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View/Open - Kenyatta University
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: …………………………… iii 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. iv 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! v 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 vi 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 vii 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 viii 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 ix 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 xii 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 xiii 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. 80 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). 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Zinc inhibition of renin and the protease from human immunodeficiency virus type 1. Biochemistry, 30: 8717-8721. 99 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 112 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 113 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 114 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 115 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 116 Appendix XIII: Map of locality of the study (Molo Sub-county) Source: Molo District development plan (GOK, 2008). 117 Appendix XIV: Research permission letter from Molo Sub-county hospital 118 Appendix XV: Ethical approval 119 Appendix XVI: Research Permit