A Study of Thyroid Dysfunction and Associated Risk

A STUDY OF THYROID DYSFUNCTION AND ASSOCIATED RISK

FACTORS AMONG PATIENTS WITH HIV/AIDS IN KANO,

NORTH -WESTERN NIGERIA

A DISSERTATION SUBMITTED TO THE NATIONAL POSTGRADUATE

MEDICAL COLLEGE OF NIGERIA IN PART FULFILMENT FOR THE

AWARD OF FELLOWSHIP OF THE COLLEGE IN INTERNAL MEDICINE

(SUBSPECIALTY; ENDOCRINOLOGY, DIABETES AND METABOLISM)

DR. ABUBAKAR USMAN IBRAHIM, MBBS (BUK), 2007

DEPARTMENT OF INTERNAL MEDICINE

AMINU KANO TEACHING HOSPITAL

KANO, NIGERIA

NOVEMBER, 2017

DECLARATION

I hereby declare that this work is original unless otherwise acknowledged. It has not been presented to any college for any award nor has it been submitted elsewhere for publication.

…………………………………………..

Dr. Abubakar Usman Ibrahim

Endocrinology, Diabetes, and Metabolism Unit

Department of Internal Medicine,

Aminu Kano Teaching Hospital,

Kano State.

CERTIFICATION 1

Certification by Supervisors

The study reported in this dissertation was carried out by DR ABUBAKAR USMAN IBRAHIM, under our supervision. We have also supervised the writing of the dissertation to our satisfaction and authorized the submission of the work for the Fellowship Examination in the Faculty of Internal Medicine.

Principal Supervisor

Prof. Andrew E Uloko, BMBCh, FMCP, FACE

Professor of Medicine, Consultant Physician and Endocrinologist Department of Medicine, Bayero University Kano Aminu Kano Teaching Hospital P.M.B 3452, Kano State, Nigeria. Year of fellowship……………………………………………. Signature and Date…………………………………………….. Co-Supervisor

Dr. I. D Gezawa, MD, FMCP, FACE, FRCP

Senior Lecturer, Consultant Physician and Endocrinologist Department of Medicine Bayero University Kano Aminu Kano Teaching Hospital P.M.B 3452, Kano State, Nigeria. Year of fellowship…………………………………………….. Signature and Date……………………………………………...

CERTIFICATION 2

Certification by Head of Department

I certify that the work reported in this dissertation was carried out in the Department of

MEDICINE by DR ABUBAKAR USMAN IBRAHIM and under the supervision of

PROF. ANDREW E ULOKO AND DR ID GEZAWA

Head of Department’s Name and Address

Prof A. A Sama’ila, MBBS, FWACP

Professor of Medicine

Consultant Physician and Gastroenterologist

Department of Medicine,

Bayero University Kano

Aminu Kano Teaching Hospital

P.M.B 3452, Kano State, Nigeria.

Signature and Date……………………………………………

DEDICATION

I dedicate this work to all patients living with HIV/AIDS as well as to all the participants of this study.

ACKNOWLEDGEMENT

This dissertation would not have been successfully and timely completed without the support of my supervisors, family, friends, my study population and the special grace of God. Worthy of mention are;

1) My parents, my wife, my children and my relations for their continuous prayers and

other logistic supports till the completion of this work.

2) My supervisors, teachers and trainers, Prof. Uloko and Dr Gezawa for their

patience, guidance, motivations and assistance throughout the work. All other

teachers in the department and the entire college of medicine, AKTH/BUK Kano.

3) My colleagues at the Endocrine, Diabetes, and Metabolism unit- Drs. Ramalan,

Abdullahi, and Raliya for their support and guidance.

4) All the individuals that volunteered to and participated in this study. I thank you all

for the work will not have been possible without your kind cooperation.

5) Ramadan, Mal Aminu Sarki of record, Matron Sarah of HIV-clinic, Halah Imam

Abubakar and my able brother Ibrahim Usman Ibrahim for your support and

assistance during data collection. A big thank you.

6) I am indebted to Drs Mukhtar Gadanya MFR and Baba Maiyaki for kindness and

help with data analysis. Thank you very much.

Original ideas by me in this work are reflected alongside previous efforts of others in the area of study, and to whom I therefore give due recognition.

Appreciation and a big thank you to those authors whose work I cited in this dissertation.

TABLE OF CONTENTS

HEADINGS PAGES

Title page ……………………………………………………………………………………...…..i

Declaration…………………………………………………………………………………...……ii

Certification 1….……………………………………………………………………………..…..iii

Certification 2..……………………………………………………………………………...……iv

Dedication……………………………………………………………………………...………….v

Acknowledgement……………………………………………………………………………...... vi

Table of content………………………………………………………………………….……...viii

List of Tables...………………………………………………………………………………….xv

List of Figures………………………………………………………………………………..….xvi

List of Abbreviations ……………………………………………….………………………....xvii

Summary………………………………………………………………………………….…..…xix

CHAPTER ONE

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

1.1 Definition of Research Problem……………………………………………………...………..3

1.2 Relevance of the Study to Clinical Practice…………………………………………...…..…..4

1.3 Justification for the Study……………………………………………………………….…….4

1.4 Research Hypothesis……………………………………………………………………….….5

1.5 Aims and Objectives……………………………………………………...………..………….6

CHAPTER TWO

2.0 Literature Review……….....……………………………………….…………....………...…..7

2.1.0 The Thyroid Gland…………………..………………………………….….……………..…7

2.1.1 Anatomy of the Thyroid Gland……………………………………………….…………….8

2.1.2 Physiology of the Thyroid Gland…………………………………………………………..10

2.1.3 Pattern of Thyroid Dysfunction among HIV-positive Individuals………………….……..14

2.1.4 Pattern and Causes of Thyroid Function Abnormalities among HIV negative……………16

2.1.5 Clinical Features of Thyroid Dysfunction among HIV Positive Individuals……………...18

2.1.6 Laboratory Evaluation of Thyroid Dysfunction…………………………….……………..19

2.1.7 Treatment of Thyroid Dysfunction …………………………...………………………..….20

2.2.0 The Human Immunodeficiency Virus/AIDS..……...……..…………………...…………..21

2.3.0 Effects of HIV/AIDS on Thyroid Function………..…………………….………………..23

2.3.1 Glandular Infections and Infiltration…………..……………………………………...…...24

2.3.2 Effect of HIV on Thyroid Status…………………………….…….……………………....24

2.3.3 Effects of Medications on Thyroid Function……………………………………..………..25

2.4.0 Epidemiology of the Thyroid Dysfunction and Risk Factors among HIV/AIDS positive and

HIV-Negative Individuals………………………………...….....……………...…………..26

2.4.1 Thyroid Dysfunction among HIV/AIDS Positive Individuals……………………………..26

2.4.2 Thyroid Dysfunction among HIV Negative Individuals………………………………...... 29

2.4.3 Risk Factors for Thyroid Dysfunction among HIV Positive Individuals……...…………..30

2.4.4 Risk Factors for Thyroid Dysfunction among HIV Negative Individuals…………………32

2.5.0 Assessment and Treatment of Thyroid Dysfunction among HIV/AIDS Patients...... 35

CHAPTER THREE

3.0 Subjects, materials and methods

3.1 Background of the study area………………………………………………………………..36

3.2 Study design……………………………………………………………………………..…...37

3.3 Study population……………………………………………………………………………..37

3.3.1 Subjects…………………………………………………………………………………….37

3.1.1 Controls…………………………………………………………………………………….37

3.4 Study Period……………………………………………………………………………….…37

3.5 Inclusion Criteria...………………………………………………………….…………….....38

3.5.1 Subjects…………………………………………………………………………………….38

3.5.2 Controls……………………………………………………………………….……………38

3.6 Exclusion Criteria…...…………………………………………………………...... 38

3.6.1 Subjects…………………………………………………………………………………….38

3.6.2 Controls…………………………………………………………………………….………39

3.7 Sample Size Determination……………………………………………….….....……………39

3.8 Sampling Technique…………………………………………………………………...….....40

3.9 Ethical Consideration………………………………………………..…….…………………41

3.10 Materials, Equipment and Reagents………….…………………………………………….41

3.11 Study Procedure.…….……………………….………………………………………….….42

3.11.1 Clinical Procedure….……...…………………………………….….………………….…42

3.11.2 Laboratory Procedure...... 45

3.12 Data analysis…………………………………….……………………………..……….…..47

3.13 Definition of Operational Terms…………………………………………………....….…...47

3.14 Outcome Measures………………………………………………………………………….49

CHAPTER FOUR

4.0 Results

4.1 Introduction to Results…………….……………………………………………………..….50

4.2 Sociodemographic Characteristics of the Subjects and Controls………………….………..50

4.2.1 Distribution of Subjects and Controls by Age and Gender ..………………….……..…..50

4.2.2 Educational Status of Subjects and Controls………………………...………………...…..52

4.2.3 Marital Status of the Subjects and Controls………………………………………...……...53

4.2.4 Occupational Status of the Subjects and Controls…………………………………………54

4.3 Clinical Characteristics of the Subjects and Controls………………………….………...…..55

4.4 Laboratory Characteristics of Subjects and Controls…………………………….………...... 56

4.5: Prevalence and pattern of Thyroid Dysfunction among Subjects and Controls…………….57

4.6 Frequency of Anti-TPO Antibody among Subjects and Controls…….…..…………………58

4.7 Factors associated with Thyroid Dysfunction among Subjects and Controls……………...... 59

4.8 Logistic regression model to determine risk factors associated with Thyroid Dysfunction

among Subjects and Controls…………………………..….……………………………....….61

4.9 Correlation of Thyroid Function test Parameters with Clinical and other Laboratory

Parameters among Subjects and Controls…………………..….………………….………..62

4.9.1 Correlation between BMI and FT4 among Subjects……………………………………….62

4.9.2 Correlation between Diastolic Blood Pressure and TSH among Subjects………………...63

4.9.3 Correlation between Total Cholesterol and TSH among Controls………………………...64

4.9.4 Correlation between Triglycerides and TSH among Controls……………………………..65

4.10 Comparison of Subclinical Thyroid dysfunction between Subjects and Controls………....66

CHAPTER FIVE

5.0 Discussion

5.1 Introduction………………………………………………………………………………...... 68

5.2 Socio-demographic characteristics of the study subjects………………………………….....68

5.3 Clinical Characteristics of the Subjects and Controls…………………………………...... 69

5.4 Laboratory Characteristics of Subjects and Controls…………………………….……...... 70

5.5 Prevalence and Pattern of Thyroid Dysfunction among Subjects and Controls………...... 72

5.6 Thyroid Autoimmune Status of the Subjects and Controls……....……….……...……….…74

5.7 Risk factors for Thyroid Dysfunction among Subjects and Controls…………………...…...74

5.8 Correlation of Thyroid Function test Parameters by Clinical and Other Laboratory

Parameters among Subjects and Controls………………………..…………………..………76

5.9 Limitations of the Study…………………………………………………………….……….77

CHAPTER SIX

6.0 Conclusion and recommendations

6.1 Conclusion……………………………………………..…………………..………….…...... 78

6.2 Recommendations……………………………………………………….…….………….....79

References……………………………………………………..………….……….…………….80

Appendix I: Consent Form for the Study Participants….……………………………………....91

Appendix IIA: Questionnaire (English)…………………………………………………………93

Appendix IIB: Questionnaire (Hausa Translated)……………………………………………....99

Appendix III; Ethical approval………..…………………………...…………………...…...….105

Appendix IV; Laboratory Procedure………………………………..………………………….106

Appendix V; Spread sheet of data entry……….………………...…………………….…...... 109

LIST OF TABLES:

Table 1: Common causes of hypothyroidism

Table 2: Common causes of hyperthyroidism

Table 3: Clinical Features of Hypothyroidism

Table 4: Clinical Features of Hyperthyroidism

Table 5: Distribution of the Subjects and Controls by Age and Gender

Table 6: Comparison of Clinical Characteristics of the Subjects and Controls

Table 7: Comparison of Laboratory Characteristics of Subjects and Controls

Table 8: Comparison of Prevalence and Pattern of Thyroid Dysfunction among Subjects and

Controls

Table 9: Comparison of Frequency of Anti-TPO Antibody among Subjects and Controls

Table 10a and b: Factors Associated with Thyroid Dysfunction among Subjects and Controls

Table 11: Univariate Logistic Regression Model to determine Risk Factors Associated with

Thyroid Dysfunction among Subjects and Controls

LIST OF FIGURES

Figure 1: Gross Anatomy of the Human Thyroid Gland (Anterior View)

Figure 2: Histology of the Thyroid Gland

Figure 3: Structure of Thyroid Hormones

Figure 4: Comparison of Educational Status of Subjects and Controls

Figure 5: Comparison of Marital Status of the Subjects and Controls

Figure 6: Comparison of Occupational Status of the Subjects and Controls

Figure 7: A Scatter Plot showing Correlation between BMI and FT4 among the Subjects

Figure 8: A Scatter Plot Showing Correlation between DBP and TSH among the Subjects

Figure 9: A Scatter Plot showing Correlation between TSH and TC among Controls

Figure 10: A Scatter Plot showing Correlation between TSH and Triglycerides (TG) among

Controls

ABBREVIATIONS

AIDS Acquired Immunodeficiency Syndrome AITD Autoimmune Thyroid disease AKTH Aminu Kano Teaching Hospital APIN AIDS Prevention Initiative in Nigeria ART Anti Retroviral Therapy BMI Body Mass Index CDC Center for Disease Control and Prevention CD4 Cluster of Differentiation 4 CMV Cytomegalovirus CT Computerized Tomography DBP Diastolic Blood Pressure EDTA Ethylene Diamine Tetra Acetic Acid FPG Fasting Plasma Glucose FNAC Fine Needle Aspiration Cytology FT3 Free Triiodothyronine FT4 Free thyroxine GOPD General Out Patient Department HAART Highly Active Anti Retroviral Therapy HBV Hepatitis B Virus HCV Hepatitis C Virus HDL High Density Lipoprotein HIV Human Immunodeficiency Virus KS Kaposi Sarcoma L Lymphocyte

LDL Low Density Lipoprotein LGA Local Government Area MCV Mean Corpuscular Volume N Neutrophils NAT Nucleic Acid Testing NNRTI Non-Nucleoside Reverse Transcriptase Inhibitor NTI Non Thyroidal Illness OIs Opportunistic Infections PCV Pack Cell Volume PEPFAR Presidential Emergency Program for AIDS Relief RBCS Red Blood Cells SBP Systolic Blood Pressure SD Standard Deviation rT3 Reverse T3 T3 Triidothyronine T4 Thyroxine TBG Thyroxine Binding Globulin TC Total Cholesterol TG Triglycerides TPO Thyroid Peroxidase TRH Thyrotrophin Releasing Hormone TSH Thyroid Stimulating Hormone TSH-R Thyroid Stimulating Hormone Receptor UNAIDS United Nations Programme on HIV/AIDS US United States VLDL Very Low Density Lipoprotein WBC White Blood Cell

SUMMARY BACKGROUND

The prevalence, pattern and risk factors for thyroid dysfunction among Nigerians with HIV/AIDS have been sparsely studied. This study aimed to determine the prevalence, pattern, autoimmune status and risk factors for thyroid dysfunction among patients with HIV/AIDS.

SUBJECTS AND METHODS

A cross sectional comparative study of HIV positive patients attending the PEPFAR clinic of

Aminu Kano Teaching Hospital, Kano, was undertaken. Data obtained included demographics, history suggestive of thyroid dysfunction, anthropometry, blood pressure measurements, thyroid and cardiovascular examination. Laboratory variables included thyroid function test (FT3, FT4, and TSH), anti-TPO antibody, fasting plasma glucose, plasma lipids, CD4 cell count, viral load and complete blood count.

RESULTS

A total of 115 HIV positive and 115 HIV negative subjects (20% males, and 80% females) in both groups were recruited and evaluated. The mean ± SD age of the subjects and controls was 38.2 ±

11.75years and 37.6 ± 13.12years respectively, p=0.138. Although the prevalence of thyroid dysfunction among the HIV positive participants [32.7% (male 9.1%, female 32.6%)] was slightly lower than the HIV negative controls [34.5% (male 10.9%, female 23.6%)], this difference was not statistically significant, p=0.775. Primary hypothyroidism, 11.8% (Male 4.5%, Female 7.3%) and isolated low FT3, 9.1% (Male 0.9%, Female 8.2%) were the predominant patterns among

HIV-infected group. While subclinical hypothyroidism, 16.4% (Male 4.6%, Female 11.8%) and primary hypothyroidism, 11% (Male 4.5%, Female 5.5%) predominate among controls (p=0.034).

Anti-TPO antibody was positive in 14.6% (Male 5.5%, Female 9.1%) among HIV-infected group and 13.6% among the controls. Positive history of diabetes in the parents of study subjects appears to confer risk for thyroid dysfunction. Among the study subjects, diastolic blood pressure was found to be positively correlated with TSH (r=0.044, p=0.2) while MCV showed a negative correlation with Anti-TPOAb (r=0.21, p=0.025). For the control subjects, lipid parameters

(TC, TG and LDL) correlated positively with TSH and Anti-TPOAb titers.

CONCLUSION

There was no statistically significant difference in the overall prevalence of thyroid dysfunction and autoimmunity among HIV/AIDS patients compared with controls. In patients with HIV, the predominant pattern of thyroid dysfunction was primary hypothyroidism while among the controls the predominant pattern was subclinical hypothyroidism. Family history of diabetes among parents was found to be the only risk factor for thyroid dysfunction among the subjects.

CHAPTER ONE

1.0 INTRODUCTION

Acquired Immunodeficiency Syndrome (AIDS) was first recognized in the United States in the summer of 1981. The U.S. Center for Disease Control and Prevention (CDC) reported the unexplained occurrence of Pneumocystis jiroveci (formerly P. carinii) pneumonia in five previously healthy homosexual men in Los Angeles and Kaposi's sarcoma (KS) with or without

P. jiroveci pneumonia in 26 previously healthy homosexual men in New York and Los Angeles.1

In 1983, the human immunodeficiency virus (HIV) was isolated from a patient with lymphadenopathy, and by 1984 it was clearly demonstrated to be the causative agent of AIDS.1

HIV infection/AIDS is a global pandemic, with cases reported from virtually every country. The highest prevalence rates are in sub-Saharan Africa and other parts of the developing world.2 The impact of HIV in some African countries has been sufficient to reverse population growth and reduce life expectancy into the mid thirties, although HIV incidence has recently declined in some of these high-prevalence countries. However, there are large-scale epidemics of HIV elsewhere, e.g. in India, the Russian Federation, and Eastern Europe.2

At the end of 2007, 33.2 million individuals were living with HIV infection (range: 30.6–36.1 million) according to the Joint United Nations Programme on HIV/AIDS (UNAIDS). More than

95% of people living with HIV/AIDS reside in low- and middle-income countries, about 50% are female, and 2.5 million are children under 15 years old.3

Two and a half million people were newly infected during that year and 2.1million died.3 An estimated 6800 people acquire HIV and 5700 persons die of AIDS daily.3 Sub-Saharan Africa remains the most seriously affected, but in some areas the numbers of new cases has stabilized.

However, in Eastern Europe and parts of central Asia infection rates are rising exponentially.3 The human, societal and economic costs are huge – 33% of 15-year-olds in high prevalence countries in Africa will die of HIV.3 Effective therapy for HIV has reduced mortality and morbidity but not in the poorer parts of the world.3 The demographics of the epidemic have varied greatly, influenced by social, behavioral, cultural and political factors.3

The thyroid gland produces two related hormones, thyroxine (T4) and triiodothyronine (T3).

Acting through nuclear receptors, these hormones play a critical role in cell differentiation during development and help maintain thermogenic and metabolic homeostasis in the adult.4Disorders of the thyroid gland can either be due to excess secretion of the hormones known as hyperthyroidism, or due to decreased secretion known as hypothyroidism.4 Clinically, no organ system is spared.

The presence of eye signs like lid lag, lid retraction, Ophthalmoplegia, pretibial myxoedema and digital clubbing is in favor of Grave’s disease.

Thyroid function may be altered in 10–15% of patients with HIV infection with both hypo- and hyperthyroidism seen, though the predominant abnormality is subclinical hypothyroidism.1

Another entity is goiter which can either be simple euthyroid goiter, solitary toxic nodule, toxic multilobular goiter, thyroiditis, malignancy or it could be due to infilteration with opportunistic infections. In the setting of highly active antiretroviral therapy (HAART) up to 10% of patients have been noted to have elevated thyroid-stimulating hormone levels, suggesting a manifestation of immune reconstitution.4

Different classes of antiretroviral drugs can affect thyroid function and produce different patterns of thyroid abnormalities. Similarly, male gender and low level of CD4 count has been identified as a risk factor for the development of thyroid dysfunction in HIV patients in some studies.4

In the early AIDS epidemic, the diverse endocrine manifestations of HIV infection were more often a consequence of opportunistic infections (OIs), neoplasms, or concomitant systemic illness.3

In advanced HIV disease, infection of the thyroid gland may occur with opportunistic pathogens, including P. jiroveci, Cytomegalovirus (CMV), mycobacteria, Toxoplasma gondii, and

Cryptococcus neoformans while Immune-reconstitution Graves' disease may occur as a late (9–48 months) complication of HAART.3

The widespread use of potent antiretroviral therapy (ART) has led to a decline in the incidence of glandular infiltration by OIs and neoplasms and has generated increased attention toward the metabolic complications of HIV therapy, including insulin resistance, dyslipidaemia, and alterations in body fat distribution.4 This study will address the prevalence, pattern, autoimmune status and risk factors of thyroid disorders in patients with HIV/AIDS in Aminu Kano Teaching

Hospital Kano, North-western, Nigeria.

1.1 Definition of the Research Problem

Both the prevalence and incidence of HIV/AIDS is increasing globally, particularly in Sub-

Saharan Africa including Nigeria.1 Likewise, mortality and morbidity is exponentially increasing despite the availability of HAART, largely due to ignorance and side effects of multiple drugs use in the treatment of these patients.1

The coexistence of HIV/AIDS and thyroid dysfunction appear complex. It is possible that the

ARTs may affect thyroid function in various ways. There may be similarities in the clinical presentation of HIV/AIDS and thyroid hyperfunction.5

There are few studies on the subject across the globe and particularly in Africa, Nigeria inclusive.

This study therefore desires to answer the following research questions:

 What is the prevalence of thyroid dysfunction among HIV/AIDS patients in Kano?

 What is the pattern of thyroid function abnormalities among HIV/AIDS patients in Kano?

 What are the risk factors associated with thyroid function abnormalities among HIV/AIDS

patients in Kano?

 Are there any differences between thyroid function test of HIV/AIDS patients and

apparently healthy HIV negative controls?

1.2 Relevance of the Study to Clinical Practice

Given the limited availability of data on ‘Thyroid dysfunction in HIV/AIDS’, it is hoped that this present study will provide insight as to whether patients with HIV / AIDS should undergo comprehensive evaluation for thyroid dysfunction and the findings may elucidate reasons for high morbidity and mortality in HIV/AIDS.

1.3 Justification for the Study

HIV/AIDS is now a common clinical condition encountered in medical practice. A determination of thyroid function abnormalities in such patients will provide a significant opportunity to reduce associated mortality and morbidity.

Globally, data on the coexistence of thyroid dysfunction in the setting of HIV/AIDS are few.

It is hoped that information from this study would further provide evidence for the care of these patients in our environment and contribute to knowledge on the subject in Nigeria.

1.4 Hypothesis Testing

The following null hypothesis would be tested in this study:

1. There is no difference in the prevalence of thyroid dysfunction among HIV/AIDS subjects

compared to apparently healthy controls.

2. There are no differences in the pattern of Thyroid function abnormalities among HIV/AIDS

subjects compared to apparently healthy controls.

3. There are no risk factors associated with thyroid dysfunction among HIV/AIDS patients in

Kano.

1.5 Aim and Objectives

The aim of the study was:

To evaluate thyroid function abnormalities among patients with HIV/AIDS in Kano, north-western

Nigeria.

The Specific objectives were to:

i. Determine the prevalence of thyroid dysfunction among patients with HIV/AIDS in

AKTH, Kano

ii. Describe the pattern of thyroid dysfunction among patients with HIV/AIDS in AKTH,

Kano

iii. Identify the risk factors associated with thyroid function abnormalities among patients

with HIV/AIDS in AKTH, Kano.

CHAPTER TWO

LITERATURE REVIEW

2.0 Preamble

This chapter reviews existing literature on thyroid function abnormalities in persons with HIV. A critical appraisal of the effects of thyroid dysfunction on HIV and vice versa is presented in this chapter.

2.1.0 The Thyroid Gland

The metabolism of virtually all nucleated cells of many tissues is controlled by the thyroid hormones.6 The thyroid gland produces two related hormones, thyroxine (T4) and triiodothyronine

(T3). Acting through nuclear receptors, these hormones play a critical role in cell differentiation during development and help maintain thermogenic and metabolic homeostasis in the adult.6

Autoimmune disorders of the thyroid gland can either stimulate the overproduction of thyroid hormones (thyrotoxicosis) or cause glandular destruction and hormone deficiency

(hypothyroidism).

The adult thyroid weighs 15 to 20 g; each lobe is around 4 cm long and 2 cm wide, although the right lobe is often larger than the left.7 The isthmus connecting the two lobes lie just below the cricoid cartilage. The blood supply on each side is derived from the external carotid artery via the superior thyroid artery and from the subclavian artery via the inferior thyroid artery

2.1.1 Anatomy of the Thyroid Gland

A. Embryology of the Thyroid Gland

The thyroid gland originates in the embryo as a mesodermal invagination in the pharyngeal floor at the foramen caecum, from which it descends anterior to the trachea and bifurcates, forming two lateral lobes, each measuring approximately 4cm in length, 2cm in width, and 1cm in thickness in adulthood.6 Ectopic thyroid tissue can be present anywhere along or beyond this thyroglossal duct, from the tongue base (lingual thyroid) to the mediastinum. The thyroglossal duct may also give rise to midline cysts lined with squamous epithelium, which can remain asymptomatic, or become infected or give rise to thyroid tumors. The caudal end of the thyroglossal duct forms the pyramidal lobe of the thyroid, which can become palpable in conditions causing diffuse thyroid inflammation or stimulation (Figure 1 below).

Figure 1: Gross Anatomy of the Human Thyroid Gland (Anterior view).

B. Histology of the Thyroid Gland

The gland is made up of lobules each comprising 20 to 40 spherical follicles.7 The follicles vary considerably in size, but average 200 µm in diameter, and are made up of a single layer of thyroid follicular epithelial cells. The cells are cuboidal when quiescent and columnar when active, and have a microvillous apical membrane. The follicular lumen contains colloid, the principal constituent of which is the glycoprotein thyroglobulin secreted by the thyroid cells. Each follicle is surrounded by a rich capillary network. Clear cells lie scattered between follicular epithelial cells or in the interstitium, and account for around 1% of the epithelial mass.8 Figure 2 below shows histology of the thyroid gland

Figure 2: Histology of the Thyroid Gland

Source: http://www.lab.anhb.uwa.edu.au/mb140/corepages/endocrines/Images/ThyrHE40.jpg

2.1.2 Physiology of the Thyroid Gland:

A. Structure and Synthesis of the Thyroid Hormones

Thyroid hormones are derived from thyroglobulin (Tg), a large iodinated glycoprotein. After secretion into the thyroid follicle, Tg is iodinated on tyrosine residues that are subsequently coupled via an ether linkage. Reuptake of Tg into the thyroid follicular cell allows proteolysis and the release of newly synthesized T4 and T3 as shown in figure 3 below.

Figure 2.3: Structure of Thyroid Hormones

(i) Iodine Metabolism and Transport

Iodide uptake is a critical first step in thyroid hormone synthesis. Ingested iodine is bound to serum proteins, particularly albumin. Unbound iodine is excreted in the urine. The World Health

Organization (WHO) recommends a daily dietary iodine intake of 150ug for adults, 200ug for pregnant and lactating women, and 50 to 250ug for children.9 According to WHO, dietary iodine deficiency, defined as a daily iodine intake less than 100ug/d, affects an estimated two billion people, which is about one-third of the world's population.10 When iodide intake is less than

50ug/d, a normal-sized thyroid cannot sustain adequate hormone production, resulting in gland enlargement (goiter) and, ultimately, hypothyroidism.11 The consequences of endemic dietary iodine deficiency are especially devastating for the developing fetus and children, who require thyroid hormone for normal neurologic development and growth.11

Thyrocytes abundantly express the sodium-iodide symporter (Na-I– symporter; NIS), which spans the cells' basal membranes and actively transports iodide from the blood. The thyroid gland concentrates and uses only a fraction of the iodide supplied to it for hormone synthesis, and the remainder returns to the extracellular fluid pool. Consequently, the normal fractional uptake of iodide, which can be quantified with a radioactive iodine tracer, is approximately 10% to 30% after 24 hours.12 This iodine provides a buffer in the event of temporary dietary iodine deficiency.

Synthesis of T4 and T3 by the thyroid gland involves six major steps: (1) active transport of iodide across the basement membrane into the thyroid cell (trapping); (2) oxidation of iodide and iodination of tyrosyl residues in thyroglobulin (organification); (3) linking pairs of iodotyrosine molecules within thyroglobulin to form the iodothyronines T3 and T4 (coupling); (4) pinocytosis and then proteolysis of thyroglobulin with release of free iodothyronines and iodotyrosines into the circulation; (5) deiodination of iodotyrosines within the thyroid cell, with conservation and reuse of the liberated iodide; and (6) intrathyroidal 5'-deiodination of T4 to T3. Thyroid hormone

31 synthesis requires that NIS, thyroglobulin, and the enzyme thyroid peroxidase (TPO) all be present, functional, and uninhibited.12

(ii) Secretion of the Thyroid Hormones

The thyroid gland secretes predominantly thyroxine (T4), and only small amount of triodothroinine

(T3); approximately 85% of T3 is produced by monodeiodination of T4 in other tissues such as liver, muscle and kidney. T4 is not metabolically active until converted to T3. T4 and T3 circulate in plasma almost entirely bound to transport protein, mainly thyroxine binding globulin (TBG).13

It is the free hormone which diffuses into tissues and exerts its metabolic actions. The free hormone is not influenced by changes in concentration of binding proteins. Production of T3 and

T4 is stimulated by thyrotrophin (thyroid stimulating hormone, TSH) release from the anterior pituitary gland in response to the hypothalamic thyrotrophin releasing hormone (TRH). There is negative feedback of thyroid hormone secretion by the TSH.

The introduction of sensitive immunoradiometric assays for detecting circulating TSH, with a detection level of 0.1 mU/liter or less, has transformed the evaluation of thyroid status.

B. Functions of the Thyroid Hormones

The transcriptional effects of T3 characteristically demonstrate a lag time of hours or days to achieve full effect.12 These genomic actions have a number of vital effects, including tissue growth, brain maturation, increased calorigenesis and oxygen consumption, as well as other specific effects on the heart, liver, kidneys, skeletal muscle, and skin. However, some actions of T3 are believed not to be genomic, including its reduction of pituitary type 2 5'-deiodinase activity and the

32 increased glucose and amino acid transport that it can induce in some tissues.13 Some specific effects of thyroid hormones are summarized below.

(i) Enhance foetal brain and skeletal development.13

(ii) Enhance oxygen consumption, heat production and basal metabolic rate.14

(iii) Enhance systolic function thereby increasing heart rate, lower peripheral vascular resistance and also increase cardiac output.15

(iv) Increase the number of adrenergic receptors in heart, skeletal muscle, adipose tissue, lymphocytes and may also amplify catecholamine action at a postreceptor site.14

(v) Maintain ventilatory responses to hypoxia and hypercapnia in brain stem respiratory center.14

(vi) Increase production of erythropoietin and therefore erythropoiesis.14

(vii) Promote gut motility.14

(viii) Thyroid hormones stimulate bone turnover, increasing bone resorption and, to a lesser degree, bone formation.14

(ix) Increase protein turnover and loss in skeletal muscle, which can lead to a characteristic proximal myopathy. There is also an increase in the speed of muscle contraction and relaxation.14

(x) Increase hepatic gluconeogenesis and glycogenolysis, as well as intestinal glucose absorption, and there may also be thyroid hormone–mediated decreases in insulin sensitivity. Cholesterol synthesis and degradation are both increased.14

(xi) Thyroid hormones alter the production, responsiveness, and metabolic clearance of a number of hormones.14

2.1.3 Pattern of Thyroid Dysfunction among HIV-Positive Population

The following patterns of thyroid abnormalities are recognized;

1. Primary Hypothyroidism

Overt hypothyroidism is recognized by low serum T4 and T3 and elevated serum level of

TSH. The prevalence of primary hypothyroidism in the general population compared to

HIV patients is 0.3% and 2.6% respectively in some studies.16 It is negatively correlated to

the level of the CD4 counts which could be as a result of opportunistic infections such as

Pneumocystis jiroveci, Cytotomegaloviral infection or neoplasm such as Kaposi

sarcoma.17

2. Subclinical Hypothyroidism

It is characterized by high level of TSH and normal T4, although patients may have subtle

features of hypothyroidism. It has a prevalence of 14.5% compared to 12.4% among the

general population.16

3. TSH Secreting Tumour / Secondary hyperthyroidism:

It is identified by high level of TSH and also high level of T4. It is common among HIV-

infected individuals with a prevalence of 3.5-12.2%. 1

4. Primary Hyperthyroidism:

It is primary because it indicates that the abnormality is from the thyroid gland itself. It is

identified by low level of TSH and high level of FT4 and FT3. Grave’s disease is the

leading cause of hyperthyroidism in both general population and HIV infected patients.18

5. Slow Conversion of T4 to T3 or Thyroid Hormone Antibody Artifact

This is indicated by the presence of high level of TSH, high level of T4 and low level of

T3.7

6. Sick Euthyroid Syndrome

It is also known as Non thyroidal illness or low T3 syndrome. It describes abnormal thyroid

function in the setting of severe illness or infections. The highest prevalence of this

syndrome was reported to be 16% in patients with terminal AIDS before the advent of

HAART.19 after recovery of the illness the thyroid function abnormalities should

completely reverse to normal. The changes seen are; reduced level of T3, TSH and T4.

7. Subclinical Hyperthyroidism

This is defined by low TSH and normal level of both T4 and T3 and absence of

thyrotoxicosis, although patients could have subtle features of hyperthyroidism.

2.1.4 Pattern and Causes of Thyroid Function Abnormalities among HIV negative

Population

Table 1 and 2 below summarized the most common causes of hypo and hyperthyroidism respectively among HIV negative individuals which are also shared with HIV positive individuals.

Table 1: Common causes of hypothyroidism Pattern Causes

Primary Hypothyroidism Autoimmune hypothyroidism: Hashimoto's thyroiditis, atrophic thyroiditis

Iatrogenic: 131I treatment, subtotal or total thyroidectomy, external irradiation of neck for lymphoma or cancer.

Drugs: iodine excess, amiodarone, lithium, antithyroid drugs, p-aminosalicyclic acid, aminoglutethimide and cytokines

Congenital hypothyroidism: absent or ectopic thyroid gland, dyshormonogenesis, TSH-R mutation and iodine deficiency,

Infiltrative disorders: amyloidosis, sarcoidosis, hemochromatosis, scleroderma, cystinosis, Riedel's thyroiditis Overexpression of type 3 deoiodinase in infantile hemangioma

Transient Silent thyroiditis, including postpartum thyroiditis, subacute thyroiditis, Withdrawal of thyroxine treatment in individuals with an intact thyroid after 131I treatment or subtotal thyroidectomy for Graves' disease.

Secondary Pituitary: Tumors, pituitary surgery or irradiation, infiltrative disorders, Sheehan's syndrome, trauma, genetic forms of combined pituitary hormone deficiencies, Isolated TSH deficiency or inactivity Bexarotene treatment, Hypothalamic disease: Tumours, trauma, infiltrative disorders

Source: Harrison’s Principles of Internal Medicine, 17th edition with modifications

Table 2: Common Causes of Hyperthyroidism Pattern Causes

Primary Hyperthyroidism Graves' disease, Toxic multinodular

goiter, Toxic adenoma, Functioning thyroid

carcinoma, metastases, Activating mutation of

the TSH receptor, Activating mutation of Gsa

(McCune-Albright syndrome) Struma ovarii,

Drugs: iodine excess (Jod-Basedow

phenomenon)

Secondary hyperthyroidism TSH-secreting pituitary adenoma, Thyroid

hormone resistance syndrome: occasional

patients may have features of thyrotoxicosis,

Chorionic gonadotropin-secreting tumors,

Gestational thyrotoxicosis.

Source: Harrison’s Principle of Internal Medicine, 17th edition with modifications

2.1.5 Clinical Features of Thyroid Dysfunction among HIV Positive Individuals

The clinical features of thyroid dysfunction are not different between HIV positive and HIV negative individuals. Tables 3 and 4 below summarize the major clinical features of both hypo and hyperthyroidism in the general population.

Table 3: Clinical Features of Hypothyroidism Symptoms Signs Tiredness, weakness Dry coarse skin

Dry skin cool peripheral extremities

Feeling cold Puffy face, hands and feet (myxoedema)

Hair loss Diffuse alopecia

Difficulty concentrating and poor memory Bradycardia

Constipation Peripheral oedema

Weight gain with poor appetite Delayed tendon reflex relaxation

Dyspnoea, Hoarse voice Carpal tunnel syndrome

Menorrhagia (later oligomenorrhea/ amenorrhoea)

Paresthesia, Impaired hearing Serous cavity effusion

Source: Harrison’s Principle of Internal Medicine, 17th edition

Table 4: Clinical Features of Hyperthyroidism Symptoms Signs Hyperactivity, irritability, dysphonia Tachycardia; atrial fibrillation in the elderly

Heat intolerance and sweating Tremors

Palpitations Goiter

Fatigue and weakness Warm, moist skin

Weight loss with increased appetite Muscle weakness, proximal myopathy

Diarrhea/hyper-defeacation Lid retraction or lag

Oligomenorrhea, loss of libido Gynaecomastia

Source: Harrison’s Principle of Internal Medicine, 17th edition

2.1.6 Laboratory Evaluation of Thyroid Dysfunction

The most frequent cause of thyroid dysfunction in iodine-sufficient areas is autoimmunity, and the simplest test for this is measurement of thyroid autoantibodies using different methods such as haemagglutination, immunofluorescence, radioimmunoassay, electrocheminoimmunoluscent and enzyme-linked immunosorbent, particularly those directed against thyroid peroxidase (the

‘microsomal’antigen). Antibodies against thyroglobulin are also easily measured but are almost always accompanied by thyroid peroxidase antibodies, so testing for the latter alone is usually adequate.

Thyroid imaging by scintiscanning is useful in determining the aetiology of thyroid disease when this is not obvious clinically, particularly in hyperthyroidism and ectopic thyroid tissue. 99Tcm pertechnetate is usually used as it has a short half-life (6 h) which allows safe administration of high activity and rapid scanning. 123I is not as readily available but is preferable to 131I, especially in children, as it has a short half-life and does not emit β-radiation. Thyroid ultrasound is being increasingly used as an alternative to scintiscanning. CT scanning is particularly valuable in determining the extent of retrosternal goitre and assessing tracheal compression. In contrast, a standard chest radiograph can be misleading in evaluating tracheal compression, particularly in the anterior–posterior plane.

2.1.7 Treatment of Thyroid Dysfunction

Treatment of thyroid dysfunction depends on the cause, severity and associated clinical conditions such as pregnancy e.t.c. When the hormone level is low (hypothyroidism) replacement may be needed. When there is excess hormone antithyroid drugs may be needed to normalize the excess hormone in circulation. Radio-iodine treatment is an option to ablate hyperthyroid goitre causing thyrotoxicosis. Surgery may be indicated in various forms of thyroid function abnormalities such as multinodular goitre and different forms of thyroid malignancies.

2.2.0 The Human Immunodeficiency Virus/AIDS

HIV is a virus that attacks the immune system. If untreated, a person’s immune system will eventually be completely destroyed. AIDS refers to a set of symptoms and illnesses that occur at the very final stage of HIV infection.

Worldwide, the principal mode of transmission is heterosexual intercourse. Other risk factors for acquisition of HIV include homosexuality, injection drug use, blood or blood products, and mother to child transmission.20

A few weeks after acquisition of HIV, many people develop a nonspecific influenza-like illness

(seroconversion illness/acute retroviral syndrome), with a transient macular or maculopapular rash affecting the upper body. Rarely, there are neurological complications and severe immunodeficiency with secondary opportunistic infections.20

Following primary infection (symptomatic or asymptomatic) a period of clinical latency follows, typically lasting eight to ten years before development of further illness. The infected person is asymptomatic, but some have persistent generalized lymphadenopathy, and they may develop minor opportunistic conditions affecting the skin and mucous membranes, e.g. viral warts, oropharyngeal candidiasis, oral hairy leucoplakia21.

A final stage is progression to symptomatic HIV disease (AIDS).22

Many complications of late HIV infections ensue and include the following;

(1) Opportunistic infections—e.g. pneumocystis pneumonia, oesophageal candidiasis, cerebral toxoplasmosis, and cytomegalovirus retinitis;

(2) Opportunistic tumours—e.g. Kaposi’s sarcoma.

(3) Direct HIV effects—e.g. HIV encephalopathy/dementia where CD4 lymphocyte count and

HIV viral load are the two laboratory markers with the best prognostic value.

(i) CD4 count— an indicator of HIV-related immune impairment, with decline to below 200/mm3 associated with the risk of life-threatening opportunistic infection; antiretroviral treatment is currently considered when it has fallen to around 350/mm3. 23

(ii) Viral load—quantitative estimation of HIV RNA in the blood plasma adds additional prognostic information before starting antiretroviral treatment, and is useful in monitoring the effectiveness of therapy, which aims to maintain suppression of viral RNA at undetectable levels

(<20 copies/ml). The choice of initial antiretroviral regimen should take into account the results of baseline genotypic resistance testing.23

The establishment of HIV as the causative agent of AIDS and related syndromes early in 1984 was followed by the rapid development of sensitive screening tests for HIV infection24. By March

1985, blood donors in the United States were routinely screened for antibodies to HIV. In June

1996, blood banks in the United States added the p24 antigen capture assay to the screening process to help identify the rare and infected individuals who were donating blood during incubation period

(up to 3 months)25. In 2002 the ability to detect early infection with HIV was further enhanced by the licensure of nucleic acid testing (NAT) as a routine part of blood donor screening. These refinements decreased the interval between infection and detection (window period) from 22 days for antibody testing to 16 days with p24 antigen testing and subsequently to 12 days with nucleic acid testing25. The development of sensitive assays for monitoring levels of plasma viraemia ushered in a new era of being able to monitor the progression of HIV disease more closely.

Utilization of these tests, coupled with the measurement of levels of CD4+ T lymphocytes in peripheral blood, is essential in the management of patients with HIV infection.26

More than 20 agents are now available for treatment of HIV: a minimum of 3, drawn from at least

2 drug classes, is required for effective treatment. Treatment regimens usually include (1) a backbone of two nucleoside analogues (inhibitors of HIV reverse transcriptase), e.g. lamivudine, emtricitabine, abacavir, or zidovudine, with either (2) a non-nucleoside reverses transcriptase inhibitor (NNRTI), e.g. efavirenz or nevirapine, or (3) a protease inhibitor, e.g. lopinavir, atazanavir, fosamprenavir or saquinavir (usually ritonavir-boosted).27 Co-infections involving

HIV and tuberculosis, hepatitis B or hepatitis C are common and require specialized treatment.28

Strategies to raise awareness and provide education, and promote risk reduction, underpin HIV control programmes worldwide. Control of coexistent sexually transmitted genital ulcers and other genital infections reduces HIV transmission. Mother to child transmission can be reduced to below

1% if antiretroviral treatment is administered to the mother during pregnancy, delivery is by planned caesarean section, and breastfeeding is avoided.29 No vaccine is available.30

The outlook for people with HIV infection in well-resourced countries was transformed in the late

1990s by the advent of highly active antiretroviral therapy (HAART), but access to antiretroviral drugs continues to be difficult in less-developed countries.31

2.3.0 Effects of HIV/AIDS on Thyroid Function

In general, the diagnosis and treatment of thyroid disorders in a patient with HIV infection does not differ from that in an immunocompetent individual. There are, however, some special considerations. HIV infection may cause changes in thyroid function that are adaptive and do not require treatment.32 Furthermore, many of the signs and symptoms of thyroid dysfunction are nonspecific and can overlap with other non-endocrine disorders that are common in HIV-infected

44 patients.32 Finally, many medications that are used to treat HIV infection and its complications can induce endocrine dysfunction, including affecting thyroid hormone metabolism.32

2.3.1 Glandular Infection and Infiltration

Infection by a diverse array of organisms, as well as HIV-associated malignancies (i.e., Kaposi's sarcoma and lymphoma), have been detected in the thyroid gland. Such occurrences were far more common prior to the widespread introduction of potent ART, although they may still be observed in patients not receiving ART or who have antiretroviral drug resistant infection.33 Histology of thyroid tissue is generally required for a definitive diagnosis. Fine needle aspiration (FNA) biopsy of the thyroid is safe, effective, and widely available. Standard functional testing should also be performed since clinically significant thyroid dysfunction may accompany glandular infection or infiltration.

2.3.2 Effects of HIV on Thyroid Status

The clinical status of the HIV-infected patient is closely linked to the presence and/or severity of thyroid dysfunction. Asymptomatic HIV-positive patients with stable body weight usually maintain clinically normal thyroid function.33 In a study of 202 stable HIV-infected patients, there were no thyroid abnormalities found in the 20 treatment-naïve patients.34 In contrast, changes similar to those seen in non-thyroidal illness (NTI) in HIV-seronegative individuals are often present in patients with AIDS.35 NTI is generally characterized by low-normal thyroid-stimulating hormone (TSH), low tri-iodythyronine (T3), high reverse T3 (rT3), and normal free thyroxine (T4) levels due to impaired peripheral T4 to T3 conversion and reduced rT3 clearance. Low T3 levels in NTI may be adaptive, by lowering metabolic rate and decreasing protein catabolism, in the setting of systemic illness or reduced caloric intake.36 However, T3 levels are often higher while

45 reverse T3 levels is lower in AIDS patients than expected for NTI, raising concern that these protective effects may be compromised.37 These differences in T3 levels are not explained by the increased levels of thyroid hormone-binding globulin (TBG) seen in HIV-infected individuals13.

Reductions in T3 levels, however, are often observed in AIDS patients with acute secondary infection and anorexia.36,37 One cross-sectional multicenter study of 350 HIV-infected patients suggested that the prevalence of hypothyroidism was high (ie, 16 percent), although there was no

HIV-seronegative control group for comparison.38 The risk of hypothyroidism was increased among patients with lower CD4 counts. Symptoms ranged from those of overt hypothyroidism

(2.6 percent) to subclinical disease (6.6 percent) to an isolated low free T4 level (6.8 percent).38

2.3.3 Effects of Medications on Thyroid Functions

By increasing clearance of thyroid hormone, medications such as rifampin, ketoconazole, and ritonavir can precipitate hypothyroidism in patients with marginal thyroid reserve. Patients receiving replacement therapy with thyroxine may also require higher doses if taking these medications. HIV patients co infected with hepatitis C who are treated with interferon-alpha (INF- alpha) may develop autoimmune thyroid diseases (AITD) such as Graves' disease and Hashimoto's thyroiditis, as well as subacute or destructive thyroiditis.39 When effective ART leads to immune reconstitution, AITD can result from the production of new immune cells targeting thyroid antigens.40 Such patients typically present with signs and symptoms of Graves' disease one to two years after beginning ART. Immune reconstitution can also lead to development of Hashimoto's thyroiditis, which may explain reports suggesting an increased prevalence of subclinical hypothyroidism among patients taking ART.37, 38

2.4.0 Epidemiology of Thyroid Dysfunction and Risk Factors among HIV Positive

Individuals

Several studies on the subject of Thyroid Dysfunction and risk factors assessment in patients with

HIV/AIDS as well as HIV negative individuals have been carried out in different parts of the world.32

2.4.1 Thyroid Dysfunction among HIV/AIDS Positive Individuals:

In a Brazilian study of 2437 HIV patients, Sen et al41 found 54 (2.2%) with abnormal thyroid function. Twenty two had hypothyroidism (52%) and 26 hyperthyroidism. He also found the prevalence of thyroid auto-antibody (anti-TPO Ab) as 4(40%) among individuals with hypothyroidism and 4(66.7%) among individuals with hyperthyroidism.

Guilherme et al42 in Rio de Janeiro, Brazil found mean age (years) of 43.69 ± 1.06, HIV duration of 112±9.1 month and the mean CD4 count of 498 ± 272. He found 34.2% prevalence of thyroid dysfunction, 12.82% subclinical hypothyroidism, 6.83% primary hypothyroidism, 11.96% isolated low FT4, 0.85% isolated high FT4, 0.85% secondary hypothyroidism, 0.85% secondary hyperthyroidism and 4.34% AITD (anti-TPO Ab positive).

In Oxford, Madeddu et al43 found abnormal thyroid function in 23/182(12.6%) on HAART, and none among naïve patients. Most common abnormality found was subclinical hypothyroidism. .

Collazos et al44 reported low free T4 levels (1.3%) and subclincal hypothyroidism (3.5%) which correlated with low CD4 counts among Spanish population.

Shujing et al45, in China found the prevalence of thyroid dysfunction of 59/178 (33%) mostly hypothyroidism. The Mean duration of HAART was 7.42±5.2 years. Thyroid dysfunction was significantly more frequent in the HAART group (41/104, 39.4%) than in the HAART-naïve group

(18/74, 24.3%). The FT4 level was significantly lower in the HAART group than in the HAART- naïve group.

In an Iranian study Rasoolinejad et al46 found 18 (20.5%) - thyroid dysfunction, 1 (1.1%) - hypothyroidism, 2 (2.2%) - subclinical hypothyroidism, low FT4- 12 (13.6%) and 3 (3.4%) –low

FT3. There was no significant relationship between age, sex, weight, CD4 count, stage of the disease, antiretroviral disease and thyroid dysfunction.

In a multi centered study Chen et al47 found prevalence of thyroid dysfunction of 17/234 (7.3%) in black African mostly female. He found 15 out of 17 to have Graves’ disease.

In a study in Nairobi, Kenya by Thaimuta et al48 the prevalence of thyroid dysfunction was 63/110

(57.3%). Sick euthyroid is the dominant abnormality found 44% among ARV (+) and 46% among

ARV (-) patients. Subclinical hypothyroidism was found in 4.8% among ARV (+) and none among

ARV (-) patients. None also among ARV (-) was found with hyperthyroid abnormalities. High level of TPO was found in 2.4% ARV (+) and 15.4% among ARV (-) patients.

There is paucity of studies on Thyroid Dysfunction among HIV/AIDS patients in Nigeria. Amadi et al49 Jos, Nigeria (mountainous iodine-deficient area) found overt hypothyroidism in all the AIDS patients.

Abbiyesuku et al50, in a study of thyroid function test in HIV seropositive patients on HAART in

Nigeria, found a total prevalence of thyroid dysfunction to be 26% (Subclinical hypothyroidism

7%, isolated low FT3 9%, isolated low FT4 9% and subclinical clinical hyperthyroidism 1%). In the control group (HIV negative), the prevalence of 28.6% (subclinical hypothyroidism 8.3%, isolated low FT3 12.5%, isolated low FT4 12.5% and subclinical hyperthyroidism 3.6%) was found. TSH was found to be significantly higher in test groups than normal HIV-negative control, p<0.001.

Another study from Zaria, Nigeria demonstrated that, autoimmune thyroid disease occurs in 3% of women and 0.2% of men. Patients with lower CD4count at baseline with greater increments in the CD4counts following HAART are more likely to develop AITD.51

Studies on HIV infection and body metabolisms in Nigeria, include;

Muhammad et al52, who found higher total cholesterol and lower HDL among HIV patients on

HAART compared to HAART naïve with no difference in LDL and TGS.

Adewole et al53, also found higher mean LDL and lower mean HDL among HIV patients compared to control.

Daniyan et al54, also found lower HDL and TGS among the HIV-infected patients but higher cholesterol LDL among the control group.

There are also studies done on hematological profile of HIV in Nigeria notably among them are;

Ofonime et al55 at Calabar, Nigeria, found anemia as the most common haematological abnormalities among HIV/AIDS patients, anaemia -129/272 (47.4%), Leucopenia -15 (47.4%),

Neutropenia -34 (18.3%) while Lymphopenia is more prevalent in those with mycotic infection.

Amegor et al56 reported anaemia and leucopenia as the most common abnormal hematological findings in Makurdi, Benue state, Nigeria.

2.4.2 Thyroid Dysfunction among HIV-negative Individuals

In a study based on clinical records only, Loida et al57 reported weighted prevalence of hyperthyroidism at 0.0043% (95% CI: −0.0021%, 0.0107%) among adult female population and weighted prevalence of hypothyroidism at 24.2% (95% CI: 19.9%, 28.4%). The increased prevalence of hypothyroidism was found in participants 70 years or older.

Alessandro et al58 in Sardinia found a low prevalence of overt thyroid dysfunction

(hyperthyroidism 0.4%, hypothyroidism 0.7%). The rates of subclinical hypothyroidism and hyperthyroidism were 4.7% and 2.4%, respectively. Almost 16% of individuals were positive for at least one antibody and 5.2% for both AbTG and AbTPO. Nodules were detected in 17.4% of the subjects, and the prevalence of goiter was 22.1%.

In Nepal India Madhukar et al59 found the prevalence of thyroid dysfunction of 25% and the prevalence was higher in females than males. Hypothyroidism (8%), subclinical hypothyroidism

(8%), subclinical hyperthyroidism (6%) and hyperthyroidism (3%). Higher prevalence of thyroid dysfunction was observed in subjects aged above 30 years.

A study in Libya, Northern part of Africa, Ali et al60 found overt hyperthyroidism (0.84%), subclinical hyperthyroidism (0.84%), overt hypothyroidism (1.12%), and subclinical hypothyroidism (6.18%). Thyroid dysfunction was more common in females than males. Higher prevalence of subclinical hypothyroidism (27%) was found among the subjects with

50 hypercholesterolemia. Also found a significant negative correlation between subjects with hypothyroidism and hypercholesterolemia (P<0.05).

Amballi et al61 in Sagamu, Ogun State, Nigeria found 25.5% with hyperthyroidism, 8.4% hypothyroidism. Overall prevalence was 34.0%. They found higher prevalence among females than males and that the age group 36-45 years are those mainly affected, there is however no statistically significant association between gender or age and T3/T4.

In a northern Nigerian study of thyroid disorders that described the clinical pattern and complications of thyroid dysfunction, Uloko et al62 described the relative prevalence of thyroid disorders in the general population. In this study, simple goiter was 6 (5.3%), Graves’ disease 56

(49.6%), Toxic Multi-nodular goiter 2 (1.8%), primary hypothyroidism 9 (7.9%) and 2 (1.8%) with drug-induced (amiodarone) hypothyroidism. Thirty three (58.9%) have thyroid opthalmopathy, hypertension 16 (28.6%), atrial fibrillation 3 (5.4%) and congestive cardiac failure in 4 (7.1%).

2.4.3 Risk Factors for Thyroid Dysfunction among HIV-Positive Individuals

The risk factors affecting thyroid function among HIV negative individuals also affect HIV positive individuals. Peculiar risk factors affecting HIV positive individuals include;

A) Level of CD4 count:

Low level of CD4 count was identified as a risk factor for the development of hypothyroidism among HIV positive patients.63 Very few studies found no association between CD4 count and

51 development of thyroid dysfunction among HIV positive patients.41 The pathogenesis of the hypothyroidism due to low level of CD4 count was not known.

B) ARVs use and their types:

Several drugs used in the treatment of HIV infection were identified to affect thyroid function through unknown mechanisms. In some studies, thyroid dysfunction was more frequent among

HAART-treated than HAART-naïve group64. HAART use was found to be positively correlated with TSH level, For instance, protease inhibitors were associated with hyperthyroidism in many studies while non-nucleoside reverse transcriptase inhibitors were associated with hypothyroidism.41 In contrast to the afore mentioned statement Efavirenz, a well known non- nucleoside reverse transcriptase inhibitor was found to be associated with both hyper and hypothyroidism.65 Similarly, stavudine and lamivudine use was found be associated with hypohyroidism63,64 as well as subclinical hypothyroidism in some studies.42

C) Duration of HIV:

Few studies have found association between HIV duration and thyroid dysfunction45,64 and have implicated the role of opportunistic infections as a cause of thyroid dysfunction in chronic HIV infection.66

D) Opportunistic infections/infiltration:

Different forms of opportunistic pathogens/Tumors such as tuberculosis, CMV and Kaposi sarcoma associated with HIV infection have been implicated in several studies for development of

52 hypothyroidism. The mechanism of which is mostly due to infiltration of the gland by the organisms/tumors.66.

E) Co-infection with HBV/HCV:

Co infection with HBV/HCV have been found to increase the probability of thyroid dysfunction.64

The effect was not very clear.64 Some studies have found co infection with HBV to be associated with lower mean FT3 while HCV co infection was found to be associated with higher mean FT3 and FT4, although very few studies have linked HCV co infection with hypothyroidism.45,64

2.4.4 Risk Factors for` Thyroid Dysfunction among HIV-Negative Individuals

Factors that contribute to the risk of developing thyroid disease or thyroid dysfunction include:

A) Gender

Women face a greater risk of developing thyroid disease than men. While experts vary in their estimates, it's said that women are anywhere from 6 to 8 times more likely than men to develop a thyroid condition.67

B) Age

Being 50 and above increases the risk of thyroid disease for both men and women.67

C) Personal History

A past history of thyroid disease increases the risk of developing another thyroid disease.

A personal history of any autoimmune disease slightly increases the risk of developing an autoimmune thyroid disease such as Hashimoto's disease or Graves' disease.67

D) Family History

A family history of thyroid disease increases the risk for developing thyroid disease. The risk is slightly greater if a first-degree female relative (mother, sister, and daughter) has thyroid disease.67

A family history of having any autoimmune disease slightly increases your risk of developing an autoimmune thyroid disease such as Hashimoto's disease or Graves' disease.

E) Pregnancy/Post-Partum Period

The risk of developing autoimmune thyroid disease or a temporary thyroiditis increases slightly with pregnancy or during the first-year postpartum67

F) Iodine Exposure/Intake

Use of iodine or herbal supplements containing iodine, in pill or liquid form, by people who are iodine sufficient increases the risk of autoimmune thyroid disease and hypothyroidism, and, less commonly, hyperthyroidism or thyrotoxicosis.68

G) Iodine Deficiency

Lack of sufficient iodine -– iodine deficiency -– increases the risk of hypothyroidism and goiter.

Iodine deficiency is more common in developing nations and countries where table salt is not iodized.68

Other identified risk factors include; thyroid surgery, radioactive iodine treatment, cigarette smoking, medications such as amiodarone, lithium, interferon e.t.c., goitrogens, radiation exposure, neck surgery/trauma, stress and major illnesses.69

A look at previous studies that investigated these risk factors;

In a study of elderly subjects in Denmark, 3.8% of subjects in the area of low iodine intake had high serum TSH, while 18% in the area of high iodine intake showed subclinical hypothyroidism.70

In another study of Japanese, the frequency of high urinary iodine correlated with hypothyroidism in the absence of autoantibodies.64

Deepthi71 in Palakkad, India found female gender as a risk factor for thyroid dysfunction particularly hypothyroidism. Type 2 Diabetes mellitus was the most common co morbidity presenting with hypothyroidism.

Safia72 in Hail, Saudi Arabia found iodine deficiency, poor nutrition, and family history of thyroid disease to be associated with hypothyroidism in women while stress, malignant tumours, diabetes mellitus and Hashmoto’s thyroiditis were the risk factors among men. Iodine deficiency was associated with hyperthyroidism among both sexes.

2.5.0 Assessment and Treatment of Thyroid Dysfunction among Patients with HIV

The characteristic changes in thyroid function tests among patients with HIV/AIDS usually do not require treatment and must be interpreted in their appropriate clinical context. If an HIV-infected patient presents with typical signs and/or symptoms of hypo- or hyperthyroidism, TSH should be the initial laboratory test ordered. If the TSH is normal, but there is still reason to suspect secondary

(pituitary) hypothyroidism, then a free T4 must also be checked. Anti thyroid anti bodies may also be checked in case of auto immunity such as Grave’s disease. Patients with clinically significant and biochemically confirmed hypothyroidism should initially be prescribed low doses of replacement therapy (levothyroxine 25 to 50 mcg daily) with gradual dose titration and monitoring of thyroid function tests to avoid exacerbation of AIDS-related cachexia.

The management of HIV-infected patients with hyperthyroidism is similar to that for non-HIV- infected individuals, with the notable exception that a tender thyroid gland in a patient with advanced HIV disease requires FNA to exclude an opportunistic infection (e.g. pneumocystis).

Teatment may involve the use of antithyroid drugs such as carbimazole, radio iodine therapy or surgery.

CHAPTER THREE

3.0 SUBJECTS, MATERIALS AND METHODS

3.1 Background of the Study Area

The study site was Aminu Kano Teaching Hospital (AKTH), established by the Federal

Government of Nigeria on 24th of August, 1988. The hospital is located at Tarauni LGA of Kano

State.

Kano is situated in the North-Western geopolitical zone of Nigeria with a population of about

10million people based on the 2006 National census.73 It serves the people of Kano state and also as a referral center for all neighboring states. Majority of Kano people are Hausa-Fulani. The hospital has a bed capacity of 500 and is still expanding. There are several clinical departments in

AKTH Kano and they are supported by well equipped laboratories (Hematology, Chemical pathology, Microbiology & Histopathology) in providing clinical care for patients as well as teaching and research at different levels.

The HIV/AIDS clinic of the hospital previously called PEPFAR clinic (now called Professor SS

Wali centre) is the centre that provides routine evaluation and clinical care of patients with HIV /

AIDS. The clinic has five consultants, ten medical officers and it is supported by residents on weekly posting from the department of medicine, community medicine and laboratory hematology. It also has separate laboratory attached for their routine tests. The clinic runs on a daily basis (Monday – Friday) with an average daily patient load of 100.

The general out-patient department (GOPD) of Aminu Kano Teaching Hospital is under family medicine department. The department has five consultants, 12 senior residents, 18 junior residents and 15 medical officers. It also runs on daily basis, weekends inclusive with an average daily patient load of 150.

3.2 Study Design

The study was a descriptive cross-sectional hospital-based comparative evaluation of patients with

HIV/AIDS and apparently healthy HIV negative controls.

3.3 Study Population

3.3.1 Subjects:

Adult HIV/AIDS patients attending the SS Wali HIV centre of Aminu Kano Teaching Hospital

who satisfied the inclusion criteria.

3.3.2 Controls:

Apparently healthy HIV-negative individuals who were age and gender matched to the subjects

attending the AKTH Kano GOPD for medical checkup.

3.4 Study Period:

The study spanned over six month period (January to June, 2016) to include data collection,

analysis and write-up.

3.5 Inclusion Criteria

3.5.1 Subjects:

 Patients diagnosed with HIV who were receiving care at the SS Wali AKTH HIV clinic;

 Patients aged 18 to 65 years;

 Patients who gave informed consent.

3.5.2 Controls:

 Apparently healthy HIV negative adults aged 18 to 65 years;

 Not on any drug that can cause thyroid dysfunction e.g. amiodarone;

 Not pregnant;

 Those who gave informed consents.

3.6 Exclusion Criteria

3.6.1 Subjects:

 Age less than 18years or more than 65years;

 Very ill patients; e.g. Those with severely compromised cardio-respiratory disease or those in

coma;

 Subjects who decline consent;

 Pregnant women;

 Patients with ailments that adversely alter body immunity e.g. diabetes mellitus, malignancies,

autoimmune disorders, and use of immune-modulating drugs;

 In-patients.

3.6.2 Controls

 Age less than 18years or more than 65years;

 Individuals who decline consent;

 Individuals who tested HIV positive;

 Pregnant women;

 Severely ill patients;

 Individuals with ailments that adversely alter body immunity e.g. diabetes mellitus, malignancies,

autoimmune disorders, and use of immune-modulating drugs;

 Individuals on drugs that affect thyroid functions e.g. amiodarone.

3.7 Sample Size Determination

The minimum sample size for the study was determined using the Cochrane’s formula

n= z2pq/d2 (Cochrane’s formula)74

Where n= minimum sample size

z= standard normal deviation at 95% confidence interval= 1.96

p= estimated prevalence of thyroid dysfunction in HIV from a previous study (7.3% in black

Africans) 47

q= alternative probability= 1- p= 1- 0.073= 0.927

d= level of precision= 5 %( 0.05)

Therefore; n= (1.96)2x0.073x0.927/ (0.05)2=103.9 subjects

Attrition rate of 10% was used.

Therefore;

Attrition rate= 10% of 103.9= 10.39

Therefore, n= 103.9+10.39= 114.29

Sample size was rounded up 115.

One hundred and fifteen (115) HIV positive patients and 115 apparently healthy HIV negative patients were recruited in each group.

3.8 Sampling Technique

Subjects who met the inclusion criteria were verified and recruited using systematic random sampling until the estimated sample size was achieved. The sampling frame was obtained from the average number of individuals who attended each clinic monthly, which was 2000 for HIV clinic and 4200 for GOPD totaling 6200. The Sampling fraction and interval was then calculated as: Sampling fraction = calculated Sample Size/ Sampling Frame i.e 230/6200 =0.037. The

Sampling Interval = Reciprocal of Sampling Fraction = 27. Every 27th eligible patient who presented at the HIV clinic and GOPD during the study period was recruited after randomly selecting the first patient by balloting.

3.9 Ethical Considerations

1) Ethical clearance was obtained from the ethical committee of AKTH , Kano before the

commencement of the study (Appendix III).

2) The provision of HELSINKI declaration was respected (appendix I).

3.10 Materials, Reagents and Equipment

Materials:

 A pretested interviewer administered structured questionnaire (appendix IIa and b)

 Consent form (appendix I)

 Cotton wool and methylated spirit

 Lithium heparin bottles

 Ethylenediaminetetraaceticacid (EDTA )bottles

 Fluoride oxalate bottles

 Plain bottles

 10ml syringes and needles

 Latex gloves

 Pipettes (BioExpress, Biotech Inc. (ISC), U.S.A.)

 Assay cups

Reagents

 Glucose oxidase

 Cholesteryl ester hydrolase

 Phosphotungstate

 Glycerokinase

 M- Streptavidin-coated microparticles (0.72mg/mL)

 R1-Anti-TSH~Biotin, Anti-T3-Ab~Ru (bpy) and Anti-T4-Ab~Ru(bpy)

 R2- Anti-TSH~Ru(bpy), Anti-T3-Ab~ Biotin and Anti-T4-Ab~ Biotin

Equipment

 Weighing scale (adult + ZT – 120 Health scale)

 Height measuring scale/Stadiometre (Surgifriend medicals, England)

 Non-stretchable tape measure (ButterflyR Tailors Tape)

 Mercury in Glass Sphygmomanometer (AccusonR)

 Stethoscope (Littmann Classic ii S.E)

 Refrigerator (-4oC) (Westinghouse, Germany)

 Centrifuge (Gallenkamp, England)

 Elecsys 2010 immunochemistry analyzer

 Freezer (-20oC) (Westinghouse, Germany)

3.11 Study Procedure

3.11.1 Clinical Procedure

A signed informed consent form (Appendix I) was obtained from each study participant before

commencement of the study. An interviewer-administered questionnaire (Appendix IIa and b) in

both English and Hausa translations was deployed to capture relevant sociodemographic and

historical details of each study participant. Details of physical examination especially

anthropometry, general physical examination, cardiovascular examination and thyroid

examination were entered into relevant sections of the questionnaire.

a) Anthropometric Measurements:

Weight: It was measured with subjects’ shoes removed and wearing light clothing using standard weighing scale placed on an even horizontal surface.75

Height: This was measured in meters using stadiometer; the patient did not wear head gears, caps and shoes.75

Body Mass Index (BMI): was calculated as the weight of the patient in kg divided by the square of his height in meters.75 It was recorded in kg/m2 to one decimal place.

Waist Circumference: This was measured to the nearest cm using non stretchable measuring tape at a point half way between the lowest rib and the top of the iliac crest with the tape parallel to the floor at the end of normal expiration while standing.75

NB: A female Nurse was properly trained to assist in anthropometric measurements of the female subjects.

b) General Physical Examination:

This include, obvious wasting, anxiety, or depressed mood, facial puffiness, or obvious eye bulging, warm sweaty palms or cold and dry palms, palpable lymph node enlargements, fine tremors of the outstretched hands, thyroid acropachy, pretibial myxoedema and pedal oedema.

c) Thyroid Examination:

With the patient seated comfortably in a chair, the region of the thyroid gland at the anterior neck was inspected from in front and side wards for obvious thyroid enlargement and confirmation was done by given some instruction to the patients.

The remaining examination was completed from behind the patient for size in two dimensions, shape, and consistency, attachment to underlying or overlying tissues, tenderness, regional lymph node enlargement, retrosternal extension and thyroid and carotid bruit were noted and recorded.

The examination was completed by examination of the eyes for lid lag, lid retraction and

Ophthalmoplegia. d) Cardiovascular Examination:

Pulse: The radial pulse was examined with particular emphasis on the pulse rate, character, volume, rhythm and arterial wall thickness using standard methods.

Blood Pressure: The blood pressure (BP) was measured with patient seated calmly in a chair with the mercury in glass sphygmomanometer (Accuson) at the level of the heart and with an appropriate cuff size. Both systolic and diastolic blood pressures were determined using phase I and iv Korotokov sound. The average of two measurements taken at the two arms was recorded as the patient blood pressure. The measurement was repeated for every patient discovered to have value within the hypertensive range (diastolic blood pressure of ≥90mmHg and/or systolic blood pressure of ≥140mmHg according to the JNC 7 protocol)76 before being classified as having systemic hypertension. The other aspects of the cardiovascular examination (cardiac apex and heart sounds) and deep tendon reflexes were adequately examined.

3.11.2 Laboratory Procedure

A total of 13ml of venous blood was drawn under aseptic condition from each patient and 3ml was put into a lithium heparin bottle with aid of a vacutainer needle from an antecubital vein. The samples were transported to the laboratory and the plasma was separated from the cells using a centrifuge at a speed of 1200revolution/sec for 5mins. The samples were analyzed using electrochemiluminescence based immunoassay using Elecsys 2010 automated analyzer (Roche diagnostics) for TSH, and free thyroid hormones and anti-thyroid peroxidase antibody (Anti-TPO

Ab).77 Samples that were not analyze immediately were stored at -20oC. Two milliliter each of the remaining sample was put into appropriate bottles and analyzed for viral load, CD4 counts,

(total cholesterol, triglycerides and high density lipoprotein), LDL, full blood count and fasting blood glucose. All these were determined using reverse transcriptase polymerase chain reaction,

78 flow cytometry, 79 auto analyzer biolabo Kenza 240TX, automated analyzer80, Freidwald’s equation, 81 Sysmex automated blood analyzer, 82 and glucose oxidase method of Trinder83 respectively. a) Electrochemiluminescence Assay for Thyroid Function Test and Anti-TPO

Assay Principle:

The three test principles used include, competitive principle, sandwich and bridging;

Competitive is used for anti-TPO and TSH while sandwich is used for FT3 and FT477 (see appendix iva)

b) Plasma Glucose Estimation Assay Principle:

Plasma glucose was estimated, using the glucose oxidase method of Trinder83, using 4-amino phenazone as Oxygen acceptor (appendix ivb) c) Plasma Lipid Assay

Total cholesterol, triglyceride and HDL-C were assayed using enzymatic reagents with an auto analyzer biolabo Kenza 240TX automated analyzer85 while LDL-C was calculated using the

Freidwald’s equation80 (appendix ivc)

3.12 Data Analysis

The data generated were collated, checked, and analyzed using a computer based Statistical

Package for Social Sciences (SPSS) version 20.0 (Chicago, Illinois, USA). Mean±SD was used to describe continuous variables while proportions and percentages for categorical variables.

Independent student t-test was used for comparison of two means. The Chi-squared (χ2) test or

Fisher’s exact test as appropriate was used to compare proportions (categorical data). Risk factors assessment was done using logistic regression (univariate and multivariate analysis). A Pearson’s correlation analysis was used to determine the relationship between two quantitative variables. A confidence interval of 95% was used and a p-value of ≤ 0.05 considered significant.

3.13 Definition of Operational Terms

1) AIDS Patient- This was defined as a person who has an advanced stage of HIV infection for

which he is vulnerable to infections which his body immune system will have difficulty

fighting off e.g. Pneumocystis jiroveci and immunologically a CD4 count of less than

200cells/mm3.84

2) Body Mass Index (BMI) -Was defined as the body weight (kg) of an individual divided by

the square of his height in meters, expressed in kg/m2. Subject with BMI < 18.5kg will be

classified as underweight and those with BMI 18.5- 24.9 as having normal weight, those with

BMI 25- 29.9 as having over weight and those with BMI≥ 30.0 as obese.85

3) HAART – Highly active antiretroviral therapy refers to the use of combinations of various

antiretroviral drugs with different mechanism of action to treat HIV. It usually involves a

combination of at least three drugs from two or more classes of ARVs.84

4) HIV Positive Patient- Defined as a person exposed to HIV and that, two HIV tests- a

preliminary enzyme immunoassay (EIA) test and a confirmatory western blot test have both

come back positive to HIV.84

5) Primary Hyperthyroidism- This was defined as overactive thyroid gland, evidenced by

raised thyroid hormone levels→ FT4 >6.8pmol/l or TSH values <0.3mU/L or both.86

6) Primary Hypothyroidism- This was defined as underactive thyroid gland, evidenced by low

levels of the thyroid hormone → FT4<3.1pmol/l or TSH >4.2mU/L or both.87

7) Normal Thyroid Function- This is defined as normal functioning thyroid gland evidenced

by→ FT3 between 3.1- 6.8pmol/l, FT4 between 12-22pmol/l, TSH values between 0.3-

4.2Mu/l.88

8) Subclinical Hypothyroidism- in which T4 is normal—usually is a laboratory diagnosis

defined in a spectrum:

TSH of 4.5 to 10 mU/L is mild subclinical hypothyroidism (80% of cases)

TSH of 10 to 20 mU/L is more severe subclinical hypothyroidism.89

9) Subclinical Hyperthyroidism- This refers to situation when the serum free T4 is normal

but the TSH was suppressed to almost undetectable level (to <0·1 mU/l).90

10) Sick Euthyroid Syndrome - Is a state of adaptation or dysregulation of thyrotropic feedback

control where the levels of T3 and/or T4 are at unusual levels, but the thyroid gland does not

appear to be dysfunctional.

This condition is often seen in starvation, critical illness or patients in intensive care unit.

The most common hormone pattern in sick euthyroid syndrome is a low total and unbound

T3 levels with normal T4 and TSH levels but all may be depressed.91

11) Hashmoto’s thyroiditis: It a form of autoimmune thyroid disease in which patient with

primary hypothyroidism (described above) has positive thyroid autoantibodies usually anti-

TPO antibody.87

12) Grave’s Disease: It is a form of autoimmune thyroid disease in which patient with primary

hyperthyroidism (described above) has positive thyroid autoantibodies.86

13) Hypertension: This was defined based on WHO/JNC – 7 criteria76 as positive history of

hypertension, use of antihypertensive drugs or systolic blood pressure equal to or greater than

140mmHg and or diastolic blood pressure greater than or equal to 90mmHg measured using

standard procedure.

3.14 Outcome Measures

1. At the end of the study we found the prevalence of thyroid dysfunction among HIV/AIDS

patients in Kano;

2. We had different patterns of thyroid function abnormalities among this group of patients;

3. We determined different risk factors associated with the development of thyroid dysfunction

among HIV/AIDS patients;

4. At the end of the study we were able to adequately compare HIV/AIDS positive and HIV

negative subjects in terms of prevalence, pattern and risk factors associated with the

development of thyroid dysfunction.

CHAPTER FOUR

4.0 RESULTS

4.1 Introduction to Results

In this study spanning over six months (January to June, 2016), a total of 115 subjects and 115 controls were recruited. Of these, the number of subjects and controls who completed the study with a complete data set was 110 each for the subjects and controls. Ten subjects (five from each group) dropped out of the study accounting for 4.3% attrition and an overall response rate of

95.7%.

4.2 Sociodemographic Characteristics of the Subjects and Controls

4.2.1 Distribution of the Subjects and Controls by Age and Gender:

Table 5 below compared the distribution of the subjects and controls by age and gender.

Table 5: Comparison of Subjects and Controls by Age and Gender AGE GENDER (Years) Subjects Controls Male n Female n Total n Male n Female n Total n (%) (%) (%) (%) (%) (%) <20 2 (1.8) 5 (4.5) 7(6.4) 3 (2.7) 6 (5.5) 9 (8.2) 20-29 3 (2.7) 17 (15.5) 20 (18.2) 6 (5.5) 21 (19.1) 27 (24.5) 30-39 7 (6.4) 29 (26.4) 36 (32.7) 4 (3.6) 25 (22.7) 29 (26.4) 40-49 3 (2.7) 24 (21.8) 27 (24.5) 7 (6.4) 12 (10.9) 19 (17.3) 50-59 3 (2.7) 8 (7.3) 11 (10.0) 3 (2.7) 12 (10.9) 15 (13.6) ≥60 4 (3.6) 5 (4.5) 9 (8.2) 0 (0.0) 11 (10.0) 11 (10.1) Total 22 (20.0) 88 (80.0) 110 23 (20.9) 87 (79.1) 110 (100.0) (100.0)

The two groups were similar with respect to age composition and gender distribution with no statistically significant difference in age (p =0.514) and gender (p =0.867) distribution. The mean age±SD of subjects and controls was 38.2±9.54 and 35.9±13.57 respectively with no statistically significant difference in the mean age of the two groups (p=0.138). The predominant age group was 30-39 years and gender ratio was m: f of 1:4 for both subjects and controls [Table 5].

4.2.2 Educational Status of Subjects and Controls:

Figure 3 below is a clustered bar chart comparing the distribution of subjects and controls by educational status.

40 40

35 33.6 34.5 31.8 30

25 22.7 20 SUBJECTS 15 PERCENTAGE 11.8 10.9 14.5 CONTROLS 10

PRIMARY SECONDARY TERTIARY INFORMAL EDUCATIONAL STATUS

Figure 3: Comparison of Educational Status of Study Subjects and Controls

There was no statistically significant difference in educational status between the subjects and controls (p=0.383). More than one-third of both subjects and controls attained secondary and tertiary education [Figure 3].

4.2.3 Marital Status of the Subjects and Controls

Figure 4 below is a clustered bar chart comparing the distribution of subjects and controls by marital status

68.2 70 62.7 60

SUBJECTS 30 24.5 PERCENTAGE CONTROLS 20 16.4 11.8 10 7.3 5.5 0.9 1.8 0.9 0 MARRIED SINGLE DIVORCE WIDOW SEPARATED MARITAL STATUS

Figure 4: Comparison of Marital Status between Subjects and Controls

There was statistically significant difference in marital status between the subjects and controls

(p= 0.001). More than half of both subjects and controls were married [Figure 4].

4.2.4 Occupational Status of the Subjects and Controls

Figure 5 is a clustered bar chart comparing the distribution of subjects and controls by occupational status

35 31.8 30 26.4 26.4 25 23.6 18.2 20 17.3 16.4 15 11.8 9.1 10 PERCENTAGE 10 SUBJECTS 6.4 5 2.7 CONTROLS 0

OCCUPATIONAL STATUS

Figure 5: Comparison of Occupational Status between Subjects and Controls

There was statistically significant difference in occupational status between the subjects and controls (p=0.017). Majority of the subjects work but majority of the controls were not employed

[Figure 5].

4.3 Clinical Characteristics of the Subjects and Controls

Table 6 below compared clinical characteristics of the subjects and controls

Table 6: Comparison of Clinical Characteristics between HIV (+ve) and HIV (-) Participants Clinical characteristics HIV+ HIV- p-value

Age ±SD (yrs) 38.2±11.75 37.6±13.12 0.086

Discriminating features of thyroid hyper function n (%) 22 (20) 19 (17.3) 0.603

Discriminating features of thyroid hypo function n (%) 4 (3.6) 3 (2.7) >0.999

Personal history of hypertension n (%) 15 (13.6) 31 (28.2%) 0.008*

Family history of hypertension n (%) 37 (33.6) 58 (52.7) 0.008*

Family history of thyroid disease n (%) 4 (3.6) 6 (5.4) 0.517

Family history of diabetes mellitus n (%) 15 (13.6) 28 (25.5) 0.027*

History of cigarette smoking n (%) 16 (14.5) 10(9.1) 0.210

History of alcohol consumption n (%) 9 (8.2) 3 (2.7) 0.075

Goitre n (%) 0 (0.0) 0 (0.0) -

Opportunistic infection n (%) 0 (0.0) 0 (0.0) -

Height ±SD (m) 1.64±0.09 1.65±0.10 0.273

Weight ±SD (kg) 67.1±15.97 67.0±12.42 0.968

BMI ±SD (kg/m2) 24.8±5.16 24.7±4.99 0.886

Waist circumference ±SD (cm) 88.5±11.85 86.7±14.66 0.320

Resting pulse ±SD ( beats/min) 87.1±14.64 78.6±13.60 <0.001*

Systolic blood pressure ±SD (mmHg) 126.2±19.11 129.5±19.34 0.208

Diastolic blood pressure ±SD (mmHg) 82.5±11.84 81.9±13.38 0.709

*statistically significant; BMI, body mass index; Discriminating features of hyperthyroidism, weight loss, anxiety, heat intolerance, excessive sweating, hyperdefeacation, irritability, wasting, warm and sweaty palm, irregular menses; Discriminating features of hypothyroidism, slow activity, weight gain getting tired easily, cold intolerance, depressed mood, body swelling; SD, standard deviation.

The mean duration of HIV±SD of the subjects in years was 6.4±4.03, the mean duration of ARVs

±SD use by the subjects in years was 5.7±4.02, 107 (97.3%) of the subjects use ARVs, 103 (93.6%) use first line ARVs and only 4 (3.6%) use second line ARVs.

Personal history of hypertension, family history of hypertension, family history of diabetes mellitus (all higher in the controls) and mean resting pulse (higher in the subjects) were the only clinical features that demonstrated statistically significant difference [Table 6].

4.4 Laboratory Characteristics of Subjects and Controls

Table 7 below compared the laboratory characteristics of the subjects and controls

Table 7: Comparison of Laboratory Characteristics between Subjects and Controls Laboratory Subjects (Mean±SD) Controls p-value characteristics (Mean±SD)

TFT

FT3 (pmol/l) 1.2±1.27 1.6±1.37 0.946

FT4 (pmol/l) 3.0±3.49 2.9±2.94 0.202

TSH (miu/l) 2.6±1.88 3.5±2.81 0.006*

anti-TPO Abs (iu/ml) 1.2±16.1 1.6±17.1 0.183

CD4 503.1±290.56 - - Count(cells/ml)

viral load (cells/ml) 27381.7±7461.13 - -

FPG (mmol/l) 4.7±0.63 4.8±0.69 0.449

FPL

TC (mmol/l) 4.9±1.03 4.9±1.19 0.935

HDL (mmol/l) 1.2±0.41 1.6±0.43 <0.001*

LDL (mmol/l) 3±1.01 2.9±0.98 0.451

TG (mmol/l) 1.2±0.54 1.6±0.61 <0.001*

CBC

Haematocrits (%) 34.6±4.08 38.3±4.49 <0.001*

Total WBC (cells/nl) 5.1±3.82 6.0±1.72 0.024*

Neutrophils (%) 46.9±10.61 56.5±7.89 <0.001*

Lymphocytes (%) 42.4±9.39 40.3±6.83 1.922

MCV (fl) 77.9±8.54 76.8±6.98 0.334

CBC, complete blood count; FPG, fasting plasma glucose; FPL, fasting plasma lipids; FT3, free triiodothyronine; FT4, free thyroxine;, HDL, high density lipoprotein; LDL, low density lipoprotein; MCV, mean corpuscular volume; T.ch, total cholesterol; TFT, thyroid function test; TG, triglyceride; TSH, thyroid stimulating hormone; WBC, white blood cells; *statistically significant. There was statistically significant difference between subjects and controls in mean TSH, triglyceride, HDL, haematocrits, white blood cell count and neutrophils [Table 7].

4.5: Prevalence and Pattern of Thyroid Dysfunction among Subjects and Controls

Table 8 below compared the prevalence and pattern of thyroid dysfunction between subjects and controls

Table 8: Comparison of Prevalence and Pattern of Thyroid Dysfunction between Subjects and Controls Pattern Subjects n (%) Controls n (%)

Male Female Total Male Female Total p-value

Subclinical hypothyroidism 2 (1.8) 4 (3.7) 6 (5.5) 5 (4.6) 13 (11.8) 18(16.4) 0.009*

Isolated low FT3 1 (0.9) 9 (8.2) 10 (9.1) 2 (1.8) 1 (0.9) 3 (2.7) 0.045*

Isolated low FT3 & FT4 2 (1.8) 5 (4.6) 7 (6.4) 0(0.0) 5 (4.5) 5 (4.5) 0.553

Primary hypothyroidism 5 (4.5) 8 (7.3) 13 (11.8) 5(4.5) 6 (5.5) 11 (10) 0.665

Subclinical hyperthyroidism 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (0.9) 1 (0.9) >0.999

Isolated high FT3 0 (0.0) 1 (0.9) 1 (0.9) 0 (0.0) 0 (0.0) 0 (0.0) >0.999

Total 10 (9.1) 26 (23.6) 36 (32.7) 12 (10.9) 26 (23.6) 38 (34.5) 0.775

FT3, free triiodothyronine; FT4, free thyroxine; *statistically significant.

The overall prevalence of thyroid dysfunction among the subjects was 32.7% (Male 9.1%, female

23.6%) while among the controls was 34.5% (male 10.9%, female 23.6%) p= 0.775. However, there was a statistically significant difference in the prevalence between male (10.9%) and females

(23.6%) among the controls (p=0.046) but not among the subjects (p=0.155).

The most prevalent thyroid dysfunction among the subjects was primary hypothyroidism (11.8%, male 4.5% and female 7.3%) followed by isolated low FT3 (9.1%, male 0.9% and female

8.2%), while among the controls, the most common thyroid dysfunction was subclinical

79 hypothyroidism (16.4%, male 4.5% and female 11.8%) followed by primary hypothyroidism

(10%, male 4.5% and female 5.5%), p= 0.034 [Table 8].

4.6 Frequency of Anti-TPO Antibody among Subjects and Controls

Table 9 below compared the frequency of Anti-TPO antibody between subjects and controls

Table 9: Comparison of Frequency of Anti-TPO Antibody between Subjects and Controls Anti-TPO Ab Positivity

Pattern of thyroid function Subjects n (%) Controls n (%)

Yes No Total Yes No Total p-value

Normal 3 (2.7) 71 (64.6) 74 (67.3) 0 (0.0) 72 72(65.5) 0.247 (65.5)

Subclinical hypothyroidism 2 (1.8) 4 (3.6) 6 (5.5) 1 (0.9) 17 18(16.4) >0.999 (15.5)

Isolated low FT3 1 (0.9) 9 (8.2) 10 (9.1) 0 (0.0) 3 (2.7) 3 (2.7) >0.999

Combined low FT3 & FT4 5(4.5) 2 (1.8) 7 (6.3) 4(3.6) 1 (0.9) 5 (4.5) >0.999

Primary hypothyroidism 5 (4.5) 8 (2.3) 13 (11.8) 10(9.0) 1(0.9) 11 (9.9) 0.181

Total 16 (14.5) 94 (85.5) 110 (100.0) 15 (13.6) 95 110 (100.0) 0.846 (86.4)

FT3, free triiodothyronine; FT4, free thyroxine

The overall prevalence of thyroid autoimmunity (Anti-TPOAb positive) among subjects was

14.6% (male 5.5% and female 9.1%), while among controls was 13.6% (male 4.5% and female

9.1%) p =0.846.

There was no statistically significant difference in the distribution of Anti- TPO Ab among the subjects and controls (p = 0.291).

Anti-TPO Ab was most prevalent with primary hypothyroidism and combined low FT3 and FT4 among the subjects (4.5% each) while among controls was most prevalent with primary hypothyroidism (9%) followed by combined low FT3 and FT4 (3.6%) [Table 9].

4.7 Factors Associated with Thyroid Dysfunction among Subjects and Controls

Tables 10a and b below compared risk factors associated with thyroid dysfunction between subjects and controls.

Table 10a: Comparison of Identified Risk Factors Associated with Thyroid Dysfunction between Subjects and Controls Clinical variables Subjects Controls

Thyroid p-value Thyroid p-value dysfunction n (%) dysfunction n (%)

Age

Young 25(22.7) 28(25.5)

Middle age 8(7.3) 0.837 7(6.4) 0.738 elderly 3(2.7) 3(2.7)

Gender

Male 10(9.1) 0.155 12(10.9) 0.046*

Female 26(23.6) 26(23.6)

Marital Status

Married 26(23.6) 21(19.1)

81 single 5(4.5) 14(12.7)

Divorce 0(0.0) 0.197 0(0.0) 0.071

Widow 5(4.5) 2(1.8) separated 0(0.0) 1(0.9)

Occupation

Civil servant 8(7.3) 8(7.3)

Business 9(8.2) 11(10.0)

Students 1(0.9) 0.893 4(3.6) 0.024*

House wife 8(7.7) 5(4.5)

Self employed 7(6.4) 8(7.3) unemployed 3(2.7) 2(1.8)

Educational Status

Primary 4(3.6) 6(5.5)

Secondary 12(10.9) 13(11.8)

Tertiary 10(9.1) 0.767 14(12.7) 0.603

Informal 10(9.1) 5(4.5)

Table 10b: Comparison of Identified Risk Factors Associated with Thyroid Dysfunction between Subjects and Controls Clinical variables Subjects Controls

Thyroid p-value Thyroid p-value dysfunction n(%) dysfunction n (%)

Family history of hypertension

Yes 8(7.3) 0.077 19(17.3) 0.677 No 26(25.5) 19(17.3)

Family history of diabetes

Yes 1(0.9) 0.020* 11(10.0) 0.541 No 35(31.8) 27(24.5)

Cigarette smoking

Yes

No 5(4.5) 0.892 6(5.5) 0.091

31(28.2) 32(29.1)

Alcohol intake

Yes 1(3.6) 0.471 3(2.7) 0.039*

No 32(29.1) 35(31.8)

BMI (kg/m2)

Underweight 4(3.6) 6(5.5)

Normal 18(16.4) 0.535 14(12.7) 0.859

Over weight 9(8.2) 13(11.8) obeise 5(4.5) 5(4.5)

Systolic hypertension

Yes 10(9.1) 0.696 10(9.1) No 26(23.6) 28(25.5) 0.880

Diastolic hypertension

Yes 18(16.4) 0.102 12(10.9) 0.969 No 18(16.4) 26(23.6)

Anaemia

Yes 19(17.3) 0.795 10(9.1) 0.238

No 17(15.5) 28(25.5)

Personal history of hypertension

Yes 7(6.4) 0.244 8(7.3) 0.227 No 29(26.4) 30(27.3)

BMI, Body mass index; *statistically significant.

The factors associated with thyroid dysfunction among the subjects and controls were assessed using χ 2 test of association. The only factor found to be significant among the subjects was family history of diabetes (p=0.020). Among controls, the factors associated with thyroid dysfunction were gender (p=0.046), alcohol consumption (p=0.039) and occupational status (p=0.024).

No association between thyroid dysfunction and use of ARVs, duration of HIV, duration of ARVs use, types of ARVs, level of CD4 count and viral load among subjects.

4.8 Logistic Regression Model to Determine Risk Factors Associated with Thyroid Dysfunction among Subjects and Controls

Table 11 compared the result of logistic regression model of factors associated with thyroid dysfunction between subjects and controls

Table 11: Univariate Logistic Regression Model Comparing Factors Associated with Thyroid Dysfunction between Subjects and Controls

Variables Subjects Controls

Odds 95% CI p- Odds ratio 95% CI p-value ratio value

Gender

Male 0.785 0.256-2.409 0.672

Female

Occupation

Civil servant 1.479 0.237-9.231 0.675

Business 1.788 0.315-10.144 0.512

Students 1.982 0.242-16.226 0.524

House wife 1.568 0.298-8.251 0.595

Self employed 0.692 0.105-4.554 0.702 unemployed - - -

Alcohol intake

Yes 0.014 - 0.999 No

Family history of 1184 34.134- * - - diabetes 0.002 - 410900

CI, confidence interval; *statistically significant

Family history of diabetes was the only significant risk factor for thyroid dysfunction among the subjects as shown in the table above [Table 11].

4.9 Correlation of Thyroid Function test with Clinical and Laboratory Parameters among Subjects and Controls.

4.9.1 Correlation between BMI and FT4 among Subjects

Figure 7 below is a scatter plot showing the correlation between BMI and FT4 among the Subjects

Figure 7: A Scatter Plot showing Correlation between BMI and FT4 among the Subjects

BMI positively correlates with FT4 (p=0.049, r=0.19) [Figure 7]

4.9.2 Correlation between Diastolic Blood Pressure (DBP) and TSH among Subjects

Figure 8 below is a scatter plot showing the correlation between DBP and TSH among Subjects

Figure 8: A Scatter Plot showing Correlation between TSH and DBP among the Subjects.

DBP positively correlates with TSH (p=0.044, r=0.19) [Figure 8]

However, MCV negatively correlates with Anti-TPOAb (p=0.025, r=-0.21) among the subjects.

No correlation was found between thyroid function test and the rest of the parameters such as CD4 count, viral load, duration of HIV, duration of ARVs used e.t.c among the subjects.

4.9.3 Correlation between Total Cholesterol (TCL) and TSH among the Controls

Figure 9 below is a scatter plot showing the correlation between TCL and TSH among the Controls

Figure 9: A Scatter Plot showing Correlation between TSH and TCL among controls

Total cholesterol positively correlates with TSH (p=<0.001, r=0.38) among controls [Figure 9].

4.9.4 Correlation between Triglyceride (TGS) and TSH among the Controls

Figure 10 below is a scatter plot showing the correlation between TSH and triglyceride among controls

Figure 10: A Scatter Plot showing Correlation between TSH and Triglycerides (TGS) among Controls

Among controls, Triglycerides positively correlates with TSH (p=0.033, r=0.2). [Figure 10]

It however negatively correlates with FT3 (p=0.048, r= -0.19) and FT4 (p=0.001, r= -0.30). LDL positively correlates with TSH (p=<0.001, r=0.41) and Anti-TPO (p=0.001, r=0.31) but it negatively correlates with FT3 (p=<0.001, r= -0.33) and FT4 (p=0.015, r= -0.23).

Age positively correlates with FT3 (p=0.049, r=0.19) but negatively correlates with Anti-TPO

(p=0.034, r= -0.20).

Height positively correlates with Anti-TPO (p=0.011, r=0.24) but negatively correlates with FT3

(p=0.041, r= -0.19) and FT4 (p=0.027, r= -0.21).

BMI positively correlates with FT3 (p=0.039, r=0.2) but negatively correlates with Anti-TPO

(p=0.028, r= -0.21).

Finally, MCV positively correlates with Anti-TPO (p=0.036, r=0.20).

No correlation between thyroid function test and the remaining parameters such as systolic blood pressure, pulse rate, waist circumference e.t.c. among the controls.

4.10 Comparison of Subclinical Thyroid Dysfunction between Subjects and Controls

There were 25 individuals with subclinical thyroid dysfunction (24 subclinical hypothyroidism and 1 subclinical hyperthyroidism). Among these individuals with subclinical hypothyroidism 6 were from the subjects while 18 were from the controls. The mean TSH between subjects and controls with subclinical hypothyroidism was 3.6±2.73 and 2.9±2.39 miu/l respectively. Among the subjects, five (83.3%) had TSH value within 5-10miu/l while the remaining one subject had

TSH value above 10miu/l and only one had discriminating features of thyroid hypofunction. While among the controls 15 (83.3%) had TSH value less than 5miu/l, 2 (11.1%) had TSH value between

5 and 10miu/l, the remaining one (5.6%) had a TSH value above 10miu/l but none of them had discriminating features of thyroid hypofunction.

The only patient with subclinical hyperthyroidism was a woman among the control groups with a

TSH<0.01miu/l and had discriminating features of thyroid hyperfunction and negative for Anti-

TPO antibody.

All participants with subclinical hypothyroidism who had TSH above 10miu/l were referred for commencement of thyroid hormone replacement with levothyroxine.

CHAPTER FIVE

5.0 DISCUSSION

5.1 Introduction

Thyroid dysfunction among individuals with HIV/AIDS may be difficult to recognize clinically due to the similarities in their clinical presentation particularly with hyperthyroidism and could have serious prognostic effects as it may lead to delay in appropriate investigations and treatment.

There is no universal recommendation for routine screening of this group of individuals for thyroid dysfunction worldwide and the subject is poorly studied worldwide especially in Africa including

Nigeria. The outcome of this study will shed more light on the prevalence, pattern of thyroid dysfunction including autoimmune thyroid disease as well as risk factors contributing to the development of thyroid dysfunction among subjects with HIV/AIDS with possibility of routine screening and also facilitates more studies on the subject matter.

5.2 Socio-Demographic Characteristics

In this study, there was no statistically significant difference in the age distribution of the subjects and controls. Majority of who were within the fourth decade of life. There were more females than males among both subjects and controls. This is similar to the findings of Thaimuta et al48 in Kenya and Ketsamathi et al66 in Bangkok who also reported more females than males among their subjects. Louise et al92 in Toronto and Rasoolinejad et al46 in Tehran, however found more men than women among their subjects. The higher proportion of females than males in the index study could be attributed to the health seeking behavior in our setting which varies among gender with more women than men visiting hospitals.

The two groups were comparatively similar in terms of their educational status. More than one- third of both subjects and controls attained secondary and tertiary education. There was significant difference in the marital status between the subjects and controls, with more widows, separated and divorcees among the subjects than controls. This is attributable to the fact that, their husbands might have died of the disease long before this study was conducted.

There was statistically significant difference in the occupation of the two groups, with majority of the controls being unemployed. A possible explanation is that female HIV/AIDS patients must have lost their husbands and have to struggle for their living.

5.3 Clinical Characteristics of the Subjects and Controls

There were clinical features of thyroid dysfunction with hyperthyroidism predominating among both the study groups. There are few studies on clinical features in literature but majority were laboratory based.41-50;66 Ketsamathi et al66 reported no clinical features of thyroid dysfunction among 200 HIV-positive patients he studied in Bangkok.

In this study, while medical and family history of hypertension and diabetes were more common among the controls, social history of cigarette smoking and alcohol consumption were not. This finding is in contrast with that of Palanisamy et al93 who reported higher incidence of hypertension, cigarette smoking and alcohol consumption in patients with HIV/AIDS when compared with controls.

The mean duration of HIV infection and anti-retroviral use found in this study was lower than that reported by Shujing et al45 in China, where he found a higher mean duration of HIV among their subjects. Also Guilherme et al42 in Rio de Janeiro, Brazil, found higher mean duration of HIV among their subjects. Earlier detection and better access to health care facilities and ARVs in these

93 countries may explain the differences observed. However, Ketsamathi66 in Bangkok and Thaimuta et al48 in Kenya found lower mean duration of HIV and ARVs use respectively, among their subjects. In this study 97.3% of the subjects use ARVs. This could be attributed to the recent guideline94 recommending commencement of ARVs at CD4 counts≤500 cell/mm, as opposed to the previous guideline that considered a lower CD4 count of ≤ 350cell/mm. Other studies have reported lower figures43, 48, 50, 66 because they were carried out long before WHO introduced a new guideline94 for commencement of ARVs at a CD4 count of ≤ 500 cells/mm.

In this study, there was no difference in the mean weight, height, BMI and waist circumference between the subjects and controls. This is similar to what was found by Abbiyesuku et al50 in

Ibadan, Nigeria reporting no significant difference in weight, height and BMI between his subjects and controls, but contrast to the findings of Madedu43 in England who found significant difference in weight and BMI between their subjects and controls and similarly Palanisamy et al93 in India found lower BMI among AIDS patient compared with HIV-infected and controls.

No difference was found in the mean systolic and diastolic blood pressures between the subjects and the controls. Abbiyesuku et al50 in a study from Ibadan, Southwestern Nigeria on their part reported significant differences in the mean diastolic blood pressures between their subjects and controls with higher values among the controls. Regional differences in the prevalence of hypertension and the varied types of instrument used for blood pressure measurements may explain the difference. The pulse rate was higher among the subjects than controls possibly because of higher frequency of anaemia and anxiety among the subjects.

5.4 Laboratory Characteristics of Subjects and Controls

In this study, the subjects had lower mean values of PCV, WBC and neutrophils than the controls.

Haematological abnormalities among HIV infected patients is multi-factorial including use of

94 myelosuppressive drugs, blood loss from gastrointestinal opportunistic infection and infiltration, nutritional deficiencies, myelosuppression and destruction of the adaptive immunity by the HIV itself. Majority of our subjects were on Zidovudine based ARVs which is probably the cause of their anaemia and the effect of HIV on adaptive immune cells could be a reasonable explanation for their leucopenia and neutropenia. This is in agreement with the findings of Ofonime et al55 in

Calabar, Nigeria who reported anaemia as the most frequent haematological abnormality followed by neutropenia and leucopenia among their patients with HIV compared with controls. Amegor et al56 also found anaemia and leucopenia as the most frequent haematological abnormalities among

HIV patients compared with controls in Makurdi, Benue State, North-central Nigeria .

Plasma triglycerides and high density lipoprotein levels were found to be lower among subjects compared with controls. Lipid abnormalities in HIV infection are fairly common especially with the advent of HAART. Many ARVs have been implicated; for instance protease inhibitors were associated with high total cholesterol and triglycerides but low HDL. Although, very few of our patients were on protease inhibitor based ARVs, this can be the explanation for lower mean value of HDL among our subjects. This finding concurs with the reports from similar studies across

Nigeria.52-54

Most of our patients had mean CD4 count and viral load of over 500 cells/ml and less than 3000 cells/ml respectively which is higher than what was reported by Ketsamathi et al66 in Bangkok and

Abbiyesuku et al50 in Ibadan Nigeria. The higher mean values of CD4 count found in the index study may be explained by better adherence to ARVs among our subjects.

In the index study, mean values of TSH, FT3 and anti-TPO were found to be lower among the subjects compared with the controls, the difference being more profound for TSH. Collazos et al44

95 also found lower TSH levels among their subjects with HIV compared with controls and also reported that HIV status had no effect on FT3 and FT4. This is in contrast to what was reported by Gagnon et al92 in Toronto, Canada where they found higher TSH levels among subjects with

HIV compared with controls, but in agreement with this study for lower FT3 among HIV compared with controls. Palanisamy et al93 in India found lower FT4 but higher FT3 and TSH among subjects with HIV compared with controls. In Ibadan, Southwestern Nigeria, Abbiyesuku et al50 found higher TSH levels among HIV patients compared with controls, but as in the index study they reported lower FT3 levels among their HIV infected patients compared with controls. The lower

FT3 found among the cases compared with controls in many of these studies including this study, may be explained by higher incidence of non-thyroidal illness (low T3 syndrome) in the setting of

HIV infection.

5.5 Prevalence and Pattern of Thyroid Dysfunction among Subjects and Controls

The prevalence of thyroid dysfunction among the subjects in this study was 32.7%, while among the controls the prevalence was 34.5%. This is similar to the report by Shujing et al45 in

Guangzhou, China where they found a prevalence of 33% among their subjects. In the latter study, similar laboratory technique was used and the mean duration of HIV infection and ARVs use by their subjects was comparable to that of the subjects of this study. The prevalence of thyroid dysfunction among subjects in this study is lower than reported from many studies across the world. 42, 45 ,49, 92 Many of those studies however, had subjects with longer duration of HIV but shorter duration of ARVs use than the subjects of this study. One of these studies by Amadi et al49, who reported prevalence of 100% (all overt hypothyroidism), was carried out in Jos, North-central

Nigeria which is a mountainous iodine-deficient region of the country. These variations in geographical location and exposure of the study population to varying degrees of iodine deficiency

96 may be the reason behind the reported higher prevalence rates from these studies. On the other hand, studies that reported lower prevalence rates of thyroid dysfunction among their subjects have shorter mean duration of HIV and lower levels of CD4 count than the subjects of the index study.65

The most common pattern of thyroid dysfunction among subjects in this study was primary hypothyroidism followed by isolated low FT3, combined low FT3 & FT4, subclinical hypothyroidism and isolated high FT3 in that order. Among the controls, the most common pattern of thyroid dysfunction was subclinical hypothyroidism followed by primary hypothyroidism, combined low FT3 & FT4, isolated low FT3 and subclinical hyperthyroidism in that order. Similar findings were reported by Ketsamathi et al66 in Bangkok. Several studies have also found primary hypothyroidism as the most frequent thyroid abnormality among their study population.41, 45, 49, 95

Gagnon et al92 in Toronto, Canada and Guilherme et al42 in Rio de Janeiro however, both reported subclinical hypothyroidism as the most common pattern of thyroid dysfunction among their subjects. The longer duration of HIV infection among subjects in those studies and the fact that many of the patients were not on ARVs may explain the difference. Association of ARVs use with overt hypothyroidism and low level of CD4 count was demonstrated by some studies.42, 62 The isolated low FT3 and isolated combined low FT3 and FT4 found with high frequency in this study were also reported by Rasoolinejad et at46 in Tehran, Iran and Abbiyesuku et al50 in Ibadan, Nigeria as the most common thyroid dysfunction among their subjects. Both abnormalities could be due to sick euthyroid syndrome in the setting of advanced HIV infection and could also be due to clinical or subclinical opportunistic infection.

5.6 Thyroid Autoimmune Status of the Subjects and Controls (Anti-TPOAb Positivity)

From this study there was no significant difference in the distribution of the subjects and controls by both prevalence and pattern of thyroid autoimmunity (Anti-TPO Positivity). The overall prevalence of thyroid autoimmunity (anti-TPOAb positive) among the subjects was 14.5% while among the control group it was 13.6%. Anti-TPO Ab was most prevalent with primary hypothyroidism and isolated low FT3 and FT4 among the subjects (4.5% each) while among controls was most prevalent with primary hypothyroidism (9%) followed by isolated low FT3 and

FT4 (3.6%).

The prevalence of thyroid autoimmunity found in this study is higher than reported from many other studies across the world.42, 48, 62, 66, 51 Nelson et al95 in England, reported 1.1% and Ketsamathi et at66 in Bangkok, reported 6.5%. Nelson found that thyroid anti-bodies were present in 40% of those with hypothyroidism and 56.7% of patients with hyperthyroidism. Our subjects had longer duration of HIV and ARVs use and higher CD4 count. This may suggest greater immune restoration with longer duration of ARVs use among our patients. This explained the higher prevalence of autoimmunity among the subjects of this study.

5.7 Risk factors for Thyroid Dysfunction among Subjects and Controls

From this study, the only factor associated with thyroid dysfunction found among the subjects was family history of diabetes. Among controls, the factors were male gender which was identified in some studies to affect thyroid function among HIV patients1,63, alcohol consumption and occupational status. Further analysis on logistic regression yielded only family history of diabetes as a risk factor for thyroid dysfunction among the subjects with odds ratio of 1,184. Although studentship, house wives and business activities appeared to be risk factors for thyroid dysfunction among the controls with odd ratios of 1.989, 1.568 and 1.788 respectively, these risk factors could result from stressful activity of studying by students, housekeeping by house wives or a more

98 stressful business environment. Stress had been described to affect thyroid function in general population.69

Rasoolinejad et at46 in Tehran, Iran and Powles et al65 in England also found no significant relationship between age, sex, weight, and CD4 count, stage of the disease, ARV use and the development of thyroid dysfunction among their subjects. However, Ketsamathi et al66 in Bangkok found history of opportunistic infection as an independent risk factor for the development of thyroid dysfunction while Collazes et al44 in Spain found low CD4 count as a risk factor of subclinical hypothyroidism. Sen et al41 in Rio de Janeiro, Brazil found protease inhibitors as risk factors for hyperthyroidism among their subjects while non-nucleoside reverse transcriptase inhibitors as risk factors for hypothyroidism. Beltran63 in multicenter study in London found male gender, low CD4 count and use of stavudine as risk factors for hypothyroidism among his cohort.

Gulherme et al42 in Sao Paulo found stavudine as a risk factor for subclinical hypothyroidism among 117 HIV-infected patients he investigated while Ji S et al65 among 178 HIV patients in

Japan found that, thyroid dysfunction is more common in HIV patients on HAART, mainly manifested as hypothyroidism and HBV/HCV co infection increases the probability of thyroid dysfunction. Many of these studies found stavudine as a risk factor which is no longer in use and different study design may also account for the differences in the risk factors found by these different studies.

5.8 Correlation of Thyroid Function test Parameters with Clinical and other Laboratory

Parameters among Subjects and Controls

In this study, BMI correlated positively with FT4, while diastolic blood pressure positively correlated with TSH. There was a negative correlation between MCV and Anti-TPOAb. No

99 correlation was found between thyroid function test and the rest of the parameters such as CD4 count, viral load, duration of HIV, duration of ARVs used and others among the subjects. Among the controls, Age positively correlated with FT3, but negatively correlated with Anti-TPO. Height positively correlated with Anti-TPO but negatively correlated with FT3. The correlation between

BMI and FT3 was positive, while that with Anti-TPO was negative.

Total cholesterol levels correlated positively with TSH and Anti-TPO antibody. The correlation with FT3 and FT4 was however negative and insignificant. Similarly, both TG and LDL-C positively correlated with TSH but their correlation with FT3 and/or FT4 was negative. Finally,

MCV positively correlated with Anti-TPO antibodies.

No correlation between thyroid function test parameters and the remaining parameters such as systolic blood pressure, pulse rate, waist circumference e.t.c.

Shujing et al45 in China, found negative correlation between FT3 and FT4 and HIV duration but

FT3 positively related to CD4 count, Madedu et al45 in England found TSH negatively correlated with CD4 counts and positively correlated with HAART duration, while Jain et al96 in India found direct correlation between CD4 count and FT4 and an inverse correlation between CD4 count and

TSH. Difference in the study design could account for these varied findings.

5.9 Limitations of the Study

1. Paucity of local studies for adequate comparison.

2. Inability to assay for other thyroid autoantibodies such as Anti-TSH antibody and Anti-TG

antibody due to cost.

100

3. Fine needle aspiration cytology (FNAC) was not done because none of the patients was

found to have goitre.

CHAPTER 6

6.0 CONCLUSION AND RECOMMENDATIONS

6.1 Conclusion

101

The prevalence of thyroid dysfunction was lower among the HIV-positive 32.7% (male 9.1%, female 23.6%) than among the controls 34.5% (male 10.9%, female 23.6%). While the prevalence of thyroid autoimmunity (Anti-TPO antibody positive) was higher among HIV-positive 14.6%

(male 5.5%, female 9.1%) than among controls 13.6% (male 4.5%, female 9.1%) but these differences were not statistically significant. There was however, statistically significant difference in the pattern of thyroid dysfunction (p=0.034) between HIV/AIDS patients and HIV-negative controls, with primary hypothyroidism followed by isolated low FT3 being the predominant patterns among HIV positive while subclinical hypothyroidism and primary hypothyroidism predominated the HIV negative controls. Anti-TPO Ab was most prevalent with primary hypothyroidism and isolated low FT3 and FT4 among the subjects (4.5% each) while among controls was most prevalent with primary hypothyroidism (9%) followed by isolated low FT3 and

FT4 (3.6%).

Family history of diabetes was the only risk factor found to be associated with thyroid dysfunction among HIV- positive but male gender, alcohol consumption and occupational status were the factors associated with thyroid dysfunction among the HIV- negative controls.

Body mass index (BMI) and diastolic blood pressure positively correlated with FT4 and TSH respectively while MCV negatively correlated with anti-TPO antibody among the subjects. Among controls, age, weight, height, and lipid profile parameters variably correlated with thyroid function test parameters and anti-TPO antibody.

6.2 Recommendations

102

1) Screening of thyroid function test may not be recommended among patients with

HIV/AIDS because there was no statistically significant difference in prevalence of thyroid

dysfunction between HIV positive and HIV negative participants.

2) Thyroid autoantibody testing may be required in patients with HIV/AIDS requiring thyroid

function test because of higher prevalence of thyroid autoantibody among participants with

HIV/AIDS.

3) There is pressing need for more studies to uncover more risk factors.

REFERENCES

103

1) Fauci AS, Braunwald E, Kasper DL, Hauser SL, Longo DL, Jameson JL et al, editors.

Disorders of the Thyroid Gland. In: Harrison’s principles of internal medicine. 17th edition.

New York: McGraw Hill; 2008. 14:1:184

2) Graz AL, Peto TE, Goulder P, Conlon PC. HIV/AIDS. David AW, Timothy MC, John DF.

Editors. Oxford Textbook of Medicine. 5th edition. USA: Oxford University Press; 2010.

7.5.23.

3) Parveen K, Micheal C. editors. Human Immunodeficiency Virus (HIV) and AIDS. In:

Kumar and Clark’s Clinical Medicine. 7th ed. London: Saunders Elsevier; 2009. 4: 184

4) Lambert M. Thyroid dysfunction in HIV infection. Baillieres Clin Endocrinol Metab 1994;

8:825.

5) Gillam MP, Kopp P. Genetic regulation of thyroid development. Curr Opin Pediatr. 2001;

13:358. [PMID: 11717563].

6) Anthony PW. The thyroid gland and disorders of thyroid function. In: David AW, Timothy

MC, John DF. Editors. Oxford Textbook of Medicine. 5th edition. USA: Oxford University

Press; 2010,1: 13.4.

7) http://www.lab.anhb.uwa.edu.au/mb140/corepages/endocrines/Images/ThyrHE40.jpg.

8) Glinoer D. Pregnancy and iodine. Thyroid. 2001; 11:471.

9) Dunn JT, Dunn AD. Update on intrathyroidal iodine metabolism. Thyroid. 2001; 11:407.

10) Kohn LD, Suzuki K, Nakazato M, Royaux IE, Green ED. Effects of thyroglobulin and

pendrin on iodide flux through the thyrocyte. Trends Endocrinol Metab. 2001; 12:10.

11) Wrutniak-Cabello C. Thyroid hormone action in mitochondria. J Mol Endocrinol. 2001;

26:67. [PMID: 11174855]

104

12) Haddow JE, Palomaki GE, Allan WC. Maternal thyroid deficiency during pregnancy and

subsequent neuropsychological development of the child. N Engl J Med. 1999; 341:549.

[PMID: 1041459]

13) Yen PM. Physiological and molecular basis of thyroid hormone action. Physiol Rev. 2001;

81:1097. [PMID: 11427693]

14) Klein I, Danzi S. Thyroid disease and the heart. Circulation. 2007; 116:1725. [PMID:

17923583]

15) Quirino T, Bongioviovanni M, Ricci E. Hypothyroidism in HIV Infected Patients Who

have and have not received HAART. Clin Infect Disease, 2004;38:596-597

16) Mollison LC, Mijch A, McBride G, Dwyer B. Hypothyroidism Due to the destruction of

the thyroid gland by Kaposi sarcoma. Rev Infect Dx, 1991;13: 826-827

17) Calza L, Manfredi R, Chiado J. Subclinical Hypothyroidism In HIV-Infected Patients

Receiving HAART. J AIDS, 2002; 31: 361-363

18) Gough SC. The genetics of Graves' disease. Endocrinol Metab Clin North Am. 2000;

29:255. [PMID: 10874528]

19) Schussler GC. The thyroxine-binding proteins. Thyroid. 2000; 10:141. [PMID: 10718550]

20) Joint United Nations Programme on HIV/AIDS. 2008 Report on the Global AIDS

Epidemic.

http://www.unaids.org/en/KnowledgeCentre/HIVData/GlobalReport/2008/2008_Global_

Report.asp

21) Altfeld M, Goulder P. ‘Unleashed’ natural killers hinder HIV. Nat Genet, 2007; 39: 708–

10.[CrossRef] [Web of Science] [Medline]

105

22) US Department of Health and Human Services (DHHS). AIDSinfo. http://www.hivatis.org

[United States of America HIV Treatment Guidelines Library; regularly updated.]

23) Benson CA. Treating opportunistic infections among HIV-infected adults and adolescents.

Recommendations from CDC, the National Institutes of Health, and the HIV Medicine

Association/Infectious Diseases Society of America. MMWR, 2004; 53(RR-15):1.

Updates available at http://www.aidsinfo.nih.gov

24) Brenner BG et al: High rates of forward transmission events after acute/early HIV-1

infection. J Infect Dis, 2007; 195:951. [PMID: 17330784]

25) HIV/AIDS Surveillance Report, 2005; 17 (Revised ed), 2007. Available at

http://www.cdc.gov/hiv/

26) Department of Health and Human Services Panel on Antiretroviral Guidelines for Adults

and Adolescents: Guidelines for the Use of Antiretroviral Agents in HIV-1-Infected Adults

and Adolescents, October 10, 2006. Updates available at http://www.aidsinfo.nih.gov

27) Hammer SM. Treatment for adult HIV infection: 2006 recommendations of the

International AIDS Society-USA panel. JAMA 2006; 296:827. [PMID: 16905788]

28) Freiberg M. The association between hepatitis C infection and prevalent cardiovascular

disease among HIV-infected individuals. AIDS 2007; 21:193. [PMID: 17197810]

29) Guay LA. Intrapartum and neonatal single-dose nevirapine compared with zidovudine for

prevention of mother-to-child transmission of HIV-1 in Kampala, Uganda: HIVNET 012

randomized trial. Lancet, 1999;354:795, [PMID: 10485720]

30) Berkley SF and Koff WC. Scientific and policy challenges to development of an AIDS

vaccine. Lancet, 2007; 370:94. [PMID: 17617277

106

31) Walensky RP et al: The survival benefits of AIDS treatment in the United States. J Infect

Dis, 2006; 194:11. [PMID: 16741877]

32) Hommes MJ, Romijn JA, Godfried MH. Increased resting energy expenditure in human

immunodeficiency virus-infected men. Metabolism 1990; 39:1186.

33) Sellmeyer DE, Grunfeld C. Endocrine and metabolic disturbances in human

immunodeficiency virus infection and the acquired immune deficiency syndrome. Endocr

Rev 1996; 17:518.

34) Wartofsky L, Burman KD. Alterations in thyroid function in patients with systemic illness:

the "euthyroid sick syndrome". Endocr Rev 1982; 3:164.

35) LoPresti JS, Fried JC, Spencer CA, Nicoloff JT. Unique alterations of thyroid hormone

indices in the acquired immunodeficiency syndrome (AIDS). Ann Intern Med 1989;

110:970.

36) Sujit KT, Ritesh KA, Anoj KB. Endocrine alterations in HIV- infected patients. Indian

Journal of Endocrinology and Metabolism. Odisha, India. 2015;19:143-147.

37) Carella C, Mazziotti G, Amato G. Clinical review 169: Interferon-alpha-related thyroid

disease: pathophysiological, epidemiological, and clinical aspects. J Clin Endocrinol

Metab 2004; 89:3656.

38) Beltran S, Lescure FX, El Esper I, Schmit JL, Desailloud R. Subclinical hypothyroidism

in HIV-infected is not an autoimmune disease. Horm Res. 2006; 66:21-6. PMID:16685132

39) Jubault V, Penfornis A, Schillo F. Sequential occurrence of thyroid autoantibodies and

Graves' disease after immune restoration in severely immunocompromised human

immunodeficiency virus-1-infected patients. J Clin Endocrinol Metab 2000; 85:4254.

107

40) Jimenez C, Moran SA, Sereti I. Graves' disease after interleukin-2 therapy in a patient with

human immunodeficiency virus infection. Thyroid 2004; 14:1097.

41) Sen. P, Scourfield A, Smythe J, Stefanovich M, Jones R, Mandilia S et at. ‘’Thyroid

dysfunction in individuals with HIV Infection; The effect of HAART’’ International AIDS

Conference Rio de Janeiro. Brazil, July 24-27 2005.

42) Guilherme AR, Mayra CT, Daniel AS, Rafael FN, Jorge FC, Walter AE et al. Association

between antiretroviral and thyroid disease. Endocrinol. Metab. Sao Paulo. 2015; 59

(2):240-44.

43) Madeddu G, Spanu A, Chessa F. Thyroid function in human immunodeficiency virus

patients treated with highly active antiretroviral therapy (HAART): a longitudinal study.

Clin Endocrinol (Oxf) 2006; 64 (5):375-383.

44) Collazos J, Ibarra S, Mayo J. Thyroid hormones in HIV infected patients in the highly

active antiretroviral therapy era: evidence of an interrelationship between the thyroid axis

and the immune system. AIDS 2003; 16:763-765.

45) Shujing J, Changzhong J, Stefan H, Wolfgang F, Tiansheng X, Xiangyun L et al.

Prevalence and Influencing Factors of Thyroid Dysfunction in HIV- infected Patients.

Biomed Research International.2016. http://dx.doi.org/10.1155/2016/3874257.

46) Rasoolinejad M, Afhami S, Izadi M, Hajabdolbaghi M, Khairandish P. Clinical and

paraclinical manifestations of thyroid dysfunction among patients with HIV/AIDS Tehran,

Iran. 2004. http://dx.doi.org/10.1155/2016/3874257 Google search on 10th august, 2017.

47) Chen F, Day SL, Metcalfe RA, Sethi G, Kapembwa MS, Brook MG et al. Characteristic

Autoimmune disease occurring as a late complication of immune reconstitution in patients

108

with advance human immunodeficiency virus (HIV) disease. Medicine; 2005; 84(2):98-

106.

48) Thaimuta ZL, Sekadde-Kigondi, C, Makawiti DW. Thyroid Function among HIV/AIDS

patients on HAART. East African Medical Journal. 2010;87(12):474-480.

49) Amadi K, Sabo AM, Ogunkoye OO and Oluwole FS. Thyroid Hormone. A Prime Suspect

in Human Immunodeficiency Virus (HIV/AIDS) Patients. In: Nigerian Journal of

Physiological Sciences. 2008; 23 (1-2): 61-66.

50) Abbiyesuku FM, Osuji KC, Kuti MO, Atiba AS. Thyroid Function Tests in Nigerian HIV

Sero-Positive Patients on Highly Active Antiretroviral Therapy (HAART). International

Journal of Medical and Clinical Research, ISSN: 0976-5530 & E-ISSN: 0976-5549,

2014;5(1):277-281.

51) Bakari AG and Sani BF. Endocrine Manifestation of HIV infection. In: INTECH Open

Science/Open minds. 2013; 11:243-259.

52) Muhammad S, Sani MU, Okeahialam BN. Prevalence of dyslipidemia among human

immunodeficiency virus infected Nigerians. Ann Afr Med, 2013; 12:24-8.

53) Adewole O, Eze S, Betiku Y, Anteyi E, Wada I, Ajuwun Z et al. Lipid profile of HIV/AIDS

patients in Nigeria. Afr Health Sci.2010; 10(2):144-9.3.

54) Daniyan C. A, Iroezindu M.O. Lipid profile in HIV/AIDS patients in Nigeria Afr Health

Sci 2010; 10:144-149.

55) Ofonime MO, Lydia NA, James E, Baki IM, Josephine A, Godwin I et al. Haematological

profile of HIV infected patients with opportunistic respiratory mycoses in relation to

immune status? A Hospital Based Cohort from Calabar, Nigeria. Tropical Medicine and

Surgery.2013; 1:122.

109

56) Amegor OF, Bigila DA. Oyesola OA, Oyesola TO and Bseni ST. Haematological changes

in HIV patients placed on anti retroviral therapy in Makurdi, Benue State of Nigeria. Asian

Journal of Epidemiology.2009; 2:97-103.

57) Loida AG, Marcos EF, Lillian H. Thyroid Dysfunction in Adult Female Population: A

population-based study of Latin American Vertebral Osteoporosis Study (LAVOS)- Puerto

Rico Site Hypothyroidism in LAVOS-Puerto Rico site. In: P R Health Sci J. 2013 ; 32

(2):57-62.

58) Alessandro PD, Maria GP, Liana F, Francesco L, Nicolò C, Lenuta B et at. Prevalence of

unknown thyroid disorder in a Sardinian cohort. Eur J Endocrinol. 2014; 171(1):143-149.

59) Madhukar A, Prabin G, Nirakar R, Pratibha A, Dipendra RP. A prevalence of thyroid

dysfunction in Kathmandu University Hospital, Nepal. Biomedical Research 2010; 21 (4):

411-415.

60) Ali MN, Ibrahim A. M. Eshnaf, Mohamed AB. Prevalence of Thyroid Dysfunction and Its

Effect on Serum Lipid Profiles in a Murzok, Libya Population. Thyroid Science 2008;

3(10):CLS1-6. www.thyroidscience.com

61) Amballi AA, Adeleye AO, Oritogun KS and Salu LO. Pattern of thyroid dysfunction in a

semi urban Nigerian population through thyroid function tests in a private laboratory,

Sagamu, Ogun State, Nigeria. International Journal of Biochemistry and Biotechnology. 2012; 1 (4):146-149. http://internationalscholarsjournals.org 62) Uloko AE, Nagoda M, Uloko AT, Reng R, Yusuf SM, Gezawa ID et al. Thyroid Disorders

in Northern Nigeria- A Report of Clinical Patterns and Complications. African Journal of

Endocrinology and Metabolism. 2015; 11(1): 22-28.

110

63) Beltran S, Lescure FX, Desailloud R. Increased prevalence of hypothyroidism among

human immunodeficiency virus-infected patients; a need for screening. Clin Infec Dis

2003; 37:579-83.

64) Ji S, Jin C, Höxtermann S, Fuchs W, Xie T, Lu X, et al. Prevalence and Influencing Factors

of Thyroid Dysfunction in HIV-Infected Patients. Biomed Res Int. 2016; 2016:3874257.

65) Powles T, Zeitlin A. Thyroid Dysfunction and relationship to Antiretroviral Therapy in

HIV-Positive Individuals in the HAART era. JAIDS Journal of Acquired Immune

Deficiency Syndromes. 2009; 50 (1); 113-114.

66) Ketsamathi C, Jongjaroen PW, Chailurkit LO, Undomsubpayaky U, Kiertiburanakul S.

Prevalence of Thyroid Dysfunction in Thai HIV-infected Patients Curr HIV Res 2006;

4(4) 465-7.

67) Braverman MD, Lewis E, Robert D, Utiger MD, Werner, Ingbar's et al. The Thyroid:

A Fundamental and Clinical Text. 9th ed. Philadelphia: Lippincott Williams & Wilkins

(LWW), 2005.

68) Elengold MA. “Statement before the committee on Government Reform, Operations

Center for Biologics Evaluation and Research, Food and Drug Administration,” U.S.

House of Representatives, October 3, 2000.

69) Mizokami T, Wu LA, El-Kaissi S, Wall JR. “Stress and thyroid autoimmunity.” Thyroid.

2004; 14(12):1047-55.

70) Nils K, Inge B, Torben J, Peter L, Lars O, Hans P et al. Comparative study of thyroid

function and types of thyroid dysfunction in two areas in Denmark with slightly different

iodine status. European Journal of Endocrinology. 2000; 143: 485-491.

111

71) Deepthi G, Dhanya CS, Sajeeth CI. Prevalence of Thyroid Disorders in Patients in Two

Tertiary Care Hospitals at Palakkad: A Prospective Study. IOSR Journal of Pharmacy and

Biological Sciences. 2013; 8, (3):33-37. www.iosrjournals.org

72) Safia M, Amal Al, Gada A, Kawther A, Maram A, Nadia A, et al. Pattern of Thyroid

Disease in Hail Region, Saudi Arabia. International Journal of Science and Research (IJSR)

ISSN (Online): 2319-7064 Index Copernicus Value 2013: 6.14.

73) http://www.onlinenigeria.com/map.gif ‘’Commercial Agriculture Development Project

CADP”. 2010

74) Bartlett JE, Kotrlik JW, Higgins C. Organizational research Determining appropriate

sample size for survey research. Information Technology, Learning, and Performance

Journal. 2001; 19 (1): 43–50.

75) National Institute of Health. The Practical guidelines to the identification, evaluation and

treatment of overweight and obesity in adults. The Evidence Rep. Obes. Res. (suppl 2).

17:515 – 2095.

76) Chobanian AV, Bakaris GL, Cushman WC, Green LA, Jones DW, Meterson BJ. Seventh

Report of the Joint national committee on prevention, detection evaluation and treatment

of High blood pressure. JNC – 7 Reports. JAMA. 2003; 289: 2560 – 2572.

77) Foster RJ, Bertoncello P, Keyes TE. Electrogenerated chemiluminescence. In: Annual

Review of Analytical Chemistry 2 (Palo Alto Calif). 2009; 359-85. [PubMed]

78) Puren, Adrian, Gerlach, Jay L, Weigl, Bernhard H et al “ Laboratory Operations, Specimen

Processing, and Handling for Viral Load Testing and Surveillance”. The Journal of

Infectious Diseases. 2010; 201:27-36.

112

79) Carella AV, Moss MN, Provost V, Quinn TC. Clinical Vaccine Immunology.

1995;2(5):623-625.

80) Folch J, Lees M, Slone Stanley GH. J. Biol. Chem. 1956; 226, 497-509.

81) Tremblay AJ, Morrissette H, Gagne JM, Bergeron J, Gagne C, Couture P. “Validation of

the Friedewald formula for the determination of the low-density lipoprotein cholesterol

compared with β-quantification in a large population.” Clinical Biochemistry.

2004;34(9):785-790.

82) Mauro Butarello MD, Mario Plebani MD. Am J Clin Pathol. 2008; 130(1):104-116.

83) Vivek NA, Sharma YV, Somani BL. Method for Estimation of Blood Glucose: A

Comparative Evaluation. Med J Armed Forces India. 1998; 54(2): 131-133.

84) Sepkowitz KA. "AIDS—the first 20 years". N. Engl. J. Med. 2001; 344 (23): 1764-1772.

85) World Health Organization. Obesity: preventing and managing the global

epidemic (WHO, 2000).

86) Geffner DL, Hershman JM. "β-Adrenergic blockade for the treatment of

hyperthyroidism". The American Journal of Medicine. 1992; 93 (1): 61–68.

87) Louise Gagnon. Thyroid function in older men who are HIV-positive or at risk for HIV.

AIDS 2006. Toronto. Canada, 2006;9(7):544-549.

88) Escobar-Morreale HF, Botella-Carretero JI, Escobar del Rey F, Morreale de Escobar

G. "Treatment of hypothyroidism with combinations of levothyroxine plus

liothyronine". The Journal for Clinical Endocrinology and Metabolism (Review).

2005; 90 (8): 4946–54.

89) Dayan CM. "Interpretation of thyroid function tests". Lancet. 2001; 357(9256):619–

624.

113

90) Robert M, McCarron DO. Subclinical Hypothyroidism: Merely monitor or time to treat?

Current Psychiatry. 2009; 8(2):47-48.

91) Jayne AF. The Thyroid – Too Much and Too Little Across the Ages. Clin Endocrinol.

2013; 78(1):1-8.

92) Rosolowska-Huszcz D, Kozlowska L, Rydzewski A. "Influence of low protein diet on

nonthyroidal illness syndrome in chronic renal failure". Endocrine. 2005; 27 (3): 283–8.

93) Palanisamy P, Perisamy M, Uma M, Mathiyalagan D. Thyroid function, cardiac risk

assessment profile and haematological changes during HIV infection and AIDS patients. J

Medicine. 2010; 11:131-136.

94) World Health Organization. GUIDELINES: HIV/AIDS. 26 Nov, 2016.

95) Nelson M, Powles T, Zeitlin A, Sen P, Scourfield A, Bower M et al. Thyroid Dysfunction

and relationship to Antiretroviral Therapy in HIV-Positive Individuals in the HAART era.

JAIDS Journal of Acquired Immune Deficiency Syndromes. 2009; 50 (1); 113-114.

96) Jain G, Devpura G, Gupta Bs. Abnormalities in the thyroid function tests as a surrogate

marker of advancing HIV infection, in infected adults. J Assoc Physicians India. 2009;57:

508-10.

APPENDIX I:

CONSENT FORM FOR THE STUDY PARTICIPANTS

Dear Participant,

114

I am Dr. Abubakar Usman Ibrahim, of the Department of Medicine, Aminu Kano Teaching

Hospital. I am conducting a study in this Hospital (AKTH), titled;

‘’THYROID DYSFUNCTION AND ASSOCIATED RISK FACTORS AMONG HIV/AIDS

PATIENTS IN KANO, NORTH-WESTERN NIGERIA’’

I would be very grateful if you volunteer to enroll in the study as a participant. If you consent, you will be asked some health-related questions, examined and blood sample taken for thyroid function tests.

Note that:

1) Your participation is voluntary.

2) You can withdraw from the study whenever you want to. The result of the tests will be

made known to you.

3) Declining to participate in the study will not be used against you in case you need care in

future.

4) Information obtained will be treated with utmost confidentiality.

5) All tests are free.

Kindly sign in the space below if you are willing to participate;

Signature of participant/thumbprint ______Date ______

Signature of Witness/thumbprint ______Date ______

Signature of the Doctor / Researcher______Date ______

115

Thank you for your cooperation.

APPENDIX IIA:

QUESTIONNAIRE (ENGLISH)

THYROID DYSFUNCTION AMONG PATIENTS WITH HUMAN

IMMUNODEFICIENCY VIRUS IN KANO, NORTH-WESTERN NIGERIA

116

SECTION A

PERSONAL DATA

SERIAL NUMBER……………………. FILE NO…………………………..

1. Age (years) ______

2. Gender______Male [ ] Female [ ]

3. Marital status______Married [ ] Single [ ] Divorce [ ] Widow [ ]

4. Occupation______

5. Educational status______

6. Phone number______

CLINICAL HISTORY

1. Do you notice any neck swelling; yes[ ] no[ ]

2. Do you feel anxious; yes[ ] no[ ]

3. Do you have excessive sweating; yes[ ] no[ ]

4. Do you have increase bowel motion; yes[ ] no[ ]

5. Do you have heat intolerance; yes[ ] no[ ]

6. Do you get angry easily; yes[ ] no[ ]

117

7. Do you have hoarseness of voice; yes [ ] no [ ]

8 Do you get easily tired; yes[ ] no[ ]

9 Are you slow in your activity; yes[ ] no[ ]

10. Do have intolerance to cold environment yes [ ] no [ ]

MEDICAL HISTORY

1. Hypertension yes[ ] no[ ]

2. Diabetes yes[ ] no[ ]

3. History of liver disease yes[ ] no[ ]

4. History of renal disease yes[ ] no[ ]

5. History of psychiatric illness yes[ ] no[ ]

6. History of thyroid disease yes[ ] no[ ]

7. Duration of the thyroid disease…………………………..

8. When was the HIV diagnosed……………………………

9. Opportunistic infections e.g. Tuberculosis etc…..yes[ ] no[ ]

DRUG HISTORY

1. Use of anti thyroid yes[ ] no[ ]

2. Use of ARVs yes[ ] no[ ]

Duration ……………………………..

118

Types of ARVS………………………….

FAMILY HISTORY

1. Thyroid disease yes[ ] no[ ]

2. Hypertension yes[ ] no[ ]

3. Diabetes yes[ ] no[ ]

Others (specify)………………………………

SOCIAL HISTORY

1. Cigarette smoking yes[ ] no[ ]

2. Alcohol intake yes[ ] no[ ]

GENERAL PHYSICAL EXAMINATION

1. Chronic illness yes[ ] no[ ]

2. Wasting yes[ ] no[ ]

3. Peripheral lymphadenopathy yes[ ] no[ ]

4. Digital clubbing yes[ ] no[ ]

5. Body swelling yes[ ] no[ ]

6. Depressed mood yes[ ] no[ ]

7. Anxious/agitated yes[ ] no[ ]

8. Weight…………………………kg

9. Height………………………….m

10. BMI…………………………….kg/m2

119

THYROID EXAMINATIION

1. Obvious anterior neck swelling yes[ ] no[ ]

2. Scars of thyroid surgery yes[ ] no[ ]

3. Moves with swallowing yes[ ] no[ ]

4. Moves with tongue protrusion yes[ ] no[ ]

5. Differential warmth yes[ ] no[ ]

6. Tenderness yes[ ] no[ ]

7. Nodular yes[ ] no[ ]

Single node ……………………. Multiple……………………

8. Diffuse yes[ ] no[ ]

9. Regional lymph node enlargement yes[ ] no[ ]

10. Attached to underlying structure yes[ ] no[ ]

11. Attached to overlying structure yes[ ] no[ ]

12. Retrosternal extension yes[ ] no[ ]

13. Any bruit over the mass yes[ ] no[ ]

14. Carotid bruit yes[ ] no[ ]

15. Any proptosis yes[ ] no[ ]

16. Lid lag yes[ ] no[ ]

17. Lid retraction yes[ ] no[ ]

18. Warm and sweaty hand yes[ ] no[ ]

19. Cold and dry hand yes[ ] no[ ]

20. Pretibial myxoedema yes[ ] no[ ]

120

21. Tremor yes[ ] no[ ]

22. Digital clubbing yes[ ] no[ ]

23. Proximal myopathy yes[ ] no[ ]

24. Reflexes normal[ ] increase[ ] slow relaxing phase[ ]

CARDIOVASCULAR EXAMINATION

1. Pulse rate……………………………….bpm

2. Regular yes[ ] no[ ]

3. Blood pressure……………………….mmHg

4. Apex palpable yes[ ] no[ ]

5. Location of the apex…………………………………..

6. Heart sounds 1st and 2nd only[ ] 1st, 2nd and added sounds [ ]

LABORATORY INVESTIGATION

1. TSH………………………

2. T4………………………….

3. T3…………………………

4. FT4……………………….

5. FT3………………………..

6. Anti TPO………………….

7. HIV TEST…………………

8. CD4 count ………………...

121

9. Lipid profile…………......

10. FBS………………………..

11. Viral load…………………...

12. FNAC (If possible)………………………..

13. FBC…………………………………………………………….

APPENDIX IIB

QUESTIONNAIRE (HAUSA TRANSLATION)

MATSALOLIN DA SUKA SHAFI AIKIN MAKOKO DA ABUBUWAN DA SUKE JAWO

SU A MUTANE MASU DAUKE DA CUTA MAI KARYA GARKUWAR JIKI A KANO

AREWA MASO YAMMACIN KASAR NIGERIA

122

SASHI NA DAYA

CIKAKKEN BAYANIN KA

1} Shekaru……………………

2} Jinsi……………………………………………….namiji { } mace { }

3} Matsayin aure……………………….mai aure { } mara aure { } an rabu { } mai takaba

{ } ba’a tare { }

4} Sana’a………………………………...

5} Matakin ilmi…………………………..

6} Lambar waya…………………………

BAYANI AKAN CUTAR

1} Ka/kin taba samun kumburin wuya…………………………………..E { } A’a { }

2} Kana/kina samun tunzira……………………………………………...E { } A’a { }

3} Yawan gumi..……………………………………………...………….E { } A’a { }

4} Yawan bahaya………………………………………………………...E { } A’a { }

5} Yawan Jin zafi………………………………………………………E { } A’a { }

6} Yawan fishi…………………………………………………………E { } A’a { }

7} Dashewar murya…………………………………………………….E { } A’a { }

8} Saurin gajiya……………………………………………………….. E { } A’a { }

123

9} Kasala……………………………………………………………….E { } A’a { }

BAYANI AKAN WATA RASHIN LAFIYAR DA KA/KI KE DA

1} Hawan jini……………………………………………………………E { } A’a { }

2} Ciwon suga………………………………………………….……….E { } A’a { }

3} Ciwon hanta………………………………………………………... E { } A’a { }

4} Ciwon koda………………………………………………………… E { } A’a { }

5} Tabin hankali……………………………………………………….. E { } A’a { }

6} Cutar makoko………………………………………………………. E { } A’a { }

7} Tsawon lokacin cutar makokon…………………………………………

8} Yaushe ne aka gano cutar mai karya garkuwa………………………….

BAYANI AKAN MAGANIN DA KA/KI KE AMFANI DASU

1} Maganin cutar makoko…………………………………………….. E { } A’a { }

2} Maganin cuta mai karya garkuwa…………………………………. E { } A’a { }

3} Tsawon lokacin da aka dauka ana sha……………………………… E { } A’a { }

4} Irin maganin………………………………………………………… E { } A’a { }

BAYANI AKAN LARURAR DA DANGIN MARA LAFIYA KE DA

1} Ciwon makoko……………………………………………………… E { } A’a { }

124

2} Hawan jini…………………………………………………………... E { } A’a { }

3} Cutar suga…………………………………………………………... E { } A’a { }

4} saura…………………………………………………………………

YADDA MARA LAFIYA KE GUDANAR DA RAYUWAR SA/SHI

1} Shan taba…………………………………………………………… E { } A’a { }

2} Shan giya…………………………………………………………… E { } A’a { }

GENERAL PHYSICAL EXAMINATION

11. Chronic illness yes[ ] no[ ]

12. Wasting yes[ ] no[ ]

13. Peripheral lymphadenopathy yes[ ] no[ ]

14. Digital clubbing yes[ ] no[ ]

15. Body swelling yes[ ] no[ ]

16. Depressed mood yes[ ] no[ ]

17. Anxious/agitated yes[ ] no[ ]

18. Weight…………………………kg

19. Height………………………….m

20. BMI…………………………….kg/m2

THYROID EXAMINATIION

25. Obvious anterior neck swelling yes[ ] no[ ]

125

26. Scars of thyroid surgery yes[ ] no[ ]

27. Moves with swallowing yes[ ] no[ ]

28. Moves with tongue protrusion yes[ ] no[ ]

29. Differential warmth yes[ ] no[ ]

30. Tenderness yes[ ] no[ ]

31. Nodular yes[ ] no[ ]

Single node ……………………. Multiple……………………

32. Diffuse yes[ ] no[ ]

33. Regional lymph node enlargement yes[ ] no[ ]

34. Attached to underlying structure yes[ ] no[ ]

35. Attached to overlying structure yes[ ] no[ ]

36. Retrosternal extension yes[ ] no[ ]

37. Any bruit over the mass yes[ ] no[ ]

38. Carotid bruit yes[ ] no[ ]

39. Any proptosis yes[ ] no[ ]

40. Lid lag yes[ ] no[ ]

41. Lid retraction yes[ ] no[ ]

42. Warm and sweaty hand yes[ ] no[ ]

43. Cold and dry hand yes[ ] no[ ]

44. Pretibial myxoedema yes[ ] no[ ]

45. Tremor yes[ ] no[ ]

46. Digital clubbing yes[ ] no[ ]

47. Proximal myopathy yes[ ] no[ ]

126

48. Reflexes normal[ ] increase[ ] slow relaxing phase[ ]

CARDIOVASCULAR EXAMINATION

7. Pulse rate……………………………….bpm

8. Regular yes[ ] no[ ]

9. Blood pressure……………………….mmHg

10. Apex palpable yes[ ] no[ ]

11. Location of the apex…………………………………..

12. Heart sounds 1st and 2nd only[ ] 1st, 2nd and added sounds [ ]

LABORATORY INVESTIGATION

14. TSH………………………

15. T4………………………….

16. T3…………………………

17. FT4……………………….

18. FT3………………………..

19. Anti TPO………………….

20. HIV TEST…………………

21. CD4 count ………………...

22. Lipid profile…………......

23. FBS………………………..

24. Viral load…………………...

127

25. FNAC (If possible)………………………..

26. FBC………………………………………………………

APPENDIX 111:

ETHICAL APPROVAL

128

APPENDIX IV:

Laboratory Procedure a) Electrochemiluminescence assay;

129

 In the first step, sample and specific anti-T3/T4/TSH/anti-TPO antibody labeled with ruthenium

complex are combined in the assay cup.

 After the first incubation, biotinylated T3/T4 and streptavidin- coated paramagnetic micro particles

are added. The still free binding sites of the labeled antibody become occupied with the formation

of an antigen- hapten complex. The entire complex is bound to the micro particle via interaction

of biotin and streptavidin. For TSH and anti-TPO antibody, there was no need for second

incubation all were added at the same time

 After the second incubation, the reaction mixture containing the immune complexes is transported

into the measuring cell. The immune complexes are magnetically entrapped on the working

electrode, but unbound reagent and sample are washed away by a system buffer.

 The electrochemiluminescent reaction, the conjugate is a ruthenium based derivative and the

chemiluminescent reaction is electrically stimulated to produce light. The amount of light

produced is indirectly proportionate to the amount of antigen in the patient sample for competitive

method but directly proportionate for sandwich.77

Finally, evaluation and calculation of the concentration of the antigen (FT3/FT4/TSH/anti-TPO)

are carried out by means of a calibration curve that was established using standards of known

antigen concentration.

Assay Precision

130

Intra-assay coefficient. Assay precision across the measurement range was TSH [CV% (coefficient of variation which is the standard deviation/ mean expressed as a percentage)] 0.46 mU/L (CV 5

1.6%) 4.7 mU/L (CV 5 1.5%) 28.8 mU/L (CV 5 1.8%); free T4 (FT4), 8.8 pmol/L (CV 5 4.2%)

21.5 pmol/L (CV 5 4.1%) 82.1 pmol/L (CV 5 8.2%. The inter-assay coefficient of variation was

2.9% for TSH, 2.5% for FT4 and 12.3% for FT3. The minimum detectable level of TSH was 0.005;

FT4 was 0.3pmol/L and 0.1pmol/L for FT3.77

b) Plasma Glucose Estimation

glucose oxidase c) Glucose + O2 >gluconic acid + H2O2

peroxidase d) H2O2 > H2O + O (nascent oxygen)

e) O + 4-amino phenazone------> red-violet solution + phenol

f) Glucose oxidase promotes the oxidation of glucose to gluconic acid with the production of

an equivalent amount of hydrogen peroxide. In the presence of peroxidase, O2 from H2O2

is transferred to an acceptor in this case 4-amino phenazone with the production of a colour

complex, the intensity of which is proportional to the concentration of glucose

concentration in the plasma sample.83

c) Plasma Lipid Assay

i) Total Cholesterol: Cholesterol ester is hydrolyzed by cholesteryl ester hydrolase liberating cholesterol. The 3 –OH group of cholesterol is then oxidized to a ketone (cholestenone) in an oxygen reaction catalyzed by cholesterol oxidase. Hydrogen peroxide produced from this reaction reacts with phenol and 4- aminoantipyrine catalyzed by peroxidase to form quinone imine dye. The colour change is proportional to the concentration of cholesterol, which was measured with a spectrophotometer.80

ii) Triglyceride:

131

Triglyceride undergoes hydrolysis to glycerol and fatty acid by lipase. Glycerol is then phosphorylated in an ATP requiring reaction catalyzed by glycerokinase producing glycero- phosphate, which is further oxidized to dihydroxyacetone and hydrogen peroxide. H2O2 reacts with phenol and 4-aminoantipyrine catalyzed by peroxidase to give a red quinone imine dye also measured spectrophotometrically.80

iii) High-density lipoprotein- cholesterol:

This is assessed by determining the concentration of cholesterol associated with HDL. HDL-C will be separated from non-HDL lipoproteins i.e. chylomicrons, LDL-C, VLDL-C by adding a precipitating agent phosphotungstate. These will sediment the lipoprotein contained in plasma and

HDL –C is then measured in the supernatant. 80

iv) Low-density lipoprotein-cholesterol:

LDL-C was measured indirectly using the Freidwald’s formula: 81

LDL-C = (Total cholesterol) – (HDL-C) - (triglyceride) / 2.2.

For triglycerides values above 5mmol/l, the samples for the LDL were analyzed separately.

APPENDIX V:

SPREAD SHEET OF DATA ENTRY

132

133