EFFECT OF MICRONUTRIENT SUPPLEMENTATION ON SOME INDICES OF HIV INFECTION PROGRESSION AMONG ANTIRETROVIRAL NAÏVE PATIENTS ATTENDING HIV CLINICS IN NIGERIA

Abstract

Nigeria has the second highest HIV/AIDS burden in the world with an estimated 3,391,546 people living with HIV and AIDS; an estimated 239,155 people acquired new infections in 2013; and about 174,253 people died from AIDS related illness (s) in 2014. HIV infection causes a chronic disease that progresses from stage one (seroconversion or asymptomatic) to stage four (advanced disease/AIDS) over a median period of 5 years. The course of the HIV infection is characterized by a persistent pro-oxidant state that leads to programmed CD4+ cell apoptosis, elaboration of proinflammatory cytokines and acute phase proteins, HIV RNA replications, hyper-catabolism, increased utilization of antioxidants and micronutrients, and subsequent micronutrient deficiencies. The aim of this study was to evaluate the effect of micronutrient supplementationon some indices of HIV infection progression among ART-naïve patients. To achieve the aim of the study, 90 ART naïve HIV infected adult patients with CD4+ cell count ≥520/µL were enrolled. After obtaining informed written consent, their sociodemographic data, CD4+ cell counts (cc), body mass index (BMI), vital signs, symptoms and signs of HIV infection, were obtained, and blood samples for plasma HIV RNA load (pVL), haemoglobin, and serum C-reactive protein (CRP), interleukin-6 (IL-6), albumin, lipids and micronutrients (vitamin B₁₂, zinc, and copper) were collected. After these, each patient was given a daily dose of one capsule of Synovit^TM supplements and two tablets of co-trimoxazole. Eligible patients were re-evaluated every 12 weeks, and data onvital signs, BMI, symptoms and signs of HIV infection, CD4+ cc, and haemoglobin were obtained; while bloodsamples for pVL, CRP, IL-6, albumin, lipids and micronutrients werecollected at 24 weeks and 48 weeks of Synovit supplementation respectively.Patients who defaulted from one scheduled clinic visit, became pregnant, or became eligible for ART before the 48 weeks of Synovit^TM supplementation were dropped. The socio-demographic characteristics, vital signs, BMI, haemoglobin, and serum albumin, zinc, copper and vitamin B₁₂ of the patients were compared with those of 90 age and sex-matched HIV negative apparently healthy volunteers from the same environments.Patients comprised 21 males and 69 females, aged 42.9±9.8 years and 34.6±8.6 years respectively (p≤0.05). They were mainly low income earners and were enrolled within a median of 4 months of HIV infection diagnosis. Ninety (90) patients were initially evaluated, 78 and 68 were re-evaluated at 12 and 24 weeks respectively, while 57 patients were re-evaluated at 36 and 48 weeks respectively. The sociodemographic factors that determined retention of patients on micronutrient supplementation were found to be the age, occupation and source of income of the patients. The patients‘ baseline median CD4+ cc (704.5/µL) was within reference range(365-1571/µL) for healthy Nigerians. Their vital signs (temperature=38.2±0.7^oC; pulse rate=78.0±10.1 beats/minute; respiratory rate=12.0±3.0 breaths/minutes; systolic blood pressure=128.0±13.7 mmHg) were higher than those of healthy controls (temperature=36.8±0.2^oC; pulse rate=72.0±21.1 beats/minute; respiratory rate=16.0±1.0 breaths/minutes; systolic blood pressure=138.0±37.4 mmHg) (p≤0.05). Although theywere overweight (median BMI of 25.8kg/m²), their baseline median haemoglobin (11.0 g/dL), serum albumin (36.0g/L), zinc (0.01ppm), copper (-0.4ppm), vitamin B12 (15.0ng/L) and total cholesterol (4.6mmol/L)were significantly lower than those of healthy volunteers (p≤0.05). Their serum CRP (27.8 ng/ml), and IL-6 (1.9 pg/ml) were also significantly higher than reference values (p≤0.05).Micronutrient supplementation significantly decreased incidence of opportunistic illnesses (OIs), pVL, CRP and IL-6; and increased haemoglobin, serum zinc, copper, vitamin B12, total cholesterol and high density lipoproteins (p≤0.05); but did not increase CD4+ cc. The pVL maintained significant negative correlations with CD4+ cc, zinc and copper (p≤0.05), and positive correlations with IL-6 and CRP (p≤0.05). Significant predictors of HIV RNA load at baseline were zinc(p=0.00) and IL-6 (p =0.05); while serum IL-6 (p=0.00) and haemoglobin (p=0.02) were the predictors at 24 weeks and 48 weeks of micronutrient supplementation respectively. In conclusion,micronutrient supplementation enhanced retention of patients on the pre-ART care and support service programme and reduced the indices of progression of HIV infection with the exception of CD4+ cell counts.

CHAPTER ONE

INTRODUCTION

1.1    Human Immunodeficiency Virus(HIV)

1.1.1 Family and types

HIV belongs to the virus family Retroviridae and subfamily Lentiviridae. Lentiviruses typically cause a slowly progressive disease with prolonged subclinical infection (Yuet al., 2005). HIV comprises HIV-1 and HIV-2. HIV-1 is closely related to a naturally occurring virus of sub-species of chimpanzees called SIVcpz, while HIV-2 is closely related to SIVsm, a virus that naturally infects sooty mangabey monkeys (Diopet al., 2002). In contrast to HIV-1, there has been little international spread of HIV-2 despite the fact that the two viruses share similar modes of transmission. Most HIV-2 infections were reported in West Africa, and only few cases were identified in other countries (Kankiet al., 1997). Co-infection of HIV-1 and 2 can occur but recombinants of the 2 types were reported to be rare (Kankiet al., 1997).

1.1.2 Structure

Electronmicroscopicstudies showed that HIV has a cylindrical structure consisting of viral genome, core proteins and lipid bilayer membrane & envelopes (Yu et al., 2005). The viral genome is diploid, with a 60-70s complex of two identical RNA copies. The genome is flanked on both sides by long terminal repeat sequences that contain gag, pol and envstructural genesthat code for capsid proteins, viral enzymes and envelopes respectively. The genome has five other regulatory & accessory genes: tat, rev, nef, vif&vpu(HIV-1) or vpx (HIV-2). The viral enzymes are the reverse transcriptase (RT), protease (PR) & integrase (IN) which are encoded by pol geneThe core protein is made up of capsid protein (p24) and nucleocapsid protein (p18) encoded by the gag genes. The envelopes surround the core proteins that enclose the viral RNA genome and enzymes. The envelopes consist of external surface (gp 120) and transmembrane (gp 41) that connects to the core proteins. The gp 120 contains binding sites for CD4+ receptor and co-receptors. The gp41 tranverses through the lipid bilayer and both arepartially acquired from the surface of host cells as mature virions are released. Figure 1 below shows a schematic structure of HIV.

Figure 1.1:    Human Immunodeficiency Virus

1.1.3 Genetic Diversity

HIV strains can be classified into three groups – major (M), non-major (N) and outlier

• (McCutchan, 2000). Group M, the most common globally, is further divided into nine subtypes (A, B, C, D, F, G, H, J and K) and several circulating recombinant forms (CRFS). Subtype C has been documented to account for more than 50% of prevalent global HIV-1 infections and a higher proportion of new infections, particularly in China and the African continent; subtypes A, C and D were found more frequently in India and Central, Southern and the Horn of Africa; subtype B strains were only isolated from the USA and Western Europe through 1994, and subtype A/E recombinant were observed in South-East Asia (McCutchan, 2000). In Nigeria and West Africa, the dominant subtype was the CRF 02/A/G; while HIV-1 N and O groups were rare and limited to Cameroon and neighbouring countries (Kanki, et al., 1997; McCutchan, 2000).

1.2Clinical Course of HIV Infection

1.2.1   Determinants of clinical course from infection to disease and AIDS

Entry of HIV into immune cells activates initial anti-inflammatory T cell response in an attempt to curtail HIV replication and viraemia. However, continued elaboration of anti-inflammatory cytokine cascade eventually becomes detrimental to the HIV infected person as HIV-1 stimulates CD40L on infected macrophages and monocytes to increase pro-inflammatory cytokines (such as interleukin-1 beta (IL-1ß), tumor necrosis factor- alpha (TNF-α) and interleukin (IL-6)), which in turn, directly increase the expression of interleukin-2 (IL-2) receptors on T cells and thymocytes leading to more T cell activation, growth, differentiation and programmed apoptosis (Poliet al., 1990). IL-6, with or without IL-1ß and TNF-α, mediate B cell differentiation, maturation and synthesis of antibodies. Antibodies indirectly stimulate T cell apoptosis, IL-6 secretion and HIV-1 replication. This vicious cycle between IL-6 secretion and HIV replication is responsible for increased viraemia, hypergammaglobulinaemia, lymphopaenia, and elevation of acute phase reactant proteins (Doueket al., 2009). This phenomenon has also been linked to the immune reconstitution inflammatory syndrome (IRIS) and HIV encephalopathy (Conollyet al., 2005). Thus the two antagonistic features of HIV infection, which are progressive increase in HIV RNA replication and a corresponding progressive decrease in the quantity and quality of the infected patient‘s CD4+ lymphocytes and other immune cells, are responsible for the cascade of events that are characterized by a profound cell mediated immune deficiency, cytokines dysregulation, inappropriately elevated acute phase proteins, gammaglobulins and superoxides, hypercatabolism and emergence of various forms of opportunistic infections (OIs) and diseases (ODs) (Longo and Fauci, 2010). The HIV infected person thus requires 10% to 15% more energy per day and approximately 50 to 100% more protein than the recommended 2070 kcal/ day of energy and 57 grams/day of protein for apparently active HIV negative adult (Piwoz and Preble, 2000; Marston et al., 2004).

1.2.2 Types of HIV infection progressors

1.2.2.1Typical and rapid progressors

In more than 80% of HIV infected persons, programmed CD4+-cell apoptosis will persist from either the direct effect of HIV, or HIV-induced chronic inflammation and concomitant antioxidant imbalances in the host cells (Lelievreet al., 2004); and the rate of CD4+- cell apoptosis will then determine whether theHIV infected individual can

4develop AIDS within the median time of ten years (typical progressors) or within 2–3 years of infection (called rapid progressors) (Kirchhoff et al., 1995).

1.2.2.2Long- term non progressors

In about 5–10 % of HIV-infected persons, the virus is contained in the lymphoid tissues with preservation of the lymph node architecture, and little or no viral replication and/or propagation of virions to the CD4+- cells (Sanchez et al., 1997). Such individuals will, therefore, have stable CD4+-cell counts, low HIV RNA load in the blood, and slow disease progression despite years of HIV infection. These individuals, who are called long- term non progressors (LTNP), can remain asymptomatic for about 7–20 years after infection with stable CD4+ cell counts and low plasma HIV RNA, without been on ART (Sanchez et al., 1997). Currently, they are divided into two groups based on viral load calculations/standards. Those with undetectable viremia (HIV RNA<50 copies/ml) are called elite controllers, while those with low but detectable viral load (HIV RNA <2000 copies/ml) are known as viremic controllers (Okuliczet al., 2009).

1.2.3Inter-and intra-population variability

The course of HIV infection has been noted to vary within a population and between populations due to a combination of socioeconomic and immunological factors. These factors, particularly, genetic make- up, nutrition, and exposure to environmental pathogens, are responsible for inter- and intra-population variability and differences in CD4+ cell levels, both in HIV infected and HIV negative populations; and are the major limitations to the universal acceptability of the CD4+ for HIV classification and staging (WHO, 2005). This variability is demonstrated by the results of the reference CD4  values of some healthy populations reported thus: 500/µl to 2,000/µl for WesternEuropean countries (Reichert et al., 1991); 559/µl to 2,333/µl for Ugandans (Tugumeet al., 1995); 430/µl to 1740/µl for Indians (Uppalet al., 2003); and 365/µl to 1571/µl for Nigerians (Oladepoet al., 2009) respectively. In sub-Saharan Africa (SSA), clinical latency and progression to AIDS have been reported to be shorter due to constant exposure to many pathogens and malnutrition (Enwonwu, 2006).

1.3       Micronutrient Supplementation in HIV infection

Micronutrients function mainly as anti-oxidants and immune-modulators in HIV infection. Vitamin A, in the form of retinol or retinoic acid, improves immunity by modulating the growth and function of T-cells, B-cells and natural killer cells and their products. Vitamin C (ascorbate) is a potent antioxidant which can resonate between the free radicals (Asc-) and ascorbate (AscH-)(Nikiet al., 1995). Ascorbate is readily regenerated from Asc- through NADH or NADPH-dependent reductases. Ascorbate can also regenerate other antioxidants such as glutathione (GS⋅) and vitamin E by neutralizing their radical form (Nikiet al., 1995; Durak, 2014). Vitamin E is also an antioxidant which inhibits CD95 (APO–1/Fas) ligand expression (part of TNF receptor which T-cell uses to undergo apoptosis) and protects T-cell from activation-induced cell death of the CD95/CD95 ligand system of T-cells (Li-Weber et al., 2002). It therefore stimulates CD4+-cell and IL-2 proliferation and completely blocks NFκB induced programmed T cell apoptosis (Li-Weber et al., 2002, Durak, 2014). Trace elements such as copper assist vitamin E in down-regulating T-cell destruction by stimulating maturation and mobilisation of granulocytes in peripheral blood and proliferation of T-cells (Percival, 1998). Selenium enhances synthesis and expression of selenoproteinsantioxidants such as thioredoxin reductase, glutathione peroxidase, and phospholipids hydroperoxide in T-cells, thereby boosting cellular immunity (Baum et al., 2000; Moghadaszadehet al., 2006; Kamwesigaet al., 2011). It binds to and activates thymulin, a thymic peptide responsible for the maturation and differentiation of immature thymocytes thereby enhancing expression of IL-2, T-cell proliferation and function (Baum et al., 2000; Moghadaszadehet al., 2006). Zinc influences gene expression by structural stabilisation of different immunological transcription factors (Prasad, 2007). For example, it induces cytokines, including IL-1, IL-6 and TNF- α and influences NK cell-mediated killing (Fleigeret al., 1995). It also inhibits NFκβ and TNF-

• thereby modulating cytolytic T-cell activity. It stabilizes the thiol groups and phospholipids in biological membranes and protects them against oxidative stress (Fleigeret al., 1995). Zinc therefore has both an enhancing and inhibiting activity depending on its concentration in the surrounding tissues.

The importance of nutritional interventions in the form of micronutrient supplementations as a tool to combat malnutrition and encourage patients to remain on treatment was recognised in 1984 by the Food and Nutrition Division, Food and Agriculture Organization (FAO) of WHO (William, 1985). This made the United Nations to add the following Millennium Development Goals (MDGs) to the HIV public health response (United Nations, 2006):

MDG 1 (reducing extreme poverty and hunger),

MDG 4 (reducing child mortality),

MDG 5 (reducing maternal mortality) and

MDG 6 (halting and beginning to reverse the spread of HIV by 2015).

At the expiration of MDGs, the following Sustainable Development Goals (SDGs) were also pursued as part of the HIV public health response (IISD, 2016; WHO/UNAIDS, 2016):

SDG 1 (end poverty in all its forms everywhere),

SDG 2 (end hunger, achieve food security and improved nutrition and promote sustainable agriculture),

SDG 3 (ensure healthy lives and promote well-being for all at all ages), and SDG 12 (ensure sustainable consumption and production pattern by 2030)

1.4 Statement of the Research Problem

The fight against HIV/AIDS has posed enormous challenges worldwide, generating fears that success in its control may be too difficult or even impossible to attain (Barnet and Whiteside, 2002; NACA, 2015). By the year 2000, more than 70% of the PLWHAs live in Africa, but less than 20% were receiving HIV treatment (WHO/UNAIDS, 2016). But between 2005 and 2014, many countries in the African Region overcame formidable constraints to build and sustain national public health programmes powerful enough to turn the tide against the HIV pandemics by bringing HIV services closer to communities and scaling up HIV treatment programmes, using the public health approach recommended by WHO (WHO/UNAIDS, 2016). Yet, major challenges remain in SSA, particularly in some large countries with high HIV prevalence, because both the coverage and quality of HIV services are still insufficient(WHO/UNAIDS, 2016). For instance, in Nigeria where large scale use of ART started in 2000 (Idigbeet al., 2003), in 2014, about 1,665,403 HIV infected persons (1,454,565 adults and 210,838 children respectively) were in need of ART using the eligibility criteria of CD4 of 350/µL (NACA, 2015). In addition, the number of persons who do not know their HIV status, or the number of pre-ART and post-ART patients who default from care, follow-up and treatment respectively remain high (WHO/UNAIDS, 2016). These gaps in the HIV prevention and treatment response have been attributed to factors such as socio-cultural beliefs and practices, low literacy level, poverty, ignorance (Obiako et al., 2010; Obiako et al., 2012) , ART-related toxicities and ART-failures (Obiako et al., 2011; Obiakoet al., 2012; Ogoinaet al., 2012).

The greatest challenges to the HIV prevention and treatment response today are: how to get individuals to do periodic HIV screening test for early diagnosis; how to persuade all HIV infected persons to start ART and remain on ART to avoid development of ARV resistance; and how to retain pre-ART HIV infected persons on care until they are ready for ART. Closing these observed gaps will require action and innovative goals such as those enunciated and crystallized into a set of global Fast-Track targets by the Joint United Nations Programme on HIV/AIDS and WHO (WHO/UNAIDS, 2016).These actions include: getting 90% of PLWHAs to know their HIV status; making 90% of those diagnosed with HIV to initiateand remain on ART, making 90% of HIV infected persons not ready to initiate ART to remain on care until they are ready to start ART, and getting 90% of the people receiving ART to have suppressed viral loads by 2020. To achieve the first target will require extensive HIV testing and counseling (HTC) services in communities through provision of free condoms and services such as sexual transmitted infection(STI) clinicsand marriage counseling. To achieve the second, third and fourth targets will require provision of facilities for pre-ART and ART services. These services which include nutritional intervention services, micronutrient supplementation, prophylaxis and treatment of opportunistic infections/ diseases, ARV adherence counseling and toxicity monitoring willincrease adherence and prevent or reduce development of ARV resistance.

1.5 Justification for the Study

Micronutrients (essential trace elements and vitamins) have been shown to be important in maintaining immune function and neutralizing the reactive oxygen intermediates produced by activated macrophages and neutrophils in their response to microorganisms (Oguntibejuet al., 2009). Their role as antioxidants and immune boosters for the optimization of health and prevention and/or treatment of chronic diseases such as HIV/AIDS was recognised from observational studies in some developed countries,which showed that deficiencies of vitamins and trace elements were common among HIV-infected patients (Sullivan et al., 1998; Tang et al., 2002). Studies also showed that diet rich in carbohydrates, proteins, fats, and antioxidant vitamins and minerals were associated with reduced risk of HIV progression to AIDS and death (de Luis Roman et al., 2001).Other studies in some developing countries such as Thailand (Jiamtonet al., 2003), Malawi (Bowie et al., 2005), and Vietnam (FANTA III Project, 2014), also supported the beneficial effects of micronutrient supplementation in HIV/AIDS. These studies revealed that HIV infected patients who were treated with a multi-micronutrient supplement (Jiamtonet al., 2003), or were given food rations (Bowie et al., 2005), or Ready-to-Use Therapeutic Food (RUTF) (Weiringaet al., 2013; FANTA III Project, 2014) showed significant improvement in nutritional status, weight gain and reduction in morbidity and mortalitythan those who were given placebo. A report of the global analysis of delays from ART eligibility to ART initiation among adults with HIV/AIDS from 1996 to 2006, revealed that nutritional intervention programmeshelped to retain many HIV positive persons who could have dropped-out of care before they became eligible for ART(CUNY, 2015). These studies,therefore, suggest that food supplementation could offer a simple and relatively inexpensive strategy to slow HIV progression, and improve quality of life of HIV infected persons, particularly in resource-limited countries where poverty and hunger are prevalent (de Lius Roman et al., 2001; Marston et al., 2004).The importance of food supplementation in HIV/AIDS was summarised by Clay William of the Food and Nutrition Division, Food and Agriculture Organization (FAO) of WHO, who said that ―Food isn‘t a magic bullet. It won‘t stop people from dying of AIDS but it can help them live longer, more comfortable and productive lives‖ (William, 1985). Although malnutrition among HIV infected patients have been documented in Nigeria (Obi et al., 2010; Sudawaet al., 2013; Anyaboluet al., 2014), and other African countries such as South Africa (Evans et al., 2012), Ethiopia (Gedleet al., 2015), and Uganda (Lwangaet al., 2015); and despite abundant evidence that nutritional intervention reverted malnutrition (Jiamtonet al., 2003; Bowie et al., 2005; Weiringaet al., 2013; FANTA III Project, 2014), and helped to retain many HIV positive persons on pre-ART care (CUNY, 2015);there is dearth of data on the effect of micronutrient supplementation on HIV infected adolescent and adults in Nigeria. Similarly, the lack of data on the effect of micronutrient supplementation on indices of HIV infection progression among antiretroviral –naïve adult patients necessitated the rationale for this study among ART- naïve patients attending HIVclinics in Zaria and Kaduna.

1.6Aim and Objectives of the Study

1.6.1Aim

The aim of the study is toevaluate the effect of micronutrient supplementation on some indices of HIV infection progression in HIV infected ART-naïve patients attending HIV clinics in Ahmadu Bello University Teaching Hospital (ABUTH) Shika Zaria and St Gerard‘s Catholic Hospital (SGCH)Kakuri Kaduna.

1.6. 2      Specificobjectives

1. To determine the sociodemographic characteristics of HIV infected ART-naïve patients and the effect of these on their retention on pre-ART care with micronutrient supplementation
2. To determine the effect of micronutrient supplementation on vital signs, BMI and incidence of opportunistic diseases and infectionsin HIV infected ART-naïve patients

• To determine the baseline nutritional status (zinc, copper, vitamin B₁₂, haemoglobin, albumin and lipids) of HIV infected ART-naïve patientsin comparison with those of age-and sex-matched HIV negative healthy volunteers

1. To determine the effect of micronutrient supplementation on some nutritional indices(zinc, copper, vitamin B₁₂, haemoglobin, albumin and lipids) of HIV infection progression in HIV infected ART-naïve patient

1. To determine the effect of micronutrient supplementation on plasma HIV RNA loadand some immunological indices (CD4+ cc, IL-6 and CRP) of HIV infection progressionin HIV infected ART-naïve patients

1. To determine the relationships between HIV RNA load and other indices of HIV infection progression before and with micronutrient supplementationin HIV infected ART-naïve patients

1.7 Research Question/Hypothesis

Micronutrient supplementationdoes not significantly affectthe progression of HIV infection in HIV infected ART-naïve patients

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