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LEVELS OF β-CAROTENE, IRON AND ZINC AMONG HIV POSITIVE
PREGNANT WOMEN ON ANTI-RETROVIRAL THERAPY IN MOLO SUBCOUNTY, KENYA
OTIENO DOMNIC ODULA (B. Ed. (Sc.)
I56/CE/11508/08
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF
SCIENCE (IMMUNOLOGY) IN THE SCHOOL OF PURE AND APPLIED
SCIENCES OF KENYATTA UNIVERSITY
JUNE, 2015
ii
DECLARATION
I declare that this thesis is my original work and has not been presented for a degree or
other awards in any other University.
Signature: ………………................ Date……………………………….
Otieno Domnic Odula
Department of Zoological Sciences
Supervisors
We confirm that the work in this thesis was carried out by the candidate under our
supervision as University supervisors.
Prof. Michael Gicheru
Department of Zoological Sciences
Signature: ………………………………. Date: ……………………………
Dr. Ruth Wanjau
Department of chemistry
Signature: ………………………………. Date: ……………………………
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DEDICATION
To my savior Jesus Christ, to my late mother Ojiga Odhiambo a shining example of
perseverance, engine of courage and fell due to HIV and AIDS, to my brother Larry who
survived MTCT of HIV, to all HIV infected mothers and to my first born Naomi.
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ACKNOWLEDGEMENTS
It is rare that milestones in life are reached alone, this was certainly not an exception. I
hesitate to single out a few names for particular mention. However, with the disclaimer
that any list of people to whom I am indebted is not meant to be and indeed can never be
complete, I must thank here the following mentors and friends: My supervisors Prof.
Michael Gicheru and Dr. Ruth Wanjau for their tireless commitment and highly qualified
support, constructive comments, friendliness, inspiration and understanding. To Mr. Joel
Juma for introducing me to this specific field of work and then providing continual
support and encouragement. My host at Molo Sub-county hospital Dr. James B. Motanya
(medical superintendent) for permitting me to carry this work in hospital facility. I greatly
appreciate the friendliness, support and encouragement of health workers at the Molo
Sub-county hospital maternal health unit whom we shared all laughs and tears. To my
friends Rachael Nambafu for the initial motivation to apply for this programme and
continuous encouragement and Fredrick Oduor, for offering prayers of faith throughout
this work. I also thank my wife Leah for her support and love. I would also like to give
thanks to my best friend George Otieno, who guided me on statistical data analysis.
Finally I would like also to acknowledge those mothers who attend prevention of mother
to child transmission (PMTCT) unit at Molo Sub-county hospital and voluntarily
participated in this study. God bless you all!
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TABLE OF CONTENTS
TITLE ................................................................................................................................. i
DECLARATION............................................................................................................... ii
DEDICATION.................................................................................................................. iii
ACKNOWLEDGEMENTS ............................................................................................ iv
TABLE OF CONTENTS ................................................................................................. v
LIST OF TABLES ........................................................................................................... ix
LIST OF FIGURES .......................................................................................................... x
ACRONYMS AND ABBREVIATIONS ........................................................................ xi
ABSTRACT .................................................................................................................... xiii
CHAPTER ONE: INTRODUCTION ............................................................................ 1
1.1 Background information ........................................................................................... 1
1.2 Statement of the problem .......................................................................................... 3
1.3 Justification of the study ........................................................................................... 4
1.4 Research questions .................................................................................................... 5
1.5 Hypothesis ................................................................................................................. 6
1.6 Objectives of the study .............................................................................................. 6
1.6.1 General objective ................................................................................................ 6
1.6.2 Specific objectives .............................................................................................. 6
1.7 Significance and output of the study ......................................................................... 7
CHAPTER TWO: LITERATURE REVIEW ............................................................... 8
2.1 Epidemiology and pathophysiology of the human immunodeficiency virus ............ 8
2.2 HIV-related clinical characteristics and their relationship in pregnancy ................ 10
2.3 Immunology of HIV ................................................................................................ 11
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2.4 Diagnosis of HIV infection ..................................................................................... 13
2.5 CD4 T-lymphocytes counts in HIV pregnant women ............................................. 15
2.6 Nutritional micronutrients in management of HIV and AIDS ................................ 17
2.7 Role of nutritional knowledge and attitudes in management of HIV and AIDS .... 19
2.8 Required daily allowances (RDAs) of Zn, Fe and β-carotene in pregnant women 20
2.8.1 Iron in relation to HIV and AIDS ..................................................................... 21
2.8.2 β-carotene in relation to HIV and AIDS ........................................................... 23
2.8.3 Zinc in relation to HIV and AIDS .................................................................... 25
2.9 Analytical methods for determination of micronutrients ........................................ 26
2.9.1 Photometric colorimetric test for iron with lipid clearing factor (LCF) ........... 26
2.9.2 Atomic absorption spectroscopy ...................................................................... 27
2.9.3 High performance liquid chromatography (HPLC).......................................... 28
2.10 Antiretroviral agents used in management of HIV in pregnant women ............... 29
CHAPTER THREE: MATERIALS AND METHODS .............................................. 33
3.1 Study site ................................................................................................................. 33
3.2 Study subjects .......................................................................................................... 33
3.3 Study design ............................................................................................................ 34
3.4 Sample size.............................................................................................................. 35
3.5 Sampling method..................................................................................................... 36
3.6 Inclusion criteria and exclusion criteria .................................................................. 36
3.7 Ethical considerations ............................................................................................. 37
3.8 Quantitative data collection procedure .................................................................... 37
3.9 Examination of clinical characteristics ................................................................... 38
3.10 Collection and pre-treatment of blood .................................................................. 38
3.11 CD4 cell counts ..................................................................................................... 39
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3.12 Determination of HIV-RNA (viral load) using system 340 bDNA analyzer ........ 40
3.13 Biochemical determination of serum nutrient profiles .......................................... 43
3.13.1 β-carotene ....................................................................................................... 43
3.13.2 Iron.................................................................................................................. 45
3.13.3 Zinc ................................................................................................................. 47
3.14 Data management and statistical analysis ............................................................. 47
CHAPTER FOUR: RESULTS ...................................................................................... 49
4.1 Demographic characteristics of HIV pregnant women study participants .............. 49
4.2 Dietary preferences and nutritional knowledge levels of study participants .......... 51
4.3 Clinical characteristics in HIV+ pregnant clients ................................................... 53
4.4 Absolute CD4 count at various stages of study....................................................... 55
4.5 Comparison of viral loads at various stages of study .............................................. 56
4.6 Levels of serum nutrients ........................................................................................ 57
4.6.1 Zinc levels ............................................................................................................ 57
4.6.2 Iron levels ............................................................................................................. 58
4.6.3 β- Carotene levels ................................................................................................. 60
CHAPTER FIVE: DISCUSSION................................................................................. 62
5.1 Demographic characteristics of study participant (HIV+ pregnant women) .......... 62
5.2 Nutritional knowledge and dietary preferences of the participants......................... 65
5.3 Clinical characteristics in HIV infected pregnant clients ........................................ 67
5.4 The CD4 cells absolute counts and viral loads at various stages of study .............. 71
5.5 Levels of micronutrients (Zinc, iron and β-carotene) in the study participants ...... 73
CHAPTER SIX: CONCLUSIONS AND RECOMENDATIONS .............................. 80
6.1 Conclusions ............................................................................................................. 80
6.2 Recommendations from the study ........................................................................... 81
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6.3 Suggestions for further studies ................................................................................ 82
6.4 Limitations of the study........................................................................................... 82
REFERENCES................................................................................................................ 83
APPENDICES ................................................................................................................. 99
Appendix I: Data management process in the study ..................................................... 99
Appendix II: Nutrient levels (µg/dL), CD4 cell/µL and viral loads copies/µL .......... 100
Appendix III: Paired samples statistics for CD4 (cells/µL) ........................................ 101
Appendix IV: Table of T-Test in relation to Zn findings (µg/dL) .............................. 102
Appendix V: Table of T-test of serum concentration of iron (µg/dL) ........................ 103
Appendix VI: A table of significant tests results for β- carotene (µg/dL) .................. 104
Appendix VII: A table of Wilcoxon signed ranks test for viral load (copies/µL) ...... 105
Appendix VIII: Test statistics of CD4, viral load and levels of nutrients ................... 106
Appendix IX: Informed consent .................................................................................. 107
Appendix X: Structured questionnaire ........................................................................ 110
Appendix XI: Clinical evaluation form (Medical details) .......................................... 114
Appendix XII: Clinical staging system for HIV infection WHO (2006). ................... 115
Appendix XIII: Map of locality of the study (Molo Sub-county) ............................... 116
Appendix XIV: Research permission letter from Molo Sub-county hospital ............. 117
Appendix XV: Ethical approval .................................................................................. 118
Appendix XVI: Research Permit................................................................................. 119
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LIST OF TABLES
Table: 2.1: Average content of β-carotene (µg/100g) in some vegetables ...................... 24
Table 3.1: Content of photometric colorimetric test for iron ........................................... 46
Table 3.2: Pipetting scheme of photometric colorimetric test for iron ............................ 46
Table 4.1: Socio-demographic characteristics of study participants................................ 50
Table 4.2: Food preference among HIV pregnant women attending the hospital ........... 52
Table 4.3. CD4 count (cells/µL) during the study period ................................................ 55
Table 4.4: Zinc levels in serum of HIV pregnant women of the hospital ........................ 58
Table 4.5: Serum concentration of iron ........................................................................... 59
Table 4.6: Levels of β- carotene in serum of of study paricipants .................................. 60
x
LIST OF FIGURES
Figure 2.1: A diagram of pathogenesis of HIV ................................................................. 9
Figure 2.2: Relationship between viral load and CD4 counts ......................................... 11
Figure 2.3: Schematic representation of bDNA assay ..................................................... 14
Figure 2.4: Flow cytometer .............................................................................................. 17
Figure 2.5: Importance of good nutrition in management of HIV................................... 21
Figure 2.6: Flame atomic absorption spectrometer ......................................................... 28
Figure 2.7: High performance liquid chromatography (HPLC) ...................................... 29
Figure 2.8: The life cycle of HIV and ARV drugs action sites ........................................ 32
Figure 3.1: Schematic of study design ............................................................................. 35
Figure 4.1: Responses of HIV pregnant mothers on service delivery at PMTCT clinic . 51
Figure 4.2: Knowledge on importance of Zinc, iron and β-carotene in health stutus ..... 53
Figure 4.3: Clinical status of the subjects at onset and end term of the study ................. 54
Figure 4.4: Comparison of CD4 cells/µL values at different time points ...................... 56
Figure 4.5: Comparison of viral loads at various stages of study .................................... 57
xi
ACRONYMS AND ABBREVIATIONS
AAS
Atomic absorption spectrophotometer
AIDS
Acquired immune deficiency syndrome
ART
Antiretroviral therapy
ARV
Antiretroviral
CDC
Centers for disease control
EDTA
ethylenediaminetetraacetic acid
ELISA
enzyme linked immunosorbent assay
FDA
Food and drug Administration
gm
gram
HPLC
High performance liquid chromatography
HIV
Human immunodeficiency virus
HIV-1
Human immunodeficiency virus type-1
IFA
Immunofluorescent antibody assay
KAIS
Kenya AIDS indicator survey
mg
Milligram
mg/L
Milligrams/liter
mm3
Cubic millimeter
mmol/mL
Millimole per milliliter
MTCT
Mother-to-child transmission
NASCOP
National AIDS and STI control programme
NARTIs
Nucleoside analogue reverse transcriptase inhibitors
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NsRTI
Nucleoside reverse transcriptase inhibitor
NtRTIs
Nucleotide reverse transcriptase inhibitors
NNRTI
Non-nucleoside reverse transcriptase inhibitor
OIs
Opportunistic infections
PCR
Polymerase chain reaction
PCP
Pneumocystis pneumonia
PI
Protease inhibitor
PLWHA
People living with HIV/AIDS
PMTCT
Prevention of mother-to-child transmission
RDA
Recommended daily allowance
RNA
Ribonucleic acid
USA
United States of America
µg/dL
Microgram per deciliter
μmol/L
Micromole per liter
VCT
Voluntary counseling and testing
WHO
World Health Organization
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ABSTRACT
Since Kenya recorded its first HIV case in 1984, AIDS epidemic has emerged to be one
of the central impediments to national health. It has deepened poverty; slowed economic
growth; reduced life expectancy; worsened other infectious diseases, with the harshest
effects experienced by women. The HIV prevalence rate in Molo sub-county stands at
5.9%. However, there has been no documentation about antiretroviral therapy (ART)
performance in relation to nutritional status of people living with HIV and AIDS.
Micronutrient status of a pregnant woman is an important determinant of foetal growth
and survival, and deficiencies of micronutrients can alter pregnancy outcome. The study
was aimed at determining levels of β-carotene, iron and zinc among HIV positive
pregnant women on ART in Molo sub-county in the context of pregnancy and HIV. A
descriptive longitudinal study design was used. The sample size was 222 HIV positive
expectant women. HIV – 1 RNA assay (bDNA) was done to determine viral load. The
CD4 count was assessed by the use of four colour flow cytometer (BD FACS Caliber).
Socio-economic and food preferences were determined using a questionnaire. Serum
levels of Zinc were measured by flame atomic absorption spectrophotometry and Iron by
photometric colorimetric test with lipid clearing factor (LCF). Sera concentrations of βcarotene was quantified by reversed-phased HPLC. The ARVs were given to all the
women at appropriate time during their routine visits at the clinic. The overall mean CD4
counts at onset were 186 cell/µL, by midterm it was 301 cells/µL and increased to
349/µL at end term. The paired sample T-Test of CD4 count at end term and at onset was
significant (p= 0.00). The mean viral load at the onset was 67374 copies/μL. This
decreased to 8427 copies/μL at midterm and decreased further to 6062 copies/μL at end
term with Wilcoxon signed-rank test statistic showing a statistically significant change
(reduction) in viral load at end term as compared to onset (Z = -13.964, P = 0.000).
Spearman's correlations were weak and non-significant between serum nutrient
concentration and CD4 counts or viral. The CD4 counts and viral loads at onset and at
end term were found to be inversely correlated (p<0.001; r = -0.5934), indicating that as
CD4 count increased, viral load decreased. Either CD4 counts or viral loads were
predictive of the benefits of ART. The mean level of serum β-carotene increased from
70.5283 µg/dL at onset to 95.849 µg/dL at end term. Mean Iron levels also increased
from 36.887 µg/dL at onset to 42.167 µg/dL at end term. Mean Zinc levels on the other
hand increased slightly from 54.133 µg/dL at onset to 55.632 µg/dL end term. The
percentage concentrations for below recommended to recommended values increased
from 20.7% to 38.3% in β-carotene and from 41.4% to 53.2% in but in Iron, those with
below recommended levels increased from 56.8% at onset to 59.9% at end term. These
findings stress the importance of the inclusion of functional periodic evaluation of serum
nutrients in infected HIV expectant mothers, but confirms importance of ARVs in
management of HIV in pregnant mothers. In addition, from this study, nutrition has a role
in management of HIV and AIDS and probably supports the view that malnutrition is a
major factor in HIV and AIDS progression. Therefore, expectant mothers living with
HIV and AIDS should be supplemented or fed with foods rich in these micronutrients.
1
CHAPTER ONE: INTRODUCTION
1.1 Background information
The steady stream of catastrophic data on human immunodeficiency virus (HIV) and
acquired immune deficiency syndrome (AIDS) rarely paints a hopeful picture. At the end
of 2011, an estimated average of 34 million people were living with HIV worldwide, up
by 17% from 2001 (UNAIDS, 2011). In Kenya, HIV prevalence among adults aged 15 to
64 years decreased nationally from 7.2% (KAIS, 2007) to 5.6% in 2012 (NASCOP,
2013). Kenya has fourth largest HIV epidemic in the world. In 2012, there were 1.6
million people in Kenya who were living with the virus country wide (prevalence =
6.1%) and about 57,000 people died from AIDS related illnesses (UNAIDS, 2013).
Gender is a factor associated with differences in prevalence of HIV and AIDS infection
with women being disproportionally affected. A higher proportion of women aged 15-64
years (6.9%) than men (4.4%) are infected with HIV (NASCOP, 2013). In fact, Kenya
has seen a very slight decline in HIV incidence in women (ages 15-49) between 2009 and
2011–from 0.58% to 0.52% (UNAIDS, 2012). Kenyan women are prone to violent sexual
contact, which is thought to contribute to the higher prevalence of HIV. In a 2003
nationwide survey, almost half of women reported having experienced sexual violence
and a quarter of women aged between 12 and 24 had lost their virginity by forced sexual
contact (UNGASS, 2010). Prevalence in young women (25-29 years) is higher at 13%
compared to 4% in older women (45-49 years). The HIV prevalence among pregnant
women was estimated at 5.6% in 2009, and in 2010, almost 87,000 pregnant women were
estimated to be living with HIV in Kenya and 67% of pregnant women with HIV
2
received efficacious ARVs for prevention of mother to child transmission (PMTCT) in
2011 (WHO/UNICEF/UNFPA, 2012).
By December 2013, there were 3667 HIV patients enrolled in HIV management
programme in Molo sub-county hospital. Out of these, 2661 (72.6%) were females. Molo
sub-county is in Nakuru County which is classified as medium incidence cluster which
contributes to 45% of new HIV infections, 7th county, with 61598 living with HIV and
AIDS (Siringi, 2013). Furthermore, most of infected people in Nakuru County live in
absolute poverty (GOK, 2005). A combination of AIDS epidemic and extreme
malnutrition have created a record low life expectancy in the county to only 44 years
(GOK, 2005).
Research in HIV has led to increased knowledge on HIV and development of life
prolonging anti-retrovirals (ARVs) used in the management of HIV. Use of ARVs has
improved the quality of life for those infected by HIV and reduced mortality. An
estimation of close to 2.5 million deaths have been averted since 1995 due to use ARVs
and through other research initiatives, there has been discovery of new ways to reduce
risk of infection such as voluntary medical male circumcision which reduce risk by 60%
(UNAIDS, 2011).
Micronutrient deficiencies may contribute to HIV and AIDS progression. Larger clinical
trials demonstrated that daily micronutrient supplementation increased survival in adults
with low CD4 cell counts (Jaimton et al., 2003), prevented adverse birth outcomes when
3
given during pregnancy (Fawzi et al., 1998), and reduced HIV mother-to-child
transmission (MTCT) in nutritionally vulnerable women with more advanced HIV
disease (Fawzi et al., 2002) but the optimal formulation of a daily multiple micronutrient
supplement for HIV positive individuals requires further study (WHO, 2003).
Major advances have been made in the understanding of the biology HIV infection as a
prelude to developing appropriate intervention tools in the cycle of the disease.
Consequently, significant progress in therapy has occurred in the last few decades. The
basic role of host nutrition in the pathogenesis of HIV infection is not yet fully
understood. It is however, well documented that the human immune function is highly
dependent on nutritional status (Ibeh et al., 2012).
1.2 Statement of the problem
Even in transition to more universal access to ARV drugs, data on deaths of HIV infected
pregnant mothers is discouraging. For instance, in 2011 alone, 67% of pregnant women
with HIV received efficacious ARVs for prevention of MTCT but maternal mortality
ratio was still high at 360/100,000 (WHO, 2012). It has been argued that the provision of
ARV prophylaxis for MTCT alone is unlikely to have a marked impact on child survival
in the absence of strengthening initiatives to address the health and wellbeing of women
with HIV infection (Temmerman et al., 1995). Despite the large number of women in
reproductive age who are HIV positive (WHO, 2005), only a few studies have
investigated the nexus between HIV infection during pregnancy, maternal nutritional
4
status and health. Indeed, women still comprise an increasing proportion of HIV-infected
persons, and most of these women are of child-bearing age (UNAIDS, 2008).
The variations of an individual’s response to changes in nutrient metabolism makes
nutritional assessment of the pregnant woman complex (King, 2000). No pregnancyspecific nutrient laboratory reference values are available for comparison, thus
assessment of micronutrient status during pregnancy is difficult (Picciano, 2003). Some
intervention studies have found that certain micronutrients including β-carotene, Fe and
Zinc in serum to be beneficial adjuncts to ARVs but the effects of these interventions on
maternal HIV disease progression have not been fully assessed (Cooper et al., 2002). A
few investigations have been done on relationship between HIV, pregnancy and
nutritional status yet poor micronutrient levels have been associated with risk of
progression to AIDS. There has been a failure to understand the trend of serum
micronutrients levels, CD4 count and viral load during pregnancy and their consequences
in HIV era and this has contributed to improper management practices. Regarding
opportunistic infections (OIs) and pregnancy, no large studies have been conducted on
manifestations of HIV associated OIs among pregnant women and no data demonstrate
that spectrum differs from among non-pregnant women with comparable CD4 counts
(CDC/NIHHMA/IDSA, 2004).
1.3 Justification of the study
In antiretroviral therapy (ART), the intervention uses the woman to deliver prevention to
the infant rather than being for her benefit (WHO, 2001) and yet women comprise an
5
increasing proportion of HIV-infected persons, and most of these women are of childbearing age (UNAIDS, 2008). In developing countries like Kenya, where nutritional
problems are common (Ibeh et al., 2012) the identification and correction of
micronutrients deficiency may provide a useful intervention tool in the cycle of HIV and
AIDS disease. The present study endeavored to evaluate serum status of β-carotene, iron
and zinc in HIV and AIDS on ARV in context of pregnancy management in relation to
their CD4 count and viral load. Molo sub county has a low prevalence of HIV at 5.9%
(GOK, 2008) compared no national prevalence of 6.1% (UNAIDS, 2013). Generally,
there are more data from high-prevalence settings than from mid/low prevalence areas.
Therefore, studies such as this are important.
1.4 Research questions
i.
What are the demographic, nutritional knowledge and dietary preferences of HIV
positive pregnant women on ART attending Molo sub-county hospital during
pregnancy?
ii.
What are the clinical characteristics of HIV positive pregnant women on ART
attending Molo Sub-county hospital during pregnancy?
iii.
What are the viral loads and CD4 counts in HIV positive pregnant women on
ART attending Molo Sub-county hospital during pregnancy?
iv.
What are the serum levels of Zinc, Fe and β-carotene and their relationship to
CD4 count and viral load in HIV positive pregnant women on ART attending
Molo Sub-county hospital during pregnancy?
6
1.5 Hypothesis
There was no significant difference in absolute CD4 count, viral load and serum nutrient
concentrations at onset of pregnancy and at end term in HIV positive pregnant women
enrolled in ART program attending Molo Sub-county hospital.
1.6 Objectives of the study
1.6.1 General objective
To determine serum levels of micronutrients β-carotene, iron and zinc and their
relationship with CD4 count and viral load during pregnancy in HIV expectant women on
ART attending Molo sub-county hospital.
1.6.2 Specific objectives
i.
To determine demographic, nutritional knowledge and dietary preferences of HIV
positive pregnant on ART attending Molo sub-county hospital during pregnancy.
ii.
To determine the clinical characteristics of HIV positive pregnant women on ART
attending Molo Sub-county hospital during pregnancy.
iii.
To determine viral loads and CD4 counts on HIV positive pregnant women who
are on ART attending Molo Sub-county hospital during pregnancy.
iv.
To determine serum levels of β-carotene, Iron and Zinc and their relationship to
CD4 count and viral load in HIV positive pregnant women on ART attending
Molo Sub-county hospital during pregnancy.
7
1.7 Significance and output of the study
This study profiles serum levels of β-carotene, iron, zinc, CD4 count and HIV RNA
pregnant women attending Molo Sub-county hospital in the course of pregnancy. The
immediate result was to help in decision on mode of delivery. This report will be used by
policy makers, VCT, PMTCT, obstetricians and other caregivers in addressing nutritional
issues among HIV pregnant mothers and add to the growing body of knowledge, the
importance of nutritional care and support to HIV positive pregnant women. Findings
obtained in this study also generates useful information on performance of ART in
management of HIV in pregnant mothers.
8
CHAPTER TWO: LITERATURE REVIEW
2.1 Epidemiology and pathophysiology of the human immunodeficiency virus
The HIV is the etiologic agent for AIDS which represents a global health crisis that
threatens to overwhelm even the best health care delivery systems (Mugwe, 2011). The
virus is transmitted mainly in three ways: through unprotected sexual intercourse,
heterosexual or homosexual; through blood or blood products, donated semen or organs;
or from an infected mother to her child. More than 70% of infections are a result of
heterosexual transmission and over 90% of infections in children result from MTCT
(Fowler et al., 1997). Although the HIV epidemic is centered in the developing world,
AIDS has also become a leading cause of death for young women even in the USA. In
developing countries, Southern Africa is the most affected region. In Namibia, South
Africa, Zambia and Zimbabwe, Rwanda, Malawi, Tanzania and Kenya, over 10% of
women attending antenatal clinics in urban areas are HIV-positive, with rates of almost
60% in some sites (UNAIDS, 1997).
Although, pathogenic mechanisms of HIV disease are extremely complex and
multifactorial (Fauci, 1993), nevertheless, depletion of CD4 cells has been recognized as
the hallmark of AIDS. Unlike other infections, it is practically impossible for the body to
naturally eliminate HIV. In the pathogenic mechanism (Figure 2.1), HIV enters the body
and binds to a Langerhans or dendritic cell, which carry the virus to CD4 cells. Infected
CD4 cells then home to the lymphoid tissue, where the infection is established. Virus
replication accelerates, and massive viremia leads to the wide dissemination of virus
9
throughout the body's lymphoid tissue. Ultimately, lymphocyte depletion occurs, along
with destruction of the architecture of the infected lymphoid tissue (Fauci, 2003).
Primary infection
(CD4+)
HIV specific
immune
response
Lymph Node (establishment of infection
in lymphoid tissue)
Massive viremia
(wide dissemination to lymphoid organs )
Partial
immunological
control of viral
replication
Trapping of virus and
establishment of chronic,
persistent infection
(lymph node)
Accelerated virus
replication
Destruction of immune
system
Figure 2.1: A diagram of pathogenesis of HIV (Fauci, 2003)
Rapid CD4+ T cell turn
over
10
2.2 HIV-related clinical characteristics and their relationship in pregnancy
Taking advantage of weakened immune system of its host with advanced HIV infection,
several infections and malignancies called 'opportunistic infections' appear and is the
main reason behind hospitalization and substantial morbidity in HIV infected patients
(Nilanjan, 2011). There are conflicting data in literature on the effect of pregnancy on
HIV disease progression and survival among HIV-infected women. Studies conducted
early in the HIV epidemic reported a possible nexus between pregnancy and accelerated
HIV disease progression (Biggar et al., 1989), particularly in developing countries, but
some have reported that there is no evidence that pregnancy accelerates progression to
HIV related illnesses (Cavert et al., 2014).
The length of time it takes for untreated and asymptomatic HIV infection to become
symptomatic disease depends on several factors, including the general health and
nutritional status of a person before and during the infection period (Stine, 2005). For
individuals with adequate food and health resources, the average time for an HIVinfected adult to develop to full-blown AIDS is approximately ten years (Rofles et al.,
1991). The WHO (2006) clinical staging (Appendix XII), has been proven reliable for
predicting morbidity and mortality in infected adults. Infection with HIV is associated
with a progressive decrease of the CD4 count and an increase in viral load, progresses
slowly and may take years before the infected person shows persistent signs of illness
(Figure 2.2).
11
Figure 2.2: Relationship between viral load and CD4 counts over the average course
of treated HIV infection (Pantaleo et al., (1993)
2.3 Immunology of HIV
Human immunodeficiency virus type1 (HIV-1) and type 2 (HIV-2) are transmitted
‘vertically’ that is from mother to infant, and ‘horizontally’ through sexual intercourse
and through infected blood (Peter and Mathew, 2001). After the virus enters the body
there is a period of rapid viral replication, leading to an abundance of virus in the
peripheral blood and the level of HIV may reach several million virus particles per
milliliter of blood (Piatak et al., 1993). This is accompanied by a marked drop in the
numbers of circulating CD4 T cells. This acute viremia is associated in virtually all
people with the activation of CD8 T cells, which kill HIV- infected cells, and
subsequently with antibody production, or seroconversion. The CD8 T cell response is
thought to be important in controlling virus levels, which peak and then decline, as the
CD4 T cell counts rebound. A good CD8 T cell response has been linked to slower
12
disease progression and a better prognosis, though it does not eliminate the virus
(Pantaleo et al., 1997). During the acute phase, HIV- induced cell lysis and killing of
infected cells by cytotoxic T cells accounts for CD4 + T cell depletion, although
apoptosis may also be a factor. HIV infects cells by a process of membrane fusion that is
mediated by its envelop glycoprotein (gp120-gp41, Env) and triggered by interaction of
gp120 with CD4+ and co-receptor CXCKR-4 and CCKR-5 (Stine, 2005). The major
target for HIV infection is cell expressing CD4+ molecule on their surface. However,
some infections also occur in T-helper lymphocytes, mononuclear phagocytes, and
dendritic cells in lymph nodes and microglia in brain (Fenyö, 1993).
HIV seeks out and destroys CCR5 expressing CD4 + cells during acute infection. A
vigorous immune response eventually controls the infection and initiates the clinically
latent phase. However, CD4 + T cells in mucosal tissues remain depleted throughout the
infection, although enough remain to initially ward off life-threatening infections.
Continuous HIV replication results in a state of generalized immune activation persisting
throughout the chronic phase (Appay and Sauce, 2008). Nevertheless, development and
full function of the immune system requires an array of essential micronutrients and
adequate micronutrients achieved through good nutrition. Generally, immune suppression
responds rapidly to nutrition intervention and maintenance of good nutrition among
People living with HIV and AIDS (PLWHA) improves survival and quality of life
(Piwoz and Preble, 2000).
13
2.4 Diagnosis of HIV infection
The HIV testing shows whether a person is infected. This can be done in three ways: first,
by HIV antibody or antigen testing prior to the signs and symptoms of AIDS, second; by
detecting the presence of HIV nucleic acid; and third, by physical examination after
symptoms occurs (Stine, 2005). The most commonly used test is enzyme linked
immunosorbent
assay
(ELISA).
Other
tests
include:
western
blot,
indirect
immunofluorescent antibody assay (IFA), polymerase chain reaction (PCR), the rapid
HIV testing KIT and HIV gene probes (Stine, 2005).
Moreover, viral load assays are good surrogate markers for virus replication and cell
killing (CDC, 1993). One such assay is branched DNA (bDNA) technology which
quantitatively detect nucleic acids. This signal amplification (Stine, 2005) technology is
built on a series of hybridization reactions that are highly amenable to full automation
and thus lessen the amount of labor required to perform this type of analysis (Gregory,
2006). An overnight incubation is a significant drawback of highly sensitive bDNA
assays. The VERSANT® HIV-1 RNA 3.0 assay (bDNA) currently used in clinical
laboratories was modified to allow shorter target incubation, enabling the viral load assay
to be run in a single day. To dramatically reduce the target incubation from 16–18 h to
2.5 h, composition of only lysis diluent solution was modified (Baumeister et al., 2012).
The Versant® HIV-1RNA 3.0 assay (bDNA) (Figure 2.3), according to Siemens
Healthcare Diagnostics, 2008, is a sandwich nucleic acid hybridization procedure for
quantification of HIV RNA in human plasma.
14
Figure 2.3: Schematic representation of bDNA assay (Siemens Healthcare
Diagnostics, 2008)
The HIV-1 is first concentrated from plasma by centrifugation. After HIV genomic RNA
is released from the virions, the RNA is captured to a microwell by a set of specific,
synthetic oligonucleotide capture probes. A set of target probes hybridize to both the viral
RNA and the pre-amplifier probes. The capture probes, comprised of 17 individual
capture extenders, and the target probes, comprised of 81 individual target extenders,
bind to different regions of the pol gene of the viral RNA. The amplifier probe hybridize
to the pre-amplifier forming a branched DNA (bDNA) complex. Multiple copies of an
alkaline phosphatase (AP) labeled probe are then hybridized to this immobilized
complex. Detection is achieved by incubating the complex with a chemiluminescent
substrate. Light emission is directly related to the amount of HIV-1 RNA present in each
sample, and results are recorded as relative light units (RLUs) by the analyzer. A standard
curve is defined by light emission from standards containing known concentration of
15
HIV-1 8E5/LAV virus. Concentration of HIV-1 RNA in specimens are determined from this
standard curve.
Potential inexpensive markers for measures of HIV disease progression in HIV-positive
pregnant women such as lymphocyte proliferation, delayed hypersensitivity skin testing
and antibody formation have not been validated (Kapiga, 2000). In fact, use of surrogate
markers of HIV disease is further complicated by potential influence of independent
factors like pregnancy, tuberculosis infection and treatment and opportunistic infections
(OIs) (Schreibman and Friedland, 2004). The World Health Organization has published
guidelines for initiation of ART based on clinical signs and CD4 count (WHO, 2006), but
clinical expression of HIV-1 infection is often a late-stage event that can be fatal.
Therefore, earlier identification of persons who are at risk for morbidity and mortality but
without clinical signs or symptoms requires laboratory assessment (Mayanja-Kizza et al.,
2005).
2.5 CD4 T-lymphocytes counts in HIV pregnant women
The type and number of ARV drugs needed, together with the foreseeable ARV treatment
duration partly depend on the mother's CD4 cell count (WHO/UNAIDS, 2004). The risk
of MTCT rises with the increasing immunosuppression of the mother, and whether a
pregnant woman meets the criteria for ARVs largely depends on her immunological
status (Leroy et al., 2004). The CD4 counts have been reported to be lower in pregnant
than in non-pregnant women in HIV-positive population (Burns et al., 1996).
Furthermore, several studies suggest that pregnancy does not necessarily accelerate the
16
normal rate of decline in CD4 count in HIV+ women (Nightingale et al., 1992) or
increase the risk of HIV disease progression (Miotti et al., 1992).
Persistent viral replication is now recognized for its central role in HIV-1 pathogenesis
and natural history (Ho et al., 1989). Viral load determinations in plasma, peripheral
blood mononuclear cells (PBMCs), and lymphoid tissue have been significantly
correlated with clinical stage and CD4 lymphocyte counts, with highest viral levels
occurring in primary (acute) and late stage infection and lower levels in early and
intermediate stages (Ho et al., 1989; Daar et al., 1991). Certain viral measurements such
as p24 antigen (p24 Ag) have been shown to have prognostic value (Allain et al., 1987),
but their utility as surrogate markers for clinical endpoints in therapy trials remains to be
proven (DeGruttola et al., 1994). The most common technique for measuring CD4 counts
is flow cytometry. Flow cytometer (Figure 2.4) use lasers to excite fluorescent antibody
probes specific for various cell surface markers such as CD3, CD4 and CD8, which
distinguish one type of lymphocyte from another (Fahey et al., 1998).
17
Figure 2.4: Flow cytometer (Sari, 2004)
2.6 Nutritional micronutrients in management of HIV and AIDS
Low serum concentrations of micronutrients, intestinal abnormalities, and an
inflammatory state have been associated with HIV progression (Ruben et al., 2010). The
impact of pre-existing malnutrition on HIV susceptibility and disease progression is
difficult to study, and knowledge in this area is still limited (WHO, 2005). Though little
has been published on the effect of malnutrition on maternal health, Tomkins (2001)
review concluded that malnutrition contributes to the high rate of maternal morbidity and
mortality in developing countries. Although literature on micronutrients and the
pathogenesis of HIV is often contradictory (Fenton et al., 2004), the role of
18
micronutrients in immune function and infectious disease is well established (WHO,
2003). Blood or serum micronutrient levels may however not reflect true nutritional
status, and measurement of dietary intake may be useful in determining nutritional status
(Fenton et al., 2004). Although information regarding micronutrient status from
developing countries during HIV infection is limited (Semba et al., 1999),
underprivileged women in these countries have been identified as particularly vulnerable
to such deficiencies (Marston et al., 2004). Some researchers have called for
micronutrient supplements as adjuncts therapy to ARVs, but no review articles to this end
have summarized studies describing micronutrient concentrations and micronutrient
intervention trials in HIV-positive persons receiving ARVs (Singhal et al., 2002).
Poor nutrition may also affect the spread of HIV in a very different way: by altering
sexual behaviour. A study in Botswana and Swaziland found that women lacking enough
food to eat were less likely to use condoms and more likely to engage in risky activities,
such as exchanging sex for money or other resources (Weiser et al., 2007).
Notwithstanding the major achievements made in knowledge of the biology of HIV
infection and significant progress in ART in the past few years, the basic role of nutrition
in pathogenesis of HIV infection still remains a lacuna. Questions such as which
micronutrient deficiencies occur commonly in HIV-infected populations in developing
countries, which factors contribute to the development of these deficiencies, and whether
HIV-infected individuals have the same nutrient requirements as healthy individuals also
have to be ascertained (Semba et al., 1999). While it is almost globally acceptable that a
malnourished individuals are more susceptible to infections, with a relatively worse
19
prognosis, it is however burdensome to demonstrate that specific nutritional deficiencies
contribute to poor clinical outcomes. The identification and correction of micronutrient
deficiencies may thus become more important in developing countries. Despite the fact
that more affordable, and in some instances even free ARV drugs have become available
(Kupka et al., 2002), nutrition evaluation and counselling must still form a fundamental
part of the clinical care of HIV-infected patients (Baum et al., 1995). Furthermore, the
importance of addressing and maintaining optimal health early in the asymptomatic stage
of HIV infection needs to be emphasized (Cimoch, 1997).
2.7 Role of nutritional knowledge and attitudes in management of HIV and AIDS
Human immunodeficiency virus (HIV) and acquired immune deficiency syndrome
(AIDS) have posed various medical, nutritional, social and economic problems, femaleheaded households being the most affected. With the advent of antiretroviral therapy
(ART), the treatment and management of HIV and AIDS has changed and created new
challenges (Dybul et al., 2002). For this reason, nutrition assessment and education based
on the needs of the individual is a critical component of any AIDS treatment program
(FANTA, 2004). A healthy diet, nutritional supplements, avoiding smoking and taking
alcohol and caffeine, adequate sleep and exercise, all play an important role in helping
the immune system to work well (Stine, 2005).
Poor nutritional knowledge and dietary practices common among the most affected
households significantly contribute to rapid progression of HIV (Bukusuba et al., 2010).
20
However, very little data exist concerning these aspects of nutrition among women living
with HIV and AIDS in resource limited settings. Addressing gaps in nutrition among
people living with HIV and AIDS (PLWHA) is essential because nutrition plays a vital
role in care and management of HIV and AIDS as it is intrinsically linked to immune
function (Tang, 2003). Therefore, consumption of proper nutrients which can be
enhanced by knowledge of importance of good nutrition for PLWHA and proper dietary
practices can support already-compromised immune system.
2.8 Required daily allowances (RDAs) of Zn, Fe and β-carotene in pregnant women
The RDA is defined as the average daily dietary intake level that is sufficient to meet the
nutrient requirements of nearly all (97-98%) healthy individuals in an age and genderspecific group (Stang et al., 2005). For pregnant women, the RDA of iron is 27 mg/day,
that of zinc is 11 mg/day and 15 mg/day of β-carotene are required (Health Canada,
2006). The RDA and tolerable good nutrition strengthens the body’s ability to fight
diseases and delays progression of HIV to AIDS (RCQHC, 2005a). Nutritional
assessment helps HIV positive people receive appropriate treatment, care and nutritional
support. Screening for nutritional status and assessment of dietary intake should be
included routinely in HIV treatment and care for adults and children (WHO, 2005).
Figure 2.5 shows role of good nutrition in the management of HIV and AIDS.
21
Good nutrition: Weight gain,
increased muscle size and
maintains body strong
Nutritional needs met
through: balanced diet,
adequate food intake and
good appetite
HIV
Strengthens body’s
ability to fight
infections
Reduced vulnerability to
infections and fighting
infections: such as enteric
infections, flu and TB and HIV
progression
Figure 2.5 Importance of good nutrition in management of HIV (RCQHC, 2005a)
2.8.1 Iron in relation to HIV and AIDS
The standard serum reference range of Fe in female adult is 41-132 µg/dL (Painter, 1994;
Slon, 2006). The presence of anaemia in pregnancy increases morbidity and mortality of
the mother and foetus. According to WHO haemoglobin level below 10 gm/dL at any
time during pregnancy is considered as anaemia (Bhide et al., 2003). The incidence of
anaemia in pregnancy ranges from 40-80% in the tropics, compared to 10-20% in the
developed countries (Dutta, 2004). Although some researchers like Moyle (2002) have
reported that anemia is a prognostic marker of future disease progression or death,
independent of CD4 count and viral load; others like Clark et al. (2001) report no
significant association between indicators of iron status and HIV disease severity in
22
African pregnant women. Iron deficiency is well known in Africa and anemia, although
not completely understood, appears widely among PLWHA (Castaldo et al., 1996).
Whereas haemoglobin and hematocrit measurements are simple to carry out and their low
values are clear indication of iron deficiency, they are not specific and sensitive enough
for mild iron deficiency (Hambidge, 2003). Although serum level poorly reflects the pool
and lacks specificity, they are widely used in clinical practice epidemiologic research
because they are easy to determine and are practical. Other biomarkers of iron status
include plasma soluble serum trasferrin receptor concentration, serum transferrin
saturation, erythrocyte protoporphyrin concentration and red blood cell indices (ESWG,
1985). Bone marrow aspirates staining is also used. For example in a study of HIV
positive U.S. adults who had diagnostic bone marrow aspirates done, iron status was
assessed through iron staining of the bone marrow (Delanghe et al., 1998). Those with
high iron stores had shorter survival from the time of diagnosis than did those with
normal or low iron stores.
Iron is a co-factor of many enzymes involved in immune functions such as catalase and
cytochromes. Catalase is antioxidant that removes reactive oxygen radical which
enhances viral replication by activating nuclear transcription factors such as NF-κB (Staal
et al., 1993; FAO/WHO, 1998). Iron deficiency reduces interleukin-1 (IL-1) production,
decreases oxidation burst of neutrophils and lowers concentration of iron-based
myeloperoxidase (Chester and Arthur, 1988). However, balanced intake of iron is
23
necessary as high intake may lead to increased oxidative stress and viral replication
(Delanghe et al., 1998).
2.8.2 β-carotene in relation to HIV and AIDS
There are several dozen carotenoids in foods that we consume. Most carotenoids have
antioxidant activity and β-carotene has been best studied, since in most countries it is the
most carotenoid in fruits and vegetables (Paiva and Russel, 1999). Carotenoids have
many physiological functions. Beta carotene, a fat-soluble antioxidant, is a well-known
scavenger of the singlet oxygen radical, enhance the vertebrate immune system and can
decrease free-radical induced lipoperoxidation damage in HIV (Favier et al., 1994).
Deficiencies of serum and plasma β-carotene and other carotenoids (including lutein and
lycopene) have been observed in multiple studies in both HIV-positive and AIDS patients
(Ullrich et al., 1994).
The standard serum concentration of β-carotene in adult female is 30-65 μg/dL (Uppsala
University Hospital, 2008). It is one of the naturally occurring carotenoids with high
vitamin A activity and is regarded as the most important nutritionally (Combs, 1998). In
Africa, vitamin A is the micronutrient that has been studied most intensely in the context
of HIV infection ((Piwoz and Preble, 2000), particularly for its role in child morbidity
and mortality, and the increased risk of MTCT associated with poor vitamin A status.
Vitamin A deficiency is associated with accelerated HIV disease progression, increased
adult mortality, and increased MTCT of HIV (Semba, 1994; Baum, 1995). The βcarotene supplementation has been shown to increase CD4 counts, and natural killer cells
24
in HIV infected patients (Coodley et al., 1993). A South African study on pregnant
women who received either preformed vitamin A or β-carotene versus placebo during the
prenatal period revealed a significant reduction in the rate of premature birth, but no
effect was noted on birth weight; the supplements had no effect on the risk of HIV
infection in infants (Coutsoudis et al., 1999).
People consuming diets rich in carotenoids from natural foods, such as fruits and
vegetables, are healthier and have lower mortality from a number of chronic illnesses
(Diplock1 et al., 1998). Meanwhile, indigenous green leafy vegetables consumed in
Kenya contain sufficient β-carotene and their β-carotene levels are indicated in (Table
2.1).
Table: 2.1 Average content of β-carotene (µg/100g) in some vegetables
Common name
Botanical name
β-carotene (µg/100g)
Cowpea leaves
Vigna unguiculata
7500
Pumpkin leaves
Cucurbita maxima
8500
Amaranthus LL
Amaranthus hybridus
7600
Miro
Crotalaria brevidens
6250
Frying spider
Gynandropsis gynandra
7600
Bean leaf
Phaseolus vulgaris
9500
Night shade
Solanun nigrum
6760
Nyambaka and Nyaga (1991).
25
2.8.3 Zinc in relation to HIV and AIDS
The standard serum concentration of Zinc in a HIV negative mother is 60-110 µg/dL
(Uppsala University Hospital, 2008) and reference ranges of zinc in serum is 70 -120
ug/dL (Painter et al., 1994). There is evidence that Zinc deficiency in PLWHA may
account for an improper maturation of CD4 cells (Ruben et al., 2010). However,
evidence for the link between Zinc status and HIV is inconclusive. Further research is
warranted to assess a potential role for zinc among HIV patients treated with
antiretroviral therapy (ART) (Mocchegiani et al., 1995).
A number of studies have investigated the role of zinc in HIV disease progression. In
recent observational study of asymptomatic HIV-infected men in the U.S (Ruben et al.,
2010), high zinc intake was accused for significant association with faster HIV
progression and an increased mortality amongst men (Tang et al., 1996). The largest
study ever on zinc supplementation on PLWHA was one aimed at assessing the potential
benefits of zinc supplementation on pregnancy outcomes (Ruben et al., 2010). The study
population consisted of pregnant Tanzanian women who were on ARV (Fawzi et al.,
2004). The results showed no differences in pregnancy outcomes, HIV transmission, CD4
count and viral load (Ruben et al., 2010). While Zinc has a fundamental impact in
sustaining cellular immunity (Villamor et al., 2006) ; it is also required for the activity of
reverse transcriptase and the production of infectious virus (Tanchou et al., 1998) and
may inhibit HIV replication through binding to the catalytic site of HIV protease (Zhang
et al., 1991).
26
Given the considerable importance of zinc in immune functions (Friis et al., 2001) and in
the prevention of diarrhoea and respiratory tract infections (Black, 2003), it is likely to
play a role in HIV infection (Friis et al., 2001). The major dietary sources of zinc include
lean red meat, whole grain cereals, pulses and legumes; whereas fish, roots and tubers
provide only modest amounts of zinc (Dunne, 1990). Levels of zinc in grains reported in
Kenya are in the range of 17.89 mg/kg in sorghum to 28.78 mg/kg in bulrush millet
(Muchemi et al., 2007), which are comparable to levels reported in India, that range
between 10.8 mg/kg to 25.7 mg/kg (Hemalanthan et al, 2007).
2.9 Analytical methods for determination of micronutrients
2.9.1 Photometric colorimetric test for iron with lipid clearing factor (LCF)
According to Shaik et al. (2014) lipemic specimens usually generate turbidity of the
sample reagent mixture which leads to false high results. This turbidity can cause
problems in the performance of photometric assays and can be avoided by employing
pre-treatment steps. Such steps, however, are time-consuming and prone to error hence
LCF, to eliminate these pre- treatment steps. LCF is integrated in the respective assay
reagent and begins working immediately in the incubation phase of the test procedure and
clearing is achieved within minutes. It does not require additional pre-treatment steps
hence allowing for easy obtaining of correct results.
The principle
behind this method is that chromogenic agents provide, without
exceptions, the setting free of the seral iron from transferrine, either by deproteination at
acidic pH values, or by using specially provided capillary-active agents. The iron which
27
has been set free from transferrine is then reduced to its ferrous state by strong reducing
agents such as sodium dithionite, ascorbic acid, and others of the sort. When the serum
sample is added, the serum iron is released from transferrin because of the low pH. The
iron from the sample then forms a complex with the remaining excess of Chromazurol B,
increasing the absorbance (Gordon et al, 2002). Iron (III) reacts with chromazurol B
(CAB) and cetyltrimethylammonium bromide (CTMA) to form a coloured ternary
complex with an absorbance maximum at 623 nm. The intensity of the colour produced is
directly proportional to concentration of iron in the sample. Calculation of the iron
concentration with standard. If a different wavelength (620 nm-640 nm) is to be used for
measurement the standard provided with the kit has to be employed for the calculation
(Shaik et al. 2014).
(µg/dL)
(µmol/L)
2.9.2 Atomic absorption spectroscopy
Flame atomic absorption spectroscopy (FAAS) is the most used of the atomic methods.
The AAS is a spectro-analytical procedure for the quantitative determination of chemical
elements. The principle of FAAS (Figure 2.6) is the absorption of light. The hollow
cathode lamp produces a spectrum of a specific wavelength. The atomizer converts the
sample into population of free atoms which absorbs part of the light. The light then
passes through the free atoms population. The detectors then detect the transmitted light
(Syed et al., 2012). It is applied for measurement of calcium and magnesium which are
poorly excited, but it is better for measurement of trace elements such as Zinc, Iron,
28
Copper, Manganese, Chromium, Cobalt, Cadmium and Lead. The drawbacks of FAAS
include: measuring only one element at a time, sample preparation is necessary,
nebulizers are frequently partially clogged with proteins or metal residues, contamination
by dust is easy and dust particles on the burner exhibit coloured glow in the flame
(Jacobson and Lockitch, 1988).
Figure 2.6: Flame atomic absorption spectrometer (Syed et al., 2012)
2.9.3 High performance liquid chromatography (HPLC)
High performance liquid chromatography formerly referred to as high pressure liquid
chromatography, is a type of liquid chromatography which is used to separate, identify,
and quantify components in a mixture relying on pumps to pass a pressurized liquid
solvent containing the sample mixture through a column filled with a solid adsorbent
(Gerber et al., 2004). The classical method for the determination of carotenoids in blood
is by the spectrophotometric method while the HPLC method is also recommended for
use (Tee, 1995). The simpler spectrophotometric assay of carotenoids is faster and
cheaper, yet it is assumed to be less accurate compared to HPLC methods. The HPLC
29
method also gives quantitative information about individual carotenoids, which cannot be
done by spectrophotometer (Michael, 2012). Reversed-phase “high-performance” liquid
chromatography (HPLC) (Figure 2.7), is a chromatographic method that uses a
hydrophobic stationary phase (McNaught et al., 1997). Reversed phase refers to liquid
rather than gas chromatography.
Figure 2.7: High performance liquid chromatography (HPLC) (Schewdt, 1997)
2.10 Antiretroviral agents used in management of HIV in pregnant women
There are about 25 single or combination of preparations in use globally which are
broadly categorized into four classes of drugs (Hoffmann, 2007). They are: nucleoside
and nucleotide analogs, non-nucleoside reverse transcriptase inhibitors (NNRTIs) and
protease inhibitors (PI) and fusion inhibitors. Although there are over 4 million people on
ART (WHO, 2008), it is not known with certainty how early to start ARVs. In subSaharan Africa, despite progress made with improving earlier access, mortality remains
markedly higher when compared with other contexts (Brinkhof et al., 2008).
30
There is currently no cure for HIV infection and treatment still consists of ARVs (DHHS,
2005). This has been highly beneficial to many HIV-infected individuals since its
introduction in 1996, when the protease inhibitor-based ARVs initially became available
(Palella et al., 1998). Current ARVs options are combinations consisting of at least three
drugs belonging to at least two types, or classes, of ARVs agents. Typically, these classes
are two nucleoside analogue reverse transcriptase inhibitors (NARTIs or NRTIs) plus
either a protease inhibitor or a non-nucleoside reverse transcriptase inhibitor (NNRTI).
There is no empirical evidence for withholding treatment at any stage of HIV infection
(Rachlis et al., 1998) and death rates are almost twice as high when therapy is deferred
(until the CD4 count falls below 500) compared to starting therapy when the CD4 count
is above 500. One study suggests that the average life expectancy of an HIV infected
individual is 32 years from the time of infection if treatment is started when the CD4
count is 350/µL (Kitihata et al., 2009). However, the timing for starting HIV treatment is
still subject to debate (Sax, 2009).
The ART neither cures the patient nor uniformly removes all symptoms; high levels of
HIV-1, often ART-resistant, return if treatment is stopped. Moreover, it would take more
than a lifetime for HIV infection to be cleared using ARVs (Blankson et al., 2002).
Despite this, many HIV-infected individuals have experienced remarkable improvements
in their general health and quality of life, which has led to a large reduction in HIVassociated morbidity and mortality in the developed world (Palella et al., 1998). In the
absence of ARVs, progression from HIV infection to AIDS has been observed to occur at
a median of between 9 to 10 years and the median survival time after developing AIDS is
31
only 9.2 months (Morgan et al., 2009). In Kenya, the standard first line regimen for adult
is a combination of staduvine, lamivudine and nevirapine or efavirenz, which are used
simultaneously with prophylaxes (cotrimoxazole) for OIs (NASCOP, 2005). Current
information may not recommend use of some ARV drugs in pregnancy. For women of
childbearing age, nevirapine is preferred over efavirenz due to the teratogenic effects of
efavirenz (MOH, 2005/2006).
According to WHO/SEARO (2006), different types of ARVs act in different ways to
prevent the replication of HIV in the human body. These different pathways are:
nucleoside reverse transcriptase inhibitors (NsRTIs) and nucleotide reverse transcriptase
inhibitors (NtRTIs). These act by incorporation into the DNA of the virus (competing
with natural nucleotides/nucleosides), thereby stopping the building process of
transcription from RNA to DNA. The resulting DNA is incomplete and cannot create a
new virus. Non-nucleoside reverse transcriptase inhibitors (NNRTIs), on the other hand
act by stopping HIV production by binding directly onto reverse transcriptase (noncompetitively) and preventing the conversion of RNA to DNA. Four NNRTIs are
available for treatment of HIV infection. Examples are efavirenz, delavirdine and
entravine (Finkel et al., 2009).
Others are protease inhibitors (PIs). Examples of PIs are lopinavir, nelfinavir, retonavir,
indinavir and saquinavir (Walker et al., 2003). These act by binding to the viral protease,
thereby preventing the correct cleavage of viral proteins. Thus, they prevent HIV from
32
being successfully assembled and released from the infected cells. Lastly, the fusion
inhibitors (peptides), act by binding to a region of the gp41 transmembrane glycoprotein
of HIV thus preventing virus–cell fusion. Enfuvirtide, a 36-amino acid peptide, binds to
gp41 to prevent the conformational change. This leads to failure of viral entry into the
host cell (Finkel et al., 2009) (Figure 2.8) (the red text show the sites of action of the
various ARV drugs).
Figure 2.8: The life cycle of HIV and ARV drugs action sites (Furtado et al., 1999)
33
CHAPTER THREE: MATERIALS AND METHODS
3.1 Study site
Molo Sub-county hospital is situated in Molo sub-county, Nakuru County, Kenya. The
Sub-county covers an area of 2,447.3 km² and is located between longitude 35º 28’ and
35º 36’ East and latitude 0º 13’ and 1º 10’ South (Appendix XIII). The total population of
the former larger Molo district served by the hospital was estimated to be 523,605 in
2008 and was expected to increase to 546,513 by 2010 and 634,388 by 2012 (GOK,
2008). The hospital is under the ministry of health. It serves Molo, Njoro, Rongai,
Kuresoi north and south constituencies. It offers services such as antenatal care,
antiretroviral therapy (ART), HIV counseling and testing, and prevention of mother to
child transmission (PMTCT) of HIV, among other services. The HIV prevalence rate in
the district stands at 5.9%, which is a decline from 10% in 2003. This has increased the
dependency rate and impacted negatively on the labour force. Molo sub-county is one of
the places that is characterized by ethnic violence and displacement of families mostly
affecting women and children. Perhaps some are raped during violence. The capture of
accurate rape statistics is known to be challenging (Kim et al., 2003), and it is generally
accepted that those cases which are reported constitute an underestimate of the actual
number that occurs.
3.2 Study subjects
The study involved all HIV and AIDS pregnant infected women in their first trimester
and throughout gestation period who came for routine antenatal care follow-ups at Molo
sub-county hospital. Subjects in this study were essentially cases detected in the antenatal
34
clinic, those detected on in-patient basis and those identified during routine communitybased screening exercises normally done by the hospital outreach services then referred
to the hospital.
3.3 Study design
A descriptive longitudinal study design was employed (information collected without
changing environment/nothing manipulated and individual mothers followed over time).
(Figure 3.1). Blood samples were collected and determination of CD4 count and viral
load done at least 2 months periodically (onset, midterm and end term) for serum levels
β-carotene iron and zinc were done at onset and at end term of pregnancy only. Viral load
was measured by performing HIV-1 RNA bDNA assay. The women’s CD4 counts were
assessed by use of flow cytometer (BD FACS Caliber). Overall mean of viral and CD4
load were determined. All women were offered antiretroviral therapy (ART) and specific
therapy for opportunistic infections. Serum concentrations of Zinc was measured by
flame atomic absorption spectrophotometry (FAAS) and Iron using Humastar 180 by a
method referred to us photometric colorimetric test with lipid clearing factor. Serum βcarotene on the other hand was quantified by high performance liquid chromatography
(HPLC). There was determination of the relationship of these factors (viral load, CD4)
with mean serum concentrations of nutrients (β-carotene, Fe and Zn) on these mothers on
administration of antiretroviral drugs (ARVs) during the period of pregnancy. Data were
collected from women with at least 3 visits in the 8 or 9 months period of pregnancy.
Primary data were collected on demographic characteristics and their knowledge on the
role of the micronutrients in health and HIV through filling in questionnaire by study
35
participants. Secondary data were collected through reviewing medical records of the
study participants. During pregnancy, nurse interviews and physician examinations were
conducted whenever the mothers appeared for routine checkup.
Prior to
Enrollment
Visit 1
(Onset)
Total N: Obtain informed consent. Potential participants screened by
inclusion and exclusion criteria; history obtained and documented.
Initial assessments
(Blood specimens collected, examinations of clinical characteristics)
laboratory assays to performed, questionnaires to be completed)
Follow-up assessments
Visit 2
(Midterm)
(Blood specimens collected, laboratory assays performed)
Visit 3
(End term)
Follow-up assessments
(Blood specimens collected, laboratory assays performed and clinical
assessment done)
End Point
Data
Figure 3.1: Schematic of study design
3.4 Sample size
The sample size for this study was determined using equation by Israel (2003) at 95%
confidence interval and at HIV prevalence rate in the sub-county which stood at 5.9% in
2007 (GOK, 2008).
36
Where:
Z =1.96 (Standard deviation)
n = Sample size
p = Estimated prevalence = 5.9% (0.59)
q = 1 – p = 1 – 0.59 = 0.41
I = Error of assumption = 5% (0.05)
Therefore, the target sample was 371.7132 ≈372.
3.5 Sampling method
Purposive sampling method was used to recruit study subjects sequentially; especially the
ones that were available and could easily access the PMTCT clinic. The selected potential
subjects were briefed on the purpose and procedure of the study. Consenting subjects
were requested to sign consent form.
3.6 Inclusion criteria and exclusion criteria
The eligible candidates for this study involved pregnant women attending Molo Subcounty maternal clinic, were over 18 years, willing to participate in this study and were in
stages 1-3 of WHO HIV/AIDS staging criteria. Those excluded were clients who were
below 18 years, severely sick, unable to speak, not given consent to participate in the
study, terminally ill and bed ridden for over 50% of study time. Pregnant women who
were lost during follow-up before the end of the study period were also excluded.
37
3.7 Ethical considerations
The study protocol was reviewed and approved by the Kenyatta University Ethical
Review Committee (Appendix XV). All responses were confidential, anonymous and
privacy of the study participants were fully guaranteed. Seeking for research
authorization was done by obtaining research permit from National Commission for
Science Technology and Innovation (Appendix XVI) and the ministry of health through
Molo Sub-county hospital management (Appendix XIV). Informed consent was sought
from all the study participants, which culminated in signing of consent form (Appendix
IX). Counselling for initiation of ARVs and adherence was offered by qualified clinicians
and counsellors to those who were eligible to start ART. Treatment for illness and
prophylaxis was offered according to the WHO and national HIV management guidelines
WHO (2006). Patients’ records were kept in the clinic in hard-cover file holders in a
lockable shelf. Access to databases was restricted. Completed questionnaires were
archived in a lockable shelf in the office.
3.8 Quantitative data collection procedure
Quantitative data such as social demographic and economic characteristic, level of food
preference, knowledge on nutritional elements under service delivery and utilization of
the hospital PMTCT facility study were collected using structured questionnaire
(Appendix XI). Structured questionnaire provides data that is objective, scientific and
reliable for hypothesis testing (Ong, 1993).
38
3.9 Examination of clinical characteristics
Study subjects were clinically examined by qualified healthcare personnel at onset and at
end term and details of HIV-associated clinical characteristics recorded. Treatment of any
condition requiring further management was taken accordingly. Opportunistic infections
(OIs) history and, finally, the variable relating to therapy was entered at the beginning of
the study and at end term (Appendix XI).
3.10 Collection and pre-treatment of blood
A 8 mL of blood samples were obtained at each visit into purple top Vacutainer (4 mL)
and red top vacutainer (4 mL) from HIV pregnant women by venipuncture of the
anticubital vein of the left arm by a registered nurse preferably in the morning but fasting
status was not ascertained. The blood samples were labeled with the participant's ID.
Blood sample from purple top vacutainer (contained EDTA) and were to be used for
plasma viral load and CD4 count. This whole blood sample were stored at room
temperature (18-30oC) for up to 4 hours. Plasma was removed from the cells within 4 hr
of collection. The separation was done by centrifugation at 1000 x g for 10 minutes.
Plasma were stored in cool box at 8oC. Blood collected in a red top vacutainer tube (does
not contain EDTA or an anticoagulant) was used for determination of levels of serum
nutrients. Soon after the collection, the tubes were inverted least 8 times for better mixing
to avoid the clots. The blood completely clotted and was ready for centrifuge within 90
minutes and were spun for 10 minutes at approximately 2,500 rpm.
39
The blood samples were collected at three different times (at the onset of the study/first
trimester, midterm/second trimester and at end term/third trimester) of gestation period.
The samples were transported within 48 hours to biochemistry and immunology
laboratories for analysis. Within arrival, blood samples were accepted into the laboratory
software to confirm that information about them tally. These samples were given
laboratory numbers that enabled tracking of results and the laboratory information. The
samples were stored at -80oC in a freezer till analysis
3.11 CD4 cell counts
A four colour flow cytometer (BD multi-check CD4 low control FACS Caliber) was
used. Beckman coulter cytometer interfaced with a detector module (BD FACS count
manufactured by San Jose CA 95131 USA B9320) was used. The BD FACSCount
system uses whole blood, eliminating lyse and wash steps suited with a software
algorithm that automatically identifies the lymphocyte populations of interest. It is easy
to use, cost effective, and reliable. A 1 mL of heparinized blood was mixed with 10 µL of
monoclonal anti bodies (AntiCD3-FITC, anti CD-PE). Red blood cells were lysed using
lysing solution. Briefly, 20 µL of BD tritest reagent was pipetted into the bottom of the
mesh in the true count tube; caution was observed by not touching the pellet. A 50 µL of
sample was added to the tubes at room temperature. The tubes were capped and vortexed
gently to mix then incubated for 15 minutes in the dark at room temperature (20 – 25
º
C). A 450 µL x BD was added by lysing solution to the tubes. Capped tubes were then
vortexed gently to mix. This was incubated for 15 minutes in the dark at room
40
temperature (25ºC) and then run in FACS count system. The data was processed and
reported on a sample print out sheet.
3.12 Determination of HIV-RNA (viral load) using system 340 bDNA analyzer
A 2 mL blood sample was taken first to processing room where the samples were
separated by the use of centrifuge. More than 1000 μL of plasma was collected into cry
vials and immediately stored at –80ºC in the freezer. The assay protocol was as described
by manufacture (Siemens Healthcare Diagnostics, 2008). The entire procedure consisted
of preparing viral pellet, hybridizing the probes and measuring light output.
The first process involved preparing viral pelleting. This was done by pre- chilling the
centrifuge and rotor at 2˚C. A vacuum system with a 2 to 4 L primary trap to contain 10%
bleach v/v final volume and secondary trap in a biological safety class II cabinet was
prepared. Then 50 L of bead suspension was added to each 1.5 mL centrifuge tube.
Specimens, standards and controls in cold H2O were completely thawed, then inverted or
vortexed to mix completely and kept on ice or at 2˚C. A 1 mL sample was added to
appropriately labeled centrifuge tube and kept on ice until centrifuged. Immediately,
unused portions of standards and controls were returned to -80˚C freezer. Centrifugation
at 23,500 x g for 1 hour at 2˚C was done and then transferred from the rotor to the safety
cabinet before opening the rotor. The supernatant was vacuum aspirated using a sterile
200 L disposable tip without filter, and approximately 20 L left on the pellet.
Immediately, the pellets were frozen at -80˚C.
41
The second process involved lysing the pellet (day 1). This was performed by turning the
dry heat block incubator on and ensuring that the temperature was at 63˚C. Lysing diluent
was placed in a 37˚C H2O bath for 10 minutes or until any visible crystals dissolved.
Lysing reagent was placed, followed by capture probes then target probes. They were put
on the bench to attain room temperature. A lysing working reagent in a 50 mL
polypropylene tube was prepared. The frozen pellets were thawed at room temperature
for 5 minutes and 120 L of lysis working reagent was added to each tube. Capture well
strips were pressed firmly into the plate until the tops of the strips were levelled.
The next procedure involved hybridizing the capture and target probes. Tubes from the
heating block were removed, cooled at room temperature for 10 minutes and centrifuged
briefly to recover from condensation then vortexed for 10 seconds. Using 200 L manual
pipette and a new tip for each sample, 100 L from each tube was transferred in the
appropriate capture wells according to the plate map. When prompted, the plates were
placed on the loading tray of the analyzer. Assembled sealing pad was placed on each
plate before incubation was done for 16 (overnight).
Hybridizing the pre-amplifier and amplifier probes (day 2) was done by ensuring that
wash A and wash B bottles had enough solution for the number of strips used in the
assay. Pre-amplifier/amplifier diluent, dextran sulfate and label diluent was placed in a
37˚C H2O bath for at least 10 minutes. The bottle was swirled with dextran sulfate and
each bottle containing diluent inverted several times to mix until homogeneous then kept
at 37˚C. The pre-amplifier/amplifier working diluent in 50 mL polypropylene tube was
42
prepared. Volume appropriate to the number of strips used in the assay was added.
Dextran sulfate using a positive displacement pipette was then added and the tube
capped, inverted for 10 times and vortexed for 20 seconds to mix. This was repeated until
diluent was thoroughly mixed and placed in 37˚C H2O bath. The pre-amplifier probes
were placed on the bench top and brought to room temperature.
The pre-amplifier working reagent in a 15 mL polypropylene tube was prepared and
volume appropriate to the number of strips used in the assay was also added. The tubes
were capped, inverted 10 times and vortexed for 5 seconds to mix. This was repeated
until thoroughly mixed then placed in 37˚C H2O bath. When incubation was completed
and the analyzer allowed to cool and wash the plates. When plates were ejected,
immediately 100 L of pre-amplifier working reagent was added in each well. Using a 12
channel pipette, the plate from row A to row H was filled. Incubation took 30 minutes
and this was followed by cool/wash cycle. Amplifier working reagent in a 15 mL
polypropylene tube was prepared and when cool/wash started, amplifier probe was placed
on the bench top to bring it to room temperature. Volume appropriate to the number of
strips used in the assay was added. The tubes were capped, inverted 10 times and
vortexed for 5 to 20 seconds to mix. This was repeated until thoroughly mixed then
placed in 37˚C H2O bath. When incubation was completed, the plates were cooled, then
ejected.
Hybridizing the label probe was done when the label diluent was placed in a 37˚C H2O
bath for at least 10 minutes and mixed by inverting the bottle several times. The label was
43
thawed in cold H2O just before using it. A label working reagent was prepared in 15 mL
polypropylene tube. Volume appropriate to the number of strips used in the assay was
added. The tubes were capped, inverted 10 times and stored at room temperature to settle.
When the plates were ejected, 100 L of label working reagent was added immediately to
each well. A 12 channel pipette was used to fill the plate from row A to row H.
Incubation took 45 minutes and was followed by cool/wash cycle. The substrate and
substrate enhancer were placed on the bench top and brought to room temperature.
Finally, measuring the light output was done when wash cycle started, a substrate
working reagent was prepared in 15 mL polypropylene tube. Volume appropriate to the
number of strips used in the assay was added, tube capped, invert 10 times and stored at
room temperature. Immediately, 80 L of substrate working reagent was added to each
well. A twelve-channel pipette was used to fill the plate from row A to H and incubated
for 30 minutes and the system analyzer read the light units of the plates and transferred
the data to data management software (DMS) for analysis.
3.13 Biochemical determination of serum nutrient profiles
3.13.1 β-carotene
The β-Carotene standard stock solution was prepared by dissolving 10 mg/mL of βcarotene in 20 mL of ethanol and topping up to 250 mL with methanol. All stock
solutions were protected from light and stored at -20ºC. The stock solution was further
diluted with methanol to give a series of working standard. The stock solutions were kept
under refrigeration conditions to be used for 2 weeks. The working solutions of different
44
concentrations were prepared daily by serial dilution of the standard. Validation
procedure involved measuring whole blood from three subjects that had been stored in
freezer before analysis. The intra (n=6) and interassay variances (n=5) were determined
during a two week period. The reliability of the method was validated through its
linearity and spike recovery. Calibration curve was used to test for linearity. A calibration
curve for the peak areas against concentration of standard was used. The blood sample
calibration curve was constructed using spiked serum with a known amount of β–
carotene. This was determined by addition of working solution of β–carotene to whole
blood individually. For all analysis, an external standard was used to obtain peak area
ratio for quantification. Recovery of serum standards spiked with a known amount of βcarotene was used to determine the extraction recovery, within day and between day
precision (n=5) of the method. The recovery of β-carotene after liquid- phase extraction
was calculated by comparing β-carotene peak areas in extracted serum, to those of nonprocessed standard solutions.
The β-carotene in whole blood was extracted by a method described by Jie et al. (2008).
A 100 μL serum, 100 μL internal standards (retinyl acetate + echinenone), 500 μL saline
(0.85%), and 4 mL of a ratio of chloroform to methanol [(C: M) 2:1, by vol.] were added
to glass tube (16 x 100- mm), mixed by using a vortex mixer for 1 min, and then
centrifuged at 800 x g for 10 min 4 ºC. The lower layer (chloroform layer) was collected
onto another glass pipette. Hexane (3 mL) was added to the aqueous layer, mixed by
being vortexed and centrifuged again. The hexane layer and the chloroform layer were
combined and dried under N2 on an N-EVAP (nitrogen evaporator). The residue was
45
dissolved in 100 μL of mixture of ethanol and tetrahydrofuran at a ratio of 2:1 (by
volume), and then transferred to a vial. The dissolved residual solution (20 μL) was
injected into HPLC and β-carotene was quantified by reversed-phased HPLC as
described by Nambafu (2011). The HPLC chromatograph used was model L-6000 with
dual plunger reciprocating pump (Hitachi Instrument Inc model L-6000). Twenty
microlitres of both the standard solutions and the sample extracts were injected into
HPLC system.
The sample was introduced into the column through a 50 μL sample injector of Rheodyne
(Model B/N 655-0890). Separation was achieved by a reversed phase column consisting
of a stainless steel (15 cm long x 4 mm internal diameter) packed with μ Bondapak C18
column (125 x 4 mm) particle size, 10 μm ). The column effluents were monitored by
ultraviolet (UV) absorption using a variable wavelength (UV-VIS) detector (Model
UV6000 LP photodiode-array UV/visible detector). The chromatographic peaks were
recorded by model D- 2500 chromato- integrator. The sensitivity used was mainly 0.02
absorbance units’ full scale (AUF’S). The detection wavelength was set at 450 nm and
the flow rate was 1.2 mL/min while the chart speed was 10 mm/min.
3.13.2 Iron
Determination of iron in serum was done by photometric colorimetric test for iron with
lipid clearing factor (LCF) (chromazurol B-CAB) method. This was performed by
HumaStar 180 chemistry analyzer as outlined by the manufacturer (Human Gesellschaft
46
fur Biochemica und Diagnostica mbH Max-Plank-Ring 21.65205 Wiebaden-Germany)
with various contents (Table 3.1).
Table 3.1: Content of photometric colorimetric test for iron
Contents
Reagents
concentration
Reagent (2×30 mL or 2×200 CAB
0.18 mmol/L
mL CAB reagent)
CTMA
2.2 mmol/L
Guanidium chloride
2.6 mol/L
sodium
acetate
buffer 45 mmol/L
(pH = 4.7)
Standard (5mL)
Iron (ionized)
100
μg/dL
or
17.9
μmol/L
The assay was carried out at absorbance maximum wavelength of 623 nm with optical
path of 1 cm at room temperature (25ºC). Only one reagent blank per series was required.
The pipetting scheme (Table 3.2), involved centrifugation serum samples in the red top
vacutainers to separate the serum. Serums from the tubes were then aspirated into
respective labelled eppendorf tubes using pipette.
Table 3.2 Pipetting scheme of photometric colorimetric test for iron
Pipette into cuvuttes
Rb
Sample/ (standard)
Sample/ STD
…….
50 μL
Distilled water
50 μL
…..
Reagent
1000 μL
1000 μL
47
The content was mixed well and incubated for 15 minutes at room temperature and the
absorbance of the sample (ΔA sample) and standard (ΔA STD) was measured against reagent
blank within 60 minutes. The method is linear up to a concentration of 895 mol/L (500
g/dL) therefore samples above this concentration were diluted to 1 part in 2 parts of
iron-free deionized water and re-assayed. Running the controls involved using vials with
already prepared controls. Humatrol N and P were placed in position
C
1
and C
2
respectively, and let to attain 37 ºC on the machine. It was also ensured that all the
calibration results were within the specified limits of the controls.
3.13.3 Zinc
Zinc in serum was determined using flame (FAAS) (AA-646) (Shimadzu, Japan) as
described by Syed et al. (2012). Reagents used included zinc granules, hydrochloric acid
(HCl) and deionized water. Preparation of stock bulk solution, was done by dissolving 1
gm of zinc granules in a minimum volume of 1:1 HCl then diluting up to 1 litre with 1%
v/v HCl which gave a 1000 ppm (part per million) of zinc stock/bulk solution.
Preparation of samples was done by adding deionized water with a ratio of 1:10 to the
collected blood samples and thus, the detection of zinc concentration done automatically
by FAAS.
3.14 Data management and statistical analysis
All data were entered in a database using Microsoft Excel software and exported to
Scientific Package for Social Science (SPSS) version 16 program. A Wilcoxon signedrank test the significant difference in viral loads between the periods of study. The Paired
48
sample T-test statistics was used to determine if there was any significant difference in
absolute CD4 count between the periods of study and it was used also to determine if
there was any significance difference between serum concentration at onset and end term
of study. Paired sample correlation was performed to determine whether there was any
significant difference between serum nutrient levels (Zn, Fe an β-carotene) at onset of the
study and at end term with CD4 count and viral load). Significance level for all the
analyses was set at a value of P < 0.05. Quantitative data was analyzed using descriptive
statistical methods such as tables, bar charts and pie charts percentages and frequencies,
generated by excel spread sheet of Microsoft office 2010 and by narrative. However, data
analysis was done after exclusion of cases (Appendix I).
49
CHAPTER FOUR: RESULTS
4.1 Demographic characteristics of HIV pregnant women study participants
The Women's ages ranged from 18 to 46 years with a peak at age group between 31 and
35 years comprising 33%. Most of the women were within the reproductive age group of
18-48 years. Married mothers were 60% among those who were interviewed. Only 10%
were single, 3% were divorced and 17% were separated, while 10% were widowed.
Majority (47%) of the study participants had primary school education and 18% were
illiterate, 30% had secondary education and only 5% completed college level education.
It was also observed that 55% of these pregnant mothers mainly purchased food for their
households. The remaining 42.2% got food from their household farms and 2.8% got
their food from relatives and friends. Income here represents average money received
directly by the pregnant woman from varied sources. It was observed that 15.6% of the
women earned less than Kshs. 2000 per month, 20.6% earn between Kshs. 2001 and 4000
per month, 10.5% earned between Kshs. 4001 and 6000 while 10.9% earned Kshs. 6001
to 1000. The highest category, 42.4% of study participants had no specified amount of
earnings. It was found that 6.3% of respondents spent less than Ksh. 2500 to buy food,
44.7% used between Kshs. 2500 and Kshs. 4000 to purchase food, 20.3% spent between
Kshs. 4001 and Kshs. 6000 for food purchase and 28.7% spent more than Kshs. 6,000 to
buy food in a month. It was discovered that most women (60%) lived 5-10 kilometers
away from the hospital. Those who lived more than 10 kilometers away were 29.5% and
only 10.5% were living in less than 5 kilometers away from the hospital (Table 4.1).
50
Table 4.1: Socio-demographic characteristics of study participants
Characteristic
Age (years)
Marital Status
Literacy levels
Distance covered to the
hospital (Km)
Source of food
Monthly income (Ksh.)
Average monthly
expenditure on food
category
18-25
26-30
31-35
36-40
41-45
>45
Total
Married
Separated
Single
Widowed
Divorced
Total
Illiterate
Primary level
Secondary level
Tertiary
Total
5-10
>10
<5
Total
purchase
household farms
relatives and friends
Total
<2000
2001-4000
4001-6000
6001-10000
No specified earning
Total
<2500
2500-4000
4001-6000
>6000
Total
Frequency Percent (%)
11
5
49
22
73
33
60
27
27
12
2
1
222
100
132
60
37
17
22
10
22
10
3
3
220
100
40
18
103
47
66
30
11
5
220
100
132
60
65
29.5
23
10.5
220
100
121
55
93
42.2
6
2.8
220
100
34
15.6
45
20.6
23
10.5
25
10.9
93
42.4
220
100
14
6.3
99
44.7
45
20.3
64
28.7
222
100
51
It was also observed that 79.5% of respondents did indicate that HIV management service
delivery at the clinic was satisfactory, 15.5% indicated that it was fair, while 5%
percentage of respondents
indicated that it was unfair (Figure 4.1).
100
50
79.5%(176)
15%(34)
5%(11)
0
satisfactory
fair
unfair
Responses to service delivery
Figure 4.1: Responses of HIV pregnant mothers on service delivery at the PMTCT
clinic
4.2 Dietary preferences and nutritional knowledge levels of study participants
The foods commonly preferred (Table 4.2) by pregnant mothers living with HIV
attending this hospital included: chapati (79%), bananas (70%), mokimo (mixture of
maize, potatoes, pumpkin leaves/stinging nettles among others) (69%), tea (50%) and
beans (65%).
52
Table 4.2: Food preference among HIV pregnant women attending the hospital
Foods
Total number of clients
Percentage (%) preferring food
(n)
Bananas
222
70
Beans
222
65
Carrots
220
28
Chapati
218
79
Eggs
222
10
Fish
221
4
Mokimo
221
69
Milk
218
71
Mrenda
220
3
Nduma
222
20
Potatoes
222
33
Sweet potatoes
222
3
Rice
222
6
Tea
220
50
Ugali
219
4
Millet porridge
222
2
Meat(goat/sheep/cow)
221
58
Most women in this study had a little knowledge on importance of various nutrients in
management of HIV and AIDS during pregnancy. The least known being β-carotene
(96%), then Zinc at 70% and lastly Iron at 62%. Only 20%, 25% and 5% knew that zinc,
iron and β-carotene respectively improve immunity. A proportion of 10%, 13% and 1%
said that zinc, iron and β-carotene respectively prevent HIV and AIDS (Figure 4.2).
53
Percentage of respondents
100
90
80
70
60
50
40
30
20
10
0
Zinc
96
Iron
Beta carotene
70
62
20
25
5
Improve immunity
(n=180)
10 13
1
Prevent HIV and AIDS
(n=180)
I do not know (n=180)
Responses on importance of micronutrients under study
Figure 4.2: Knowledge on importance of zinc, iron and β-carotene on health status
4.3 Clinical characteristics in HIV+ pregnant clients
The study reported significant frequencies of the subjects at onset with opportunistic
infections (number of cases are in brackets). At onset there was no case pelvic
inflammatory disease (PID), sub-acute encephalitis (2), trichomoniasis (4), candidiasis
(6), weight loss (38), cough (7), TB (3), piles (2), boils (2), pneumonia (17), fatigue (56),
Cryptococci, (0), kaposi’s sarcoma (0), herpes zooster (4), heart burn (31), fever (22),
diarrhoea (6), pallor (27), headache (40), oedema (37), vomiting (48), loss of appetite
(30) and skin rush (17). However the study reported low significant frequencies of the
subjects with opportunistic clinical manifestations at end term. There were no cases of
PID and sub-acute encephalitis but trichomoniasis (1), candidiasis (0), weight loss (1),
cough (1), TB (3), piles (0), boils (0), pneumonia (2), fatigue (4), cryptococci (0),
54
Kaposi’s sarcoma (0), herpes zooster (1), heart burn (19), fever (1), diarrhoea (6), pallor
(2), headache (5), oedema (2), vomiting (9), loss of appetite (5) and skin rush (5) (Figure
4.3).
Sub-acute encephalitis
PID
Clinical characteristics
Trichomonas
candidiasis
Weight loss
Cough
TB
Piles
Boils
Pneumonia
Fatigue
Cryptococci
kaposi's Sacoma
Herpes
Heart burn
Fever
Diarrhea
Pallor
Headache
Oedema
Vomiting
Appetite loss
Skin rash
0
10
20
30
40
50
Frequency of cases
End term
0nset
Figure 4.3: Clinical status of the subjects at onset and end term of the study
60
55
4.4 Absolute CD4 count at various stages of study
The subjects were classified into three groups according to CD4 levels; Less than 200
(AIDS victim), 200 to 499 (HIV positive stage 2) and 500 and above (HIV positive stage
1). The mean CD4 counts at onset was 185.59 cells/µL, but increased to 300.9 cells/µL
and 323.53 cells/µL at midterm and end term respectively (Appendix II). The study
revealed that 51.1% of the subjects had CD4 counts less than 200 cells/µL of blood,
while 45.9% of subjects had CD4 count of between 200-499 cells/µL at onset. At
midterm, 26.1% of the subjects had CD4 counts less than 200 cells/µL of blood, while
64.4% of subjects were having a CD4 count of between 200-499 cells/µL and the
proportion having CD4 counts above 500 were 9.5% having risen from zero at onset. At
the end of the study, 19.4% of the subjects had a CD4 counts less than 200 cells/µL of
blood, while 64.4% of subjects were having a CD4 counts of between 200 and 499
cells/µL and the proportion having CD4 cell above 500 increased to 16.2% (Table 4.3).
Table 4.3. CD4 count (cells/µL) during the study period
Study period
HIV WHO staging
Onset
Less than 200
120
54.1
200 to 499
500 and above
Total
Less than 200
200 to 499
500 and above
Total
Less than 200
200 to 499
500 and above
Total
102
0.0
222
58
143
21
222
43
143
36
222
45.9
0.0
100.0
26.1
64.4
9.5
100.0
19.4
64.4
16.2
100.0
Mid term
End term
Frequency
Percent (%)
56
Paired sample t-test statistics showed a strong significance (p=0.00). A comparison of
CD4 values at different time points (box plot) (Figure 4.4) demonstrated a dramatic
increase in absolute CD4 counts from onset (185.59 cells/µL) through the midterm
(300.93 cells/µL) to the end term (348.53 cells/µL) respectively.
Onset: First appearance; midterm: Midterm of pregnancy; End term: End of
pregnancy
Figure 4.4: Comparison of CD4 count (cells/µL) values at different time points
4.5 Comparison of viral loads at various stages of study
It was observed that (Figure 4.5), the overall sum total of viral load at onset was
14308402 copies/μL. At midterm it was 320237 copies/μL and at end term the viral load
reduced further to 109107 copies/μL. The mean viral load was 67374 (82%), 8427 (10%)
57
and 6062 (8%) copies/μL at onset, midterm and end term respectively. A Wilcoxon
signed-rank test statistic showed that there was a statistically significant change
(reduction) in viral load of the clients at end term as compared to that at onset (Z = 13.964, P = 0.00) (Appendix VII ).
Onset: First appearance; midterm: Midterm of pregnancy; End term: End of
pregnancy
Figure 4.5: Comparison of viral loads at various stages of study
4.6 Levels of serum nutrients
4.6.1 Zinc levels
The most positive significant observation was the paired sample difference between
serum zinc at onset of the study and at end term (p = 0.00) (Appendix IV). On the other
hand, correlations of CD4 counts with Zinc levels in serum were insignificant (p = 0.662
and p = 0.725 at onset and at end term respectively) (Appendix VIII). At onset, 56.8% of
participants had below normal levels (< 60 μg/dL) and those with normal serum zinc
58
levels (60-120 μg/dL) were 42.3%. Only 0.9% had above normal levels (>120 μg/dL). At
end term there were variations as follows: A proportion of 59.9% had below normal
levels (< 60 μg/dL) and those with normal serum zinc levels (60-120 μg/dL), were
39.6%. Only 0.5% of participants had above normal levels of zinc (>120 μg/dL) (Table
4.4). The mean concentration was 54.133 μg/dL at onset and increased slightly to 55.632
μg/dL at end term (Appendix II) and paired sample T-test was significant (p=0.00).
Table 4.4: Zinc levels in serum of HIV pregnant women of Molo sub-county hospital
Study period
µg/dL
Onset
Below 60
126
56.8
End term
60 to 120
Above 120
Total
Below 60
60 to 120
Above 120
Total
94
2
222
133
88
1
222
42.3
.9
100.0
59.9
39.6
.5
100.0
Frequency
Percent (%)
Comparison of viral load with serum concentrations of Zinc using Chi-Square test
statistic at onset was significant (p =0.00) but was insignificant at end term (p = 0.855)
(Appendix IX).
4.6.2 Iron levels
The results of iron levels (Table 4.12) indicates that at onset, 58% had below normal (<
41 μg/dL) and those with normal serum iron levels (41-132 μg/dL), were 42%. At end
59
term, there were variations as follows: It was found that 45.9% had below normal (< 41
μg/dL) and those with normal serum iron levels (41-132 μg/dL) rose to 54.1%. The mean
concentration was 36.887 μg/dL at onset and increased to 42.167 μg/dL at end term
(Appendix II).
Table 4.5: Serum concentration of iron
Study period
µg/dL
onset
Below 41
127
57.2
41 to 132
Total
92
219
3
41.4
98.6
1.4
100.0
100
118
218
4
45.0
53.2
98.2
1.8
Frequency
Missing system
Total
end term
222
Below 41
41 to 132
Total
Missing system
Total
222
Percent (%)
100.0
The paired sample T-test between serum iron at onset of study and at end term was
significant (p = 0.005) (Appendix V). On the other hand correlations of CD4 counts with
iron concentrations in serum were insignificant (p = 0.662 and p = 0.725 at onset and at
end term respectively) (Appendix IX). Chi-Square statistical analysis of viral load with
serum concentrations of Fe at onset was not significant (p=0.410) and insignificant too at
end term (p = 0.580) (Appendix VIII). There were no significant relationship between
iron status and CD4 count at onset (p= 0.772) and at end term (p= 0.76) by Chi-Square
60
test. On the other hand, a significant correlation in paired samples T-test for iron
concentration at onset and at end term (p = 0.005) was observed (Appendix VIII).
4.6.3 β- Carotene levels
From the data collected, the value for β- carotene levels of the clients at the onset of the
study with below normal levels (< 30 µg/dL) were 20.7%. Those with normal serum
concentration of (30 and 65 µg/dL) were 38.3% and those with above normal levels (> 65
µg/dL) represented 41%. At end term, those with concentration below normal levels (<30
µg/dL) were 5.4%. Those with normal levels (30 and 65 µg/dL) were 30.6% and those
with above normal levels (65 µg/dL) represented 64% (Table 4.6).
Table 4.6: Levels of β- carotene in serum of HIV+ pregnant women of Molo subcounty hospital
Study periods
µg/dL
Onset
Below 30
46
20.7
End term
30 to 65
Above 65
Total
Below 30
30 to 65
Above 65
Total
85
91
222
12
68
142
222
38.3
41.0
100.0
5.4
30.6
64.0
100.0
Frequency
Percent (%)
There was an increase in mean concentration of β- carotene from 70.52837 µg/dL at
onset to 95.849 µg/dL at the end of the study (Appendix II). Paired samples T-test for
comparison of β- carotene at onset and at end of study was significant (p = 0.00)
(Appendix VI). Pearson Chi-Square test indicated significant relationship between viral
61
load and beta carotene with was significant at onset but insignificant at end term (p =
0.03 and p=0.539 respectively). Pearson's R Spearman correlation did not show any
meaningful significance with CD4 count at onset and at end term (p =0.502 and p= 0.465
respectively) (Appendix VIII).
62
CHAPTER FIVE: DISCUSSION
5.1 Demographic characteristics of study participant (HIV+ pregnant women)
Participants for this study were a sample of HIV positive pregnant women on ART
residing within Molo sub-county. The Womens’ peak age group was between years 31
and 35. This is the most reproductive age group. A study carried out by Ministry of
Health (MOH) (2005) indicated that age group between 18 and 35 years is the most prone
age group in HIV and AIDS prevalence in Kenya. Most women (60%) were married,
with only 10% being single, 3% were divorced, and 17% were separated, while 10%
were widowed. The present study seems to support the Kenya AIDS indicator survey
(KAIS) of 2012 (NASCOP, 2013), that reported that both women and men who said that
they were widowed had a high HIV prevalence of (20.3% and 19.2% respectively). Those
who reported that they had never married or cohabited had the lowest HIV prevalence
(2.7% and 1.3% respectively).
Majority of the women (47%) had primary school education and 18% were illiterate. This
suggests that most of the participants were literate enough to be able to adequately
respond to questionnaire items. Education seems to play no role in mitigating HIV
prevalence in this population. The 2012 KAIS (NASCOP, 2013), also reported that HIV
prevalence is higher among women than men (4.0% and 2.4% respectively) at all
education levels and lowest among both women and men with no primary education. In
Chicago, women who remained at risk following AIDS education were found to have
lower levels of formal education (Grey et al., 1992). Ugandan secondary school students
63
around 17-18 years of age demonstrated that they had an inaccurate perception of the risk
of HIV infection, despite their education level (Dente et al., 2002). The best evidence of
education status and HIV infection in developing countries comes from a systematic
review by Glynn (2002) where 27 articles were included that showed findings from the
general populations of six developing countries. In three African countries (Tanzania,
Uganda and Zambia) studies showed a statistically significant increase in HIV infection
in those of higher educational status.
A substantial proportion (36%), of the study subjects had an income of not more than Ksh
6000 per month which indicate that they were living below poverty line. Basing on the
Kenya Central Bureau of Statistic’s classification, over 57.6% the pregnant women were
from low economic classes, with monthly income below Kenya shillings 10,000 (CBS,
2003). This is in line also with NASCOP (2005) which reported that more than 56% of
Kenyans live below the poverty line and have less than a dollar per day to spend on
everything needed to live. The intricate relationship between poverty and HIV continues
to be a vicious cycle in the national response to the pandemic. While increasing poverty
levels continue to fuel the spread of HIV, the pandemic itself exacerbates those levels in
households and families with people living with HIV and AIDS (PLWHA). The low
income by women could put them at a risk of being infected with HIV. The challenge is
therefore to identify the mechanisms that facilitate HIV transmission in the presence of
poverty.
64
The current study also discovered that a large number of people had minimal
affordability to basic need like food. Socio- economic status of a person dictates greatly
the food affordability, though other factors such as nutritional awareness could have
significant effect on feeding habits (MOH, 2005). However, rich people will obviously
secure more nutrients in boosting immunity enhancing performance of ARV drugs in
management of HIV and AIDS. The present study found out that most subjects had food
insecurity because, they obtained food through buying. This is discouraging because,
food insecurity among PLWHA leads to migratory livelihood strategies and high risk
sexual behaviours, which increases risk of HIV re-infection (NASCOP, 2005). Good
nutrition can play a part in preventing weight loss and other AIDS complications (Stine,
2005). All persons with HIV infection should be screened for nutritional problems and
concerns at the time of their first contact with a health care professional, and routine
monitoring should be performed on an ongoing basis (Mahan et al., 2000).
Since majority (60%) of women live not far away from the health facility, the findings
thus justified the results of the efforts put by the government to ensure access to health
care facilities by all. It has been pointed out that one of the ongoing reforms in health care
system in Kenya is to ensure that every Kenyan lives within 4 km of a health facility
(Njoroge, 2010). In 2008, there were 6,190 health facilities in Kenya, the equivalent of 16
facilities per 100,000 people, or 11 facilities per 1,000 Km2 (Annual Health Sector
statistics, 2008).
65
The present study sought to know whether these women were satisfied by services
offered at HIV PMTCT clinic of the hospital. It revealed that a large proportion of the
mothers (79.5%), were satisfied with services delivery due to friendly environment
provided by staff at the clinic. This showed that the hospital personnel were living to
expectation of service charter displayed at the hospital entry. The charter expresses the
ministry’s vision of improving the performance of the health sector to achieve the
following: Increased access to equitable health services, improved quality of services
offered in the health sector, improved efficiency and effectiveness of service delivery
among others. A study by Otieno et al. (2010) reported that health care facilities are
frequently short staffed and health care workers are overburdened. As a result, many
women experience long wait times and that these issues have led some women to report a
lack of faith in their health care services which contributes to lack of continued care but
this study reports otherwise. Pregnant mothers reported in the current study had sufficient
faith in this facility. The finding were in line with what World Bank (2010) reported; that
pregnant women in Kenya generally have higher rates of uptake in prenatal services than
women in comparable sub-Saharan African countries.
5.2 Nutritional knowledge and dietary preferences of the participants
A vast majority of the clients had a little knowledge on the importance of these nutrients
in management of HIV and AIDS. These findings could perhaps explain why these
micronutrients were found to be below literature values in this population in some study
participants. This could be due to lack of nutritional education. There is substantial
evidence, including a number of authoritative reviews, which suggest that agricultural
66
and homestead food production, behaviour-oriented nutritional education to be critical in
achieving nutritional outcomes (Laurie and Faber, 2008) and conversely that increasing
food production alone may have little or no effect on nutritional status. According to an
on-the-ground survey of nutrition services by Hampshire et al. (2004), routine dietary
counselling and screening for pregnant and post-partum women is often not done or is
poorly done in that most of the women do not understand the purpose of supplements and
nearly half are unlikely to comply with unfamiliar advices.
The clients showed preference to certain foods than others. Consumer preferences are
influenced to an extent by culture, traditions and income available to the household
(Ihekoronye, 1992). However, good nutrition can play part in preventing weight loss and
other AIDS complications (Stine, 2005). In order to maintain health it is necessary to eat
a variety of foods since different foods protect the body in different ways. Keeping of
animals such as rabbits for instance should be encouraged since they are cheap source of
protein other than meat from cow or goat. For pregnant women, the required daily
allowances (RDA) of zinc is 11 mg/day (Health Canada, 2006). Levels of zinc reported in
Kenya is 28.78 mg/kg in bulrush millet (Muchemi et al., 2007) yet millet porridge was
least preferred. This perhaps could explain why those below recommended level of serum
Zinc in this population increased (56.8% to 59.9%) in the course of study.
Based on FAO/WHO classification, more than half (58%) of the mothers had preference
for consumption of dietary iron (approximately >50g of flesh/meat (FAO/WHO, 1998)
which was good but they did not have purchasing power because of low income. Factors
67
such as income could have had more influence on food choice in spite of knowledge
regarding appropriate food selection. The women might as well have opted for their usual
diets with less animal based food and more vegetables. Proper income increases dietary
diversity and hence improve the quality of nutrient content including iron. This improve
iron status in expectant mothers.
A ½ cup of cooked carrots contain 8,268 μg of β-carotene (Love et al., 2001). However,
only a small proportion of study participants preferred carrots, a major source of βcarotene which is abundant in this region and form part of most meals therefore highly
consumed. This could explain the elevated levels observed through the period of study.
Therefore barriers to consumption of foods rich in β-carotene such as household taste
preference should be addressed. However, consumption of foods rich in vitamin C (from
vegetables) which this population is known to consuming a lot is known to increase
absorption of iron by reducing ferric ion to more absorbable ferrous form (FAO/WHO,
1998). This could also help to explain why these women had significant increase in iron
levels between onset and end term.
5.3 Clinical characteristics in HIV infected pregnant clients
Clinical status was evaluated by assessment of opportunistic infections (OIs). Findings of
clinical assessment were obtained from women files done through evaluation of common
HIV related OIs which included: tuberculosis, malaria, pneumonia, skin rash, diarrhea,
fever, oral thrush, fatigue, Kaposi’s sarcoma, anorexia, head ache, vomiting, constipation
and Pneumocystis carinii pneumonia (PCP) (Stine, 2005). These diseases do also affect
68
people with normal immune systems, but with HIV they occur at a much higher rate. It
also takes longer for a person with HIV to recover than it takes for someone with a
healthy immune system. When the immune system is very weak due to advanced HIV
disease or AIDS, OIs such as PCP, toxoplasmosis and cyptococcosis develop (Dybul et
al., 2002).
High percentage increases in CD4 counts observed at end term when compared with
onset and reduction in frequency of OIs among the participants, could be attributed to
improvement of health status and hence immune system that was mounting a fight
against OIs. The observation of high frequency of the women suffering from fatigue that
appeared to subside at the end term of study was in tandem with several studies which
seem to suggest that most people with HIV experience fatigue at some point during
illness, with estimates ranging from less than 50% to more than 80%. The rate of fatigue
increases as HIV disease progresses (WHO, 2002).
The high significant weight loss reported in this study, could have been caused by several
factors including: loss of appetite during pregnancy, medications that women take to
control the disease, OIs which increase the calorie needs and mouth and tooth infections
which make it difficult to eat. Diarrhea which is a common problem for HIV positive
people and may be directly related to weight loss. With or without diarrhea, malfunction
of the intestine may lead to inability to absorb nutrients and may also contribute to weight
loss (Macallan, 1999). Metabolism, including the number of calories patients expend at
rest or physical activity also affect weight loss. However, weight loss could be reversed
69
by increasing energy intake by 40% to 50% over the usual requirements (Woods, 1999).
Secondary infections also increase oxidative stress and rate of cellular turnover of the
immune system, resulting into depletion of immunity boosting nutrients, such as vitamins
and trace metals (Fracker, 1994). Weight loss late in the course of HIV disease is
common and increased energy expenditure in response to OIs, as well as infection itself
(WHO, 2005). Therefore, the subjects needed to increase intake of calories, vitamins and
the trace elements.
There was no change in frequency of tuberculosis (TB) at onset and end term of the
study. It is one of the leading cause of death in women in the reproductive age group
worldwide (Diwan et al., 1999). The obvious implication of this observation was that
clinical diagnosis is usually made late in our setup and therefore TB develops resistance
to drugs. Kenya Ranks 13th in the list of 22 high burden countries in the world and has the
5th highest burden in Africa (NACC/NASCOP, 2012). These findings are similar to
previous findings in the same target population (western Kenya) by Kosgei et al. (2013)
although their study had a larger sample with only 8% of the HIV-infected women being
TB positive.
The most prevalent pregnant-related problems reported in this study were headache, loss
of appetite and heartburn. However, it is completely normal to have noticeable conditions
of this nature even in normal pregnancy although they commonly occur within the first 24 weeks of a healthy pregnancy. A past study like that of waweru et al. (2009) on anemia
70
in context of pregnancy and HIV/AIDS, a case of Pumwani maternity Hospital in Nairobi
Kenya, also found that these problems are more pronounced among HIV-infected
pregnant women.
In general, the present study hypothesised a decline in OIs at the end of study or
pregnancy. This study interestingly matched an observational cohort study by Jennifer et
al. (2012) on HIV-infected women during the ARVs era; explanations advanced in her
study were that durable virologic suppression, nutritional supplements and clinic
attendance rate may affect the course of HIV disease and differed between pregnant and
non-pregnant women. A study conducted by Tai et al. (2007) on women receiving care in
the ARVs era, also found that pregnancy was associated with a lower risk of HIV disease
progression.
Pregnant women who are attending Molo sub-county hospital were observed to have
intensive health care intervention, including regularly scheduled visits (sometimes called
on phones) with a health care provider. They were well attended to by nutritionist, and
social workers as well as being offered nutritional supplements and they adhered to
counseling hence the great success observed in reducing the magnitude of OIs in this
population. Hence the current study suggest that with proper management, there is no
reason of discouraging HIV- infected mothers from becoming pregnant.
71
5.4 The CD4 cells absolute counts and viral loads at various stages of study
Treatment decisions are often based on viral load and CD4 counts. Normal counts range
of CD4 counts in a man without HIV infection is approximately 400-1200 cells/µL of
blood and 500-1600 cells/µL of blood in women (American Association for Clinical
Chemistry, 2009). If the CD4 cells are in the range of 200-499 cells/µL of blood, the
client is said to be HIV positive and if it is less than 350 cells/µL then the individual is
regarded as AIDS client and are usually advised to be enrolled for ARVs (Annalisa,
2006). In pregnant women with confirmed HIV serostatus, initiation of ARV for her own
health is recommended for all HIV infected pregnant women with CD4 cell count <350
cells per µL, irrespective of WHO clinical staging; and for all HIV infected pregnant
women in WHO clinical stage 3 or 4, irrespective of CD4 cell count (WHO, 2009).
The CD4 cell count of majority (54.1%) of the subjects at onset were below 200 cells
/µL, indicating that they had significant immunological suppression (WHO, 2005).
However, this changed because at end term, only 19.4% had CD4 count below 200 cells
/µL. The significant increase of CD4 count from onset to midterm and to the end term,
was is a manifestation that participants were responding well to ARVs and were
achieving significant improvement in immunologic functions. Earlier studies such as one
done by O’Brien (1996) and Mugwe (2011) did also show an overall improvement of
mean CD4 counts before commencement of ART and after initiation of ART. Although
low CD4 count (below 200 cells/µL) associated with severe immune suppression were
observed in a very large proportion of subjects at onset, some PLWHA have low CD4
count but are healthy, therefore clinical observation is used to confirm immunological
72
status (WHO, 2005). Paired samples correlations at end term and at onset gave
meaningful correlations. This indicated that mean CD4 count increased with
chemotherapy, an indication of an improvement in immunologic function. The
interpretation of this finding was that ART for the participants were effective and feasible
in management of HIV in this hospital. The key factor could be the availability of the
ARV drugs in the hospital which is given freely and therefore all HIV infected pregnant
women should receive ART regardless of HIV viral load or even CD4 count. This could
go a long way of reducing even the risk of perinatal transmission.
Viral load is the amount of HIV in a body fluid and is monitored as part of routine
clinical care for PLWHA. The mean viral load in this study decreased significantly
between onset and end term with Wilcoxon signed-rank test statistic showing a
statistically significant change. This could mean that there was improved immune
competence in these individuals as a result of better responses to treatments of
opportunistic OIs. The obvious implication, therefore was that the use of ARV drugs
during pregnancy was effective in reducing viral load significantly in HIV pregnant
mothers and does indeed guarantee survival during pregnancy. The current study is
therefore in congruent with KAIS (2012) which pointed out that among all HIV-infected
persons aged 15-64 years, 40% had achieved viral suppression. However, among these
mothers on ARVs, viral suppression was 76%. This was similar to another study on
routine viral load testing among pregnant HIV-infected women on antiretroviral therapy
(ART): implications for prevention, Nyanza province, Kenya by Ng'an’ga et al. (2011).
Their study observed that in overall, 79% of all the pregnant HIV infected women on
73
ARVs had suppressed viral load of <400 copies/mL. The present study reported a
decrease in viral load from onset to end term. This is also in agreement with a study by
Mugwe (2011) in Nakuru general hospital, Kenya on the effects of antiretroviral drugs on
CD4 cells and viral load in HIV positive patients. In her study, the overall mean viral
load also decreased after commencement of ART.
5.5 Levels of micronutrients (Zinc, iron and β-carotene) in the study participants
The aim of the current study was to determine levels of these micronutrients and their
relationship with CD4 count and viral load among HIV pregnant women attending Molo
sub-county Hospital using standard procedures. The findings indicated a higher
prevalence rate of zinc deficiency (56.83%) at onset and 59.9% at end term. However,
these findings were in agreement with studies from Kenya that have evaluated the role of
RDA compliant micro-nutrients in management of HIV and AIDS subjects (Muriuki,
2009; Mburu et al., 2010). Nevertheless, Ouma (2011) reported elevated levels of Zinc in
female (non-pregnant) subjects in a study of bioavailability of trace elements in selected
indigenous foods and their potential use on management of HIV and AIDS. But in the
present study the mothers were found to have a mean serum zinc level of 55.632 µg/dL at
end term as compared to 54.133 µg/dL at onset. This represented a slight increase but
was still low. It has been estimated that 82% of pregnant women in the world may have
inadequate intake of dietary zinc (Caulfield et al., 1998) leading to poor nutritional status
of the mineral and that zinc progressively decline
in serum zinc level in normal
pregnancy with each trimester (Ashraf et al., 1999). In another study in which HIVinfected adults were treated with ART there was a significant increase in plasma zinc
74
concentrations (mean increased from 78 to 96 µg/dL), CD4 cell counts, reduced
total/active thymulin ratio and decreased quantitative RNA over a 4 month period without
zinc supplementation (Mocchegiani et al., 1999). Another prospective study reported an
inverse association between zinc consumption and survival of HIV (Villamor et al.,
2006). Among HIV-positive persons not receiving ARVs, an observational studies such
as one done by Graham et al. (1991) showed low or deficient serum concentrations of
several micronutrients, including zinc, to be individually associated with low CD4 cell
counts, advanced HIV-related diseases and faster disease progression, just like what is
reported in the present study.
Since there was significant correlations between CD4 cells and zinc, these findings
suggest that zinc could be used as biomarkers of the progression of HIV infection
alongside CD4 cell count. However, the weak correlation could be explained by the fact
that zinc metabolism is affected by facilitators and antagonists which act from the level of
intestinal absorption, distribution and activation for catabolism including utilization as
prosthetics groups of enzymes (Nwokenna et al., 2003). Infection with HIV may impair
the utilization of zinc by disturbing protein synthesis and its stability, prevention of
intestinal absorption and imposition of low activation of zinc for metabolism. This may
account for the lower cationic Zinc element observed in the blood of HIV infected
expectant mothers screened in the present study. It may also imply that the rate of
consumption of zinc is lower in HIV infection than in normal individuals. Zinc level may
be normal or high in asymptomatic HIV-infected patients and a steady decline is
expected as immunodeficiency sets in (Lyn et al., 2000). In present study, there was also
75
no major improvement in serum Zinc levels in HIV pregnant subjects. The finding could
point to what Kedzierska et al. (2001) reported that HIV infection has been associated
with increased proinflammatory cytokines, which lower the plasma levels of Zinc.
The current study showed that 56.8% of these women had low serum Zinc concentration
at onset compared to recommended serum levels. Another study by Koch et al. (1996)
among hospitalized HIV-infected adults, showed that 50% of those with plasma zinc
measurements had low zinc levels. Arinola et al. (2004) also found lower levels of trace
elements Iron and Zinc in HIV-infected mothers, compared to literature values. It is
important to remark that Wellinghausen et al. (2000) also demonstrated a decrease in
Zinc levels in 23% of a HIV infected population, which is in agreement with the results
of the current work. Similarly marked Zinc deficiency has been observed in HIV
infection at various stages of illness (Fabris et al., 1988). Many studies have reported that
Zinc deficiency is not a common contributory factor for HIV and AIDS or clinical
expression and that HIV infection doesn't induce Zinc deficiency (Walter et al., 1990).
The current study interestingly showed that at end term, there was no significant
relationship between viral load and serum Zinc. Another study by Villamor et al. (2006)
also found that Zinc supplementation was not related to increase in viral load. A plausible
explanation is the direct effect of Zinc on cellular immunity. Zinc depletion in previously
deficient subjects and appears to up regulate production of Th-1 systemic cellular host
defense cytokine like tumor necrosis factor (TNF)-α (Villamor et al., 2006).
76
Zinc has been a hot topic in HIV and AIDS studies and the results have been conflicting;
it was known for quite some time that HIV requires zinc-containing structures called zinc
fingers to reproduce (Tanchou et al., 1998). This perhaps could explain why the low
concentration of Zinc in the current study led to reduction in viral load at the end of the
study period as reported elsewhere in this report. In conclusion, the apparent
contradictions could partly be explained by differences in study designs, populations,
limitations of observational studies which include; residual confounding factors, the
potential for reverse casualty as sick subjects are prone to diet change and the fact that
zinc plasma protein are influenced by acute phase response (Villamor et al., 2006).
Iron deficiency is the most common nutritional deficiency in the world, and is a condition
mainly affecting women and 52% of pregnant women in developing countries are anemic
(WHO/UNICEF, 2001). Among PLWHA, anaemia, a symptom of iron deficiency
(defined as low haemoglobin), is highly prevalent and is associated with increased
mortality and enhanced HIV progression (O'Brien et al., 2005). In the present study, iron
deficiency stood at 58% at onset but reduced to 42% at end term yet viral load reduced
although the deficiency was still significant. The increase in serum iron concentration in
the present study could perhaps be explained by the fact that intestinal absorption of
dietary iron increases during the second and third trimesters to accommodate for
expansion of red cell mass (Food and nutrition board, Institute of medicine, 2001).
77
However, these data are in line with studies which suggest that anemia is extremely
common during pregnancy, occurring among an estimated 35% – 75% of pregnant
women in developing countries (WHO, 1992). A study by Waweru et al. (2009) also
found that iron deficiency anemia was more prevalent and severe among pregnant HIV
cases and the relative risk of being anaemic was twice higher for HIV-infected than for
uninfected pregnant women. The data from present study therefore suggest that there is a
need for pre-natal nutrition care system that emphasizes improved consumption of
essential nutrients with iron of high biological value; and implementation of interventions
such as supplementation to achieve normal iron status tailored to check anemia
prevalence and severity among pregnant women within the context of management of
HIV and AIDS pandemic in low socio-economic settings.
It was observed from this study that iron status was not related to markers of HIV disease
severity (plasma HIV load and absolute CD4 lymphocyte count) in HIV infected
pregnant mothers. Both iron deficiency and overload increase rate of progression of HIV
and AIDS. Elevated iron status induces production of reactive oxygen species which alter
and suppress immune response. High levels of reactive oxygen species are associated
with increased mRNA levels of pro-inflammatory Th2 response cytokines and
chemokines and decreased anti-inflammatory cytokines IF-y and IL-2 (Ouma, 2011). A
study of iron status and indicators of HIV disease severity among pregnant women in
Malawi by Semba et al. (2001) also reported near similar results that the only significant
correlation was an inverse correlation between hemoglobin level and plasma viral load.
The study added that prevalence of iron deficiency anemia was not significantly different
78
across quartiles of HIV viral load or CD4 count. In contrast to previous studies, their data
also suggested that iron status was not related to markers of HIV disease severity in
pregnant women in Africa.
Beta carotene, a fat-soluble antioxidant, is a well-known scavenger of the singlet oxygen
radical (Bendich et al., 1998) and can decrease free-radical induced lipoperoxidation
damage in HIV (Favier et al., 1994). The recommended range of β-carotene in blood is
50 µg/dL -250 µg/dL (Nguyen et al., 2002). The standard serum concentration of βcarotene in adult female is 30 to 65 μg/dL (Uppsala University Hospital, 2008). From the
data collected in the present study, those suffering from β-carotene deficiency were
relatively lower (20.7%). This finding seemed to be closer to results of a similar study by
Phuapradi et al. (1996), who also measured serum β-carotene and vitamin A levels in
HIV positive pregnant women in the first trimester and compared it to pregnant HIVwomen, also in the first trimester. In that study HIV infected women with CD4 counts
below 200 had 37% lower mean serum β-carotene levels when compared to controls. The
present study reported lower mean of CD4 count at onset. And also low β-carotene
compare to end term. Moreover, deficiencies of serum β-carotene and other carotenoids
(including lutein and lycopene) have been observed in multiple studies in both HIV
positive and AIDS patients (Lacey et al., 1996). Depression of serum β-carotene levels is
usually indicative of fat malabsorption and diarrhea, common complications of AIDS,
secondary to general malabsorption, infection, and altered gut barrier function (Keating et
al., 1995).
79
The value for β- carotene levels of the clients at the end of the present study increased in
the period of pregnancy. This elevated levels could be attributed to good nutrition with βcarotene rich food such as pumpkin or carrots which are abundant in this region. These
results are comparable to a study carried out by Wachira (2008) who reported an increase
in β-carotene levels after a 3 months intervention study with PLWHA using a food
supplement consisting of cowpea leaves, pumpkins and carrots (rich in β- carotene). The
present study also discovered a significant nexus between viral load and β-carotene at
onset but not at end term. The low levels of β-carotene could be accused of high viral
RNA. This could emphasize the effect of taking foods containing immune nutrients
increase in levels. Nutritional awareness should therefore be enhanced in order to enable
HIV+ pregnant mothers boost their immunity. Slightly similar results have been reported
elsewhere. No correlation was found between pre and post-supplementation with βcarotene with CD4 counts or plasma viral load in a study of effect of oral β- carotene
supplementation in HIV RNA level and CD4 cell counts in infected patients by
Nimmagada et al. (1998). Among cross-sectional studies, concentrations of α-carotene
and β-carotene have been significantly higher in HIV-positive persons receiving ARVs
(Tang et al., 2000), just like in the present study than in HIV-positive persons not
receiving. This finding could mean that antioxidant activity could be higher in HIV
positive pregnant women receiving ARVs.
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CHAPTER SIX: CONCLUSIONS AND RECOMENDATIONS
6.1 Conclusions
i.
Majority had primary education (46%), living below poverty line (57.6%) and many
participants did not know the importance of micronutrients under study in
management of HIV and AIDS (β-carotene, 96%, Zinc, 70% and Iron at 62%).
ii.
Increments in mean CD4 counts from onset (185.59 cells/µL) to end term (348.53)
and viral load suppression rate of 76% from onset to end term were observed during
the period of study, an indication that the women were responding well to
antiretroviral therapy (ART) and achieving an improvement in immunologic
functions. The ARVs prophylaxis must be made available as part of the standard of
pre-natal care for pregnant women who are diagnosed with HIV and be considered as
a key element of managing of HIV in combination with proper nutrition.
iii.
Fatigue and weight loss emerged as the most frequent characteristics to be associated
with HIV infection during pregnancy among the spectrum of clinical characteristics
observed but opportunistic manifestations reduced as pregnancy progressed when
expectant mothers were on antiretroviral therapy (ART).
iv.
There was a general increase in mean serum levels of the micronutrients from onset to
end term, but existence of Zinc deficiency was established, and this study does add to
growing body of knowledge that zinc depletion might occur within HIV pregnant
mothers during disease progression.
81
6.2 Recommendations from the study
i.
All pregnant women should be encouraged to go for voluntary counseling and testing
(VCT) for the early detection of HIV and the commencement of the administration of
ARVs for management of HIV. The Government and all other key players should
strengthen the existing policy and structures to support mothers in HIV management
programs. They need to see to it that there is an increase in the access to such services
all over the country.
ii.
Maintaining an appropriate level of micronutrient serum levels and stores should be a
mainstay of support for HIV infected pregnant clients. This is particularly important
for pregnant mothers who have an increased physiological demand as a result of both
HIV infection and pregnancy. HIV positive pregnant women may need
supplementation of some micronutrients during pregnancy, especially zinc. Zinc
supplementation should be considered in management of HIV in pregnant women.
iii.
There should be routine viral load testing and CD4 count determination during
pregnancy to help identify women requiring additional intervention to maximize care
and prevention of mother to child transmission (PMTCT) outcomes.
iv.
Pregnant HIV+ women attending Molo sub-county hospital need to be encouraged to
farm and feed on food rich in micronutrients under this study. Nutritional education
for pregnant women with HIV infection warrants a special session as early in the
pregnancy as feasible.
82
6.3 Suggestions for further studies
i.
Prompt treatment of opportunistic infections (OIs) contributes to delaying the
progression of HIV to AIDS. These findings further provide important
background information that can form the basis of future, more elaborative and
systematic studies of relative frequency of OIs.
ii.
There is need for determination of impact of serum nutrient profiles in modulating
pregnancy and birth outcomes of maternal HIV infection.
iii.
Further studies need to be undertaken with matched control samples as case
control analysis with respect to all OIs in HIV pregnant women.
iv.
Micronutrients status under study are not related to makers of HIV disease
severity (viral load and CD4 count). There is a need for investigation of definitive
evidence linking these serum nutrient status and markers of HIV disease severity
(viral load and CD4 count) during pregnancy in mothers under ART.
6.4 Limitations of the study
Treatment compliance such as adherence to ART in research settings could have been
considerably greater than the level of compliance that can be expected in a normal
setting. Selection bias and response bias were inevitable to some degree, although steps
were taken minimize response bias by having trained medical personnel, administering
questionnaire. Moreover, only literate-speaking women could have responded. Lastly
there was lack of a concurrent control group. This decision was based on cost constraints
and anticipated difficulty in enrolling adequate numbers for a randomized trial.
83
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APPENDICES
Appendix I: Data management process in the study
372 women targeted for
enrolment (by December
2012)
311 women viable for study (May 2013November 2013)
40 women whose
information were in
hospital database
15 women included; correct data entry,
consistency data, and follow-up
information available/some womens’
folders could not be retrieved.)
326 women
12 excluded; transferred to other
regions (no follow up)
314 women
3 excluded; know where about (might
have died)
311 women
31 excluded; moved away (June 2013).
280 women
50 excluded; follow-up impossible by
July 2013)
230 women
8 excluded ;( follow-up impossible by
October 2013).
222 women for statistical analysis
100
Appendix II: Nutrient levels (µg/dL), CD4 cell/µL and viral loads copies/µL
n
Minimum
Maximum
Mean
Zn at onset
222
8.2
87.2
54.133
Std.
Deviation
17.3704
Fe and onset
222
4.3
100.5
36.887
23.4273
Beta carotene at onset
222
9.1757
402.9480
70.5283
56.2444
Zn at end term
222
12.9
89.2
55.632
16.1173
Fe at end term
221
5.4
85.8
42.167
19.1949
Beta carotene end term
217
9.2996
808.379
95.849
88.5821
CD4 at onset
222
2
373
185.59
102.897
CD4 at onset categories
222
1
2
1.17
.374
CD4 Midterm
222
34
845
300.93
139.578
CD4 Midterm Categories
222
1
2
1.51
.501
CD4 end term
222
58
878
348.53
156.409
CD4 end term
Categories
VL onset Categories
222
1
2
1.62
.486
222
1
2
1.97
.175
VL Midterm Categories
222
1
2
1.18
.385
VL end term Categories
222
1
2
1.09
.287
Valid N (listwise)
216
101
Appendix III: Paired samples statistics for CD4 (cells/µL)
Mean
(cell/µL)
Pair 1 CD4 at end term
348.53
CD4 at onset
185.59
Paired Samples Correlations
N
222
222
N
Pair 1
CD4 at end term and
CD4 at onset
Std. Deviation
Std. Error Mean
156.409
102.897
10.498
6.906
Correlation
222
Sig.
.675
.000
Paired Samples Test
Paired Differences
Mean
Pair
CD4 end 162.94
1
term and 6
CD4 at
onset
t
Std.
Std.
95% Confidence
Deviation
Error
Interval of the
Mean
Difference
115.456
7.749
Lower
Upper
147.675
178.217
df
Sig. (2tailed)
21.0
22
28
1
.000
102
Appendix IV: Table of T-Test in relation to Zn findings (µg/dL)
Paired Samples Statistics
Mean
(µg/dL)
55.632
Pair 1 Zn at end term
Zn at onset
54.133
Paired Samples Correlations
N
Std. Deviation
222
222
N
16.1173
17.3704
1.0817
1.1658
Correlation
.352
Zn at end term and Zn at
222
onset
Paired Samples Test
Paired Differences
Mea
Std.
Std.
95% Confidence
n
Deviation Error
Interval of the
Mean
Difference
Lower
Upper
1.49
19.0849 1.280 -1.0257
4.0230
Pair Zn at
end term
86
9
1
and at
onset
Pair 1
Std. Error Mean
Sig.
.000
t
df
1.1
70
22
1
Sig. (2tailed)
.243
103
Appendix V: Table of T-test of serum concentration of iron (µg/dL)
Paired Samples Statistics
Pair 1
Fe at end term
Mean
(µg/dL)
42.167
Fe at onset
36.923
Paired Samples Correlations
N
Std. Deviation
221
19.1949
1.2912
221
23.4744
1.5791
N
Pair 1
Fe at end term and Fe at
onset
Std. Error Mean
Correlation
221
Sig.
.201
.003
Paired Samples Test
Me
an
Pair
1
Fe at
end
term
with Fe
at onset
5.2
439
Paired Differences
Std.
Std.
95% Confidence
Deviation Error
Interval of the
Mean
Difference
Lower
Upper
27.1718 1.827
1.6417
8.8461
8
t
df
Sig. (2tailed)
2.8
69
22
0
.005
104
Appendix VI: A table of significant tests results for β- carotene (µg/dL)
Paired Samples Statistics
Mean
(µg/dL)
β- carotene end
term
N
Std.
Deviation
Std. Error Mean
95.8499001
217
8.85821491E
1
6.01334811
β-carotene onset
70.927792
Paired Samples Correlations
217
56.5714165
3.8403180
Pair
1
N
Pair 1
Β-carotene end term and βcarotene onset
Correlation
217
Sig.
.524
.000
Paired Samples Test
β-carotene
at end term
and beta
carotene at
onset
Chi-Square Tests
Pair
1
Paired Differences
Mean
Std.
Std.
95% Confidence
Deviati Error
Interval of the
on
Mean
Difference
Lower
Upper
2.49221 7.6145 5.1690 1.4733 3.51104
078E1 3447E
8281 8069E
086E1
1
1
Value
Pearson Chi-Square
Likelihood Ratio
Linear-by-Linear
Association
N of Valid Cases
df
a
7.001
7.784
.045
222
t
df
Sig.
(2tailed)
4.8
21
21
6
.000
Asymp. Sig. (2-sided)
2
2
1
.030
.020
.832
105
Appendix VII: A table of Wilcoxon signed ranks test for viral load (copies/µL)
Wilcoxon signed ranks test
Ranks
N
Mean Rank
Sum of Ranks
Viral
Negative
0a
0
load at
Ranks
onset Viral
load at
end
term
Positive
2.E2b
98
Ranks
Ties
27c
Total
222
a. Viral load at onset < Viral load at end term
b. Viral load at onset > Viral load at end term
c. Viral load at onset = Viral load at end term
Test Statisticsb
Viral load at onset - Viral load at end term
Z
-13.964a
Asymp.
0
Sig. (2tailed)
a. Based on negative ranks.
b. Wilcoxon Signed Ranks Test
0
19110
106
Appendix VIII: Test statistics of CD4, viral load and levels of nutrients
Coefficients (r)
P = value
CD4 at onset
Zn onset (µg/dL)
0.03
0.662
Fe onset (µg/dL)
0.02
0.772
β -carotene onset
(µg/dL)
CD4 end term
0.823
0.502
Zn end term
(µg/dL)
Fe endterm
(µg/dL)
β-carotene end
term (µg/dL)
viral Load (copies/µL)
-0.024
0.725
-0.021
0.76
-0.05
0.465
Zn onset (µg/dL)
-0.122
0.00
Fe onset (µg/dL)
0.410
0.05
β- carotene
(µg/dL)
0.03
0.031
107
Appendix IX: Informed consent
My name is Otieno Domnic I am a Master of Science (Immunology) student from
Kenyatta University. I am conducting a study entiled “LEVELS OF β-CAROTENE,
IRON AND ZINC IN SERUM OF HIV POSITIVE PREGNANT WOMEN ON ANTIRETROVIRAL THERAPY IN MOLO SUB-COUNY, KENYA”. The information that
will result from the study will be used by the health care providers and in helping
management of HIV among pregnant women in this hospital and beyond.
Procedures to be followed
Participation in this study will require that I ask you some questions. Blood sample will
be
drown by the clinical staff of the hospital for further tests. I will record the
information from you in a questionare. You have the right to refuse participation in this
study. You will get the same care and medical treatment whether you agree to join the
study or not and your decision will not change the care you will receive from the clinic
today or that you will get from any other clinic at any other time. Please remember that
participation in the study is voluntary. You may ask questions related to the study at any
time. You may refuse to respond to any questions and you may stop an interview at any
time. You may also stop being in the study at any time without any consequences to the
services you receive from this clinic.
Discomforts and risks
Some of the questions you will be asked are on intimate subject and may be embarrassing
or make you uncomfortable. If this happens, you may refuse to answer these questions if
108
you so choose. Blood withdrawal may be painful, but to minimize discomfort, a licensed
nurse will draw your blood approximately 4mls in the entire study
Benefits
If you participate in this study you will be clinically monitored very closely and will
receive prompt treatment. You will also contribute to better understanding on
management of HIV during pregnancy. At the end of the study you will be provided with
1 page summary of the study findings.
Reward
There shall be no reward in the study.
Confidentiality
I assure you that the information you give will be handled with total confidence and at no
time will you be required to identify yourself by name. You will not be identified in any
public report or publication or to any other party. The questionnaires will be destroyed
after analysis. Everything will be kept private.
Contact information
If you have any questions you may contact Molo sub-county hospital authorities or
investigator on P.O BOX 156, Molo.
Participant’s statement
The above information regarding my participation in the study is clear to me. I have been
given a chance to ask questions and my questions have been answered to my satisfaction.
My participation in this study is entirely voluntary. I understand that my records will be
kept private and that I can leave the study at any time. I understand that I will still get the
109
same care and medical treatment whether I decide to leave the study or not and my
decision will not change the care I will receive from the clinic today or any other time.
Do you agree to participate in the study?
No [ ] Yes [ ]
If Yes,
Signature or thumbprint……………………………….Date…………………………
Investigator’s statement
I, the undersigned, have explained to the volunteer in a language she understands, the
procedures to be followed in the study and the risks and benefits involved.
Name of Interviewer………………………………………………………..
Interviewer signature ……………………
Date………………………..
110
Appendix X: Structured questionnaire
Structured interview guide for study of serum levels of β-carotene, Iron and Zinc in HIV
positive pregnant women on antiretroviral therapy in Molo Sub-county, kenya.
Name of Interviewer:……………………………………………
Date of interview: ………………………………………………
Section A: Bio data
1. What is your age? Tick (√) appropriately
Age category (years) Tick (√)
18-25
26-30
31-35
36- 40
41- 45
>45
2. Education background: Tick (√) appropriately
 Primary
 Secondary
 Tertiary
 No formal education
3. Religion: Tick (√) religion you belong to  Catholic Muslim SDA
protestants traditional No religion.
111
4. Marital status: single married  separate divorced  widowed
Section B: Economic characteristics
1. What is your monthly income? Tick (√) in the table below where your income
falls in.
Income level
Tick (√)
(Ksh)
0- 2000
2001- 4000
4001- 6000
6001 -10000
2. How much does your family spend on food? Tick (√) in the table below where your
expenditure falls in.
Expenditure in food
Tick (√)
(Ksh)
Less than 2500
2500 - 4000
4001- 6000
6000 and above
Section C: Dietary pattern
Which food do you prefer? Tick (√) in the table that follow if you like or dislike.
Foods
Bananas
peas
Like
Dislike
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Beans
Chapati
Eggs
Fish
Githeri
Milk
spinach
Nduma
English Potatoes
Sweet potatoes
Rice
Tea
mokimo
Millet porridge
Meat (meat/goat/sheep/cow)
3. Knowledge levels of importance of Zinc, iron and β-carotene in management of
HIV during pregnancy
What is your view on the role of the following in management of HIV and AIDS?
Tick (√) appropriately your suggestion
nutrients
Zinc
Iron
Beta carotene
Improve immunity
Prevent HIV and
AIDS
I do not know
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4. Distance of travel and use of maternal and quality care services offered at Molo
sub-county hospital.
Tick (√) appropriately
How far is the nearest health facility where MCH services are provided?
a) Less than 5 Kms.

b) 5- 10 Kms.

c) More than 10 Kms. 
During these visits, how can you rank the services delivered? Tick (√) appropriately
Satisfactory [ ] Unsatisfactory [ ]
Good [ ]
Thank you so much for taking time to participate in this interview
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Appendix XI: Clinical evaluation form (Medical details) (To be filled by physician)
CLINICAL CHARACTERISTICS
PRESENT
Onset
End term
YES
Skin rash
Appetite loss
Vomiting
Oedema
Headache
Pallor
Diarrhoea
Fever
Heart burn
Herpes
Kaposi’s Sarcoma
Cryptococci
Fatigue
Pneumonia
Boils
Piles
TB
Cough
Weight loss
candidiasis
Trichomonas
PID
Sub-acute encephalitis
NO
YES
NO
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Appendix XII: Clinical staging system for HIV infection WHO (2006).
Clinical Stage I:
 Asymptomatic
 Generalized lymphadenopathy
Clinical Stage II:
 Herpes zoster
 Moderate unexplained weight loss (under 10% of presumed or measured body
weight)
 Recurrent respiratory tract infections (otitis media, sinusitis, tonsillitis and
pharyngitis)
 Angular cheilitis
 Oral ulcerations
 Papular pruritic eruptions
 Seborrhoeic dermatitis
 Fungal nail infections
Clinical Stage III:
 Weight loss >10% of body weight
 Persistent fever (intermittent or constant for longer than 1 month)
 Oral hairy leukoplakia
 Pulmonary tuberculosis
 Severe unexplained weight loss
 Severe bacterial infections (e.g. pneumonia, empyema, meningitis, pyomyositis,
bone or joint infection, bacteraemia, severe pelvic inflammatory disease)
 Unexplained chronic diarrhea for longer than 1 month
 Unexplained anaemia.
 Acute necrotizing ulcerative stomatitis, periodontitis or gingivitis.
 Oral candidiasis.
Clinical Stage IV:
 HIV wasting syndrome
 Pneumocystis jiroveci pneumonia
 Brain toxoplasmosis
 Cryptosporidiosis with diarrhoea >1 month
 Extrapulmonary Cryptococcosis,
 Cytomegalovirus disease of an organ other than liver, spleen or lymph node (e.g.
retinitis)
 Herpes simplex virus infection, mucocutaneous (>1month) or visceral
 Progressive multifocal leucoencephalopathy
 Disseminated endemic mycosis
 Candidiasis of oesophagus, trachea, bronchi
 Lymphoma (cerebral or B cell non-Hodgkin
 Non-typhoid Salmonella septicaemia
 Extrapulmonary tuberculosis
 Kaposi’s sarcoma
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Appendix XIII: Map of locality of the study (Molo Sub-county)
Source: Molo District development plan (GOK, 2008).
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Appendix XIV: Research permission letter from Molo Sub-county hospital
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Appendix XV: Ethical approval
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Appendix XVI: Research Permit