sufficient exposure to all local strains of malaria
parasites. This is difficult to quantify as there is still no good in-vitro correlate of anti-
toxic or strain specific immunity to malaria1. In controlling the acute infection, non-
specific host defence mechanisms and the later development of more specific cell-
mediated and humoral responses are both important. Protective antibodies inhibit
parasite expansion by agglutinating merozoites and by binding to parasitized
erythrocytes. The opsonized parasitized red cells activate the Fc receptors of the
monocyte-macrophage series resulting in splenic clearance. The systemic and splenic
monocyte-macrophage series appear to be the most important immune effector cells
in the direct attack on parasitized erythrocytes and merozoites, although neutrophils
may also play a role73, 74. Non-specific effector mechanisms include non-opsonic
phagocytosis via direct binding monocyte-macrophage CD36, pro-inflammatory
cytokine release, and activation of phagocytic cells (including neutrophils) to release
toxic oxygen species and nitric oxide, both of which are parasiticidal. The reaction of
this oxygen intermediates with lipoproteins produces lipid peroxides. These are more
stable cytotoxic molecules and are unaffected by antioxidants. There is also
augmentation of splenic clearance function: the splenic thresholds for both filtration
and Fc receptor-mediated phagocytosis are lowered. P. falciparum-infected
34
erythrocytes are both more rigid and more opsonized than uninfected red cells as they
express both host and parasite derived neoantigens on the erythrocyte surface.
However, the parasite proteins expressed on the red cell surface undergo antigenic
variation which prevents complete immune clearance and thereby sustains the
untreated infection.
Following natural infection there is a transient humoral response to
sporozoites antigens; sporozoite antibodies decline then with a half-life of 3-4 weeks.
In areas of high transmission, sporozoite antibody levels tend to Plateau between 20
and 30 years of age, and do not correlate with premonition1, 75. Cytotoxic T cell
immune responses cannot be directed against the blood stage parasite as red cells do
not express human leucocyte (HLA) antigens, but the pre-erythrocytic liver stages of
the parasite are vulnerable to T cell attack1. Several line of experimental evidence in
animal malarias, and the observation that certain HLA types are relatively protected
from severe malaria, indicate that class I restricted CD + T cells play an important
role in immunity. There is evidence supporting a role for both alpha-beta and gamma-
delta CD4 + cells in the immune response to malaria.
Strain-specific immunity to the asexual blood stage parasites develops slowly
during natural untreated infections, but it then provides good protection against re-
challenge76. However, parasite populations are diverse, and cross-strain protection is
initially weak or negligible. The development of immunity in endemic areas may
represent the gradual acquisition of a repertoire of immunological memory for the
range of local parasites. This involves strain-transcending immunity sufficient to
ameliorate disease (antitoxic immunity) and a more strain specific immunity, which
protects from or attenuates the infection.
35
The immune response to malaria is clearly complex, and the relative
importance of humoral and cellular immunity in man has not been defined clearly1.
Infusions of hyperimmune serum to patients with acute malaria have reduced or
eliminated parasitaemia through opsonization and activation of phagocytic and
cytotoxic effector functions by cytophilic IgG antibodies, augmentation of ring-form
infected erythrocyte clearance, and agglutination of merozoites. In addition to the role
of cellular immunity in preventing pre-erythrocytic development, the increase in
malaria severity in patients living in endemic areas with the acquired immune
deficiency syndrome (HIV-AIDS) suggest that CD4+ cells play a significant role in
modulating the severity of falciparum malaria1.
2.4.2.3 Pregnancy:
Pregnancy increases susceptibility to malaria1. This is probably caused by a
suppression of systemic and placental cell-mediated immune responses77, 78. There is
intense sequestration of P. falciparum infected erythrocytes in the placenta, local
activation of pro-inflammatory cytokine production, and anaemia79, 80. This leads to
cellular infiltration and thickening of the syncytiotrophoblast and placental
insufficiency with consequent foetal growth retardation1.
2.4.2.4 Age:
In areas of intense P. falciparum malaria transmission, severe malaria never
occurs in adults; it is confined to the first year of life and becomes progressively less
frequent with increasing age75. In Africa overall, the average age of children admitted
to hospital with severe malaria is three years which corresponds with peak mortality
in the third years of life1. In areas with a constant high-level P. falciparum
36
transmission (e.g. average infected anopheline biting frequencies of daily up to
monthly), severe malaria occurs predominantly between six months and three years of
age; milder symptoms are seen in older children, and adults are usually asymptomatic
and have low parasitaemias1.
While asymptomatic Human Immunodeficiency Virus (HIV) infection has
little impact on malaria, with increasing immunosuppression in HIV, immune control
of malaria is impaired1. There is an increasing risk of parasitaemia, increasing risk of
illness, and in low transmission settings an increased risk of severe malaria. HIV
infection compounds malaria associated reduction in birth weight. Therapeutic
responses to antimalarial treatment are impaired, so treatment failure rates are
increased81.
2.5 PATHOLOGY OF MALARIA
As the benign human malarias are rarely fatal, there is very little information
available on the pathology of these infections1. Unfortunately, this is not the case in P.
falciparum malaria. In fatal malaria, the microvasculatures of the vital organs are
packed with erythrocytes containing mature forms of the parasite. There is abundant
intra-and extra-erythrocytic pigment in affected organs such as the liver, spleen and
placenta. These organs may be grey-black in colour. Sequestration is not uniformly
distributed; it tends to be greatest in the brain and heart followed by the gut, kidney,
adipose tissue, liver, lungs and least of all, in bone marrow and skin. There is
remarkably little extravascular pathology in malaria1, 82.
2.6 CLINICAL FEATURES OF MALARIA
37
The clinical manifestations of malaria are dependent on the previous immune
status of the host. The clinical features are divided into acute uncomplicated malaria
and severe malaria. Adults living in areas of high transmission generally come down
with uncomplicated malaria, while non-immune individuals and children below three
years of age are more likely to suffer from severe malaria. The clinical features of
uncomplicated malaria are non-specific and include the following: Fever, chills and
rigors headache, malaise, muscular discomfort, lack of appetite and bitter taste in the
mouth. Severe forms of malaria include: cerebral malaria, severe anaemia, renal
failure, pulmonary oedema, respiratory distress, hypoglycaemia, circulatory collapse
or shock, spontaneous bleeding from orifices, repeated generalized convulsions,
acidaemia/metabolic acidosis, macroscopic haemoglobinuria, jaundice, hyperpyrexia
(temperature ≥41.5), prostration and hyperparasitaemia (>250,000/μL of blood)1.
2.7 LABORATORY DIAGNOSIS OF MALARIA
The methods for the laboratory diagnosis of malaria83-85 can be broadly
classified into:
(a) The use of conventional light microscopy
(b) The use of fluorescent microscopy
2.7.1.1 CONVENTIONAL LIGHT MICROSCOPY:
38
This is used to diagnose malaria by viewing thick and thin films that have
been stained with Romanowsky stains such as Giemsa, Leishman or Field stains.
The thick film is the “gold standard” for parasite detection. This is because the
blood is concentrated 20-40 times during the preparation of film. This gives it a
detection limit of 10-50 trophozoites per micro liter of blood83. At optimum thickness,
small print should just be readable through the unstained film. If the film is too thick,
it might flake off while drying or fall off while staining or have too many leucocytes
in the fields for easy microscopy. If it is too thin, the amount of blood is insufficient
to detect low parasitaemia. The film is allowed to air dry thoroughly, preferably
overnight. It is not fixed. Fixing prevents de-haemoglobinization from taking place.
The method depends on the leaching out of the haemoglobin in the erythrocytes so
that the erythrocytes stain minimally and remain transparent, thus allowing stained
organisms lying deep in the thick layer of blood cells to be seen. The leaching takes
place during the staining with Giemsa, when the haemoglobin dissolves in the watery
solution of the stain. It is then allowed to dry after staining and viewed using the oil
immersion objective. Samples that can be used in thick film preparation include:
peripheral blood, cord blood and capillary blood from finger prick, earlobe prick or
heel prick in neonates1.
The thin film is the “gold standard” for species identification. It also aids in
the diagnosis of mixed infections, in the quantification of parasitaemia and the
assessment for the presence of schizonts (meronts) gametocytes and melanin pigments
in neutrophils and monocytes. The smear is made; air dried, fixed with absolute
alcohol, stained with a Romanowsky stain, allowed to dry and viewed using the oil
immersion objective. Samples that can be used for thin film preparation include:
peripheral venous blood, capillary blood from finger prick, earlobe prick and heel
39
prick in neonates, cord blood, intradermal smears, placental smears, cerebral grey
matter obtained by needle necropsy through the foramen magnum, superior orbital
fissure, ethmoid sinus via the nose or through the fontanelle in young children in post
mortem diagnosis83.
(i) Merozoites, Schizonts or Gametocytes. Merozoites have a “signet-
ring” appearance due to a large vacuole that forces the parasite’s
nucleus to one pole. Schizonts are round to oval inclusions that contain
the deeply staining merozoites. Gametocytes are “halter-shaped.”
(ii) The presence of haemozoin.
2.7.1.2 FLUORESCENT MICROSCOPY:
Malaria parasites contain DNA and RNA unlike mature red cells. These
nuclear materials can be stained with fluorescent dyes like acridine orange and viewed
using the fluorescent microscope or with the ordinary light microscope using
appropriate filters.
In the quantitative Buffy Coat technique, blood samples are taken into a
specialized capillary tube containing acridine orange and a float. Under high
centrifugal forces (1400g) the infected cells become concentrated around the float.
Using a modified lens adaptor with its own light source, the acridine orange
fluorescence from the malaria parasite can be visualized using an ordinary light
microscope, or it can be viewed using a fluorescent microscope. Though this system
is more sensitive than conventional light microscopy, it is expensive1.
2.7.2 Non-Microscopic tests85
antibodies and plasmodial nucleic acids.
2.7.2.1 PLASMODIAL ANTIGENS DETECTION:
The antigens detected are:
i. Histidine-Rich Protein-2 (HRP-II), which is a water-soluble protein produced
by trophozoites and young (but not mature) gametocytes of P. falciparum. The
Rapid Diagnostic Tests that use HRP-II detect P. falciparum only.
ii. Plasmodium lactate dehydrogenase (pLDH), which is produced by both
asexual and sexual stages (gametocytes) of malaria parasites. Three different
pLDH tests are available: pan-specific tests, tests that are specific to P.
falciparum, and more recent tests that are specific to P. vivax.
iii. Pan-specific aldolase, which is an antigen common to all four species of
human malaria. It is used in conjunction with HRP-II to distinguish
falciparum/mixed infections from non-falciparum infections, or in single
antigen tests to detect malaria infections of unspecified origin.
The techniques used in antigen detection include:
a. Gel diffusion
2.7.2.2 ANTIPLASMODIAL ANTIBODIES DETECTION:
While antibodies detection has no place in the diagnosis of acute malaria, it is
useful in epidemiological surveys, in the investigation of transfusion malaria and in
41
retrograde confirmation of malaria. The sample of choice is serum. The techniques
used here include:
The polymerase chain reaction (PCR) is the commonest molecular technique
used in plasmodial nucleic acid detection. Tests based on the PCR for species specific
plasmodium genome are more sensitive and specific than are other tests, detecting as
few as 10 parasites per micro liter of blood83, 87, 88. The polymerase chain reaction is a
laboratory technique used to obtain multiple copies of specific DNA fragments even
from samples containing only minute quantities of DNA or RNA89. All specimens
from which plasmodial nucleic acids can be extracted can be used for the diagnosis of
malaria by polymerase chain reaction. This includes: whole blood, serum, plasma,
bone marrow, placenta etc.
2.7.2.3.1 Essential components of polymerase chain reaction:
I. Template DNA that contains the target sequence of interest to be amplified.
II. Primers- A pair of synthetic oligonucleotides (forward and reverse primers)
that are complementary to the 3’ ends of each of the two strands of target
DNA.
isolated from thermophilic bacterium Thermus aquaticus) is a vital ingredient
of a PCR to catalyse the template-dependent synthesis of DNA.
42
IV. Divalent cations, usually Mg2+ are required in optimum concentration for the
activity of most thermostable DNA polymerases as well as for several other
steps in PCR.
V. Deoxynucleoside triphosphates (dNTPs): Equimolar amounts of each dNTP
(dATP, dCTP, dGTP, dTTP), which are building blocks used by the DNA
polymerase enzyme to synthesize a new strand of DNA.
VI. Buffer solution to maintain suitable ionic environment for optimum activity
and stability of the DNA polymerase.
2.7.2.3.2 Steps of PCR:
The PCR typically consists of three basic steps:
I. Denaturation: The first step of a PCR where the sample is heated to separate
or denature the two strands of the DNA (>900C).
II. Annealing: Following the denaturation step, the reaction temperature is
lowered (usually 3-50C below the Tm of primer) to allow the oligonucleotide
primers to bind to the single strands of the template DNA.
III. Extension: The final step of the PCR where the temperature is raised, typically
to 72°C, allowing specific enzymes to synthesize a new DNA strand
complementary to the DNA template.
One thermal cycle of these three steps theoretically doubles the amount of
DNA present in the reaction. Typically about 25 to 45 cycles of PCR are performed
depending upon the type of PCR used, the amount of initial template DNA and the
number of amplicon copies desired for post-PCR processing89. The PCR is commonly
performed in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml
43
volumes) in a thermocycler that heats and cools the reaction tubes to achieve the
temperatures required at each step of the reaction.
2.7.2.3.3 Post-PCR analysis/processing:
Post PCR detection system must accurately and reproducibly reflect the nature
and quantity of the starting DNA template. Specialized methods used in post PCR
analyses are usually tailored depending on specific applications. The simplest method
uses agarose gel electrophoresis. After the electrophoresis, PCR products can be
visualized by staining the gel with fluorescent dye such as ethidium bromide which
binds to DNA and intercalates between the stacked bases. Confirmation of size of the
DNA product is done by comparing the size with DNA ladder. The appearance of
discrete band of the correct size may be indicative of a successful PCR
amplification89.
1. Sequencing of the PCR product:
This is the gold standard but is expensive and not widely available. PCR
product may be sequenced directly or cloned before sequencing. However, it is the
test of choice in outbreak situations where there are serious public health and/or
medico-legal implications. Sequencing can be used to confirm results of other
molecular epidemiological assays. As a matter of fact, all other assays can be
considered as simpler screening assays89.
2. Restriction Fragment Length Polymorphism (RFLP):
This is a very simple, rapid and economical technique but the result may be
difficult to read.
44
There a wide variety of formats available for hybridization with a specific
oligonucleotide probe e.g. dot-blot, Southern blot, reverse hybridization, and DNA
enzyme immunoassay.
1. Reverse transcriptase PCR (RT-PCR):
In Reverse Transcriptase or RT-PCR, a strand of RNA is initially reverse
transcribed into its complementary DNA or cDNA using the reverse transcriptase
enzyme. The resulting cDNA is further amplified by PCR. The reverse transcription
step can be performed either in the same tube with PCR (one-step PCR) or in a
separate one (two-step PCR) depending on the properties of the reverse transcriptase
enzyme used. The RT-PCR is used for detection of RNA viruses in clinical samples
and in gene expression studies89.
2. Multiplex PCR:
Multiplex PCR refers to the simultaneous amplification of multiple selected
target regions in a sample using different pairs of primers. In this version, multiple
primer pairs are employed in the amplification mix so as to facilitate detection of
multiple targets. Amplification products are finally differentiated by gel
electrophoresis, sequence specific oligoprobes or in a real-time format, by melting
curve analysis. Since multiplex PCR can be used to detect multiple genes of interest
in one specimen, it can minimize the number of separate reactions and help
conservation of time, reagents and samples that are of limited volume89.
3. Nested PCR:
Nested PCR involves two successive PCRs, where the amplification product
from the first PCR reaction is used as the template for the second PCR. Either one of
45
the primers (semi-nested PCR) or both the primers (nested PCR) used in the second
PCR may be different from the primers used in the first PCR. It has been employed to
detect organisms present in low copy numbers in specimens, and has the benefits of
enhanced sensitivity and specificity, the latter resulting also from a cleaner template
provided by the first amplification89.
4. Real time PCR:
The Real Time PCR method is used for the detection and quantitation of an
amplified PCR product as the reaction progresses in ‘real time.’ This new approach of
PCR is based on the incorporation of a fluorescent dye where the increase in
fluorescence signal, generated during the PCR, is in direct proportion to the amount of
the PCR product. This modification avoids the requirement of a separate amplicon
detection step, by employing fluorescent amplicon detection technology (using DNA-
intercalating dyes such as SYBR Green or sequence-specific oligonucleotide
chemistry such as TaqMan probes). Here, the fluorescent molecules added to the PCR
mixture produce fluorescent signals which are detected simultaneously with the
progress in amplification. Use of a closed system, reduced turnaround time, dynamic
range of target detection, and feasibility for quantitation is a few of the advantages of
this method89.
5. In-situ PCR:
The PCR amplification reaction takes place within the cell which is often fixed
on a slide. It can be employed for the detection of nucleic acid in small tissue samples.
The PCR master mix is directly applied onto the sample on a slide, and then both are
covered using a cover-slip, and the latter is subjected to amplification in a thermo-
cycler with a slide adaptor or in-situ adaptor89.
46
Genetic markers that have been used for the diagnosis of P. falciparum
malaria include: the Csp gene, Stevor gene, Rifin gene, RESA gene, EBA-175 gene
and the 18S ribosomal DNA, the merozoite surface protein-1 (MSP-l), the merozoite
surface protein-2 (MSP-2) and the glutamate-rich protein-2 (GLURP-2) 90-94. In order
to examine the genetic diversity and complexity of P. falciparum populations in
patients with malaria infections as well as study the transmission dynamics in natural
isolates of P. falciparum, it is the polymorphisms in the MSP-1, MSP-2 and GLURP-
2 genes that are usually analysed 95.
The GLURP-2 gene which is located on chromosome 10, encodes a
polypeptide of 1271 residues with a predicted molecular mass of 145 kDa. The
GLURP-2 protein is an exoantigen expressed at all stages of development in the
parasite life cycle in human hosts, including on the surface of newly released
merozoites91. The gene is a stable, single copy gene that is highly conserved90. The
GLURP-2 alleles are commonly being differentiated by size polymorphisms alone
after separation by electrophoresis95. Thus, this gene can be studied without the need
for elaborate sequencing techniques and the use of sophisticated bioinformatics
software.
Due to widespread resistance to monotherapy96-101, the World Health
Organization recommends Artemisinin Combined Therapy for the treatment of
malaria. One of the reasons is that the artemisinin drugs have the broadest time
window of action on the asexual malaria parasites, from young rings to early
47
schizonts (meronts), thus explaining why they produce the most rapid therapeutic
responses. Also, resistance has been slow in developing against this class of drugs1.
2.8.2 Supportive Therapy
Supportive therapy in uncomplicated malaria includes the use of antipyretics
like acetaminophen and tepid sponging in children. In severe malaria, the patient is
cared for as an unconscious patient with therapies like blood transfusion, vitamin K
administration, oxygen, dialysis, correction of hypoglycemia, administration of
diuretics and antimicrobials being given depending on the manifestation of the severe
malaria1.
2.8.3 Intermittent Presumptive Treatment
Studies conducted in high transmission areas of Africa have shown that
administration of treatment doses of sulphadoxine-pyrimethamine two or three times
during pregnancy was associated with reduced placental parasitization, reduced
anaemia, and increased birth weight. 1, 102-107
2.9 COMPLICATIONS OF MALARIA
Malaria is a major cause of chronic ill health in the tropics, particularly in
childhood1. Acute complications include: Shock, heart failure and convulsions.
Chronic malaria is associated with the following specific syndromes: Nephrotic
syndrome of quartan malaria, chronic hepatosplenomegaly, Burkitt’s lymphoma,
chronic anaemia, chronic ill-health, failure to thrive, vulnerability to other infections
and retardation of educational development1.
2.10 PREVENTION OF MALARIA
48
Insecticide spraying and insecticide-treated bed nets are the main methods of
attacking the vector and controlling malaria. The chances of being bitten by a malaria-
infected female anopheline mosquito can also be reduced considerably by simple
measures e.g. covering exposed skin surfaces and remaining indoors or under a net at
peak biting times1.
Other preventive measures include the application of permethrin or
deltamerthrin to clothing or the use of insect repellents such as diethyltoluamide
(DEET) on exposed skin surfaces1.
2.10.3 Chemoprophylaxis
regions to malarious regions. The dihydrofolate reductase inhibitors (pyrimethamine,
proguanil and chlorproguanil) and atovaquone inhibit parasite development in the
liver (pre-erythrocytic activity) and in the erythrocytes. They are sometime called
causal prophylactics. These drugs also inhibit development in the mosquito
(sporontocidal activity). Chloroquine and mefloquine inhibit asexual blood stage
development of the liver stages. Thus, the parasites emerge from the liver but cannot
multiply in the red cells. Drugs with this action are called suppressive prophylactics.
These drugs also have gametocytocidal activity against P. vivax, P. ovale and P.
malariae but not P. falciparum. Atovaquone-proguanil, doxycline and primaquine
have been added to the list of antimalarial prophylactics. Each is active against
resistant P. falciparum but each must be taken daily. Antimalarial prophylaxis must
be taken regularly to ensure that therapeutic antimalarial concentrations are
maintained. Recommendations vary considerably, depending on risk, prevalence and
49
drug resistance. Up to date recommendations are provided by the World Health
Organization1.
2.11 CONTROL OF MALARIA
The objective of a malaria control program will depend on the prevailing
epidemiological situation, the availability of resources, and feasibility108. The main
arms of malaria control are:
i. Vector control through use of insecticides and destruction of breeding grounds
of mosquitoes.
ii. Deployment of insecticide treated bed nets or other household materials e.g.
window and door screens.
CHAPTER THREE
MATERIALS AND METHODS
3.1 STUDY DESIGN
This study was a hospital based, prospective, cross sectional study. Specimens
were collected from children and adult patients of both sexes from whom consent had
50
been received to participate in the study. Demographic and clinical data were
collected from the patients by means of a questionnaire (Appendix IV). Specimens
were collected from patients who met the inclusion criteria at the out-patient
department while working with family physicians, at the collection centre of the
medical microbiology department and from the wards and emergency units during
call hours.
At the out-patient department, the patients were interviewed and examined
alongside family physicians and those who met the inclusion criteria and gave consent
were recruited into the study. At the sample collection centre of the microbiology
department, patients who came for their samples to be collected for malaria
microscopy were interviewed and those that met the inclusion criteria and gave
consent were recruited into the study. During call hours, patients whose samples were
sent to the laboratory for malaria microscopy from the wards and emergency units
were traced, interviewed and examined and those who met the inclusion criteria and
gave consent were recruited into the study. Specimens were not collected from those
who were on anti-malarial therapy or had taken anti-malarial treatment in the two
weeks preceding the study.
The specimens were processed in the laboratory by standard techniques. In
this study, the sensitivity, specificity and predictive values of rapid diagnostic test and
microscopy were determined using polymerase chain reaction as the standard. Rapid
diagnostic test was also compared with microscopy.
3.2 STUDY AREA
Sample collection was carried out at the Jos University Teaching Hospital.
Microscopy and rapid diagnostic test was carried out in the Microbiology and
51
Parasitology Department of the Jos University Teaching Hospital. The polymerase
chain reaction was carried out at the Malaria Research Laboratories, Institute for
Advanced Medical Research, University of Ibadan.
The Jos University Teaching Hospital is a 600 bedded tertiary health
institution serving Plateau State and majority of the States in the North Central
Geopolitical Zone of Nigeria. The Malaria Research Laboratories is involved in
research aimed at a better understanding of malaria infection which includes
molecular diagnosis of malaria, parasite fingerprinting and studies on the molecular
mechanisms of anti-malarial drug resistance in the parasite.
Jos which is the administrative capital of Plateau State is divided
administratively into Jos North, Jos South and Jos East Local Government Areas and
the teaching hospital is located in Jos North Local Government Area. Jos is bounded
by Bauchi state to the north and the east, Riyom and Barkin Ladi Local Government
areas to the west and part of Barkin Ladi and Mangu Local Government Areas to the
south. Jos has a population of 900,000 people based on the 2006 national population
census109. The population is made up of civil servants, students, traders and farmers.
It is located at 9056’N 8053’E/ 9.9330N 8.8830E. With an altitude of 4,062 feet
(1,217m) above sea level, it enjoys a more temperate climate than the rest of Nigeria
(average monthly temperatures ranging from 210 to 250C. From mid-November to late
January night time temperatures drop as low as 110C resulting in chilly nights109.
The city of Jos receives about 1,400 mm (55.1 in) of rainfall annually. There
are several surface water dam which are used to provide water for the city’s
industries, for irrigation and for generation of hydroelectric power. These include the
surface dams in the Lamingo area and the Jos-Bukuru dam and reservoir on the Shen
River. Mining activities have left numerous ponds where people live, farm and work.
These dams and ponds serve as favorable breeding sites for mosquito vectors.
3.3 STUDY POPULATION
Adults and children of both sexes from whom consent had been received were
enrolled into the study.
3.4 INCLUSION CRITERIA
i) Adults complaining of fever or with an axillary temperature of more than
37.20C with or without any other symptoms and for whom a clinical
diagnosis of acute uncomplicated malaria had been entertained.
ii) Children with history of fever or with an axillary temperature of 37.50C or
above for whom a clinical diagnosis of acute uncomplicated malaria had
been entertained.
3.5 EXCLUSION CRITERIA
i) Patients with none of the above clinical features
ii) Patients who were on anti-malarial treatment or who have received anti-
malarial therapy in the two weeks preceding the study.
iii) Patients with complicated malaria.
iv) Refusal to give consent to participate in the study.
3.6 STUDY SAMPLE SIZE
The minimum sample size was calculated using the following formula 110
53
Z = The standard normal deviation corresponding to 95% levels of
significance (1.96).
p = the overall prevalence of malaria on the Jos plateau was set at 13%111, 112.
q = l-p.
Calculation:
0.052
= 0.4345
0.0025
= 173.8
Approximately =174
To take care of invalid samples, 10% of the minimum sample size of 174
was added to the minimum sample size.
10% of 174= 17.4
Therefore 191 patients were required. However, 200 hundred subjects
were recruited into the study to improve and strengthen the power of the
statistical analysis.
i) Standard precautions were adhered to throughout the procedure113.
ii) The specimen was collected by pricking the ball of the middle finger or
heel in neonates.
iii) The ball of the finger or heel was cleaned with cotton wool dampened with
70% alcohol.
iv) The finger or heel was dried with a clean piece of cotton wool, using firm
strokes to stimulate blood circulation.
v) A sterile lancet was used to puncture the ball of the finger or heel using a
quick rolling action.
vi) Gentle pressure was applied to the finger or heel to express the first drop
of blood which was wiped away with dry cotton wool, making sure that no
cotton strands remained that might later be mixed with the blood.
vii) A single drop of blood was collected on the middle of the slide for the thin
film. Using a second clean slide as a spreader and the slide with blood
drop on a flat firm surface, the drop for the thin film was touched with the
spreader allowing the blood to run along its edge. Firmly pushing the
spreader along the slide and keeping the spreader at an angle of 450 a thin
film was be made.
viii) Two drops of blood were collected on the slide intended for the thick film.
Using the corner of the spreader, the two drops of blood for the thick film
were joined to make an even thick film about 1cm in diameter.
ix) 5µL of blood was collected using the calibrated pipette provided in the
RDT pack and placed on the pad in the specimen window of the rapid
diagnostic test cassette for rapid testing.
55
x) Two drops of blood were spotted onto 3MM filter paper (Whatman
International, Ltd., Maidstone, United Kingdom), air- dried for five hours
and stored at room temperature in plastic envelopes for subsequent DNA
extraction for the polymerase chain reaction95.
xi) The slides were labeled with a soft lead pencil by writing across the
frosted end of the slide.
xii) The films were allowed to air dry.
xiii) Each thin film was fixed by flooding it with absolute methanol with a
Pasteur pipette. The thick films were not fixed.
3.7.3 Staining with Giemsa
PRINCIPLE OF THE TEST:
Giemsa stain is an alcohol-based Romanowsky stain. It is a mixture of eosin
and methylene blue. The eosin stains parasite chromatin and stippling shades of red
and pink while the methylene blue stains parasite cytoplasm blue86.
TEST PROCEDURE86:
i) The slides were placed in a staining rack and covered with 10% Giemsa
stain.
ii) The stain was left on the slides for 10 minutes.
iii) The stain was then washed off the slides with distilled water from a wash
bottle.
iv) The slides were removed one after the other and placed on a drying rack,
film side downwards, to drain and dry making sure that the thick film did
not touch the edge of the rack.
v) The films were viewed using a compound light transmission microscope
with x10 ocular and x100 objective lenses.
56
vi) In the thick films, though white blood cells were seen, no red blood cells
were seen. The trophozoites of the parasites were seen as signet rings with
the red stained knob being the chromatin and the remainder of the circle
which stained blue as the cytoplasm. In the middle, clear vacuoles were
seen. Sometimes comma shaped forms were seen and at other times the
chromatin dots were the most prominent diagnostic feature. 200 fields of
the thick film were viewed at a magnification of x1, 000 before a thick
film was declared negative.
vii) In the thin films, the parasites were looked for inside the red blood cells.
P. falciparum rings remained small, with no obvious effect on the host red
cell, apart from the presence of Maurer clefts. The red cells were also
examined for multiple infections. The films were examined until the
presence and species of the malaria parasite had been identified, or up to at
least 800 fields before declaring the slide negative.
viii) When a slide was positive for P. falciparum the parasite count was carried
out on the thick film using two manual cell counters.
ix) The numbers of parasites seen were counted on one counter and the
number of white blood cells on the other, oil immersion field by oil
immersion field.
x) If after 200 white blood cells had been counted, 100 or more parasites
were found, the counting ended there. If after 200 white blood cells had
been counted, the number of parasites were 99 or fewer, counting
continued up to 500 white blood cells.
57
xi) When counting was completed, the number of parasites relative to the
number of leukocytes was calculated and expressed as ‘parasites per
microlitre of blood’ from the formula:
Number of parasites counted x 8000
Number of leukocytes
= parasite count/µL
The 8000 used in the formula above represents the number of leukocytes. It is the
mean of the range of the white blood cells count in individuals (4000 -12000/mm3).
Though the white blood cell count of the individual should be used, the figure is
accepted internationally as reasonably accurate.
3.7.4 Rapid Diagnostic Test
The SD BIOLINE Malaria Ag P.f brand of rapid diagnostic test with Lot
number 082034 was used for the rapid diagnostic test. This test detects the Histidine
rich Protein-II antigen (HRP-II) of Plasmodium falciparum only.
PRINCIPLE OF THE TEST 114, 115
This is shown in figure 3.1 (p. 48). The figure is explained below:
a. Dye-labeled mouse monoclonal antibodies specific for P. falciparum HRP-II,
is present in a plastic well provided with a nitrocellulose strip. Antibody,
specific for another epitope on the target antigen, is bound to the strip in a thin
(test) line, and antibody specific for the labeled antibody, is bound at the
control line.
58
b. Blood and buffer, which have been placed in separate wells, are mixed with
labeled antibody and are drawn up the strip chromatographically across the
lines of bound antibody.
c. If antigen is present, some labeled antibody will be trapped on the test line.
Excess-labeled antibody is trapped on the control line. If sufficient labeled
antibody accumulates, the dye labels will become visible to the naked eye as a
narrow line.
d. The test is positive when two lines are seen. It is negative when only the
control line is seen. The result is invalid when no line is seen.
59
Figure 3.1: Principle of histidine rich protein II P. falciparum malaria RDT
60
TEST PROCEDURE115:
1. 5µL of blood was placed on the pad in the specimen well.
2. Four drops of buffer solution were placed in the buffer well.
3. The cassette was placed on a flat surface for 15 minutes and then
read.
4. The rapid diagnostic test cassettes each carried a control line for
procedural control which always appeared if the test was
performed correctly.
3.7.5 Polymerase Chain Reaction
PRINCIPLE OF THE TEST:
The basic principle of the polymerase chain reaction is as follows: One DNA
molecule is used to produce two copies, then four, then eight and so forth. This
continuous doubling is accomplished by polymerases, enzymes that are able to string
together individual DNA building blocks to form long molecular strands. To do their
job, polymerases require a supply of DNA building blocks, i.e. the nucleotides
consisting of the four bases adenine (A), thymine (T), cytosine (C) and guanine (G).
They also need a small fragment of DNA, known as the primer, to which they attach
the building blocks as well as a longer DNA molecule to serve as a template for
constructing the new strand. If these three ingredients are supplied, the enzymes will
construct exact copies of the templates.
TEST PROCEDURE:
The methanol-fixation heat-extraction method for DNA extraction was used.
Parasite genomic DNA was extracted from blood samples collected on filter paper
61
using Qiagen DNA extraction kit according to the manufacturer’s protocol. The
following steps were taken:
i) A small 40 mm2 snippet of blood impregnated filter-paper was cut using an
individual surgical blade for each sample.
ii) The snippet was cut into several small pieces into a 1.5 ml eppendorf tube.
iii) Buffer AL was added to the blood impregnated filter paper in the
eppendorf tubes and incubated at 85º C for 10mins.
iv) Proteinase K was added to the mixture and incubated at 56º C for one
hour.
v) Buffer AL was added again, vortexed and incubated at 70º C for 10mins.
vi) 100µL of absolute methanol was added and centrifuged for 30secs.
vii) The content was transferred into the Qiamp spin column provided in the
kit and centrifuge at 6000rpm for 1min.
viii) 500 µL of buffer AW1 was added and centrifuged at 8000 rpm for 1
minute and the flow-through was discarded.
ix) 500 µL of buffer AW2 was added and centrifuged at 13000 rpm for 3
minutes and the flow-through was discarded.
x) The spin column was placed in a 1.5ml eppendorf tube and 200µL of
buffer AL was added to elute the DNA from the spin column. The set up
was allowed to stand for 1 minute.
xi) The tube containing the spin column was centrifuged at 8000 rpm for 1
minute and the flow- through was collected.
xii) 200µL of buffer AL was again added a second time to elute the DNA from
the Qiamp spin column and allowed to stand for 1 min and step xi was
repeated.
62
xiii) 5µL of the flow through from steps xi and xii was used as the DNA
template for the polymerase chain reaction and the rest stored at -20oC.
B) Stage 2: Preparation of the PCR mix95.
This was prepared using the following table per sample in a PCR tube.
Table 3.1: Master Mix table for malaria PCR
PCR COMPONENTS INIT