i

DISTRIBUTION OF HAEMOGLOBIN PHENOTYPES AND THE

RELATIONSHIP WITH CLINICAL CHARACTERISTICS AND

NUTRITIONAL STATUS IN THE NEWBORN AND OLDER CHILDREN

LESS THAN 60 MONTHS IN ETHIOPE WEST LOCAL

GOVERNMENT AREA OF DELTA STATE.

“THIS DISSERTATION IS SUBMITTED IN PART FULFILLMENT

OF THE REQUIREMENT FOR THE AWARD OF THE

FELLOWSHIP OF THE NATIONAL POSTGRADUATE MEDICAL

COLLEGE OF NIGERIA IN THE FACULTY OF PAEDIATRICS”.

GLORIA .A. NWAJEI

(M.B;B.S ;2002)

NOVEMBER, 2015

ii

DECLARATION

“It is hereby declared that this work is original unless otherwise acknowledged. This work has

not been presented to any other College for a Fellowship, nor has it been submitted elsewhere

for publication”.

…………………………………………..

GLORIA ATIM NWAJEI

iii

ATTESTATION

The study reported in this dissertation was done by the candidate under our supervision.

We also supervised the writing of this dissertation.

NAME OF FIRST SUPERVISOR Professor Angela. A. Okolo

SIGNATURE :

STATUS OF SUPERVISOR Consultant Paediatrician/Professor of Paediatrics

DATE

NAME OF SECOND SUPERVISOR Dr. Moses. Diakparomre

SIGNATURE

STATUS OF SUPERVISOR Consultant Paediatrician

DATE

NAME OF THIRD SUPERVISOR Professor. Mohamed Cherif Rahimy

SIGNATURE

STATUS OF SUPERVISOR Consultant Paediatrician/Professor of Paediatrics.

Coordinator of Newborn Screening for Sickle Cell

Disease, National University of Cotonou, Republic

of Benin.

DATE :

iv

TABLE OF CONTENTS

Title Page i

Declaration ii

Attestation iii

Table of Contents iv

Dedication v

Acknowledgement vi

List of Abbreviations used in this study vii

Definition of Terms viii

Summary x

Introduction 1

Literature Review 4

Aims and Objectives 29

Subjects and Methods 30

Results 41

Discussion 60

Conclusions 73

Recommendations 74

Limitations 75

Lines of Future Research 76

References 77

Appendixes 90

v

DEDICATION

This book is dedicated to children with sickle cell disease all over the world.

vi

ACKNOWLEDGEMENTS

My deep and sincere gratitude goes to my supervisors, Professor. Angela. A. Okolo, Dr. Moses

Diakparomre and Professor. Mohamed .C. Rahimy whose scholarly influence, discipline and

depth of knowledge contributed immensely to ensuring the successful execution of this project.

I acknowledge with thanks, all the children who participated in the study, as well as their

caregivers.

My sincere gratitude also goes to Marielle Guonongbe, Gloire Gbedji and Gladys Cadete, the

laboratory technologists in charge of the sickle cell research laboratory in Cotonou, the Republic

of Benin, for all the assistance they rendered in ensuring that the work was done.

I thank my husband, Dr. Charles Nwajei for being my pillar of support, providing the moral and

financial support throughout the residency training period and my little angels, Anita and

Alexandra Nwajei, for encouraging and supporting me in their own little way and for bearing

with me during the difficult times.

I thank all those who contributed in one way or another in ensuring that this work was done.

May God reward all your endeavours.

Finally and most of all, I thank God for everything.

vii

LIST OF ABBREVIATIONS

Hb Haemoglobin

SCD Sickle cell disease

SS Homozygous sickle cell disease

SC Heterozygous sickle cell haemoglobin C disease

Sβ° Heterozygous sickle cell - beta° thalassaemia

AC Haemoglobin C trait

AS Haemoglobin S trait

AD Haemoglobin D trait

AA Normal haemoglobin phenotype

LGA Local Government Area

CHEW Community Health Extension Worker

PHC Primary Health Center

DELSUTH Delta State University Teaching Hospital, Oghara

WHO World Health Organization

CAC Cellulose acetate electrophoresis

CAG Citrate agar electrophoresis

EDTA Ethylenediamine tetraacetic acid

MUAC Mid upper arm circumference

viii

DEFINITION OF TERMS

Genotype The genetic makeup of the haemoglobin type.

Phenotype Haematological (laboratory) diagnosis inferred by identification of

the haemoglobin produced by the normal or altered genes.

SCD Connotes all the phenotypes of sickle cell disease.

Homozygous Having identical alleles for a single trait.

Heterozygous Having two different alleles for a single trait.

Sickle cell anaemia Homozygous sickle cell disease.

Carrier Frequency The proportion of individuals in a population who inherited a

single copy of a specific recessive gene mutation.

Haplotype A set of DNA variations or polymorphisms that tend to be

inherited together.

Cohort A group of subjects who have shared a particular event

together during a particular time span.

Thalassaemia A group of inherited disorders of haemoglobin metabolism in

which there is impaired synthesis of one or more of the

polypeptide chains of globin.

Balanced Polymorphism A situation in which two different versions of a gene are

maintained in a population of organisms because individuals

carrying both versions are better able to survivethan those who

have two copies of either version alone.

Holoendemicity A state of high prevalence of a condition,where transmission,

and intensity throughout the year are high,without significant

seasonal or periodic variation.

Newborn A baby in the first 28 days of life.

ix

Infancy The period from birth to one year of age.

Older Child Any child one month to less than 60 months of age.

Electro-osmotic Flow When a voltage is applied across a tube filled with an electrolyte

solution (a solution that conducts electricity), the solution begins

tomove toward the cathode.

x

SUMMARY

In an effort to describe the haemoglobin phenotype distribution in the respective age cohorts of

newborn and older children less than sixty months of age, subjects were recruited into this study

starting from the 1st of April, 2013 to the 30th of September, 2013. Two hundred and eighty eight

newborn and 1,263 older children were recruited from randomly selected homes in three health

wards of the communities of Ethiope West. These were studied for their haemoglobin

phenotype distribution by respective age cohorts. Iso-electrofocusing and capillary

electrophoresis techniques were used forsample analysis for the newborn, while capillary

electrophoresis was used for the older age cohorts. Physical examination was carried out and

anthropometric measurements taken for each subject.

One hundred and eleven blood samples were lost to testing. Of these,42 belonged to the

newborn, while 69 to older children.

One thousand, four hundred and forty results were available following sample analyses. Two

hundred and forty six were for the newborn, while 1,194were for older children less than sixty

months.

Five haemoglobin phenotypes consisting of HbAA (73.6%), HbAC (0.4%), HbAD (0.1%),

HbAS (23.7%) and HbSS (2.2%) were identified.

Haemoglobin phenotype distribution in the respective age cohorts was varied, however, beyond

24 months of age, the prevalence of HbAA, HbAS and HbAC remained relatively constant.

The observed prevalence of HbSS ranged from 1.9% to 3.2% among the cohorts aged 1 month

to less than 60 months. In the newborn,the prevalence of HbSS was 1.2%, while the specific

distributions for the other haemoglobin phenotypes were HbAA (78.5%), HbAC (0.8%) and

HbAS (19.5%).

The presence of abnormal clinical signs was significantly higher among HbSS subjects than in

children of other haemoglobin phenotypes. (p< 0.001)

xi

Normal nutritional status was documented in 86.9% of the children aged one month to less than

60months. Of the subjects with HbSS aged one month to less than 60months, 20.7% were

underweight, while 13.8% were stunted..There was no significant difference in the nutritional

status among the various haemoglobin phenotypes. (p = 0.691)

These findings support the case for early diagnosis of sickle cell disease,comprehensive care

management and follow up of these cases as well as integration of growth monitoring in the

comprehensive care package so as to enhance nutritional support for the affected children.

1

INTRODUCTION

About 800 structural variants of haemoglobin have been described, the vast majority of which

are not associated with clinical manifestations.1The inherited disorders of haemoglobin are the

commonest single gene disorders known to man. The World Health Organisation estimates that

about 7% of the world population are carriers.1

If untreated, many of the clinically significant inherited haemoglobin disorders result in death

during the first few years of life. Their effect on the burden of disease has only recently become

more apparent following an epidemiologic transition owing to improvements in hygiene,

nutrition and control of infections that have seen a reduction in childhood mortality in countries

such as India and much of Asia.2 A similar transition is expected in sub-Sahara Africa and

hence, the need to understand the full extent of the problem and to develop programs to control

and manage these diseases.

The adult haemoglobin molecule is made up of two pairs of polypeptide (globin) chains, α and

β, with each chain having an iron-containing porphyrin ring (heme group)

attached.3Abnormalities in the globin chains give rise to haemoglobinopathies. Such abnormal

haemoglobins are produced by the activity of abnormal genes which may arise by spontaneous

mutation or may be inherited.3One of such mutations that occurred in the beta polypeptide chain

is the sickle gene, giving rise to haemoglobin S.

Sickle cell haemoglobin is the commonest single gene defect of the black race. Each year, there

are about 275,000 births of babies with a sickle cell disorder, and of these, 150,000 are born in

Nigeria alone.2 Thus, 55% of sickle cell disease (SCD) patients born each year are Nigerians.

Nigeria therefore has the biggest burden of SCD worldwide. Most of these children are

homozygous SS.2

2

It is noted, however, that the Asian haplotype of SCD is shared by people throughout central

India.4 SCD of the Asian haplotype is associated with high levels of fetal haemoglobin and

frequent deletional alpha thalassaemia, both factors likely to ameliorate the disease and change

the clinical features. Malaria is another factor that influences expression of the disease in this

region.4In predicting the prevalence of SCD in India,where the population currently stands at

1.22billion, many assumptions have been made.4 With a 20% trait frequency, 1% of births

would have homozygous SCD or 10,000 cases per million population. This would equate to

500,000 cases among a population of 50 million or 1million cases for an at risk population of

100 million. These figures suggest that the disease in India is at least, as prevalent as in

Equatorial Africa.4 However, most reports attest to the fact that three-quarters of sickle cell

cases occur in Africa where the carrier frequency ranges between 10% and 40%.5 Studies in

Nigeria show that 1- 3% of the population are homozygous SS and 25% are AS.6 This high

carrier frequency ensures propagation of the S gene among the Nigerian population.

Early studies suggested that mortality rate from SCD was highest in the first five years of life

with 50% of deaths occurring in the second half of the first year of life.7-12For most of them, the

diagnosis had not been made at the time of death. Approximately 80% of young children with

SCD in Africa die from this disorder by five years of age.13

The public health implications of sickle cell anaemia are thus,significant. Its impact on human

health may be assessed against the yardsticks of infant and under-five mortality. As not all

deaths occur in the first year of life, the most valid measure is underfive deaths. Sickle cell

anaemia contributes the equivalent of 5% of under-five deaths on the African continent, more

than 9% of such deaths in West Africa and up to 16% of under-five deaths in individual West

African countries.14

3

JUSTIFICATION FOR THE STUDY

In Nigeria, over 66% of babies are born outside the formal health care system.15 Majority of the

studies that have been done on haemoglobin phenotypes in children are hospital-based.

Community-based studies would give a more precise picture of the problem than hospital-based

studies, hence the need for a community based study.16

The age cohort, zero to less than sixty months, is the most vulnerable to SCD and it is a well

known fact that about 50% of affected children die in their first year of life7-12 without ever

being diagnosed. Infant mortality on its own, contributes significantly to under-five mortality,

while neonatal mortality contributes 40% of overall under-five mortality.17 With an annual

infant death rate of 100,000, SCD contributes 8% of infant mortality in Nigeria.18 Therefore, to

aim at reducing infant and under-five mortality, it would be beneficial to identify early the

affected child so as to apply, early in life, the principles of comprehensive care and parental

education.

Ideally, early identification of abnormal haemoglobins will aid in early and prompt institution of

measures to control severe manifestations of the condition through comprehensive target

healthcare. All parents, caregivers, as well as the children, would be made to become aware of

the child’s phenotype and the nature of the disorder. Such information would be very useful for

the planning of health care preventive services and life choices by the affected children and their

families. This will also enable the affected persons make informed decisions when they reach

adulthood.

Data generated from such a study could aid governmental and non-governmental institutions in

planning effective management strategies for SCD and other haemoglobinopathies, through the

provision of comprehensive healthcare such as parental education and counseling, provision of

health facilities, regular medical follow up and adequate immunizations. In communities where

this is practiced, the mortality rate has dropped from between 15% and 30% to less than 1%.19-21

4

LITERATURE REVIEW

Over one hundred abnormal haemoglobin variants have been described, however, only a few are

common and clinically significant.22 The common abnormal haemoglobin variants are HbS,

HbCand HbE, while less common ones include HbD, HbG, HbJ, HbM, Hb Constant Spring,

HbH and Hb Barts. Some are clinically silent causing no signs or symptoms, whereas others

such as HbS, HbC and HbE, affect the function and/or stability of the haemoglobin

molecule.22In different regions of the world, their spectrum and distribution vary. Therefore,

knowledge of the pattern and distribution of haemoglobin phenotypes is important, both for

public health purposes and for genetic counseling.

Globally, haemoglobin S is the most common abnormal structural haemoglobin variant.The

haemoglobin S gene is distributed throughout sub-Sahara Africa, the Indian Subcontinent, the

Middle East, and the Mediterranean region.2 The carrier frequency ranges between 10% and

40% across Equatorial Africa, decreasing to 1-2% on the North African coast and less than 1%

in South Africa.2 In Saudi Arabia, about 4.2% of the population carry the sickle cell trait,2the

highest frequency being found in the Eastern province where approximately 17% of the

population carry the gene. In India, the carrier frequency varies from 0 to 40% in different

population groups.23Several distinct β-globin gene haplotypes are associated with the sickle

mutation, and their distribution provides evidence for the origin of the mutation in several

locations within Africa (the Senegal, Benin and Bantu haplotypes) and Asia (the Arab-Indian

haplotype).2

Various HbAS trait frequencies have been reported; 1 in 100 among Cypriots, Pakistanis and

Indians; 1 in 10 among African-Carribeans; 1in 4 among West Africans.6

Hb E is found in the eastern half of the Indian Subcontinent and throughout Southeast Asia.2

Thalassaemia is frequent in a broad band stretching from the Mediterannean basin and parts of

5

Africa to the Pacific islands. The α+ thalassaemias occur right across the tropical zone reaching

extremely high frequencies in some populations, whereas the α° thalassaemias are restricted to

parts of Southeast Asia and the Mediterannean basin.2 Three to eight percent of Americans of

Italian or Greek ancestry and 0.5% of African Americans carry a gene for beta

thalassaemia.3The regions that are most affected by beta-thalassaemia in Africa include Western

and Northern Africa respectively with 63.88% and 22.17% of the total annual affected

conceptions. The major forms of alpha-thalassaemia are almost absent in Africa.2Weatherall et

al found a prevalence of 0.2% in Lagos, Nigeria; 1.3% in Southern Ghana; and 1.7% in Northern

Ghana.2

There are 270 million carriers and 300,000 to 500,000 annual births of infants with sickle cell

anaemia or serious forms of thalassaemia worldwide.2Among them, 60,000 are born annually

with beta-thalassaemia major, the remaining (83%) with SCD. Southeast Asia, where the

thalassaemias and Hb E predominate, appears to be the most severely affected. Sub-Sahara

Africa has the second highest burden reflecting the high incidence of HbS.

In Nigeria, as in most of sub-Sahara Africa, the phenomenon of balanced polymorphism (as

regards malaria), has made the prevalence of the HbS very high.6 Nigeria, with its high

population of 165 million, a geographical location in the tropical region and holoendemicity for

malaria, has a very high burden of S haemoglobinopathy. The relatively poor healthcare

structure results in a very high mortality among these children.6

Haemoglobin C trait (HbAC) has a frequency of 1 in 30 among African-Caribbeans and 1 in 6

among Ghanaians, while the D trait (HbAD) is 1 in 100 among Pakistanis and Indians, and 1 in

1,000 Caucasian British carriers.6 About 16 variants of HbD exist.The variant of HbD found

amongst Indians and Pakistanis is HbD Punjab. It differs in its clinical manifestation from other

less common variants of HbD such as HbD Korle Bu. In combination with HbS, HbD

6

Punjabresults in SCD with severe clinical manifestations, whereas others do not result in a

disease state and the affected individuals are clinically normal.3

The HbC is believed to have originated from Northern Ghana and Burkina Faso, then spread to

other parts of Africa by population migration.24 In Nigeria, the spread to the eastern part of the

country appeared to have been limited by the River Niger.25Cabannes24 in 1965, in support of

this observation noted that there were no cases of HbC in the eastern part of Nigeria in

comparison to the western part, where the HbC was quite prevalent. An explanation for this

finding was proffered by Nwokolo and Lehmann,25 who postulated that the ‘’C’’ gene originated

from Burkina Faso and Northern Ghana. They conjectured that migration of these peoples into

parts of Nigeria was inhibited by the difficulty to cross the River Niger;25 hence, the paucity of

the ‘’C’’ gene across the Niger. This prevalence decreases as one moves from the west towards

the east. In a population study of HbC in Akwa Ibom State in 1996, Usanga et al26found an

incidence of 0.4% for HbAC and 0.07% for HbSC.In Northern Nigeria, Fleming et al (1972)

noted some foci of the HbC gene.27 These studies from Nigeria were undertaken at different

locations and different times and none of them explored the respective age cohorts of under-

fives in a given population at the same time.

In the USA, the HbC and HbS genes have been shown to have spread there through the slave

trade24 and the reported frequency of C phenotypes is 1 in 50 for HbAC and 1 in 5,000 for

HbCC amongst African Americans.3

DISTRIBUTION OF HAEMOGLOBINPHENOTYPES IN THE NEWBORN

The ‘newborn’, reflects a population group that reflects the genetic distribution of the different

haemoglobin variants, considerably uninfluenced by the environment. Hence, they represent a

unique cohort to be studied. In Italy, Ballardini et al28 found that 1.2% of newborns were carriers

of abnormal haemoglobin variants. 0.8% had HbAS, 0.2% HbAC and 0.1% HbAE while 0.05%

7

were HbAD Punjab and HbAD Ouled Rabah, respectively. One other study in England by

Allison et al29 showed a prevalence of 0.05% for SCD (HbSS 0.04% and HbSC 0.01%). In that

study, 1.2% had HbAS, 0.2% were HbAC , 0.1% were HbAD and 0.2% were HbAE.

These European studies were carried out on populations thatcomprised people of diverse racial

and ethnic groups, hence the wide range of haemoglobin phenotypes described.

In Ghana, Ohene-Frempong et al30 screened a total of 202,244 newborn infants and found

HbAA in 76.3%, HbAC in 8.5%, HbAS in 13.3% and SCD in 1.9% (HbSS in 1.1% and HbSC in

0.8%).Tshilolo et al31 in the Democratic Republic of Congo, Central Africa screened 4116

neonates, and found HbAA in 77.3%, HbAS in 20.4% and HbSS in 2.3%.In the newborn

screening program in Angola, Southern Africa,of 36,453 infants screened, HbAA was seen in

77.3%, HbAS in 21.0%, HbSS in 1.5% and HbSC in 0.2%.32

These studies conducted in Africa utilized the same method for determination of haemoglobin

phenotype, thus documenting prevalence rates and pattern that are similar. However, the lower

prevalence of HbAS in Ghana, may be attributed to the higher prevalence of HbC there.24The

lower prevalence of SCD in Angola may on the other hand be due to the low carrier frequency

for HbS in the southern part of Africa.2

Few Nigerian studies have included the neonatal population. Odunvbun et al33 (2008) found a

prevalence of 75.3% for HbAA, 20.6% for HbAS, 2.8% for HbSS, 0.2% for HbSC and 1.1% for

HbAC in a hospital-based study on newborns in Benin city, Southern Nigeria.This study

involved 647 consecutively delivered newborns and being a hospital-based study, fails to reflect

the situation in the general population.Fleming et al27 (1979) found HbAA in 73.2%, HbAS in

24%, HbSS in 2.1% and HbAC in 0.7% in a community-based study in Kano State, Nigeria.

This study involved the 534 neonates born within the Garki community over the five year period

within which the study was conducted.

8

With regards to the spectrum of haemoglobin phenotypes identified and the prevalence rates of

the various haemoglobin phenotypes, Odunvbun et al33 and Fleming et al,27 documented a

similar pattern and similar rates for all the haemoglobin phenotypes identified. This may be

related to the fact that both studies utilised the same method, isoelectrofocusing electrophoresis

for haemoglobin phenotype determination. Also, the similar pattern described in both studies

may be due to the fact that they were both carried out in the same country with similar

geographic characteristics.

On the other hand, Abhulimhen-Iyoha et al34 in a community-based study involving the under-

fives in Ekosodin community, Benin city, Nigeria, found HbAA in 100% of the newborns

screened.34The absence of the ‘’S’’ gene in the neonatal population in that study is noteworthy.

However, only fourteen newborn babies were screened, hence the small sample size compared

with those of Odunvbun et al and Fleming et al, could have accounted for the observed

difference.

The prevalence of HbAA documented by other workers30-32 outside Nigeria was similar to that

in the Nigerian studies alluded to above.27,33,34. In Ghana,30the prevalence of HbAS was much

lower compared with the studies done in Nigeria27,33 and this may be attributable to the higher

prevalence of HbC in Ghana as proffered earlier.24 The prevalence of SCD was lower in the

Ghanaian30and Angolan32 studies than that found in Nigeria. These studies30-32 outside Nigeria

were all hospital-based and neonates delivered outside the formal healthcare system were not

included in the studies. Analysis of several Demographic and Health Surveys15reveals that two

thirds of women in sub-Sahara Africa give birth outside the formal healthcare system.

9

DISTRIBUTION OF HAEMOGLOBIN PHENOTYPES AMONG OLDER CHILDREN

UNDER 60 MONTHS OF AGE

Among older children under 60 months of age, Kaine and Udeozo35 in Enugu, Eastern Nigeria

found HbAA in 75.8%, HbAS in 22.6% and HbSS in l.6%, while Adewuyi and Akintunde36 in

Ilorin, North Central Nigeria, found HbAA in 69.3%, HbAS in 21.5%, HbSS in 3.1%, HbSC in

1.7%, HbAC in 4.1% and HbCC in 0.3%. Kaine and Udeozo35 studied under-five children

presenting at the Institute of Child Health, while Adewuyi and Akintunde36 studied children

aged between one month and fourteen years in both the rural and urban communities of Ilorin,

Nigeria.The much lower prevalence of HbAA (69.3%) in Adewuyi and Akintunde’s study36 was

perhaps due to the much higher prevalence of the ‘’C’’ gene in that locality. This study was

conducted in Ilorin, which shares its boundary to the south with Oyo state, where the prevalence

of HbC is known to be high. Also, the indigenes of Ilorin consist mainly of the Yorubas who are

known to have a high prevalence of the HbC gene.24

Ogunkunle et al37 in Ibadan, South Western Nigeria, found HbAA in 75.6%, HbAS in 16.7%,

HbSS in 1.8% and HbAC in 5.9% in a community-based study involving under-five children.

South Western Nigeria is known for its high prevalence of HbC because in the past, individuals

who had migrated from Ghana where the prevalence is high, settled there.25

Abhulimhen-Iyoha et al34 in Ekosodin, Benin city, Southern Nigeria, found HbAA in 72.0% and

HbAS in 28.0%.There was no case of SCD in that study. This was a community-based study.

However, the sample size was small, as only one hundred and sevensubjects were studied and

subject selection was neither systematic nor randomized.

In Ghana, West Africa, Amoako38 found HbAA in 75.9%, HbAS in 9.2%, HbAC in 14.9%,

HbSC in 0.3%, HbCC in 0.3% and HbSS in 0.9%. This was a hospital-based study involving

10

only children aged 6 to 59 months who presented with fever to the Kintampo North Municipal

Hospital. In addition, the sample size was small (341) and the study period short (3months).

The prevalence of HbAS in the Nigerian studies alluded to ranged from 16.7% to 28.0%, 34-

37while Amoako38 in Ghana, found a prevalence of 9.2% among under-fives. In the Northern

part of Ghana where Amoako’s study38 was conducted, the C trait is known to be more prevalent

(20-25%) and the S trait less prevalent (10%),24 hence the comparatively higher prevalence of

HbAC and the lower prevalence of HbAS in that region.

Among under-fives, the prevalence of SCD in the studies alluded to above,34-37 ranged from

0.0% to 4.8%. The higher prevalence of SCD documented in Ilorin by Adewuyi and Akintunde36

was characterized by HbSS 3.1% and HbSC 1.7% and could have beenattributable to the high

prevalence of HbC in that environment.36 In that study, the prevalence of HbC trait was 4.1%.36

Kaine and Udeozo35, Ogunkunle et al37 and Abhulimhen et al,34 documented prevalence rates for

HbSS of 1.6%, 1.8% and 0.0%, respectively.In the study by Kaine and Udeozo35, the ‘’C’’ gene

was not observed and SCD was due mainly to homozygous haemoglobin S.35 Also, unlike

Adewuyi and Akintunde’s study,36 this was a health facility-based study and hence, may not

have given the true prevalence of SCD in the under-five population. In the study by

Abhulimhen-Iyoha et al,34 it is possible that had the sampling procedure been systematic and

randomized, and the sample size larger, SCD may have been documented in that community. In

the study by Ogunkunle et al,37 in Ibadan, South Western Nigeria, the prevalence of SCD was

1.8%. Being that this was a community-based study, it has probably given a concise picture of

the prevalence of SCD in that community.

11

HISTORY OF SICKLE CELL DISEASE

Africanus Horton, in 1874, described the fever crises, the recurrent joint pains, and their

exacerbation during the rainy season and the constant abnormality of blood associated with this

condition.39 Several communities in West Africa have long recognized the disease and had given

it various names signifying a chronic recurrent condition.24,39 In Ghana, the Ga tribe call it

‘’Chwechweechwe’’, the Twi tribe, ‘’Ahotutuo’’, the Fante, ‘’Nwiiwii’’ and the Ewe,

‘’Nuidudui’’.24,39 Similarly, in Nigeria, various names were given to children who tended to die

in early infancy such as ‘’Abiku’’ in the Yoruba land and ‘’Ogbanje’’ in the Ibo.40The Urhobos

call it ‘’Eda’’.

However, the first generally accepted modern report of SCD was that of Dr. James Herrick in

North America. In a 1910 publication in the Archives of Internal Medicine, he described the

disease as a ‘’peculiar elongated and sickle shaped red blood corpuscle in a case of severe

anaemia’’ in a Jamaican student.41 Mason,42 in 1922, summarized all subsequent cases and

concluded that it was a new disease entity. He was the first to use the term ‘’sickle cell

anaemia’’.42 In 1925, Castana reported the first case of SCD in a European and four years later

(1929), Cooley and Lee described the disease in a Greek family.24 Edington and Lehmann at

Accra, first described HbC outside the United States of America in 1954,24 most HbC occurring

in West Africa, with the highest frequency in Northern Ghana at 20 to 21%.24,39

PREVALENCE OF SICKLE CELL DISEASE

The commonest abnormal haemoglobin phenotype affecting the black race is sickle cell disease.

Its prevalence varies within different geographic locations. In the United States of America, and

indeed worldwide, SCD is the most common genetic disease identified through the State’s

Mandatory Screening Program, occurring in 1/2,647 births.3 SCD in the U.S occurs among

African Americans at a rate of 1/396 births and among Hispanics at a rate of 1 in 36,000 births.

Among those of Middle Eastern descent, no cases were identified among 22,000 screened by Al

12

Hosani, but at a rate of 1/16,000 subjects screened among Asian Indians.3In Jamaica, an African-

Caribbean population, the incidence is 1 in 300 live births. 43,44

In West Africa, the incidence is estimated to be about 20 per 1,000 live births(2%).6 This is in

consonance with the reports of Odunvbun et al33 and Fleming et al27, who documented incidence

rates of 30 per 1,000 (3.0%) and 21 per 1,000 (2.1%), respectively, among newborns in the

Southern and Northern parts of Nigeria, respectively. Among under-fives, Kaine and Udeozo,35

in Enugu, Eastern Nigeria found a prevalence of 16 per 1,000(1.6%). In addition to studying the

newborn, Fleming et al27 also studied the haemoglobin phenotype pattern of the entire

population sample of Garki District in Kano State. Of note, is the fact that in that study, in the

age cohort of one to four years the prevalence of SCD was only 0.4% and 0.05% in the cohort

over the age of nine years. Results from target screening of newborn babies of mothers with

sickle cell trait in Cotonou, Republic of Benin, indicate a prevalence of 9.9%.45 It would appear

that there is an age related prevalence of SCD.

More recent studies, however,by Adewuyi and Akintunde36 in Ilorin, North Central Nigeria, and

Abhulimhen et al34 in Ekosodin, Southern Nigeria, indicated prevalence rates of SCD among

under-fives of 4.8% and 0.0%,respectively. Of note is the fact that in Adewuyi and Akintunde’s

study,36 the prevalence of SCD was also found to vary with age. In that study, the highest

prevalence of HbSS was at age four years (3.75%) and of HbSC at age five years (3.45%). From

the age of eight years to fourteen years, the prevalence of sickle cell anaemia remained low at

0.0% to 1.1%, while HbSC prevalence ranged from 0% to 2.5%.The fall in prevalence of HbSS

among children from age 8years in that study may suggest that mortality and/or morbidity is

significantly increased at this age. These findings may represent a new trend of improving

survival in the under-five age group, perhaps, a result of better medical care in early childhood

which has not been extended to middle childhood, occupied by the stressful years of primary

and junior secondary school.

13

Earlier studies showed that mortality from SCD was highest during infancy, especially in the

second six months of life.9,11-13 In the USA, Porter and Thurman46 in 1963, reported a mortality

of 16% in SCD patients in the first decade of life despite the better socioeconomic environment.

Lambotte-Legrand and Lambotte-Legrand,47 in a study in Republic of Zaire in 1955, followed

up 300 children diagnosed with SCD at an average age of one year five months. In that study,

120 (40%) had died by the age of two years. According to Sergeant,39 even as at 1975, Van Ros

still claimed that the majority of Zairian children with SCD died in infancy.39Vandepitte (1995)

estimated a 1% survival into adult life. 39 In Zambia, Barclay (1970) found that 80% of SCD

patients died, also in early infancy. 39 In about 30% of these patients, the parents were not even

aware of the presence of the disease.9

Such works as these contributed to highlight the most vulnerable age for mortality in these

children and buttressed the need for introduction of earlier diagnosis and further preventive care

measures.

Improvements in survival of children with SCD during the last thirty years in the industrialized

nations have been well established, as a result of improved access to medical care, socio-

economic development and better education. More recent studies indicate that though mortality

in SCD is reducing steadily, it is still high in Africa, with the most vulnerable period being

under-five years of age.6,50 Most affected children born in low-resource settings still die

undiagnosed.48 On the other hand, in the 40% of Africa that is now urbanized, improved access

to healthcare is leading to increased survival and rising demands for hospital services.48

In a hospital cohort study in Tanzania, Makani et al48 followed up 1,725 SCD patients from

2004 to 2009. A mortality rate of 1.9% was recorded.

In one study in Lagos, South Western Nigeria, Akinyanju et al49examined the outcome of a

comprehensive care program for SCD from 1988 to 1995. Over the study period, the number of

14

subjects rose from 290 in 1988 to 1,223 in 1995, while the mortality rate fell from 20.6% in

1988 to 0.6% in 1995. Also, the number of hospital admissions fell from 350 (119%) to 30 (4%)

and the number of blood transfusions fell from 260 (90%) to 25 (2%).49

Similarly, a study in the United States of America examined the trends in mortality rates from

SCD from 1979 to 2005.50The paediatric mortality rate dropped by 3% each year during the time

period studied.

Hence, unarguably, the provision of well-organized comprehensive care helps better knowledge

of the disease by the sufferers and significantly reduces illness and death and improves the

quality of lives of children living with SCD in the developing world as they receive better and

prompt attention.

PATHOPHYSIOLOGY OF SCD

Under low oxygen conditions, the absence of a polar amino acid at position six of the β globin

chain promotes the aggregation of haemoglobin, which distorts the red blood cells into a sickle

shape and decreases their elasticity. The loss of red blood cell elasticity is central to the

pathophysiology of SCD. Normal red blood cells are quite elastic, which allows the cells to pass

through capillaries. In SCD, low oxygen tension promotes red blood cell sickling and repeated

episodes of sickling damage the cell membrane and decrease the cell’s elasticity. These cells fail

to return to normal shape when normal oxygen tension is restored. As a consequence, these rigid

blood cells are unable to deform as they pass through narrow capillaries, leading to vessel

occlusion and ischaemia.

The actual anaemia of the illness is caused by haemolysis, the destruction of the red cells,

because of their misshape. Although the bone marrow attempts to compensate by creating new

red blood cells, it does not match the rate of destruction. Healthy red blood cells typically live 90

to 120 days, but sickle cells only survive for 10 to 20 days. The bone marrow thus, undergoes a

15

compensatory hyperplasia which in these patients causes depression of the nasal bridge,

midfacial overgrowth and malocclusion.

CLINICAL FEATURES IN CHILDREN WITH SCD

A body habitus characteristic of severely affected patients with SCD is well recognized.

Pallor, jaundice, skull bossing, flattened nasal bridge and gnathopathy were common

presentations in African children with SCD.51 This is because of marrow expansion in these

areas of the flat bone of affected children.The spleen is often palpable in early life due to

extramedullary haematopoiesis, congestion and sequestration. Subsequent episodes of recurrent

vaso-occlusion and infarction lead to gradual autosplenectomy, and in most patients, the spleen

is no longer palpable by the age of ten years.52

In a study by Ambeet al53 in Northern Nigeria, it was seen that hand and foot swelling was the

main symptom at diagnosis in the under-five age group (38.7%) and was more common in the

age group, 6 to 11 months, followed by jaundice (16.1%), then pallor (13.6%).

George et al54in Port Harcourt, South- South, Nigeria, studied 169 sickle cell anaemia patients

aged 6 months to 18 years over a one year period. The most common presenting symptoms were

pallor (90%) and jaundice (58%). 19% had splenomegaly and 13.6% had bossing of the skull

bones.

In another study in Kenya,East Africa involving 124 children with SCD aged 0 to 14 years, who

were followed up for 13.8months, Sadarangani et al55 found splenomegaly in 33% and

hepatomegaly in 20%, with both being common in all age groups. The peak prevalence for both

however, occurred in the 6 to 8 year age group.

Swankar et al56 in a hospital-based study in India, involving 131 children with SCD attending a

specialist outpatient clinic in a rural hospital, found that fever was the most common presenting

16

symptom (51.1%), while splenomegaly was the most common sign (87.9%) followed by

hepatomegaly (77.6%). Pallor was documented in 56.9% and jaundice in 51.7%.

All the studies cited above were hospital-based and hence, a depiction of only the SCD cases

utilizing health facilities; not inclusive of those yet to be diagnosed in the community or of those

with clinically mild disease, who have remained in the steady clinical state.

The clinical spectrum of SCD varies widely between patients. Factors contributing to this

variability include environmental and social circumstances, geographical variation, alpha-

thalassaemia, whose co-existence with SCD has been noted to reduce haemolytic rate, and

persistence of high haemoglobin F levels which also ameliorates the clinical severity of SCD.51

NUTRITIONAL STATUS IN CHILDREN WITH SCD

It is generally believed that SCD has an adverse effect upon the physical growth and

development of affected children.57 In Nigeria, affected children are believed to be of lower than

average body weight and height than their unaffected peers.58,59 Such growth deficits in sickle

cell patients have been reported by earlier studies59,60,62-68 to be more pronounced with

increasing age. This may be because of the chronic anaemia state. An earlier report has shown

that even African American children with SCD were shorter, with lower weights and thinner

body build than unaffected children.60 Several studies61,66,68 from the United States of America,

Jamaica and Italy have similarly shown that children and adolescents with SCD have impaired

growth as compared to normal controls. Such studies61,66,68 had even shown some nutrient

deficiencies in such children, further buttressing the need for extra care in these individuals.

In a study by Oredugba and Savage62in Lagos, South Western Nigeria in 2002, the height and

weight values in 177SCD subjects aged 1 to 18 years were not significantly different from those

of controls (unaffected children). This was a hospital-based cross sectional study and the cases

17

and controls were mostly from the lower socioeconomic class, which might explain the lack of

significant difference in anthropometric measurements between both groups.

In one study by Mukherjee and Gangakhedkar63 in India, involving 58 SCD (HbSS) children

aged 2 to 14 years compared with normal (HbAA) children, the SCD children, both male and

female, showed statistically significant lower values of all the measurements except the

upper/lower segment ratio as compared to normal children of the same age and sex groups. This

was also a hospital-based cross sectional study and majority of the SCD subjects in this study

were noted to have severe disease in terms of their clinical manifestations.

Ebomoyi et al64 in 1989, studied 719 SCD subjects aged 2 to 13 years, and found all (100%) to

be below the 50th percentile of the Harvard standards for height and weight. This study was

hospital- based. As such, it assessed only SCD cases presenting in hospital. The study

population included both acutely ill and stablechildren, hence, the effects of acute illness on the

nutritional state were not accounted for.

Oyedeji65 in 1991 also investigated 102 SCD subjects aged 9 months to 17 years. For weight, all

(100%) were below the 3rd percentile, while for height, they were around the 3rd percentile.

In 1992, Caruso-Nicolettiet al66reported moderate growth delay among 76 white Sicilian

children (1 to 17 years) with SCD. Weight and height were less than the 3rd centile of reference

values for white British children in 16% and 10.5%, respectively. The majority of these children

had Benin haplotypes and normal levels of somatomedin C.66 Compared with the studies in

Africa62,64,65 and India,63 growth indices appeared to be better in these European children with

SCD. This observation is likely due to the better socioeconomic circumstances and medical care

available in the more developed parts of the world.

18

Similarly,in a longitudinal study in 1981,Mann67 reported 61 HbSS patients (3 months to 19

years) in England, 16% of whose heights were greater than 2SD below the mean Caucasian

reference value. This finding further confirms the fact that European children with SCD showed

better growth than those in Africa and elsewhere, probably indicating better nutrition and quality

of care. The varied clinical manifestations compared with reports from Jamaica and North

America led the author to conclude that the variations depended on many factors including

geographic location, endemic infection and the general standard of nutrition and medical care.

In a longitudinal study of children with SS and SC disease, Stevens et al68 in 1986, followed 455

subjects from birth to 9 years of age. Compared with normal AA controls, they showed no birth

weight differences for either gender. The weight deficit for the HbSS children commenced

before the end of the first year of life. The deficit appeared to be relatively more marked in girls

and a similar trend was observed for height.68The children with HbSC disease showed no growth

deficit.68

Most of these studies were observational and hospital-based, with wide variations in sample size

and selection of reference growth data, which limited comparability. They however, all showed

a consistent pattern of growth failure among affected children from all geographic areas,

although the severity varied with location and was most marked in low-resource settings.

External and internal factors are likely to act together to a different degree against a variable

genetic, environmental and socio-economic background.

Improving the nutritional status and growth of these children could have a favourable impact on

their clinical course and prognosis. Hence, growth monitoring with appropriate nutritional

support as part of the comprehensive care of children with SCD , when promoted, might

influence better outcomes in the affected subjects.

19

SCREENING FOR SICKLE CELL DISEASE

Whitby69 suggested use of the following criteria for screening for a genetic disorder.:

Is the abnormality clearly defined? In the case of SCD as noted by Sergeant,39 it is the

first genetic disorder in which the molecular abnormality was precisely defined.

What population should be screened? In areas where SCD is prevalent, it has been

suggested that the whole neonatal population should be screened.70-73 Universal screening,

however, may be valuable epidemiologically in the description of prevalence rates.

What is the incidence of the condition? In Nigeria, from studies conducted in infancy and

the under-fives, the incidence ranges from 1.6 to 4.8% (16/1,000 – 48/1,000). 27,33-37,74 These

values are higher than those for congenital diseases like phenylketonuria, galactosaemia

and hypothyroidism for which screening programs exist.

How do screening methods compare in efficacy and cost? The two most commonly

used screening methods are haemoglobin electrophoresis and high performance liquid

chromatography. There are several established methods of electrophoresis: cellulose acetate,

citrate agar, thin layer iso-electrofocusing and capillary electrophoresis. Thin layer iso-

electrofocusing and capillary electrophoresis have beenadvocated to be the most sensitive

methods.75-78 They are able to separate distinctly the different types of haemoglobin.

Compared with the cost of doing both cellulose acetate and citrate agar electrophoresis,

thin layer iso-electrofocusing is thought to be cheaper. 75 Citrate agar usually has to be

done to confirm abnormal phenotypes when using cellulose acetate. Capillary

electrophoresis technique, in addition, detects minor variants of haemoglobin very well and also

quantifies the amounts of the various haemoglobins present in each individual sample.78

Are diagnostic facilities available for follow up of abnormalities shown by the screening

procedure, and are there acceptable treatments for the condition? Citrate

20

agarelectrophoresis can be used to confirm abnormal phenotypes. 75 Where this is not

available, cellulose acetate electrophoresis and solubility test can be done.There are also

acceptable treatments for the varied manifestations of the disease.

Is the natural history of the disease favourably influenced by the screening program? In

the case of SCD, in communities where newborn screening is practiced with

comprehensive health care program, the mortality rate has dropped from 15-30% to less

than 1%. 79,80-84

Is the screening cost effective? With the high mortality associated with this disease in

early infancy and childhood, there is eventual loss of potential manpower in the society.

In addition, early diagnosis, parental counseling, anticipatory guidance and regular follow

up will decrease the need for frequent hospitalizations. This in effect, would reduce the

burden on health facilities posed by this disease. It will also reduce the socioeconomic

burden of the illness on the family and society.

What should be done about findings that are neither clearly normal nor obviously

abnormal? These are the trait (carrier) cases. These persons are normal physiologically.

However, as carriers, they would be offered counseling, including genetic counseling, at

the point of diagnosis and on a continuous basis to ensure that at the time the need for

this information arises, they have fully grasped the notion of their status.85

EFFORTS TO REDUCE THE DISEASE BURDEN

Varied efforts have been made in different places to reduce the burden of sickle cell

disease. In an early study in Greece, people’s knowledge of their genetic makeup had no

effect on marriage decisions; 2 however, a recent study in the Islamic Republic of Iran

21

noted that about 50% of affected couples who decided to separate, resulted in reduction

in births with severe β thalassaemia to about 30% of the expected. 2

Newborn screening for SCD was first started at the Government Maternity Hospital,

Kingston, in Jamaica in July 1973.43,44 In 1975, New York and California States in the

USA added this to their pre-existing newborn screening programs.80In Europe, this

program was commenced in Birmingham in 1978.81 With the benefits of this exercise

widely documented, newborn screening for haemoglobinopathy is now widely practiced

in over 30 states in the USA and in other parts of Europe.86-89 The first newborn

screening centre in the Middle East, was established at the King Faisal University

Hospital in Damman, Eastern province of Saudi Arabia in 1982. 90

In Sub-Sahara Africa where this disease is prevalent, very few screening centres exist.

The first newborn screening program was established in Cotonou in the Republic of

Benin in May 1993. 45In February 1995, a second centre was established in Kumasi,

Ghana. 91

For newborn screening to be effective, it should be followed by comprehensive

healthcare.79,80,87The need for effective intervention in children with SCD should provide

a major impetus for screening the under-five population. One well established intervention

for children for children with SCD has been the use of prophylactic penicillin

therapy.Prophylactic penicillin therapy in a setting of comprehensive care has been found

to significantly reduce the morbidity and mortality of patients with SCD from

pneumococcal sepsis.48 Also, the recently introduced Pentavalent conjugate vaccine offers

hope for the future wellbeing in a setting where children are diagnosed at birth and

recruited into the comprehensive care and follow up program. Besides, reliable, simple and

cost effective techniques for mass screening are available and have demonstrated

validity.71,72

22

Given the fact that the benefits of screening and early institution of comprehensive care

are so compelling, universal newborn screening should be introduced into our welfare

program at the primary healthcare level.

CURRENT TREND OF NEONATAL SCREENING IN NIGERIA

Available literature indicates that Nigeria does not have a systematic program for mass and

newborn screening for SCD. Comprehensive health care service which is the foundation

for mitigating the burden of the disease is generally not widely available. 6

In addition, awareness of this condition, as well as what could be done for affected

children appear to be low in both our rural and urban centres. 6

METHODS OF DETECTING THE ABNORMAL HAEMOGLOBINS

The following methods are available for the detection of the presence of abnormal

haemoglobins:

1. Sickling test

2. Solubility test

3. Haemoglobin electrophoresis - Cellulose acetate

- Citrate agar

- Thin layer iso-electrofocusing

- Capillary electrophoresis

4. Micro-column chromatography

5. High performance liquid chromatography

1. SICKLING TEST

This test detects the presence of sickle haemoglobin. It is unable to detect the small

amounts of sickle cells that are present in the newborn.92

23

2. SOLUBILITY TEST

This test is based on the relative insolubility of deoxygenated sickle haemoglobin in

solutions of high molarity. This method is unable to differentiate the traits from those

with the disease.39

3. CELLULOSE ACETATE ELECTROPHORESIS

This method depends on the different mobilities of the different haemoglobins (because

of their different electrical charges) in an electric field. It is based on the charge change

in the haemoglobin molecule which occurs following certain amino acid changes.

In the case of sickle cell disease, the replacement of the negatively charged glutamic

acid by the neutral valine, results in two net positive charges per molecule relative to

the haemoglobin A1. Thus, HbS moves more slowly than HbA1 towards the anode. This

method requires a source of current, a buffer system which is borate at pH 8.4, and a

supporting medium which is the cellulose acetate membrane. It is not a highly sensitive

method of newborn screening. It is unable to separate the small amounts of HbA

distinctly from the large amounts of HbF present at birth and it also gives poor

resolution between HbF and HbS. 39,75 It is also unable to differentiate abnormal

haemoglobins with the same charge like HbD (Korle Bu and Punjab) from HbS and HbO

Arab from HbC. It is however, able to detect Bart’s haemoglobin (a gamma tetramer)

which when present, may suggest the presence of alpha-thalassaemia. 75

4. CITRATE AGAR ELECTROPHORESIS

This method is similar to the cellulose acetate electrophoresis (CAC). It enables one to

distinguish HbC, S, F, D and E. Its use in places where it is available is limited to

confirmation of abnormal patterns of haemoglobin detected by other methods used for

screening. 75

24

5. THIN LAYER ISO-ELECTROFOCUSING

This method has been reported to have the advantages of increased accuracy and

sensitivity compared to cellulose acetate (CAC) and citrate agar (CAG)

electrophoresis.75,76 It detects variants not apparent on CAC and CAG, 76and enables an

easier distinction of individual haemoglobin bands. It is also able to detect Bart’s

haemoglobin if the test is performed within two weeks of collection of the blood sample.

Compared to the cost of having to do both CAC and CAG, it is reported to be a

cheaper method of newborn screening.

It has a specificity of 99.5% to detect SCD and requires only five microliters of blood.

PRINCIPLE OF THE TEST

This method is based on the fact that each haemoglobin type has a zero net charge in solution at

a specific pH called the iso-electric point (pI). The charge on the support medium on which each

haemoglobin is being analysed (in this case agarose plate) is not uniform; i.e. there is a charge

gradient along the agarose plate unlike citrate agar and cellulose acetate that have a uniform

charge throughout their membranes. The haemoglobins being analysed migrate towards their

iso-electric points in this pH-gradient gel. A band called the ‘focusing band’ is formed by this

process. The iso-electric focusing technique has greater resolving power than either of the two

electrophoresis techniques currently used in most Nigerian laboratories (i.e. CAC and CAG).

The greatest advantage of iso-electric focusing technique seems to be in the detection and

separation of those mutant haemoglobins that migrate with HbA in cellulose acetate.75,76

THE PROCEDURE

A. PREPARATION OF THE AGAROSE PLATE

The agarose plate is prepared by mixing 400gm of agarose powder with 35mls of distilled water

in a beaker. This mixture is heated until it boils; then it is allowed to cool in room air to 70°Ϲ.

25

1.2mls each of Ampholine A solution at pH 6–8 and Ampholine B at pH 7–9 are then added to

this mixture. The mixture is immediately poured on a transparent cellophane plate that has been

mounted on another glass plate measuring about 12 by 25cm. This mixture forms a thin layer of

gel on cooling. This prepared agarose gel plate is stored in the refridgerator until when it is

needed.

It should be noted however, that there are ready-made plates of agarose gel which can be

purchased from the manufacturer.

B. PREPARATION OF THE HAEMOLYSATE

Using a paper clip, two slices of the blood stained Whatmanns paper are collected into a mini

test tube. 0.5mls of 0.05M solution of potassium cyanide is added to the paper in the test tube to

elute the haemoglobin from the Whatmann’s paper.

C. PREPARATION OF THE ELECTROPHORETIC STRIPS

Two strips of electrophoretic paper measuring 0.5 by 20cm are used. One is coated with 1 M

solution of sodium hydroxide (NaOH), while the other is coated with 0.05M solution of

sulphuric acid (H2SO4). The strip containing the NaOH is placed on the cathodal end of the

prepared agarose plate, while the H2SO4 strip is placed on the anodal end of the agarose plate.

D. RUNNING OF THE TEST (MIGRATION)

The agarose plate is placed on the electrophoretic machine. A rubber separator plate containing

52 small square spaces is then placed on the agarose plate close to the electrophoretic strip

containing the NaOH; i.e.the cathodal end. With separate pipettes, 0.05mls of each of the eluted

blood samples is dropped into the space contained in each of the small squares until all the

spaces are completed. The electrodes which are part of the electrophoretic machine are

connected with the negative electrode overlying the NaOH coated electrophoretic strip and the

positive electrode overlying the H2SO4 coated electrophoretic strip. The electrophoretic machine

itself is connected to two other machines. One is a pharmacia LKB multidrive XL machine. It is

26

a very high voltage machine supplying energy of 1500-3000 volts needed for the electrophoretic

movement. The second machine is a pharmacia LKB Multitemperature II which controls the

overall temperature of the electrophoretic machine preventing it from being destroyed by the

high voltage that is supplied by the multidrive machine. It maintains the temperature of the

electrophoretic machine at 13–13.5°Ϲ. Following the connection to these two machines, the

electrophoresis is allowed to run for 45-60 minutes until a clear separation of the various

haemoglobin types is achieved. The agarose plate is then removed from the electrophoresis

machine and is fixed in a 15% solution of trichloroacetic acid. The haemoglobin phenotypes are

read from the plate.

6. CAPILLARY ELECTROPHORESIS

This method is similar to chromatography in terms of separation science, but the main

principle is to separate compounds according to their charge, based mainly on electro-

osmotic flow. In capillary electrophoresis, every ion migrates at a different rate, hence it

has the efficiency to separate similarly structured compounds with a sensitivity comparable

to that of high performance liquid chromatography. 78

The main advantages of capillary electrophoresis over thin layer iso-electrofocusing

electrophoresis are that it detects minor variants very well and achieves good quantitation of

HbA2 and HbF. In addition, the system is easy to use and specimens may be run singly rather

than in batches like thin layer iso-electrofocusing.93

CAPILLARY ELECTROPHORESIS INSTRUMENT

The typical capillary electrophoresis instrument uses the following components to achieve both

electroosmotic flow and electrophoretic mobility and therefore, separations.93

1. Cathode (a negatively charged electrode)

2. Anode (a positively charged electrode)

27

3. Power supply to generate voltage/current

4. Catholyte (buffer solution at the cathode end)

5. Anolyte (buffer solution at the anodic end)

6. Capillary (25mm to 100mm internal diameter)

7. A detection method

8. A data acquisition method

Samples are introduced into the capillary for separation by electrokinetic injection.

Electrokinetic injection works when the capillary is placed into the catholyte on one end and into

the anolyte, containing the sample to be analyzed, on the other end. A voltage is applied and

electroosmotic flow moves from the tip of the capillary to the end of the capillary. A siphoning

effect occurrs, dragging a representative sample into the capillary. Also, ions begin moving into

the capillary from the buffer solution due to electrophoretic mobility as part of the sample

loading. These injections usually last for 1 to 5 seconds.93

After injection, the capillary injection end is moved into a sample vial containing capillary

electrophoresis grade water. A water plug is injected in the same manner that the sample is

injected. Then the capillary is moved into a different anolyte solution that did not contain the

sample. Voltage is applied across the capillary and the separation takes place as the separated

samples moved electrophoretically and with electroosmotic flow, past the detector. The detector

is linked to a data collection and storage system which displays the results graphically on the

monitor of a computer attached to the machine.93

7. MICRO-COLUMN CHROMATOGRAPHY

This method has been used successfully to screen large numbers of newborn. 94 It

requires a great deal of technical expertise. In addition, it requires the collection of a

large quantity of blood from the subjects.

28

8. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

This is the most sensitive method of detecting haemoglobin phenotypes. It also quantifies

the amounts of the various haemoglobins present in each individual sample. 39,95 The cost

of this machine is very high and its use requires more expertise.

FURTHER JUSTIFICATION FOR THIS STUDY

Given the various gaps in the literature reviewed, and the fact that these studies did not evaluate

the different frequencies of haemoglobin phenotypes for the various age groups of the under-

fives at the same time in a given community, it was worthwhile conducting a comprehensive

survey, taking into consideration as many of the various factors that describe and influence

disease outcomes in a holistic manner.

29

AIMS AND OBJECTIVES OF THE STUDY

BROAD OBJECTIVE

To study the distribution of haemoglobin phenotypes and the relationship with clinical

characteristics and nutritional status in newborns and older children aged <60 months.

SPECIFIC OBJECTIVES

1. To determine the overall and age-specific prevalence of the different haemoglobin

phenotypes among children less than 60 months of age in Ethiope West LGA.

2. To describe the specific haemoglobin phenotype distribution in the newborn.

3. To describe the clinical characteristics and anthropometric parameters of the study

population and relate these to the different haemoglobin phenotypes.

4. To determine the nutritional status of subjects aged 1 to less than 60 months and relate

this to the haemoglobin phenotypes.

30

SUBJECTS AND METHODS

STUDY DESIGN

This was a descriptive cross sectional study.

STUDY PERIOD

1st April, 2013 to 30th September, 2013.

STUDY LOCATION

The study was conducted in Ethiope West LGA of Delta State, situated in the rain

forest belt where malaria is holoendemic and the average annual rainfall is 266.5mm.

The LGA is bounded by Edo state to the North East, Warri North LGA to the West,

Sapele, Okpe and Ethiope East LGAs to the South.

Ethiope West has a population of 203,592 (2006 National Census) and occupies 536

square kilometers. It is inhabited mostly by the Urhobos, with pockets of other ethnic

groups like the Igbos and Hausas.

Their major occupations are farming, trading and civil service. The population is 75%

rural and about 25% urban/semi-urban. The literacy level is 47.7%

The main languages are Urhobo and pidgin English.

STUDY SITES

Ethiope West has eleven health wards and by a process of simple random sampling, three of

these health wards were selected for this study.

These wards are:

JESSE 1 (SELECTED)(Under- five population=9,793 of 48,964 total population)

JESSE 2

31

JESSE 3

JESSE 4

MOSOGAR 1

MOSOGAR 2

OGHAREFE 1

OGHAREFE 2 (SELECTED)(Under-five population=7,722 of 38,612 total population)

OGHAREFE 3

OGHAREKI 1 (SELECTED)(Under-five population=5,236 of 26,179 total population)

OGHAREKI 2

Each of these health wards has one health center. There is a General Hospital for the entire LGA

located in Oghareki 1.

The health centers are manned by a Senior Nursing Officer and at least two Community Health

Extension Workers (CHEWs). The CHEWs have been trained to provide home-based outreach

services and conduct health promotional activities for maternal, newborn and child health in the

communities.

ETHICAL CONSIDERATION

Prior approval of the Ethical Committee of DELSUTH was obtained.

Permission of the PHC coordinator of Ethiope West LGA was also obtained.

Parents of children diagnosed with SCD were counseled by the researcher, and such children

were enrolled in the DELSUTH comprehensive care programme.

Parents of children diagnosed with sickle cell trait, HbC and HbD traits were also counseled

appropriately by the researcher.

Children found with acute illness or any abnormalities were referred to the paediatric clinic of

DELSUTH.

32

Mobilisation Of The Population For The Study

Prior to accessing the population, pre-survey activities at the initial phase included visits to the

community leaders with the assistance of the LGA Chief Medical Officer and the PHC

coordinator to inform them of the purpose of the study and mobilize them for the study.

The CHEWs at the health centers were enlightened on the purpose of the study and the age

cohorts to be involved. They were also trained on carrying out physical examinations and

anthropometric measurements by the researcher.

Intensive mobilization activities were conducted to additionally sensitize the target communities.

SAMPLE SIZE DETERMINATION

The sample size was calculated using the formula:

Sample size, S = Z² x p(1-p)

d²

Z = confidence interval = 1.96

p = true proportion/prevalence in the population or the anticipated population prevalence

d = absolute precision = 0.03

For the neonates, the anticipated population prevalence was estimated to be 3-5%. For this work,

the 5% was used.

A prevalence of 50% was utilized for the cohort 1 month to <60months.

Accomodation for 13.5% attrition was made in both estimates.

Therefore, the sample size for neonates was:

S = 1.96² x 0.05(1 – 0.05) = 203 + 13.5% attrition (27) = 230

0.03²

33

The sample size for the 1 month to less than 60 months age cohort was :

S = 1.96² x 0.5(1 – 0.5) = 1,067 + 13.5% attrition (144) = 1,211

0.03²

Therefore, sample size for the entire study was 1,441.

SAMPLING PROCEDURE

A multistage random sampling procedure was used to select communities from the three

selected health wards. Selection of the households in these target communities was determined

by the ages of the children, as only children aged 0 to less than 60 months were targeted.

Enumeration of all the children aged 0 to less than 60 months in the selected communities was

conducted by the principal researcher with the assistance of the CHEWs.

Timing for the enumeration was 6p.m to 7p.m daily, and market days (every fourth day) were

excluded.

A complete listing of the households and enumeration of children per age cohort was done as

follows:

0 to < 1 month

≥ 1 month to <12 months

12 months to < 24 months

24 months to < 36 months

36 months to < 48 months

48 months to < 60 months

Jesse 1 is made up of nine villages. Three were selected by systematic random sampling. They

are Irhodo, Boboroku and Ejenesa.

Ogharefe 2 comprises six villages of which two, Otefe and Ijomi, were selected by systematic

random sampling.

34

Oghareki 1 comprises five villages. Two villages namely, Apapa and Uduaka, were similarly

selected.

In these selected villages of the health wards, enumeration of all children aged 0 to less than 60

months was done. The number of households from which these children were enumerated was

also noted.

Proportionate sampling based on the total number of children less than 60 months enumerated in

each selected health ward and the total number from the three health wards was used to

determine the number of subjects selected per health ward. This was derived as follows:

Number enumerated from health ward x Total sample size

Total number enumerated

660, 567 and 438 children less than 60 months were enumerated from the three selected health

wards, respectively. The study households were then selected by the use of a sampling interval

calculated as follows:

Sampling interval , N = Total population of children less than 60 months

Number of households

For households that had more than two children under 60 months of age, a simple random

sampling procedure was used to determine which two children to recruit for the study.

Hence, the calculated projected sample size from each health ward was 571, 491 and 379

subjects respectively, to achieve the calculated total sample size of 1,441.

A stratified randomization process was applied to accommodate for the respective age

cohorts.Based on the total number of children enumerated in each age cohort per health ward,

proportionate sampling was used to determine the proportions of each age cohort to select from

each of the three selected communities/health wards.

35

Projected Sample Size Per Age Cohort

Age (in months) Jesse 1 Ogharefe 2 Oghareki 1

0 - <1 110 86 34

1 - < 12 84 81 75

12 - < 24 83 65 53

24 - < 36 96 84 79

36 - < 48 113 82 84

48- < 60 85 93 54

Total 571 491 379

The enumeration and selection procedures were done over a period of three weeks, one week per

health ward. All days of the week were utilized.

Heads of selected households were visited in their homes by the researcher and the CHEWs for

further education on the purpose of the study and to enlist their support. The benefits of the

study were explained to them to enable them reach an informed decision. Consent for

participation of their wards in the study was sought and consenting parents were provided with a

consent declaration form to sign. Consenting parents were then given follow up dates and time

to bring their children/wards to the health centers for the physical examination and blood

sampling procedure.

36

INCLUSION CRITERION

All live borns aged 0 to less than 60 months in the selected households; a maximum of 2

children were sampled per household.

EXCLUSION CRITERIA

1. Blood transfusion within the preceding three months.

2. Permanent residence outside Ethiope West LGA. (Children on holidays or children of

mothers who had come from a different location to deliver their babies and receive

maternity care in the immediate postnatal period.Also, households that had domiciled

less than six months in the location).

DATA COLLECTION

Blood sampling and anthropometric measurements were commenced the week after completion

of the enumeration and subject selection. Study days for the older children were Mondays to

Fridays. For the newborn, who were recruited consecutively from the health centers and the

General Hospital following delivery and for those delivered at home, from their homes (with the

assistance of the CHEWs), recruitment and sampling were done everyday of the week for the

study period. The study time was from 8a.m to 4p.m daily. Babies recruited from the health

centers were tagged by the CHEWs for follow up in the community. This enhanced the

possibility of not recruiting them twice.

A questionnaire was administered to the parents of enrolled children following which a thorough

physical examination was conducted and anthropometric measurements (height, weight and mid

upper arm circumfence) using standardized procedures96 were taken. These procedures were

carried out by the researcher and trained assistants (four doctors and six CHEWs). The

examination findings for each subject were confirmed by at least two observers. Where there

37

was a significant difference in the observations, the subject was further examined by another two

observers.

Children 24 months and above, were weighed in their underwear, using a well calibrated

stadiometer weighing equipment that was also used in measuring the height, while the child was

standing erect, without shoes. The subject was made to stand up straight against the vertical

backboard of the stadiometer with their body weight evenly distributed and both feet on the

platform, with the heels together and the toes pointing slightly outwards at approximately 600

angle. The examiner then checked that the back of the head, shoulder blades, buttocks and heels

were in contact with the backboard before aligning the head in the Frankfort horizontal when the

horizontal line from the ear canal to the lower border of the orbit was parallel to the floor and

perpendicular to the vertical backboard. The subject was instructed to look straight ahead and

then the stadiometer head piece was lowered to rest firmly on top of the subject’s head, with

sufficient pressure to compress the hair. The examiner would then capture the result, verify the

correct value and then ask the subject to step away from the stadiometer, slide the head piece to

the top of the measurement column and secure it in place with the brake lever, in preparation for

the next participant. Infants were weighed in an infant weighing scale, while their lengths were

measured using an infantometer as follows. The subject was laid on the infantometer on top of

the horizontal backboard with the feet toward the foot piece and the head against the fixed head

piece. One examiner supported the child’s head and ensured that the head was aligned in the

Frankfort horizontal plane, while an assistant positioned the feet, aligning the child’s legs by

placing one hand gently, but with mild pressure over the knees and using his other hand to slide

the foot piece to rest firmly at the child’s heels. The measurement was then read off, verified,

and then the parent or guardian asked to remove the child from the infantometer and the foot

piece slid to the end of the measurement column in preparation for the next subject.The MUAC

38

was measured on the left arm, with the elbow extended, using a non-stretch measuring tape at

the mid-point between the acromion of the scapula and the olecranon process of the ulnar.

Findings on physical examination and anthropometric measurements were recorded on a

proforma that included the age, sex and address of the subject.

In children aged one month to less than 60 months, anthropometric measurements were later

assessed using z-scores calculated using WHO child growth standards. Levels of malnutrition

were defined as z-scores less than -2 and greater than +2 standard deviation for the age and sex

for undernutrition and overnutrition, respectively.

BLOOD SAMPLING

Blood sampling was done by the researcher and four doctors, spread across the three health

centers and the General Hospital.

For children less than 6 months, blood samples were collected by heel prick after cleaning with

70% alcohol, using a sterile medpoint lancet, onto a 3 by 7.5 centimetre Whatmann’s paper with

four one centimeter diameter circles. A drop of blood was placed at the center of each circle.

Prior to the sample collection, the paper was labeled with the child’s names, sex, age, date of

birth and date of sample collection. Each sample was allowed to dry in room air before sealing

in individual polythene self-sealing envelopes and then stored in the freezer at -4°Ϲ. Every two

weeks, samples were transported by the researcher in a cold chain box to the National Sickle

Cell Disease Center, Cotonou, where thin layer isoelectrofocusing and capillary electrophoresis

were done.

For children 6 months old and above, 2milliliters of blood was collected from any visible vein

on the dorsum of the hand using a gauge 22 Terumo needle, into an EDTA sample bottle after

swabbing the site with 70% methylated spirit. The blood was then stored in the refridgerator,

39

and then transported in a cold chain box to the National Sickle Cell Disease Center, Cotonou

within two weeks of collection by theresearcher . All samples were analysed by capillary

electrophoresis technique.

Parents were given follow up dates of four weeks following the sampling procedure to return to

the health centers for collection of their results.

DISCLOSURE OF RESULTS

Results were made available to parents four weeks after the sampling procedure.

Parents of those children with haemoglobinopathy were counseled on the nature of their childs’

condition, the clinical presentation, complications and management. Arrangements were then

made for their child to be immediately referred to the sickle cell clinic of the teaching hospital

by the researcher.

Parents of children with sickle cell trait, HbC and HbD traits, and normal haemoglobin

(HbAA)were also counseled and made aware of the implications of possessing these

haemoglobin phenotypes.

LABORATORY METHODS

A single method for haemoglobin analysismay not be sufficient for definitive identification of a

haemoglobin variant, particularly for the more uncommon haemoglobin

variants.97Complementary methods are useful because haemoglobin variants that do not separate

on one method, may show separation on another method. Combinations of methods are useful

also to confirm the common variants and help to narrow down the identification of unusual

variants, as these typically show varying mobilities by different methods. The choice of methods

depends on individual laboratories.97As a standard laboratory procedure, the research laboratory

at the National Sickle Cell Disease Center in Cotonou employs the use of both

40

isoelectrofocusing and capillary electrophoresis for newborn haemoglobin phenotype analysis.

This procedure was,as such, employed for the present study. Blood samples for the newborn

were first subjected to thin layer isoelectrofocusing electrophoresis and then, were confirmed

with capillary electrophoresis.

The researcher was present in the laboratory during the process of sample analysis and

participated in the preparation of the samples for the assay which was done fortnightly.

However, for acceptance of results with a specific measure of accuracy, the laboratory

technologists conducted the bulk of the tests.

STATISTICAL ANALYSIS

The statistical analysis were done using the IBM SPSS statistics version 20.0 to produce the

means, standard deviations and chi square. Categorical variables were compared using Fisher’s

exact tests. P values <0.05 were considered significant. Nutritional status was assessed using z-

scores calculated based on WHO child growth standards.

41

RESULTS

One thousand, five hundred and fifty one children were enrolled for this study. Two hundred and

eighty eight were neonates, while 1,263 were older children less than sixty months of age. One

hundred and eleven blood samples were lost to testing due to poor quality of blood samples as a

result of denaturation of the haemoglobin during storage of the samples and poor resolution of

results on capillary electrophoresis.. Following sample analysis, 1,440 results were valid. Two

hundred and forty six belonged to neonates, while 1,194 belonged to older children less than

sixty months.

SOCIO-DEMOGRAPHIC CHARACTERISTICS OF THE STUDY GROUP

Table I shows the socio-demographic characteristics of the study group. Of the 1440 children

studied, 744(51.7%) were male and 696(48.3%) were female, giving a male:female ratio of

1.07:1.

The age distribution of the study group is shown in Figure 1. Three hundred and eighty eight

(26.9%) of the children were in the age bracket 1-<12months; while 184(12.8%) were in the age

group 48-<60months. The mean age of the study group was 24.3± 17.0months.

EDUCATIONAL QUALIFICATION OF THE PARENTS/CAREGIVERS

The educational qualifications of the parents/caregivers of the children enrolled in the study are

as shown on Table I. Six hundred and forty eight (45.0%) of the parents/caregivers in this study

had primary education, while only about1.0% (14) had no formal education.

42

SOCIOECONOMIC STATUS OF THE PARENTS/CAREGIVERS

The socioeconomic status of the study group is shown in Table I.Majority of the study

subjects(65.5%) belonged to the lower socioeconomic class based on the social classification

system described by Oyedeji.98 For the purpose of this study, social class I and II were grouped

as the upper class, social class III as the middle class, while social class IV and V were

categorized as the lower class.

MARITAL STATUS OF PARENTS/CAREGIVERS

Almost all, 1360 (94.3%) of the parents/caregivers in this study were married. Twenty one

(1.5%) were single, 47(3.3%) cohabiting, 4(0.3%) separated or divorced and 8(0.6%) widowed.

43

Figure 1: Distribution of Study Subjects By Age

17.1%

26.9%

15.8%

14.4%

12.9%

12.8%

0 - <1mth

1 - <12mth

12 - <24mth

24 - <36mth

36 - <48mth

48 - <60mth

44

Table I : Socio-Demographic Characteristics of the Study Group

Variables Frequency Percentage (%)

Sex

Male 744 51.7

Female 696 48.3

Educational Qualification of Parents/Caregivers

No formal education 14 1.0

Primary 648 45.0

Secondary 635 44.1

Tertiary 143 9.9

Socioeconomic Status of Parents/Caregivers

Upper class 172 11.9

Middle class 325 22.6

Lower class 943 65.5

Total 1440 100.0

45

46

HAEMOGLOBIN PHENOTYPES BY AGE

The overall and age specific prevalence of the haemoglobin phenotypes identified in the study

population are shown in Table II.

The phenotypes identified were HbAA, HbAC, HbAD, HbAS and HbSS. The overall prevalence

of these phenotypes was 73.6%, 0.4%,0.1%, 23.7% and 2.2%, respectively.

HbAA, HbAS and HbSS were documented in all the age group cohorts, whereas, HbAC was

not identified in the 1-<12months age group and HbAD was identified in only one age cohort,

36-<48months.

HbAA phenotype was more prevalent in the neonatal age group (78.5%) and the 12-<24months

age group (79.5%) compared to the other age cohorts.

HbAC phenotype was also more prevalent in the neonatal age group (0.8%). The prevalence was

approximately the same (0.4%- 0.5%) in the other age cohorts where this phenotype was

documented.

HbAS phenotype was least prevalent (17.5%) in the 12-<24months age group and most

prevalent in the 1-<12months age group.

The prevalence of HbSS was highest (3.2%) in the 36-<48months age bracket and lowest (1.2%)

in the neonatal age group.

For all the haemoglobin phenotypes, the difference in prevalence among the various age cohorts

of children less than 60months was not statistically significant (p=0.213).

47

Table II: Haemoglobin Phenotypes By Age

Haemoglobin Phenotype - n (%)

Age (months) AA AS AD AC SS Total (%)

Neonates 193 (78.5) 48 (19.5) 0 (0.0) 2 (0.8) 3 (1.2) 246 (100.0)

≥1 - < 12 274 (70.6) 105 (27.1) 0 (0.0) 0 (0.0) 9 (2.3) 388 (100.0)

12 - < 24 181 (79.5) 40 (17.5) 0 (0.0) 1 (0.4) 6 (2.6) 228 (100.0)

24 - < 36 148 (71.2) 55 (26.4) 0 (0.0) 1 (0.5) 4 (1.9) 208 (100.0)

36 - < 48 133 (71.5) 45 (24.2) 1 (0.5) 1 (0.5) 6 (3.2) 186 (100.0)

48 - < 60 131 (71.2) 48 (26.1) 0 (0.0) 1 (0.5) 4 (2.2) 184 (100.0)

Total 1060 (73.6) 341(23.7) 1 (0.1) 6 (0.4) 32 (2.2) 1440(100.0)

Fisher’s exact test = 15.228; df = 12; p = 0.213

48

RELATIONSHIP BETWEEN HAEMOGLOBIN PHENOTYPE AND SPECIFIC

CLINICAL SIGNS

Table III shows the prevalence of specific clinical signs in the general study population and the

relationship of these signs with haemoglobin phenotype.

Two point two percent (23/1,060) of children with HbAA, 1.4% (5/348)of those with trait status

and 15.6%( 5/32) of those with HbSS were jaundiced. The prevalence of jaundice was

significantly higher in those with HbSS compared to the others (p<0.001).

Pallor was seen in 73(5.1%) of the general study population. 34.4% (11/32) of HbSS individuals

had pallor compared to 4.6% (49/1060) of HbAA and 3.7% (13/348) of individuals with trait

status . This difference was statistically significant (p<0.001).

Hepatomegaly and splenomegaly were observed in 15(1.1%) and 65(4.6%) of the total study

population, respectively. Among HbSS subjects, hepatomegaly was observed in 15.6% (5/32)

and splenomegaly was observed in 15.6% (5/32). Zero point eight percent (0.8%)( 8/1060) of

HbAA and 0.6% (2/348) of trait individuals had hepatomegaly and 4.4%( 47/1060) of HbAA

and 3.7% (13/348) of traits had splenomegaly. For both signs, the difference was statistically

significant (p<0.001,p<0.05, respectively).

Nine(0.6%) of the total study population had bossing of the skull bones while gnathopathy was

observed in 24(1.7%). Among HbSS individuals, bossing of the skull bones was observed in

6.3% (9/32) and gnathopathy in 9.4% (3/32). Zero point four percent, 0.4% (4/1060) of HbAA

and 0.9% (3/348) of trait individuals had bossing of the skull and 1.6% (17/1060) of HbAA

individuals and 1.2% (4/348) of trait individuals had gnathopathy.The difference in prevalence

of both signs between HbSS individuals, HbAA and traits was statistically significant (p<0.05

and p<0.05 respectively).

Eight (0.6%) of all the children had a flat nasal bridge. Among HbSS subjects, this sign was

observed in 12.5% (4/32), compared with 0.3% (17/1060) among HbAA subjects and 1.2%

49

(4/348) among trait individuals. The difference in prevalence between HbSS subjects compared

with HbAA and trait individuals was also statistically significant (p<0.001).

50

Table III: Relationship Between Haemoglobin Phenotype and Specific Clinical Signs

Haemoglobin Phenotype

HbAA Traits HbSS Total

n(1060) n(348) n(32) N(1440)

Clinical Signs n(%) n(%) n(%) n(%) p value

Jaundice 23(2.2) 5(1.4) 5(15.6) 33(2.3) <0.001

Pallor 49(4.6) 13(3.7) 11(34.4) 73(5.1) <0.001

Hepatomegaly 8(0.8) 2(0.6) 5(15.6) 15(1.1) <0.001

Splenomegaly 47(4.4) 13(3.7) 5(15.6) 65(4.6) <0.05

Bossing of the skull 4(0.4) 3(0.9) 2(6.3) 9(0.6) <0.05

Gnathopathy 17(1.6) 4(1.2) 3(9.4) 24(1.7) <0.05

Flattened nasal bridge 3(0.3) 1(0.3) 4(12.5) 8(0.6) <0.001

Traits = HbAC, HbAD , HbAS

51

PREVALENCE OFCLINICAL SIGNS ACCORDING TO AGE

The prevalence of clinical signs according to age is shown on Table IV.

Seventy three of 1440 subjects studied were clinically pale. Twenty one (28.6%) of them were in

the age bracket 1-<12months, while only 4 (5.5%) were of the neonatal age group.

Jaundice was observed in 33 study subjects. The prevalence was highest in the neonatal age

group; 25 (75.7%) and was lowest, 0(0.0%) in the 1-<12months age group.

Bossing of the skull bones was not observed until 12-<24months of age. Nine individuals had

this sign. Three (33.3%) were in the age bracket 12-<24months and another 3 (33.3%) amongst

24-<36months, while 1(11.1%) was in the 36-<48months age bracket.

The onset of gnathopathy was from 24months of age. Twenty four study subjects had this sign

of which 14(58.3%) were in the 48-<60months age group, while 2(8.4%) were in the

24-<36months age group.

Hepatomegaly was observed in 15 subjects. Four (26.7%) individuals each from age groups 24-

<36months and 36-<48months presented with this sign, while none, 0 (0.0%) from the neonatal

age group exhibited hepatomegaly

Sixty five study subjects had splenomegaly. Sixteen (24.6%) were in the 24-<36months age

group, while 4(6.2%) were of neonatal age

52

Table IV: Prevalence of Clinical Signs According to Age.

Age (months) n (%)

Clinical Signs 0-<1 1-<12 12-<24 24-<36 36-<48 48-<60 Total

Pallor 4 (5.5) 21(28.6) 15(20.5) 13(17.8) 11(15.1) 9(12.5) 73 (100.0)

Jaundice 25(75.7) 0(0.0) 3(9.1) 2(6.1) 1(3.0) 2(6.1) 33(100.0)

Bossing of skull 0(0.0) 0(0.0) 3(33.3) 3(33.3) 1(11.1) 2(22.2) 9(100.0)

Gnathopathy 0(0.0) 0(0.0) 0(0.0) 2(8.4) 8(33.3) 14(58.3) 24(100.0)

Flat Nasal Bridge 0(0.0) 1(12.5) 3(37.5) 1(12.5) 1(12.5) 2(25.0) 8(100.0)

Hepatomegaly 0(0.0) 2(13.3) 3(20.0) 4(26.7) 4(26.7) 2(13.3) 15(100.0)

Splenomegaly 4(6.2) 9(13.8) 15(23.1) 16(24.6) 10(15.4) 11(16.9) 65(100.0)

53

CLINICAL SIGNS (GNATHOPATHY) ACCORDING TO AGE AND HAEMOGLOBIN

PHENOTYPE

Table V shows the prevalence of gnathopathy according to age and haemoglobin phenotype.

Twenty four subjects had gnathopathy and this sign was documented in all the phenotype

groups. Fourteen (58.4%) were in the 48-<60months age group, while 2 (8.3%) were in the 24-

<36months age group. Below 24months of age, the prevalence was 0(0.0%). Thereafter, it

showed a progressive increase with age.

54

Table V: Gnathopathy According to Age and Haemoglobin Phenotype

Haemoglobin Phenotype Age (Months) P

<1 1-<12 12-<24 24-<36 36-<48 48-<60 Total

n=246 n=388 n=228 n= 208 n=186 n=184 N=1440

HbAA 0(0.0) 0(0.0) 0(0.0) 2(11.8) 6(35.3) 9(52.9) 17(100.0)

Traits 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(25.0) 3(75.0) 4(100.0)

HbSS 0(0.0) 0(0.0) 0(0.0) 0(0.0) 1(3.1) 2(6.3) 3(100.0)

Total 0(0.0) 0(0.0) 0(0.0) 2(8.3) 8(33.3 ) 14(58.4) 24(100.0)

Traits = HbAC,HbAD, HbAS

55

RELATIONSHIP BETWEEN HAEMOGLOBIN PHENOTYPE AND NUTRITIONAL

STATUS IN CHILDREN AGED 1 TO LESS THAN 60 MONTHS

Table VI shows the relationship between haemoglobin phenotype and nutritional status in

children aged 1 to less than 60months based on the weight for age.

One thousand and thirty-seven (86.9%) were well nourished, while 146 (12.2%) were

underweight and 11 (0.9%) were overweight.

Among the phenotype groups, normal nutritional status was found in 789(87.4%) of HbAA

individuals compared with 225(85.9%) of trait individuals and 23(71.9%) of HbSS subjects.

Six (18.8%) of HbSS individuals, 35 (13.4%) of trait individuals and 105 (11.6%) of HbAA

subjects were underweight.

Nine (1.0%) of HbAA subjects were overweight compared with 2 (0.7%) of trait subjects and 0

(0.0%) of HbSS subjects.

There was no statistically significant difference in the nutritional status among the various

haemoglobin phenotypes. (p=0.691)

56

Table VI: Relationship Between Haemoglobin Phenotype and Nutritional Status in

Children Aged 1 to Less Than 60 Months

Nutritional Status

Haemoglobin Phenotype Underweight Well-nourished Overweight Total

(<2SD) (-2 to +2SD) (>2SD)

n(%) n(%) n(%) N(%)I

HbAA 105 (11.6) 789 (87.4) 9 (1.0) 903 (100.0)

Traits 35(13.4) 225(85.9) 2(0.7) 262 (100.0)

HbSS 6 (18.8) 23 (71.9) 0(0.0) 29 (100.0)

Total 146 (12.2) 1037 (86.9) 11(0.9) 1194 (100.0)

x² = 8.126; p = 0.691

Traits = HbAC, HbAD, HbAS

57

RELATIONSHIP BETWEEN HAEMOGLOBIN PHENOTYPE AND NUTRITIONAL

STATUS IN CHILDREN AGED 1 TO LESS THAN 60 MONTHS

Table VII shows the relationship between haemoglobin phenotype and nutritional status based

on height/length for age in children aged 1 to less than 60 months.

One thousand and sixty four (89.1%) of the study population were well nourished, while 125

(10.5%) were stunted and 5 (0.4%) had tall stature.

Among the haemoglobin phenotype groups, 803(88.9%) of HbAA children and 236(90.1%) of

trait individuals were well nourished (of normal height) compared with 25(86.2%) of HbSS

subjects.

Four (13.8%)of HbSS individuals were stunted compared with 24(9.2%) of trait individuals and

97(10.7%) of HbAA subjects.

Three (0.4%)of HbAA subjects had tall stature compared with 2(0.7%) of trait individuals and

0(0.0%) of HbSS subjects.

There was no statistically significant difference in the nutritional status among the various

haemoglobin phenotypes. (p=0.873)

58

Table VII: Relationship Between Haemoglobin Phenotype and Nutritional Status Based on

Height/Length for age in Children Aged 1 to Less Than 60 Months

Nutritional Status

Stunted Well nourished Tall Stature Total

Haemoglobin Phenotype (<2SD) (+2 to -2SD) (>2SD)

n(%) n(%) n(%) N(%)I

HbAA 97(10.7) 803(88.9) 3(0.4) 903(100.0)

Traits 24(9.2) 236(90.1) 2(0.7) 262(100.0)

HbSS 4(13.8) 25(86.2) 0(0.0) 32(100.0)

Total 125(10.5) 1064(89.1) 0(0.0) 1194(100.0)

x² = 6.143; p = 0.873

Traits = HbAC, HbAD, HbAS

59

Table VIII: Haemoglobin Phenotype and Nutritional Status Based on MUAC (children

aged 12 to less than 60 months)

Haemoglobin

Phenotype

Undernourished

(<12.5cm)

n(%)

Borderline

(12.5cm-

13.5cm)

n (%)

Well nourished

(13.5cm -

17.5cm)

n (%)

Obese

(>17.5cm

)

n (%)

Total

N (%)

HbAA 76(12.8) 61(10.3) 444(74.9) 12(2.0) 593(100.0)

HbAC, HbAD, HbAS 34(17.6) 22(11.4) 132(68.4) 5(2.6) 193(100.0)

HbSS 3(15.0) 3(15.0) 14(70.0) 0(0.0) 20(100.0)

Total 113(14.0) 86(10.7) 590(73.2) 17(2.1) 806(100.0)

𝑥2 = 7.784, 𝑝 = 0.703

There was no statistically significant difference in the nutritional status among the various

haemoglobin phenotypes

60

DISCUSSION

The haemoglobin phenotypes identified in this study were HbAA, HbAC, HbAD, HbAS and

HbSS. Overall, this pattern of haemoglobin phenotypes is similar to what has been previously

described for the under-five age group in Nigeria.34-37This study has additionally described

HbAD which hitherto has not been reported in Nigeria. This finding may be related to the

sensitivity of the newer capillary electrophoresis technique used in the haemoglobin phenotype

determination in this study. The capillary electrophoresis technique has a greater separation

power of globin chains than other commonly used methods of electrophoresis.93

Homozygous haemoglobin A was seen in 73.6% of the subjects, while the prevalence of the

sickle cell trait (HbAS) was 23.7%. These findings are consistent with those of Fleming et

al27who in 1979, documented a HbAA prevalence rate of 73.8%, and also with those of Kaine

and Udeozo35 in 1981 and Abhulimhen-Iyoha et al34 in 2006 in which the corresponding

prevalence rates were 75.8% and 72.0% respectively. Fleming and coworkers27 studied infants

from northern Nigeria, while Kaine and Udeozo35and Abhulimhen34 studied under-five children

in Eastern and Southern Nigeria respectively. Furthermore, Umoh et al in a 2007 survey of

8,097 Nigerians (adults and children) documented a prevalence of 78.7% for HbAA and 19.6%

for HbAS in the same study.100 In contrast, a lower prevalence rate was recorded by Omotade et

al74 in 1998 who reported a 67.2% prevalence for HbAA amongst infants presenting for measles

vaccination at the infant welfare clinic of the Institute of Child Health, University College

Hospital, Ibadan, Nigeria. Similarly, among 1,296 children aged between one month and

fourteen years at Ilorin (in North Central Nigeria), Adewuyi and Akintunde36had earlier, in

1990, recorded a prevalence of 68.9% for HbAA.

With regard to the prevalence rates of sickle cell trait (HbAS), the earlier Nigerian reports 27,34-37

alluded to above, documented values ranging from 19.6% to 28.9%. The observed HbAS

prevalence of 23.7% in this study corresponds to 20% to 30% documented for Nigeria and 20%

61

to 40% for Africa in general.101 Variations in the prevalence of sickle cell trait and disease in

children in different parts of the tropics may be determined partly by the intensity and mode of

falciparum malaria transmission in the region.27 Thus, the reported prevalence of sickle cell trait

in the Sudan Savanna Region of Nigeria, where falciparum malaria is hyperendemic was slightly

higher (28.9%), than in the rainforest belt of Nigeria (23.7%) where the parasite is

holoendemic.27This has been attributed to the more rapid acquisition of partial immunity in a

holoendemic situation, and the consequent quicker disappearance of the survival handicap

among children with normal haemoglobin.27

The prevalence of HbAC in this study was low with a trait frequency of only 0.4%. This is

consistent with the observations made by other workers from the Eastern and Southern parts of

Nigeria, where the HbC gene was infrequent or absent.35 In contrast, studies from Western

Nigeria describe a higher prevalence of the ‘’C’’ gene.36,37,74 Nwokolo and Lehman25 explained

that the ‘’C’’ gene might have originated from Burkina Fasso(formerly Upper Volta) and Ghana.

They argued that these people were hindered from migrating to the Eastern part of Nigeria

because of difficulty crossing the River Niger; hence the low frequency of the ‘’C’’ gene across

the Niger. On the other hand, the western region of Nigeria which was their first area of

settlement, had a higher prevalence of HbC. This prevalence decreases as one moves from the

west towards the east. Ethiope West,the study site, is on the western bank of the River Niger.

This may thus explain the low prevalence of HbAC and the absence of HbSC in this study.

In this study, the prevalence of HbAD was 0.1%. This finding is not consistent with those from

other studies done on underfives in Nigeria27,34-37 and in Africa30-32,38which confirms the rarity of

this haemoglobin phenotype in the African population. Among a population of 202,244 infants

screened by Ohene-Frempong et al32 in Ghana over a ten year period, no case of HbD was

documented. Similarly, among 10,115 infants screened by Omotade et al74 in Western Nigeria

over a twelve-year period, no cases of HbD were identified. In Gambia, Facer et al102screened

62

343 children aged two months to ten years and found no cases of HbD. Among 180 sickle cell

disease patients aged two to fifteen years in Western Nigeria, Adeyemoet al103 found a

prevalence of 0.6% for HbSD. The haemoglobin D trait is known to have its highest frequency

of 1 in 100 among Pakistanis and Indians.3This variant is known as HbD Punjab. Other less

common variants such as HbD Korle-Bu, are found much less frequently and more often in

individuals not of Pakistani or Indian descent.3 The variant of HbD reported in the present study

was HbD Korle-Bu.

The overall prevalence of SCD in this study was found to be 2.2%. This is higher than the 1.6%

reported by Kaine and Udeozo at Enugu, Eastern Nigeria. The higher prevalence of SCD in the

present study may possibly be due to the fact that it was a randomized community-based study,

incorporating infants of the neonatal age group as well as those below four months of age, unlike

Kaine and Udeozo’s study. In one other study of under-fives by Abhulimhen-Iyoha et al34 in

Ekosodin community, Benin city in Southern Nigeria, the prevalence of SCD was estimated to

be 0%. In that study, 107 children aged 0 to less than 60 months were studied. The relatively

smaller sample size and also, the non-systematic sampling technique utilized for the study may

have accounted for the absence of the homozygous state. Adewuyi and Akintunde36 in a 1990

study in Ilorin (North Central Nigeria) found a prevalence of 2.8% for SCD among 1,296

children between the ages of one month and fourteen years. This prevalence is higher than that

documented in the present study. This observation may be attributable to the fact that the authors

studied children one month to fourteen years of age, rather than children less than sixty months

of age as in the present study. The high prevalence of HbSC (1.3%) which was absent in the

present study may also have accounted for this observation. Omotade et al74 found a prevalence

of 4.4% for SCD in their 1998 study conducted at Ibadan, Western Nigeria. HbSS accounted for

3.3% of the SCD prevalence, while HbSC accounted for 1.1%. This prevalence is also higher

than that from the present study. This may be attributable to the much higher prevalence of HbC

63

gene in that locality. With respect to the African sub-region, Rahimy et al45in Cotonou, West

Africa documented a prevalence rate of 9.9% for SCD. The apparently higher prevalence may be

due to the fact that their study recruited newborns of mothers with sickle cell trait. In 2004,

Amoako38, in a study of 341 children aged 6 to 59 months in Kintampo North municipality,

Ghana, found a prevalence of 75.9% for HbAA, 14.9% for HbAC ,9.2% for HbAS, 0.3% for

HbSC, 0.3% for HbCC and 0.9% for HbSS. In the Northern part of Ghana where this study was

conducted, the C trait has been found to be more prevalent (20%-25%) and the S trait less

prevalent (10%),24 hence explaining the relatively higher prevalence of HbC trait and the lower

prevalence of sickle cell trait in that study.

No case of alpha or beta thalassaemia was seen in this study. In infancy, the presence of

Haemoglobin Barts in large amounts is taken to suggest the presence of alpha thalassaemia.

However, this haemoglobin undergoes denaturation when blood samples are stored for longer

than two weeks.72Most blood samples were stored longer than this period, hence a possible

explanation for its absence in the present study.

The capillary electrophoresis technique used in this study is able to differentiate heterozygous

sickle cell-beta thalassaemia from homozygous SS after infancy.97HbA2 is almost non-existent

in the newborn. Levels rise steadily until the age of one year when normal adult levels are

attained. 20,45 In beta thalassaemia, the levels of HbA2 are higher than the normal levels of

between 2.5 to 3.2% seen after the age of one year.3,104 In the present study, HbA2 levels ranged

between 2.1% and 3.2%.

The observed prevalence for HbAA by age cohorts in this study, as can be seen in Table II,

ranged from 70.6% to 79.5%. This finding is in keeping with other prevalence reports in

Nigeria, which show that normal haemoglobin (HbAA) ranges between 55% to 75%.100Beyond

24 months, the prevalence of HbAA remained relatively constant.

64

The prevalence of HbAS in this study ranged from 17.5% to 29.1%. These values also

correspond with those from other prevalence reports in Nigeria. 27,34-37

The HbAA to HbAS ratio in this study was 3.5-4.1:1 for all the age cohorts. This is in keeping

with previous reports which show that the frequency of the abnormal ‘’S’’ gene in our

population is about 25%.6This finding may merely imply that the gene frequency for HbS has

remained constant over time. The phenomenon of balanced polymorphism explains the high

prevalence of HbS in this study. Children who are HbAS are protected from severe attacks of

malaria than their HbAA counterparts.6 These HbAS individuals survive childhood better and

thus, propagate the ‘’S’’ gene.

The prevalence of HbD trait (HbAD) in this study ranged from 0% to 0.5% among the various

age cohorts. In a study by Adeyemo et al103in Lagos, Western Nigeria involving 180 SCD

patients aged 2 to 15years, a prevalence of 0.6% was documented for HbSD.103 This

haemoglobin variant and its co-inheritance with HbS are very rarely seen in the African

population.103

The prevalence of HbC trait (HbAC) among study subjects ranged from 0% to 0.8%. This

haemoglobin phenotype was documented in all the age group cohorts studied except the 1 to less

than 12month age cohort, with higher prevalence in the newborn. Odunvbun33 in Benin city,

Southern Nigeria, established a newborn prevalence of 1.1% for HbAC, while Fleming27 found

HbAC in 0.7% of infants in Garki, Northern Nigeria and Kaine and Udeozo35 in Eastern Nigeria

established a zero percent prevalence for HbC among underfives. The relatively constant

prevalence of HbC trait in all age groups is noteworthy and buttresses the fact this haemoglobin

phenotype has minimal clinical significance. In addition, like HbS, HbC has been shown to also

provide partial protection against falciparum malaria infection105.

65

HbSS was observed in 1.2% to 3.2% among the various age cohorts in this study. The frequency

was lowest in the newborn and highest at 36 to <48months. Mortality due to SCD is minimal

below the age of six months,27 and the frequencies of haemoglobin phenotypes below the age of

six months reflect the relative frequencies at birth. Thus, in the present study, it is apparent that

2.1% of babies born in the population have SCD. This value is in keeping with

Fleming’s27observation in Northern Nigeria where 2.1% of newborns were found to have SCD.

This observation may be related to the fact that a similar method of haemoglobin phenotype

determination was utilized in both studies, and that both studies were community-based, hence

reflecting the true prevalence of the condition in the population. This value is however, much

lower than was reported by Odunvbunet al33 in Southern Nigeria (3.0%) and Omotade et al74 in

Western Nigeria (4.4%). Odunvbun et al33 found a prevalence of 0.2% for HbSC,while in

Omotade’s study74, HbSC accounted for 1.1% of the SCD cases. HbSC was not documented in

the present study and its absence possibly accounts for the lower prevalence of SCD in the

present study. In addition, the studies by Odunvun et al33 and Omotade et al74 were hospital-

based unlike the present study which was community based, on a randomly selected study

population. Hence, it is likely that this study has given a precise description of the prevalence of

SCD in the various age cohorts of children less than 60months of age in this locality.

For the neonates, the haemoglobin phenotypes identified in this study were HbAA, HbAC,

HbAS and HbSS. This pattern was described by Fleminget al27 in their 1979 study in Kano

State, Northern Nigeria. In the Benin city hospital-based survey by Odunvbun et al33and the

large hospital-based study in Koumasi, Ghana by Ohene-Frempong et al30, a similar pattern of

haemoglobin phenotypes was observed.

Hitherto, there is paucity of data on community-based neonatal surveys. This work therefore,

fills a knowledge gap in providing such information on community-based patterns for the

newborn.These cited studies did not utilize the newer capillary electrophoresis technique for the

66

haemoglobin phenotype determination, though the technique has been available since the early

1990s.

In this study however, the prevalence of the different haemoglobin phenotypes in the newborn

varies from what was found by Odunvbun et al’s hospital-based survey33, in which a much

higher prevalence of SCD (3.0%) was described. This was because they found both HbSS

(2.8%) and HbSC (0.2%). Again, the cited study was hospital-based. In Fleming’s work27 in

1979 in Kano, Northern Nigeria, he found HbSS in 2.1% of newborns and HbAC in 0.7%. The

observed prevalence for HbAC, although similar to the finding of this study, may just be by

chance, as the observed prevalence of HbSS was much lower and the study communities were in

different geographic locations in Nigeria. The observed prevalence of HbSS in this cohort, may

just be due to the fact that this was a real population based survey where there has not been

published population surveys in the newborn.

In this study, 5.1% of the study population manifested with clinical pallor. This finding is in

agreement with those of Omokhodion106 in Ibadan, South Western Nigeria, who documented a

prevalence of 5.5% for pallor among 451 underfive children and Getaneh107 in Ethiopia,

Northern Africa, who found pallor in 5.7% of 628 children aged 6 to 59 months. On the other

hand, Akorede108in Akure, Ondo State and Fabunmi109in a rural community in Oyo State,

observed pallor in 9.3% and 20% of underfive children, respectively. In these studies, pallor was

shown to correlate positively with undernutrition, which was documented in 27.3% and 55% of

the underfive population respectively. In the present study, only 12.2% of the study population

were undernourished, hence, a better nutritional status in the study community may be the

reason for the lower prevalence of pallor observed. As expected, the presence of pallor was

significantly higher among HbSS subjects (p<0.001) than the other haemoglobin phenotypes.

This finding is consistent with those of Ambe et al53 in Maiduguri, North Eastern Nigeria and

Sadarangani et al55 in Kenya, Eastern Africa.

67

Jaundice was observed in 2.3% of the study subjects. It was most prevalent among the newborn

as shown in Table IV. It is expected that within the first week of life, as high as 60% of term

newborns and 80% of preterms will have jaundice.110 The observation of the present study is in

keeping with this expectation as majority of the newborns were recruited in the first week of life.

Of the older subjects with HbSS, 15.6% had jaundice, while only 2.2% of HbAA and 1.4% of

trait individuals presented with this feature. The observed prevalence of jaundice among HbSS

subjects in the present study is in agreement with the findings of Ambe et al53 in Northern

Nigeria, who documented a prevalence of 16.1% for jaundice among 333 sickle cell anaemia

patients at first diagnosis. Lysing of the sickled red blood cell as it passes through the

reticuloendothelial system and blood vessels may explain the chronic haemolytic anaemia and

jaundice seen in this condition.

Of the study population, 1.1% had hepatomegaly, while 4.6% had splenomegaly. This is in

agreement with Omokhodion’s106 finding in Ibadan, Western Nigeria, in which hepatomegaly

and splenomegaly were documented in 1.1% and 4.0% of under-fives respectively. In the

present study, splenomegaly was found in all the age cohorts, with peak prevalence among

children aged 24 to less than 36 months of age while hepatomegaly, which was not found

amongst the newborns, peaked at 36 to less than 48 months of age. The splenic rate in the

present study was lower than that documented by Adeyemo et al111among under-fives in

Erunmu village in Oyo state.This could be attributed to better knowledge of the preventive

measures against malaria and the increased use of antimalarial drugs which are readily available

in the health centres and the General hospital. Hepatomegaly and splenomegaly were

significantly more prevalent (p<0.001 and p<0.05, respectively) among subjects with SCD

compared with the other haemoglobin phenotypes. In a cohort of 125 SCD patients in Kuwait,

Adekile and Najwa112 found hepatomegaly in 15.2% and splenomegaly in 24%, while

Sadaranganiet al55 documented these features in 20% and 33%, respectively, of SCD subjects in

68

Kenya, Eastern Africa. The prevalence of splenomegaly among SCD subjects in the present

study was much lower than in the cited studies, unlike the prevalence of hepatomegaly which

compared favourably with those in the cited studies. A palpable spleen is expected to be an

uncommon finding in SCD because of autosplenectomy that occurs early in life. However, in

several cohorts conducted in sub-Sahara Africa, splenomegaly in SCD has been shown to persist

until late childhood and adolescence, and even into adulthood.113-115This finding has been

attributed to the effects of malaria.113-115The children in Adekile et al’s112 study were aged

between 3 and 16 years, while Sadarangani et al55 studied children 6months to 14 years of age.

In Ibadan, South Western Nigeria, Brown116 documented much higher prevalences of 88.4% and

31.8% respectively for hepatomegaly and splenomegaly among SCD patients. Brown’s study

involved 415 subjects aged 6 months to 17 years. Whether the same finding of splenomegaly

persisting until late childhood and adolescense will be true in this population is as yet,

unknown.In addition, the cited studies,55,112,116 were all hospital-based. Hence, they may have

studied mainly severely affected SCD subjects unlike the present study that was community-

based and identified children who had hitherto not been diagnosed with SCD. The much higher

prevalence of hepatomegaly documented by Brown116may signify a more intense chronic

haemolytic process in that population of SCD patients. Earlier workers had reported chronic

hepatomegaly in SCD patients and some, had singled out this feature as a severity index in

SCD.116-118 The variability in hepatomegaly and splenomegaly may be in consonance with the

diversity of various genetic and environmental factors governing the clinical course of SCD.

One of such factors is the coexistence of alpha thalassaemia which has been noted to reduce

haemolytic rate in SCD.119

Of the study population, 1.7% had gnathopathy, characterized mainly by a prognathic maxilla

and protruding anterior teeth. Of the subjects with SCD, 9.4% exhibited this feature and this was

significantly higher than the observations among other phenotypes (p<0.05). The prevalence of

69

gnathopathy among SCD subjects in the present study washowever, lower than documented by

Oredugba and Savage62 in Lagos, Western Nigeria. The gnathopathy in SCD has been found to

be progressive and more common in those patients with the most severe chronic haemolysis. In

a similar study by Fawehinmi120 in Port Harcourt, Southern Nigeria, features such as

gnathopathy, frontal bossing and depression of the bridge of the nose were observed to be more

prevalent among SCD subjects in the age group 11 to 14 years. Gupte and Suraj121 had earlier

documented that this characteristic facial profile is expected to be more prevalent in the age

group 11 to 14 years. In the present study, gnathopathy was observed in 3 HbSS subjects , one of

whom was in the 36 to less than 48months age group and two of whom were between 48 and

less than 60 months of age as shown in Table V. The lower prevalence of gnathopathy in the

present study compared with the cited studies62,120,121 may thus, be a reflection of the age

incidence of this feature in SCD. However, among the other phenotypes in which gnathopathy

was documented (HbAA,HbAS), it also showed a similar trend of increasing prevalence with

age. This observation is most likely due to the fact that dental development in children is

progressively achieved up until about 36months of age for the primary (deciduous) teeth.122

Bossing of the skull bones and depression of the nasal bridge were each observed in 0.6% of the

study population. 6.3% and 12.5% of HbSS subjects respectively, had these features and their

presence was also significantly higher among HbSS subjects compared with the other

haemoglobin phenotypes (p<0.05 and p<0.001, respectively). George54 in Port Harcourt,

Southern Nigeria, found a prevalence of 13.6% for bossing of the skull bones among SCD

affected subjects. This was a hospital based study involving 169 subjects aged between 6months

and 18 years of age. All the HbSS subjects in the present study who exhibited these features

were over 36 months of age. As earlier stated, the characteristic facial profile in SCD is thought

to be progressive with its highest prevalence in early to middle adolescence,121 hence the lower

prevalence in the present study.

70

Although abnormal clinical findings were more commonly found in HbSS subjects compared

with the other haemoglobin phenotypes, two hundred and thirty eight (238) of one thousand,four

hundred and eight (1,408) (16.9%) study cases who were not SCD affected also had similar

abnormal clinical findings. This emphasizes the value of laboratory evaluation in the diagnosis

of children, since physical findings alone have been found not to be sufficient in the

identification of cases of SCD in early life.

Nutritional status was assessed for study subjects based on their weight-for-age and

height/length-for-age. Z-scores were calculated based on WHO standard growth charts (Tables

VI and VII). Based on the weight-for-age, 86.9% were well nourished. The prevalence of

underweight was 12.2%, while overweight was observed in 0.9%.Based on the height/length-

for-age, 89.1% of the study population were well nourished, while the prevalence of stunting

and tall stature were 10.5% and 0.4%,respectively. The prevalence of normal nutritional status

in this study was much higher than has been documented in earlier reports.34,107-109 Among

underfive children in Akure, Southwestern Nigeria, Akorede and Abiola108 documented

undernutrition in 27.3% and normal nutritional status in 72.7%, while Abhulimhen-Iyoha34 in

Ekosodin community, Edo state, Southern Nigeria, found that 25.2% of the underfive population

were undernourished and 74.8% were well nourished.34,108 Getaneh et al107 in Ethiopia, observed

that 36.5% of the underfive children were undernourished, while 63.5% were well

nourished.107Fabunmi et al109documented undernutrition in as high as 55% of the underfive

children in rural Oyo state, Western Nigeria.These studies were all community-based. The

higher prevalence of normal nutritional state in the present study may be due to the impact of the

CHEWs who have been trained to deliver home-based health services in the communities.123

Their services include dissemination of information and demonstration of exclusive

breastfeeding and appropriate complementary feeding.123Also, perhaps, because the major

71

occupation in the LGA is farming, this may have a direct and positive impact on household food

security.

Based on the weight for age and the height/length for age, there was no significant difference in

the nutritional status among the various haemoglobin phenotypes (p>0.05). These finding

contrast with those of Oyedeji65, Ebomoyiet al64 and Kaine and Udeozo35, who demonstrated

significantly lower mean values for weight and height for SCD subjects when compared with

controls. These studies were all hospital-based, utilizing hospital cohorts of SCD subjects,

differing from the present study that assessed previously undiagnosed SCD subjects.The severity

of the disease in these subjects may have strongly impacted on their nutritional status unlike the

undiagnosed subjects whose manifestation of the disease still seemed as yet, mild. Oredugba and

Savage62 on the other hand, found no significant difference in the mean weights and heights of

SCD subjects aged under-five years compared with controls in a hospital-based study. In that

study, both SCD subjects and controls were from the lower socio-economic class which might

have attributed to the similarity in their anthropometric values.In the present study, the better

nutritional status of SCD subjects may be as a result of the generally better nutritional status of

the under-five population.

A 2009 study in England by Chawla et al124 examined the weight statusamong 675 children and

adolescents (2 to 19 years) with SCD, 60% of whom had HbSS disease and 26% HbSC disease.

Based on their body mass index percentiles, 22.4% were overweight or obese, while 6.7% of the

subjects were underweight of whom three quarters had HbSS or HbSβ°. Perhaps, because SCD

has historically been associated with growth delay or failure, therefore, children with SCD and

their families are counseled about the need for increased energy intake to prevent growth

problems, including underweight.124 As more children with SCD mimic the lifestyle of their

non-SCD peers, they may thus, experience similar trends of normal weight because of increased

72

caloric intake, and of increased weight because of the imbalance between energy intake and

expenditure.

73

CONCLUSIONS

1. Five haemoglobin phenotypes were identified in this study with HbAA being the most

common, followed by HbAS, HbSS, HbAC and HbAD.

2. In the newborn, four phenotypes were described and these included HbAA, HbAS,

HbSS, HbAC in decreasing order of frequency.

3. A fifth phenotype,HbAD, hithero unpublished,was documented in a 36month old child.

4. The clinical signs were more prevalent among HbSS subjects than among those who had

other haemoglobin phenotypes.

5. There was no difference in the nutritional status among study subjects regardless

of their haemoglobin phenotype.

74

RECOMMENDATIONS

Based on the findings of this study, it is recommended that:

1. Newborn screening for SCD and screening of all under-five children at any contact with

a healthcare facility be routinely conducted in all the nation’s health facilities.

2. Dedicated SCD centers should be established nationwide where access to follow up care

and target comprehensive care can be assessed following the newborn screening

program.

3. The Primary Health Centers and CHEWs should be empowered through appropriate

health education and training to administer prompt and appropriate primary care and

counselling to the SCD affected child, and refer to the appropriate specialist/center.

4. Growth monitoring and nutritional support be promoted within the comprehensive care

program for SCD.

75

LIMITATIONS

At the time of commencement of this study, there was no center in Nigeria equipped with

facilities for newborn/early infancy haemoglobin phenotype determination. Consequently, the

study was conducted with the support of a laboratory outside the country. This could probably

explain why Bart’s haemoglobin was not seen, being a gamma tetramer present in small

amounts in newborns. Its detection in large amounts would suggest the presence of alpha

thalassaemia. Bart’s haemoglobin denatures easily and thus cannot be detected by the methods

used for this study after the blood sample has been stored for more than two weeks.

76

LINES OF FUTURE RESEARCH

A Study:

1. Of the outcome of morbidity and mortality in early childhood following newborn

screening and early initiation of comprehensive health care in an African setting.

2. To determine the presence of other forms of haemoglobin in the newborn.

3. To compare the growth pattern of early diagnosed infants in a comprehensive health care

program to those diagnosed outside the newborn period and not enrolled in a

comprehensive health care program.

77

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APPENDIX I

Map of distribution of SCD

91

APPENDIX II

SURVEY INSTRUMENT: THE QUESTIONNAIRE

CHILD’S DATA:

1. NAME

2. AGE in months

3. SEX

4. PREVIOUS BLOOD TRANSFUSION YES/NO

5. IF YES, NUMBER OF TIMES AND DATE OF MOST RECENT

TRANSFUSION

6. YELLOW EYES IN THE PAST OR PRESENT YES/NO

7. PAINFUL HAND AND FOOT SWELLING IN THE PAST OR PRESENT

YES/NO

8. DATE OF BLOOD COLLECTION

PHYSICAL EXAMINATION:

SIGNIFICANT CLINICAL FINDING: INDICATE THE PRESENCE OR

ABSENCE OF THE FOLLOWING

SIGNS.

PALLOR

JAUNDICE

BOSSING OF SKULL BONES

GNATHOPATHY

FLATTENED NASAL BRIDGE

HEPATOMEGALY

SPLENOMEGALY

ANTHROPOMETRY:

HEIGHT/LENGTH in cm

WEIGHT in kg

MID UPPER ARM CIRCUMFERENCE in cm

92

PARENT’S DATA:

1. PARENT’S/GUARDIAN’S HOME ADDRESS

2. PARENT’S/GUARDIAN’S PHONE NUMBER:

3. NAME

4. AGE in years

5. SEX Male ( ) Female ( )

6. MARITAL STATUS

Single ( )

Married ( )

Cohabiting ( )

Divorced/Separated ( )

Widow/Widower ( )

7. EDUCATIONAL LEVEL

No formal education ( )

Primary ( )

Secondary ( )

Tertiary ( )

8. OCCUPATION

9. EDUCATIONAL LEVEL OF SPOUSE

No formal education ( )

Primary ( )

Secondary ( )

Tertiay ( )

10. OCCUPATION OF SPOUSE

11. AGE AND CAUSE OF DEATH OF ANY CHILD/CHILDREN

12. HAVE YOU HEARD OF GENOTYPE TEST IN THE PAST?

YES/NO

13. WHAT IS YOUR GENOTYPE?

93

APPENDIX III

STATEMENT OF INFORMED CONSENT

I am Dr.Gloria.Nwajei of the children’s department of DELSUTH, Oghara. I have come

to tell you about the work I want to start.

I plan to test your children so that I can find out whether they have got some special

things in their blood that can cause frequent sickness. What I am checking for in their

blood is called sickle cell disease. If I find that they have this thing in their blood, I will

begin to look after them in time so that they don’t develop any serious sickness .

To check the blood, I will have to take a small amount of blood from the foot or hand of

the child, and I will still check his body. The children may be afraid or cry from pain when I

am taking the blood, but outside this, it will not cause any problem.

I will give you the result of this test after 1 or 2 weeks, so I will need your address

and telephone number to call you when the result is ready. When I give you the result, I

will discuss the matter again with you.

If you know the type of blood your child has, then you will be able to look after the

child properly.

If you agree to have your child in this group that I will check, then you will sign this

paper for me so that I will include your child. Even where you don,t agree, I can still check

your child’s body, as the child will benefit from this check up , and you may like to know

the state of your child’s health.

I,…………………………………………………………………., give consent for my

child/ward,……………………………………,

to participate in this research work.

Signature:…………………………………………

Date:…………………………………………………

94

APPENDIX IV

95

APPENDIX V

96

APPENDIX VI

CAPILLARY ELECTROPHORESIS MACHINE SHOWING RESULTS

97

APPENDIX VII

RESULT OF THIN LAYER ISOELECTROFOCUSING

98

APPENDIX VIII

EXAMINING A CHILD AT THE HEALTH CENTRE IN JESSE 1

99

APPENDIX IX

CHILDREN AT THE HEALTH CENTRE FOR THE SCREENING PROCEDURE

100

APPENDIX X

STUDYING RESULTS OF ISOELECTROFOCUSING WITH THE LABORATORY

TECHNICIANS AT THE RESEARCH LAB IN COTONOU

101

APPENDIX XI

MOTHERS AND CHILDREN AT THE HEALTH CENTRE FOR THE

SCREENING PROCEDURE