EFFECT OF MATERNAL VITAMIN A SUPPLEMENTATION ON INFANT MALARIA PARASITAEMIA AND MORBIDI

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction

Malaria is a major public health problem and cause of suffering and premature deaths in tropical and sub-tropical countries (Cheesbrough, 1998). It is endemic in 91 countries with about 40 % of the world’s population at risk (WHO, 1995, 1996).

Annually, 300-500 million clinical cases of malaria occur. Ninety per cent of these are found in Africa. This results in 1.5 – 2.7 million deaths, mostly children under 5 years (WHO, 1995, 1996). There are at least 500 million cases of clinical malaria by Plasmodium falciparium each year in the world (Snow et al., 2005), that result in 1 million deaths in Africa alone (WHO, 2003; Taylor and Molynuex, 2003).

On the average, each African child experiences one clinical attack of malaria per year and this represents about 200 million episodes of clinical malaria annually (Taylor and Molyneux, 2003).

Malaria in pregnancy is a common cause of severe maternal anaemia and low birth weight babies (Taylor and Molyneux, 2003). Low birth weight is one of the main risk factors for infant mortality (McCormick, 1985) and this is seen in all parities of non-immune women. In the area of moderate or high transmission, the association between placental malaria and a reduction in birth weight is most apparent in primigravidae, the effect decreasing as parity increases, though an increased risk in grand multiparae has also been described (Morley et al., 1964; Greenwood et al., 1989).

Maternal Vitamin A deficiency is increasingly being recognized as a major public health problem in many developing countries, but its consequences have so far been assumed to be mainly related to infant health status, morbidity and mortality. Vitamin A deficiency disorder affects large numbers of young children and women of child- bearing age throughout the developing world.

Older reports (Sommer, 1982., Dixit, 1996) and recent surveys (Katz et al., 1995; Christian et al., 1998) indicate that night blindness from vitamin A deficiency is common among pregnant women in India, Indonesia, Bangladesh, Nepal and elsewhere, particularly during the later half of pregnancy.

However, Vitamin A supplementation has become a successful remedy to vitamin A deficiency especially in many developing countries with poor dietary habits. Reports of reduction in infant malaria parasitaemia and morbidity by vitamin A supplementation are scanty. However, childhood diseases such as measles, diarrhea and respiratory diseases are being reported as being taken care of by the supplementation. Shankar et al., (1997) reported a significant vitamin A reduction of malaria attack of 20 – 50 % in a randomized placebo-controlled clinical trial in New Guinea while Barreto et al., (1994) reported a reduction in the severity of diarrhoea disease by vitamin A supplementation. Because of the dearth of information in this topic, this research aims at bridging the information gaps.

1.2: Aims and Objectives of the Study

The study aims to determine the effects of the supplement (vitamin A), on malaria parasitaemia and morbidity of:

- Infants born by women at different months of pregnancy, starting from the 6^th month.

- Infants born by women of the above – mentioned ,at different parities and

(b) - To determine the effects of the supplements on the weights of the infants mentioned above.

1.3 Justification of the Study

The study is worth carrying out because if the supplement (vitamin A) is efficacious in reducing infant malaria parasitaemia and morbidity, it can be recommended to be included as one of the prophylactic measures in malaria control programmes in children.

The supplement will render dual purpose services, since apart from its effects on malaria parasitaemia and morbidity; it will be nutritionally beneficial to pregnant mothers in reducing night blindness which occurs in many poor rural villages; as a result of poor dietary habits.

1.4: LITERATURE REVIEW

Vitamins are complex organic compounds which must be present in the food in minute amounts to enable growth, health and life to be maintained (Keele and Nail, 1971). They are found in plants and animals and in foods made from them and are needed in small amounts for the proper functioning of organism’s chemical process, or metabolism (Graham et al., 1997). Depending on solubility properties, vitamins are divided into two groups – the ones that are soluble in fats (Vitamin A, D, E and K), called the fat soluble vitamins and those soluble in water (vitamin B and C) known as the water soluble vitamins.

Vitamin A, a fat soluble vitamin, is a term comprising two forms: a retinoid, a preformed vitamin A originating from animal tissue, and, Beta –carotene, a provitamin A compound originating from plant tissue. Beta – carotene is a caroteniod that occur naturally in foods such as carrots, meats and dark green leafy vegetables. Beta carotene is converted in the body to the retinol form of vitamin A and this conversion does decreases substantially when large amounts of Beta carotene are ingested (Eugene et al., 1996). This therefore implies that beta-carotene should not lead to toxicity or teratogenicity because the conversion to retinol form is actually decreased with increasing amount of beta – carotene.

Vitamin A is transferred from the mother to the embryo across the placenta; vitamin A concentrations in fetal blood are approximately half of those in the mother (Azais – Braesco and Pascal, 1998). Retinol binding protein (RBP) is involved in this transfer from mother to embryo; nevertheless, its specific metabolism and the existence of other yet unknown binding proteins in maternal blood, the placenta, and foetal blood requires further study (Sklan et al., 1985; Dancis et al., 1992; Torma and Vahlquist, 1986).

Vitamin A is essential for the normal functioning of the retina and for growth and differentiation of epithelial tissue as well as necessary in embryonic development, reproduction and bone growth (Eugene et al., 1996). Vitamin A is transferred in two ways from mother to offspring: via the placenta during gestation, and via the mammary gland (breast milk) during lactation. Adequate transfer of Vitamin A is essential during both of these periods of development. Animal models of Vitamin A deficiency have shown that maternal Vitamin A deficiency during pregnancy results in placental dysfunction, stillbirth, and congenital malformations (Stoltzfus, 1994). Maternal Vitamin A deficiency during lactation rapidly disposes the nursling to severe Vitamin A deficiency (Stoltzfus, 1994).

Vitamin A deficiency is defined by the WHO as the tissue concentration of Vitamin A low enough to have adverse health consequences even if there is no evidence of clinical xerophthalmia (WHO, 1996). It is estimated that Vitamin A deficiency, defined as a low serum retinol concentration (<0.70 µmol/L), affects 190 million pre-school age children and 19.1 million pregnant women, the majority in Africa and South-East Asia (WHO, 2009). Sub-clinical Vitamin A deficiency (low plasma retinol or liver stores in the absence of ocular signs is associated with increased mortality from infection (McLaren and Frigg, 2001).

Improving Vitamin A stores during infancy will help to protect children from xerophthalmia during weaning. The weaning period is typically when young children are at risk of xerophthalmia because breast milk is replaced by foods that are often low in Vitamin A (Stoltzfus, 1994). Furthermore, weaning food may be contaminated with infectious agents, putting the weanling child at the risk of infectious diseases that may precipitate xerophthalmia. Vitamin A stores accumulated during breastfeeding provide a margin of safety during this nutritionally vulnerable transition period (Stoltzfus, 1994)

Vitamin A deficiency in animal models of malaria infection is associated with increased parasitaemia and increased mortality (Krishnan et al.,1976; Stoltzfus et al., 1989) and in apparent agreement with this, a randomized controlled trial of Vitamin A supplementation showed a decreased risk of clinical malaria in children in Papua, New Guinea (Shankar et al., 1999) although no effect was reported in an earlier trial in Ghana (Binka et al., 1995) and one study observed that even low baseline Vitamin A status was associated with increased risk of parasitaemia (Sturchler et al., 1987).

Vitamin A has been reported to be often deficient in individuals living in malaria endemic areas, and is known to be essential for normal immune function and has been suggested to play a part in potentiating resistance to malaria (Shankar et al., 1999). Pregnant women are at particular risk of Vitamin A deficiency (West, 2002). And are also at increased risk of pregnancy associated complications including maternal anaemia, stillbirth, and low birth weight (McGregor et al.,1983; Steketee et al., 2001). Vitamin A seems to decrease the severity of some episodes of diarrhoea in children especially when the administration is in combination with zinc (Rahman et al., 2001). A slight significant reduction of active placental malaria infection at delivery was reported in a study from Ghana, when Vitamin A supplementation was administered in pregnancy (Cox et al., 2004). Micronutrient supplementation may cause an increase in maternal appetite, which may lead to increased food intake and/or reduced morbidity. Deficiency in one or more micronutrients may be due to inadequate food intake, poor dietary quality, or when micronutrients are not readily released from foods, not absorbed efficiently, or a combination of these factors (Pojda and Kelly, 2000).

Since Vitamin A deficiency can occur sub-clinically, and could at that level, render the risk groups (pregnant women and children) susceptible to various infectious diseases, supplementation with high doses was recommended. As at 1997, supplementation of women with 200,000 IU Vitamin A postpartum came into action in greater than or equal to 15 countries, as a national programme, recommended by the WHO for all areas where Vitamin A deficiency is a public health problem (WHO/UNICEF/IVACG, 1997). Recently, the WHO through its informal technical consultation recommended that the postpartum dose be increased to 400,000 IU administered twice as 200,000 IU each, within the safe infertile postpartum period (Ross, 2002).

Recent experiments using animal and various cell lines suggest that vitamin A and related retinoids modulate many different immune response elements, including expression of keratins and mucins, lymphopoiesis, Apoptosis, cytokine production, function of neutrophils, natural killer cells, monocytes or macrophages, T-lymphocytes and B lymphocytes, and production of immunoglobulin (Ig) (Semba, 1998).

Liver vitamin A stores are usually sufficiently high to withstand low or no vitamin A supply for a limited period, provided that usual intake is adequate. Therefore, more emphasis should be placed on vitamin A status than on vitamin A intake, yet this is difficult to do because of lack of a satisfactory biomarker for accessing the vitamin A status of individual or population except in the case of extreme hypovitaminosis A (Underwood, 1994).

Vitamin A deficiency has great consequences during pregnancy and early childhood. Vitamin A deficiency disorders (VADD) encompass the full spectrum of clinical consequencies associated with sub-optimal vitamin A status. These disorders include reduced immune competence, resulting in increased morbidity and mortality (largely from increased severity of infectious diseases); night blindness, corneal Ulcers, Keratomalacia and related ocular signs and symptoms of xerophthalmia; exacerbation of anaemia through sub- optimal absorption and utilization of iron, and other conditions not yet fully identified or clarified (e.g retardation of growth and development) (Sommer and West, 1996).

Clinical and sero-epidemiological studies and surveys indicate that vitamin A deficiency is widespread throughput the developing world. Studies in Africa where it has been less well recognized indicated that a large proportion of paediatric blindness was due to acute deterioration in vitamin A status during measles and similar childhood infections (Chirambo and Ben Ezra, 1976; Foster and Sommer, 1986). The extent and distribution of vitamin A deficiency and its consequences are remarkedly well established. Numerous local and nutritional surveys have been conducted. In countries where they have not been conducted, data from nearby countries with similar characteristics (under – 5 year mortality, poverty, diet) allow for judicious extrapolation (Sommer, 2001). Vitamin A deficiency is a serious public health problem in Nigeria. Nigeria has been identified by the World Health Organization as one of the countries with the highest risk of vitamin A deficiency (Adelekan and Adeodu, 1998).

The prevalence of marginal Vitamin A deficiency is reported to be 50 % in Nigeria, compared with 33 % in South Africa, 66 % in Zambia and 20 % in Namibia (Vitamin Information Centre, 2002).

Intervention trial shows that vitamin A deficiency poses a significant problem in more than 70 countries (Grant, 1995). Recent calculations suggest that roughly 150 million children are deficient and that 10 million children develop xerophthalmia, while 500,000 children are permanently blinded from xerophthalmia and 1 to 2 million children die unnecessarily (Humphrey et al., 1992). In 1995 the WHO estimated that 250 million children were at risk of vitamin A deficiency.

Children begin life with an urgent need for vitamin A. Full term infants – even those of well nourished mothers in wealthy countries are born with barely enough vitamin A to sustain them during the first few days of life. During the first six months of life, they need at least 125 mg of retinol equivalent(RE) daily to prevent xerophthalmia and about 300 mg to thrive (and accumulate adequate liver stores of 20 mg per gram of liver) (Humphrey and Rice, 2000).

Breast milk (or equivalent formulas) is the only significant source of vitamin A to young infants. Except when mothers suffer from severe protein energy malnutrition, the quantity of breast milk is similar around the globe, but the concentration of vitamin A in that milk varies dramatically with the vitamin A status of the mother (Humphrey and Rice, 2000; Stoltzfus et al., 1993). When mothers are vitamin A deficient, breast milk concentrations will be low and without supplementation with vitamin A, their children will become deficient.

Children in developing countries are at risk of consuming a vitamin A deficient diet throughout life, not just during early infancy. The reason is that as their counterparts in the developed countries receive abundant preformed vitamin A from animal products, they rely on beta-carotene, a precursor of vitamin A found in dark green leafy vegetables, carrots, and colour fruits (mangoes and papaya). The beta -carotenes are poor substitutes for animal source of the preformed vitamin. In addition to being poor sources, many children do not like to eat vegetables; fruits are often costly or highly seasonal, and many vegetables bind beta – carotene tightly to their cellular matrices, yielding little during digestion. It has been recently reported that the bio-availability and bio- conversion of dark green leafy vegetable sources of beta – carotene is lower than previously supposed (de Pee et al., 1995) with perhaps no more than 2 % to 4 % being absorbed, converted to vitamin A, and made available to meet metabolic needs. Children in developing parts of the world need more vitamin A than do their counterparts. This is because diarrhea, childhood exanthematous diseases and respiratory infections are more common in poor rural population, thereby enhancing the reduction of Vitamin A absorption (diarrhea) while increasing utilization (measles) and excretion (respiratory infections). Women in developed countries are often vitamin A deficient because they feed on unvaried diets that are deficient in good source of preformed vitamin A. Pregnancy and lactation place additional burden on their meager vitamin A store. Other consequences of pregnancy probably explain why deficiency is more severe and night blindness most common during the later half of pregnancy (Azais –Braesco and Pascal, 1998). Correcting mild to moderate vitamin A deficiency at the community level is thought to lead to at least 23 % reduction in mortality rates among young children (Beaton et al., 1993). To access community risk of vitamin A deficiency, the Hellen Keller International Food Frequency Method is the simplest and most innovative method (Rosen et al., 1993). This method is based on weekly intakes key foods among pre-school children (i.e. diet of children, 1-6 years of age). A score is assigned to each child based on the number of animal or plant sources rich in vitamin A that were consumed during the past week, ignoring amounts. Nearly all the food taken into consideration contain at least 100 retinol equivalent (RE) per 100g. Food frequency methods have been shown to have predictive power in relating the intake of food to risk of disease (Willet et al., 1995).

To amend vitamin A deficiency in pregnancy, vitamin A supplementation should be made available to the pregnant mothers according to the World Health Organizations (WHO, 1998) Recommended Dietary Allowances (RDAs). According to WHO (1998), during pregnancy a daily supplementation should not exceed 10,000 IU (International Unit) or (3,000 RE) (Retinol Equivalent) and a weekly supplementation should not exceed 25,000 IU (7,500 RE). The foetus starts to accumulate Vitamin A during the third trimester of pregnancy, and needs several months of sufficient intake after birth to build up an adequate hepatic store (Ortega et al., 1997) . When the baby is breast fed, the vitamin A content of the breast milk is of primary importance. The composition of breast milk is influenced by the vitamin A status and serum concentration of the mother during the last trimester of pregnancy (Ortega et al., 1997). Colostrum and early milk are extremely rich in vitamin A and even the milk of a mildly undernourished woman may meet the physiological needs of the new born during the first weeks (West et al., 1986; Humphrey et al., 1992). After this time, however, a rapidly- growing infant may exhibit negative vitamin A balance, with severe consequences for health.

Malaria is prevalent in all countries extending from 40 ⁰S to 60 ⁰N of the equator, covering a large proportion of the tropical and sub-tropical regions. Highly endemic areas are often seen in the tropical regions where humidity and temperature are favourable for breeding of the anopheline mosquitoes and growth of the parasite in the insect vector (Ichhpujani and Bhatia, 2005). Malaria, the most important parasitic disease in the world today, is disproportionately prevalent in tropical Africa, where approximately 10 % of the world’s population suffers more than 90 % of the world’s malaria infections (Anderson and May, 1991).

The sub-Saharan African region has the greatest number of people exposed to malaria transmission and the highest malaria morbidity and mortality rates in the world (WHO, 1996). Estimates from the WHO in 2008 showed that 243 million cases of malaria (around 90% caused by P. falciparum) resulted in 863,000 deaths of which more than 80% occurred in children younger than 5 years of age in sub-Saharan Africa (WHO, 2009). Malaria affected 3.3 billion or half of the world’s population, in 106 countries and territories (US Embassy in Nigeria, 2011). Thirty countries in sub-Saharan Africa account for 90% of the global malaria deaths. Nigeria, Democratic Republic of Congo (DRC), Ethiopia and Uganda account for nearly 50% of the global malaria deaths. Malaria ranks second to HIV/AIDS as being cause of mortality, resulting in 1 out of every 5 child deaths. It accounts for 60% of outpatient visits and 30% of hospitalizations among children under five years of age in Nigeria (US Embassy in Nigeria, 2011).

Plasmodium falciparum is responsible for most malaria- related deaths worldwide and is the predominant Plasmodium species in sub-Saharan Africa. Young children have the highest morbidity and mortality in malaria stable transmission areas because they do not have acquired protective immunity that is sufficient against severe disease. Immunity to malaria occurs over several years of exposure to repeated infections and it may take years to develop immunity to the full range of antigenic variations of the parasite (Baird, 1998). The immune responses against the parasite consists of antibody- mediated immunity, cell- mediated responses, and non- specific immunity. But complete immunity against re-infection is unknown (Semba, 1999).

The life cycle of Plasmodium begins with the inoculation of the sporozoites in the saliva of a biting anopheline mosquito, into the blood of the human host, and within minutes, the sporozoites reach the liver and invade the hepatocytes. During the pre-erythrocytic cycle in the liver, a sporozoite can develop into a mature schizont, which can burst, releasing thousands of merozoites into the bloodstream. The merozoites infect human red blood cells and initiate the erythrocytic cycle. Also, in the erythrocytic cycle, the merozoites mature into blood schizonts which burst, producing eight to thirty two new merozoites that can invade more erythrocytes. The rupture and destruction of the red blood cells lead to the development of symptoms, such as headache, fever, and malaise. Within 10-12 days, the sexual forms of the parasite (male and female gametocytes) appear in the peripheral blood and can be taken up by the mosquito during a blood meal. In the mosquito, the gametocytes undergo development into zygotes, ookinetes and oocysts, and finally sporozoites. The cycle is completed by the inoculation of the sporozoites in the saliva of the mosquito during another blood meal.

Malaria infection in pregnancy has been reported to be highest among the primigravidae and secundigravidae with lower rates in later pregnancies (Brabin, 1991). Malaria infection in pregnancy leads to parasite sequestration in the maternal placental vascular space, with consequent maternal anaemia (Shulman, 1996) and infant low birth weight (McGregor, 1984; Sullivanet et al., 1999) due to both prematurity (Steketee et al., 1996) and intrauterine growth restriction (Brabin, 1991).

Malaria in infants aged under six months is not a rare occurrence in endemic areas and its burden may be underestimated (D’Alessandro et al., 2012). Awareness by health professionals should increase so that any infant aged under six months brought to a health facility in a malaria endemic area, with unexplained fever should be systematically screened for malaria. Current policy for malaria diagnosis dictates that all fevers in all age groups and settings should be tested for malaria before treatment is commenced (D’Alessandro et al., 2012). Young infants infected with malaria parasites may have different clinical manifestations (Sehgar et al., 1989) and lower parasite densities than older infants. But even low parasite density infections (1-500 parasites/µL) in infants can result to anaemia and may become life threatening if untreated (Afolabi et al., 2001).

In Nigeria, malaria is hyper endemic with stable transmission (Ofovwe and Eregic, 2001). The pattern of the disease in Nigeria is that of intense transmission and remarkable stability. Malaria transmission is intense all year round in the forest belt but in the dry Savannah, transmission is relatively low during the dry season (November to April). Malaria symptoms appear about 9-14 days after the infective mosquito bites; although this varies with different Plasmodium species. The clinical features of malaria vary from mild to severe and complicated malaria, according to the species of parasites present, the patient’s state of immunity, the intensity of infection and also the presence of concomitant conditions (Ichhpujani and Bhatia, 2005).

Malaria in pregnancy is a common cause of severe maternal anaemia and low birth weight babies, and these are more common in primigravidae than in multigravidae (Shulman and Dorman, 2003). The reasons for the increased susceptibility of primigravidae compared to multigravidae are poorly understood. Parasites isolated from pregnant women and their placentae cyto- adhered to chondroitin sulphate A (CSA), a ligand present at a high concentration on the surface of the syncytiotrophoblast which covers the placental villi and is the equivalent of endothelium in the maternal vascular compartment of the placenta. Parasites isolated from non-pregnant women and from men in the same geographical area do not express binding to CSA. This raises the possibility that a clone of parasites capable of binding to CSA is selected for during pregnancy. Incubation with sera from multigravidae inhibits the binding of these parasites to CSA in vitro, providing a possible explanation for why multigravidae are less susceptible to malaria infection than primigravidae. (Shulman and Dorman, 2003). Malaria infection of the placenta seems to result in a higher susceptibility of infants to the parasites (Hesran et al., 1997).

Infants born with low birth weight (less than 2500g) suffer from extremely high rates of morbidity and mortality from infectious diseases, and are underweight, stunted, or wasted beginning in the neonatal period through childhood (ACC/SCN,2000). The two main causes of low birth weight are prematurity and intrauterine growth retardation. Most low birth weights in developing countries are caused by intra-uterine growth retardation. The causes of intrauterine growth retardation are complex and multiple, but centre on the foetus, the placenta , the mother and combination of all three. Maternal environment is the most important determinant of birth weights and factors that prevent normal circulation across the placenta cause poor nutrient and oxygen supply to the foetus, thereby restricting growth. Such factors may include maternal undernutrition, malaria anaemia (Cameron and Hofvander, 1983). At least, 17 million infants are born every year with low birth weight, representing about 16% of all newborns in developing countries (ACC/SCN,2000). About 15% and 11% are born full term with low birth weight and intrauterine growth retardation in Middle East and Africa respectively, and approximately 7% in the Latin America and the Carribean regions (ACC/SCN, 2000). Infants born with low birth weight are at risk to develop acute diarrhoea, or to be hospitalized for diarrhoea episodes at a rate almost two to four times greater than their normal birth weight counterparts (Bukenya et al., 1991). They are also two times more than their normal birth weight counterparts in contracting respiratory infections (Victoria et al., 1989; Victoria et al., 1990).

Evidence exists that suggests that infant outcomes can be improved by improving the maternal nutritional status. Vitamin A has been reportedly stated to be effective in increasing the infant birth weights. Reports of the effects of Vitamin A supplementation on birth weights have been published by Jaya and Shatrugna, 1976; Panth et al., 1991, and Kumwenda et al. 2002.

Infant malaria can be categorized as uncomplicated, moderate and severe, depending on the presenting symptoms. Parasitic patients with symptoms such as fever or history of fever, headache, cough, rapid breathing, myalgia, vomiting and diarrhea (in the absence of altered consciousness, respiratory distress, repeated convulsions, hypoglycaemia, acidosis, vomiting, prostration and severe anaemia are defined as having uncomplicated malaria (Newton and Krishna, 1998).

Children who have the symptoms of the uncomplicated malaria and require parenteral treatment but are unlikely to progress to severe disease are said to have moderate malaria (Newton and Krishna, 1998). The following features characterize severe malaria in African children: altered consciousness, convulsions, hypoglycaemia, acidosis and anaemia (Taylor and Molyneux, 2003). Hypoglycaemia, (blood glucose concentration of less than 2.2 mmol/l, or 40 mg/dl) occurs in many paediatric illnesses. In falciparum malaria, it is associated with poor outcomes (Taylor et al., 1988) and should be considered in any comatose or convulsing child. It is recommended that intravenous fluid that contains dextrose in concentrations equal to or more than 5 percent should be administered to the children.

Anaemia is a serious symptom of malaria especially in pregnancy and infancy. Anaemia is defined as a haemoglobin concentration lower than the established cut-off value defined by the World Health Organisation (WHO). This cut-off ranges from 110 g/L for pregnant women and for children 6 months to five years of age, to 120 g/L for non- pregnant women, to 130 g/L for men (WHO, 2001). Worldwide, anaemia is the commonest red cell disorder that occurs when the concentration of haemoglobin falls below what is normal for a person’s age, gender, and environment, resulting in the oxygen- carrying capacity of the blood being reduced (Cheesbrough, 2010). It is graded as mild when the haemoglobin concentration is 10-11 g/dL, morderate, when it is 7-<10 g/dL and severe when <7 g/dL.

Anaemia can result from malaria infection even at an assymptomatic level (Douamba et al., 2012). In Kano metropolis, a positive correlation between malaria and anaemia was reported by Imam (2009) and a high prevalence of malaria- induced anaemia was more among under 5 years old children. Anaemia was related to parasites density, with direct relationship between severity of anaemia and higher parasite density (Imam, 2009). Abanyie et al. (2013) reported that anaemia was highly prevalent among Nigerian pre-school children and that it correlated with malaria. Maternal Vitamin A supplementation can influence the haemoglobin status of unborn infants. Kumwenda et al. (2002), reported that Vitamin A supplementation to Malawian women reduced anaemia in their infants when born. Malaria causes anaemia through cytokine-mediated suppression of haematopoesis, and in addition when infected with P. falciparum, the erythrocyte changes and becomes vulnerable to clearance (Biggs and Brown, 2001).

Proper nutrition is very important in protection against many parasitic diseases and is vital in protective immunity. When a child is undernourished, he or she may be unable to mount an appropriate immune response to malaria parasite due to the reduction in T-lymphocytes, impairment of antibody formation, and atrophy of thymus and other lymphoid tissues, among others (Scrimshaw and SanGiovanni, 1997).

Vitamin A plays an essential role in the immune response and eye health (Sommer and West, 1996). Severe vitamin A deficiency is rare and most vitamin A- associated morbidities result from mild to moderate deficiency. Vitamin A supplementation has been shown to improve general eye health as well as decrease diarrhoea, and all-cause mortality (Sommer and West, 1996; Beaton et al., 1993). Vitamin A deficiency is common in malaria endemic regions of the world. Studies in rats indicated that vitamin A- deficient rats were significantly more susceptible to the rat malaria parasite, P.berghei than were those rats with adequate vitamin A intake and overall animal studies suggest that vitamin A- deficient animals are more vulnerable to malaria morbidity and mortality (Krishnan et al., 1976). The fraction of malaria morbidity attributable to vitamin A deficiency was determined to be 20 % worldwide and more than 90 % of the 187,000 malaria deaths worldwide attributable to vitamin A deficiency occur in Africa. (Rice et al., 2004). Current intervention strategies include supplementation, fortification of a variety of foods, and education regarding the importance of vitamin A- rich foods in the diet.

CHAPTER TWO

MATERIALS AND METHODS

2.1 The Study Area

The research was conducted at some Health Centres and rural villages within the communities of Ebonyi State. The study cut across the three senatorial zones of the state.

The vegetation characteristic of the state is that of tropical rain forest. Two distinct seasons – the wet and dry exist. The former takes place between April and October, while the later occurs from November to March. Artisans, petty traders and subsistence farmers dominate the population of the communities. The communities are of low socioeconomic status with few people having attended tertiary institution, while the majority of the educated inhabitants are within the primary education level.

2.2 Study Population

The study population were pregnant women of varying age groups who were malaria parasitaemic and seronegative to HIV infections, whose pregnancies had mature up to the sixth month, and their seronegative infants when delivered. The participants were divided into two study groups: the trial group and the control group. For any woman to be enrolled in the study she must have tested positive to the malaria parasite, negative to HIV infections, and must also be at the sixth month of pregnancy. The infants of the women that were seronegative at the third month postnatally , were followed up. A total of 152 mother-infant pairs, 76 in the trial group and 76 in the placebo group were followed up to the end of the study.

2.3 Design of the Study

The studied population was divided into two – the trial group and the placebo group. Trained health personnel including medical laboratory scientists, nurses and village-based health workers (VBHWs) were involved in the execution of the study.

2.4 Ethical Considerations

The study protocol was approved by the Department of Zoology of the University of Nigeria, Nsukka and by the Primary Health Care Unit of Ebonyi State Ministry of Health. Informed consents were obtained from the participants (pregnant women), who also consented for their infants after birth before their enrolment in the study.

2.5 Design of Placebo

A syringe of 1 ml capacity was used to pierce and drain the active ingredients of the soft gels of both 10,000 IU and 100,000 IU of vitamin A that were administered antenatally and postnatally respectively. The same syringe was also used to introduce drinkable water into the gels, so as to rinse them properly. This was repeated so as to properly get rid of the active ingredients. The gels were then inflated with water to regain their shapes and make them appear identical with the ones containing the active ingredients.

2.6 Supplement (Vitamin A) Administration and Monitoring

Vitamin A supplements within a safe dosage of 10,000 IU was administered to the pregnant women of the trial group, three times weekly, starting from their sixth months into pregnancy. Placebo was also given to the placebo group thrice weekly. The regimen was continued until the women delivered. On delivery of the babies the trial group received 200,000 IU (2 gels of the 100,000 IU) between the 6^th and 8^th weeks postpartum. This was continued every other 3 month, until the study was over. Placebo was administered at equal strength to the placebo group. Village-based health workers (VHBWs) were involved in the administration of the supplements. Direct observed therapy (DOT) was in most cases ensured especially, when supplements were being administered to the illiterate women. Also, to ensure appropriate drug administration and compliance where DOTs were not possible, the dosage of the supplements were also boldly indicated on the drug envelops. The illiterate women had their own supplements dispensed into different envelops, indicating dosages. Compliance with the study regimen by each participant was determined by adding the number of tablets taken by each participant during visitations and dividing them by the total number of tablets the participants should have taken throughout the period of the study.

2.7 Estimation of Risk of Vitamin A Deficiency of Infants

A food frequency questionnaire (FFQ) modified from Hellen Keller International (HKI) food frequency questionnaire was used to estimate the risk of vitamin A deficiency among infants in the study area. The questionnaire was administered through the mothers or caregivers of infants within 1-5 years of age. The infants were infants born by the same mothers (secundigravids or multigravids) who are the subjects of this study or infants born by other mothers.

2.8 Infant Monitoring and Follow- up:

Follow up was carried out monthly at health centres, village squares and at homes. At birth, midwives or other trained health workers determined the weight of each infant. This was recorded to the nearest 0.1 kg. At each monthly follow - up, enquiries were made concerning the health and the breast feeding status of the infants during preceding periods. The axillary temperatures and physical examinations of each infant was taken and observed respectively at each visit.

Blood films were obtained from the infants every three months. The presence of peripheral blood Plasmodium parasitaemia was assessed by thick and thin blood smears stained with Giemsa. The mothers were instructed to always take their infants to the health centres any time they develop febrile episodes or feel ill. On such out of schedule visits, blood films were made from such infants and their Plasmodium parasitaemia status determined and the appropriate treatment given.

2.9 SAMPLING TECHNIQUES/LABORATORY ANALYSIS

2.9.1: Preparation and Examination of Thin and Thick

Blood Smears:

Thin and thick blood films were made and examined for parasitaemia. The method followed Cheesbrough (1998).

The. Thin and thick blood smears were prepared in situ. For the preparation of thin film, one drop of blood was smeared on to a frosted end grease-free microscope slide and a smooth-edged spreader was used to spread the blood. The slide was labeled with the infant’s name, sex and date of collection. The film was air-dried with the slide held in a horizontal position. Thereafter, the blood film was fixed with two drops of methyl alcohol for 1-2 minutes. After fixation, the alcohol was tipped off and the film was allowed to dry.

Following drying, the films were stained with Giemsa and were placed in racks and transported to the Department of Medical Laboratory Science Laboratory, Ebonyi State University, Abakaliki, for examination. At the laboratory, a drop of immersion oil was applied to the film and it was then examined under the microscope, using x40 and x100 objectives respectively. The Plasmodium species present were identified and recorded.

The procedure was the same for the preparation and examination of thick films, except that a larger quantity of blood (about 2 drops) was used, that fixation was not carried out, and that longer staining period is required,.

2.9.2 Determination of Malaria Parasite Density

Parasite number per microlitre (µl) of blood was estimated from number of parasites per 200 leucocytes with a standard leucocyte count of 8,000 (Cheesbrough, 1998; WHO, 1991; WHO, 2010).

A part of thick film where the white blood cells (WBC) were evenly distributed and parasites well – stained was selected. With the use of oil immersion objectives, 200 white blood cells (WBC) were systematically counted. At the same time, the number of parasites (asexual) in each field was estimated. The procedure was repeated in two other areas of the film and an average of the three counts was taken. The number of parasites per microlitre (µl) of blood was calculated as:

WBC count X parasites counted against 200 WBC

200

2.9.3 Haemoglobin (Hb) Estimation

The haemiglobincyanide (cyanmethaemoglobin) method was used to estimate the heamoglobin concentration of the infants every three months. The procedure followed Lewis et al. (2006). A one (1) in 201 dilution of well-mixed EDTA anticoagulated venous blood was made by adding 2 0 µl (0.02 ml) of blood to a test tube containing 4 ml of Drabkin’s neutral diluting fluid (pH 7.0-7.4). The tube was stoppered and inverted several times to allow the solution to mix well. The test sample was left standing at room temperature for 5 minutes. Thereafter, it was poured into a cuvette and the absorbance was read in a spectrometer at a wavelength of 540 nm against a reagent blank. The absorbance of a commercially available HiCN standard was also compared to a reagent blank in the same spectrometer and their values recorded. The heamoglobin (Hb) concentrations were calculated and expressed in g/dl as follows:

Hb (g/dl)= A⁵⁴⁰ of test sample X Concentration of standard X Dilution factor (201)

A⁵⁴⁰ of standard 1 1000

Where A = Absorbance

2.9.4 Blood Glucose Concentration Determination

The blood glucose concentration of infants was determined in situ with the use of a glucometer (ACCU-CHEK^® Advantage). This is a glucometer with strip test. About 25 µl of blood was applied to cover the test strip area as instructed by the manufacturer. Thereafter, the strip was inserted in the meter. The result was digitally displayed after 12 seconds and was expressed in mg/dl (Cheesbrough, 1998).

2.10 Statistical Analysis:

Regression models will be used to analyze the relationship between parasite densities in the infants and months of pregnancies of the mothers, and parasite densities in the infants and the gravida of each participant mother.

The Chi square analysis will be employed to test whether there is a significant difference in levels of parasitaemias and morbidities between the infants of mothers who took the vitamin A supplement and the controls.

The Chi square analysis will also be used to determine whether there is significant difference between parasite densities and gender of the infants.

Correlation analysis will be used to determine the association between age (in months) and Parasite densities, blood glucose concentrations and parasite densities, and haemoglobin concentrations and parasite densities respectively. The relationship between vitamin A and the birth weight of the infants will be determined by the use of relative risk.

RESULTS

The study populations were malaria parasitaemic and HIV sero-negative pregnant women of varying age groups, who were at least at the 6^th month into the gestation period, and their infants when delivered.

A total of 195 women with the above mentioned inclusion criteria were enrolled in the study. Ninety eight (98) of them were in the trial group while ninety seven (97) were in the placebo group. Before delivery, fourteen (14) (14.29%) from the trial group withdrew through relocation and voluntarily. Among the placebo group, 11(11.34%) withdrew because of relocation and voluntarily too (Table 1).

Table 1: Prepartum baseline population and withdrawal reasons

Trial Group (N=98) Placebo Group (N=97)

Number withdrawn (%) withdrawal reason Number withdrawn (%) withdrawal reason

5(5.10) relocation 6(6.19) relocation

9(9.18) voluntary withdrawal 5(5.16) voluntary withdrawal

(Reasons unspecified) (Reasons unspecified)

TOTAL = 14(14.29) 11(11.34)

Eighty four (84) and 86 from the trial and placebo groups respectively were followed-up until delivery. Eight children, 1(1.19%) neonatal death, 2(2.38%) infantile mortality, 3(3.57%) cases of relocation to unknown residences and 2(2.38%) cases of HIV seropositivity reduced the trial group number of children to 76. The number was followed up to the end of the study. Ten (10), 1(1.16%) case of stillbirth, 2(2.33%) neonatal death, 3(3.49%) infantile death, 1(1.16%) case of relocation and 3(3.49%) cases of HIV seropositivity, reduced the number of placebo group infants to 76 also.The number was also followed up to the completion of the study.

Table 2: Postpartum baseline population and withdrawal reasons

Trial Group (N=84) Placebo Group (N=86)

Number withdrawn (%) Reason Number withdrawn (%) Reason

1(1.19) Neonatal death 1(1.16) still birth

2(2.38) Infantile mortality 2(2.33) Neonatal death

3(3.57) Relocation 3(3.49) Infantile death

2(2.38) HIV seropositivity 1(1.16) Relocation

3(3.49) HIV seropositivity 3(3.49) HIV seropositivity

TOTAL 8(9.52) 10(11.63)

When the number of women who were followed up to the delivery of their infants was stratified by gravidity, 21(27.63%), 24(31.58%), and 31(40.79%) that represented primigravids, secundigravids and multigravids respectively, were in the trial group.

Within the placebo group, 21(27.63%), 21(27.63%) and 34(44.74%) respectively, represented the primigravids, the secundigravids, and the multigravids respectively (Table 3)

Table 3: Population of follow-up mothers stratified by gravidity

Gravidity Trial group (N=76) Placebo group (N=76)

Primigravids 21(27.63%) 21(27.63%)

Secundigravids 24(31.58%) 21(27.63%)

Multigravids 31(40.79%) 34(44.74%)

TOTAL 76(100%) 76(100%)

Among the infants whose mothers received the supplement (the trial group), a total of 3(4.7%) had low birth weight. This was observed in the 7^th month 3(21.4%). Within the placebo group, low birth weight was observed in all the months, with the highest being observed among infants whose mothers took the supplement at the 8^th month. The overall low birth weight was 13(20.3%) Generally, the percentage of low birth weight was more in the placebo group than in the trial group.

Within the primigravids, no low birth weight, 0(0.0%) was recorded among the trial group. In the placebo group, 1(33.3%), 0(0.0%), and 3(42.9%) and 1(25.0%) low birth weights were recorded among infants whose mothers received the supplements in the 6^th, 7^th, 8^th and 9^th months respectively. The total low birth weight was 5(26.3%).

In the trial group of the secundigravids, 1(16.7%) low birth weight was observed only among infants whose mothers started taking the supplement at the 7^th month. Within the placebo group, low birth weights 1(33.3%), 1(20.0%), and 1 (16.7%) were observed in the 6^th, 7^th and 8^th months respectively. No low birth weight was observed in the 9^th month. The total low birth weight was 3(15.8%).

Among the multigravids, low birth weight 2(28.6%) was observed among the infants of mothers who received the supplement at the 7^th month. No low birth weight was observed in any other month. But low birth weights of 1(50.0%), 2(22.2%), 1(12.5%) and 1(14.3%) were observed respectively in the 6^th, 7^th, 8^th and 9^th months within the placebo group. The overall low birth weight of the supplemented and placebo groups in the multigravids were 2(8.7%) and 5(19.2%) respectively.

Table 4: Percentages of infants with low birth weights (BW˂ 2.5 kg)

OVERALL

Trial Placebo

Month Number examined LBW (%) Number examined LBW (%)

6^th 7 0(0.0) 8 3(37.5)

7^th 14 3(21.4) 19 3(15.8)

8^th 23 0(0.0) 21 5(23.8)

9^th 20 0(0.0) 16 2(13.3)

TOTAL 64 3(4.7) 64 13(20.3)

PRIMIGRAVIDS

6^th 4 0(0.0) 3 1(33.3)

7^th 2 0(0.0) 5 0(0.0)

8^th 9 0(0.0) 7 3(42.9)

9^th 6 0(0.0) 4 1(25.0)

TOTAL 21 0(0.0) 19 5(26.3)

SECUNDIGRAVIDS

6^th 1 0(0.0) 3 1(33.3)

7^th 6 1(16.7) 5 1(20.0)

8^th 6 0(0.0) 6 1(16.7)

9^th 8 0(0.0) 5 0(0.0)

TOTAL 21 1(4.8) 19 3(15.8)

MULTIGRAVIDS

6^th 2 0(0.0) 2 1(50.0)

7^th 7 2(28.6) 9 2(22.2)

8^th 8 0(0.0) 8 1(12.5)

9^th 6 0(0.0) 7 1(14.3)

TOTAL 22 2(8.7) 26 5(19.2)

The frequencies of malaria fever episodes in the overall trial group were 12(9.8%), 37(30.1%), 38(30.9%) and 36(29.3%) for infants whose mothers started receiving the Vitamin A supplement at the 6^th, 7^th, 8^th and 9^th months of pregnancy respectively. Within the placebo group, the frequencies were 28(16.0), 54(30.9), 52(29.7), and 41(23.4) respectively for the 6^th, 7^th, 8^th and 9^th months. The total frequencies were 123 and 175 for trial and placebo groups respectively (table 5). The mean malaria fever episodes were also higher in the placebo group when compared with the supplemented group (table 16). Frequencies of malaria fever episodes were higher among infants of the placebo group than in those of the trial group.

Within the trial group of the primigravids, the frequencies of malaria fever episodes were 6(16.7), 7(19.4), 12(33.3) and 11(30.6), for infants whose mothers started receiving the supplements from the 6^th, 7^th, 8^th and 9^th months of pregnancy respectively. In the placebo group, the frequency of the malaria fever episodes were 13(23.6), 14(25.5), 19(34.5) and 9(16.4) for the 6^th, 7^th, 8^th and 9^th months respectively. The frequencies of the fever episodes were higher in the placebo group in the 6^th, 7^thand 8^th months. But for those that started the supplement at the 9^th month, the frequency of fever episodes was higher in the trial group than in the placebo group.

Among the secundigravids, the frequencies of the malaria fever episodes in the trial group were 1(2.5), 15(37.5), 9(22.5) and 15(37.5) for the infants of mothers that started the supplement in their 6^th, 7^th, 8^th and 9^th months of pregnancy respectively. In the placebo group, the frequencies were 8(17.4), 13(28.3), 12(26.1) and 13(28.3) for the 6^th, 7^th, 8^th and 9^th months. The frequencies were higher among infants of the placebo group at the 6^th and 8^th months but were lower in the placebo group at the 7^th and 9^th months respectively.

In the multigravids, infants of mothers that started receiving the supplement from the 6^th, 7^th, 8^th and 9^th months had fever frequencies of 5(10.6), 15(31.9), 17(36.2) and 10(21.3) respectively. Among the placebo group, the frequencies were 8(10.7), 27(36.0), 21(28.0) and 19(25.3) for 6^th, 7^th, 8^th and 9^th months respectively. There were generally higher frequencies in the placebo group than in the trial group.

Table 5: Frequencies of malaria fever episodes among infants

OVERALL

Trial Placebo

Months

6^th 12(9.8) 28(16.0)

7^th 37(30.1) 54(30.9)

8^th 38(30.9) 52(29.7)

9^th 36(29.3) 41(23.4)

TOTAL 123(100) 175(100)

PRIMIGRAVIDS

6^th 6(16.7) 13(23.6)

7^th 7(19.4) 14(25.5)

8^th 12(33.3) 19(34.5)

9^th 11(30.6) 9(16.4)

TOTAL 36(100) 55(100)

SECUNDIGRAVIDS

6^th 1(2.5) 8(17.4)

7^th 15(37.5) 13(28.3)

8^th 9(22.5) 12(26.1)

9^th 15(37.5) 13(28.3)

TOTAL 40(100) 46(100)

MULTIGRAVIDS

6^th 5(10.6) 8(10.7)

7^th 15(31.9) 27(36.0)

8^th 17(36.2) 21(28.0)

9^th 10(21.3) 19(25.3)

TOTAL 47(100) 75(100)

Key: Values in parenthesis indicate percentages.

The total frequencies of coughs were 60, 15, 20, and 25 respectively for the overall, primigravids, secundigravids and multigravids in the supplemented group, and 65, 23, 18 and 24 respectively for overall, primigravids, secundigravids and multigravids of the placebo group

The overall frequencies of malaria- associated cough among infants from the trial group women indicates 10 (16.9), 13 (20.3), 18 (30.5) and 19 (32.2) for women who commenced the Vitamin A administration from the 6^th, 7^th, 8^th and 9^th months respectively. In the placebo group, the frequencies of 11 (16.9), 16 (24.6), 24 (36.9) and 14 (21.5) were observed for the 6^th, 7^th, 8^th and 9^th months groups respectively. The result indicates that there were greater frequencies of cough from the 6^th month through the 8^th month among the placebo group in comparison with the trial group. However, in the 9^th month, a higher frequency was observed in the trial group when compared with the placebo group.

Among the primigravids, the frequencies of malaria- associated cough among the trial group were 4 (26.7), 1 (6.7), 3 (20.0) and 7 (46.7) from the 6^th month through the 9^th month groups respectively. In the placebo group, the frequencies of cough among the infants were 6 (26.1), 1(4.3), 11(47.8) and 5 (21.7). In comparison, greater frequencies of cough were observed in all the months except at the 9^th month, to be in the placebo group than the trial group.

Within the secundigravids, the frequencies of malaria- associated coughs recorded in the infants of the trial group women were 1 (5.0), 7 (35.0), 5 (25.0) and 7 (35.0), respectively for the 6^th, 7^th, 8^th and 9^th month groups. Among the placebo group, the frequencies were 3 (16.7), 4 (22.2), 6 (33.3) and 5 (27.8) for the 6^th, 7^th, 8^th and 9^th month groups respectively. The frequencies were higher in the placebo group in infants whose mothers started the supplement in their 6^th and 8^th months of pregnancy. On the other hand, the frequencies were higher among the trial group in infants of women who received the supplement in the 7^th and 9^th months.

The frequencies of the malaria- associated cough were 5 (23.1), 5 (19.2), 10 (38.5) and 5 (19.2), for the trial group infants whose mothers started taking the vitamin from the 6^th, 7^th, 8^th and 9^th months respectively in the multigravids. Within the placebo group, the frequencies of 2(8.3), 11(45.8), 7(29.2), and 4(16.7) were observed among infants whose mothers commenced the Vitamin A supplement intake from the 6^th, 7^th, 8^th and 9^th months respectively. Throughout the months, with the exception of the 7^th month, the frequencies of malaria- associated cough were greater in the trial group than in the placebo group.

Table 7: Frequencies of malaria- associated cough among infants.

OVERALL

Months Trial Placebo

6^th 10(16.9) 11(16.9)

7^th 13(20.3) 16(24.6)

8^th 18(30.5) 24(36.9)

9^th 19(32.2) 14(21.5)

TOTAL 60(100) 65(100)

PRIMIGRAVIDS

6^th 4(26.7) 6(26.1)

7^th 1(6.7) 1(4.3)

8^th 3(20.0) 11(47.8)

9^th 7(46.7) 5(21.7)

TOTAL 15(100) 23(100)

SECUNDIGRAVIDS

6^th 1(5.0) 3(16.7)

7^th 7(35.0) 4(22.2)

8^th 5(25.0) 6(33.3)

9^th 7(35.0) 5(27.8)

TOTAL 20(100) 18(100)

MULTIGRAVIDS

6^th 5(23.1) 2(8.3)

7^th 5(19.2) 11(45.8)

8^th 10(38.5) 7(29.2)

9^th 5(19.2) 4(16.7)

TOTAL 25(100) 24(100)

Key: values in parenthesis indicate percentages.

Among the overall, the frequencies of malaria- associated diarrhea among the infants of women in the trial group were 1 (4.3), 10 (43.5), 8 (34.8) and 4 (17.4) for the mothers that started taking the vitamin A supplement from the 6^th, 7^th, 8^th and 9^th months respectively. Within the placebo group, the frequencies were 9 (19.1), 10 (21.3), 18 (38.3) and 10 (21.3). This is also respectively for the 6^th, 7^th, 8^th and 9^th month groups. Although the frequencies were equal in both groups in infants whose mothers received the supplement at the 7^th month, there were higher frequencies in the placebo group than in the trial group across the other months.

The frequencies were 0 (0.0), 1 (14.3), 4 (57.1) and 2 (28.6) in the trial group of the primigravids that started taking the supplement at the 6^th, 7^th, 8^th and 9^th months respectively. Within the placebo group, the frequencies of diarrhea episode were 6 (40.0), 5 (33.3), 3 (20.0), and 1 (6.7) for the infants whose mothers started receiving the supplement from the 6^th, 7^th, 8^th and 9^th months respectively. The episodes of the diarrhea was higher among the placebo group whose mothers received the supplement at the 6^th and 7^th months of pregnancy respectively but was higher in the trial group for those that their mothers commenced the supplement at the 8^th and 9^th months respectively.

The episodes of malaria- associated diarrhea among the trial group of the secundigravids were 0(0.0), 5(71.4), 0(0.0), and 2(28.6), for infants that their mothers commenced the vitamin A supplement at the 6^th, 7^th, 8^th and 9^th months of pregnancy respectively. Among the placebo group, the infants of mothers that commenced the vitamin A supplement intake at the 6^th, 7^th, 8^th and 9^th months of pregnancy had the frequencies of 1(7.7), 1(7.7), 6(46.2), and 5(38.5) respectively. Apart from the infants of mothers who took the supplement at the 7^th month that had higher episodes in the trial group, the placebo group had higher episodes of diarrhea across other months when compared with the placebo group.

Within the multigravids, the frequencies of diarrhea were 1 (16.7), 4 (66.7), 0 (0.0), and 0 (0.0) for the trial group whose mothers started taking the supplement at the 6^th, 7^th, 8^th and 9^th months respectively. The frequencies were 2 (10.5), 4 (21.1), 9 (47.4) and 4 (21.4) among the placebo group for the months of 6^th, 7^th, 8^th and 9^th respectively. There was generally higher episodes of diarrhea within the placebo group when compared with that of the trial group.

Table 8: Frequencies (%) of malaria- associated diarrhea among infants.

OVERALL

Months Trial Placebo

6^th 1(4.3) 9(19.1)

7^th 10(43.5) 10(21.3)

8^th 8(34.8) 18(38.3)

9^th 4(17.4) 10(21.3)

TOTAL 23(100) 47(100)

PRIMIGRAVIDS

6^th 0(0.0) 6(40.0)

7^th 1(14.3) 5(33.3)

8^th 4(57.1) 3(20.0)

9^th 2(28.6) 1(6.7)

TOTAL 7(100) 15(100)

SECUNDIGRAVIDS

6^th 0(0.0) 1(7.7)

7^th 5(71.4) 1(7.7)

8^th 0(0.0) 6(46.2)

9^th 2(28.6) 5(38.5)

TOTAL 7(100) 13(100)

MULTIGRAVIDS

6^th 1(16.7) 2(10.5)

7^th 4(66.7) 4(21.1)

8^th 0(0.0) 9(47.4)

9^th 0(0.0) 4(21.1)

TOTAL 9(100) 19(100)

Key: Values in parenthesis indicate percentages.

Within the overall, the frequencies of malaria- associated vomiting among the infants of the trial group were 3(37.5), 2(25.0), 0(0.0), and 3(37.5), for the 6^th, 7^th, 8^th and 9^th month groups respectively. Among the placebo group, the infants of mothers that started taking the supplement at the 6^th, 7^th, 8^th and 9^th month had the frequencies of 8(20.0), 13(32.5), 6(15.0), and 13(32.5) respectively. Generally, there were higher frequencies of vomiting episodes among the placebo group in comparison with the trial group.

Among the primigravids, the frequencies of vomiting within the trial group were 0(0.0), 0(0.0), 0(0.0), and 1(100). The placebo had the frequencies of 3(30.0), 3(30.0), 2(20.0) and 2(20.0) for infants of mothers that started taking the supplement at the 6^th, 7^th, 8^th and 9^th months respectively. There was also a generally higher frequency of malaria- associated vomiting among the placebo group in comparison with the trial group.

The frequencies of 1(50.0), 0(0.0), 0(0.0) and 1(50.0) were observed from the trial group of the secundigravids among the infants that their mothers started receiving the supplement at the 6^th, 7^th, 8^th and 9^th months respectively. Within the placebo group, 3(21.4), 4(28.6), 2(14.3), and 5(37.5) were the frequencies of the vomiting episode among the infants of the women that started taking the supplement at the 6^th, 7^th, 8^th and 9^th months. Very lower frequencies of vomiting were observed in the trial group when compared with the higher frequencies observed in the placebo group.

Within the multigravids, the frequencies of malaria- associated vomiting among the trial group were 2(40.0), 2(40.0), 0(0.0) and 1(20.0) for infants of mothers that started receiving the vitamin A supplement at the 6^th, 7^th, 8^th and 9^th months of pregnancy respectively. The frequencies of the vomiting episodes were 2(12.5), 6(37.5), 2(12.5) and 6(37.5) for infants born by mothers who started taking the vitamin A supplement at the 6^th, 7^th, 8^th and 9^th months respectively. Apart from those that started the supplement at the 6^th month that had equal frequencies in both the trial and placebo group, the frequencies of the episodes were generally higher among the placebo group.

Table 9: Frequency (%) of malaria- associated vomiting among infants.

OVERALL

Months Trial Placebo

6^th 3(37.5) 8(20.0)

7^th 2(25.0) 13(32.5)

8^th 0(0.0) 6(15.0)

9^th 3(37.5) 13(32.5)

TOTAL 8(100) 40(100)

PRIMIGRAVIDS

6^th 0(0.0) 3(30.0)

7^th 0(0.0) 3(30.0)

8^th 0(0.0) 2(20.0)

9^th 1(100) 2(20.0)

TOTAL 1(100) 10(100)

SECUNDIGRAVIDS

6^th 1(50.0) 3(21.4)

7^th 0(0.0) 4(28.6)

8^th 0(0.0) 2(14.3)

9^th 1(50.0) 5(37.5)

TOTAL 2(100) 14(100)

MULTIGRAVIDS

6^th 2(40.0) 2(12.5)

7^th 2(40.0) 6(37.5)

8^th 0(0.0) 2(12.5)

9^th 1(20.0) 6(37.5)

TOTAL 5(100) 16(100)

Key: Values in parenthesis indicate percentages.

Across the months and gravidities, there was no record of malaria- associated rapid breathing among the trial group. Within the placebo group of the overall, 2(10.5), 5(26.3), 4(21.1) and 8(42.1) episodes of malaria- associated rapid breathing were observed among infants whose mothers started receiving the vitamin A placebo at the 6^th, 7^th, 8^th and 9^th months respectively.

Within the primigravids, 0(0.0), 1(16.7), 3(50.0) and 2(33.3) episodes were observed among infants whose mothers started the supplement at the 6^th, 7^th, 8^th and 9^th months respectively.

Episodes with 0(0.0), 2(33.3), 1(16.7) and 3(50.0) frequencies were observed among infants whose mothers started taking the vitamin A placebo at the 6^th, 7^th, 8^th and 9^th months respectively, among the secundigravids.

Within the multigravids, 2(28.6), 2(28.6), 0(0.0) and 3(42.8) episodes of malaria- associated rapid breathing were observed among infants whose mothers started the placebo from the 6^th month through the 9^th month respectively.

Table 10: Frequencies (%) of malaria- associated rapid breathing among infants.

OVERALL

Month Trial Placebo

6^th 0(0.0) 2(10.5)

7^th 0(0.0) 5(26.3)

8^th 0(0.0) 4(21.1)

9^th 0(0.0) 8(42.1)

TOTAL 0(0.0) 19(100)

PRIMIGRAVIDS

6^th 0(0.0) 0(0.0)

7^th 0(0.0) 1(16.7)

8^th 0(0.0) 3(50.0)

9^th 0(0.0) 2(33.3)

TOTAL 0(0.0) 6(100)

SECUNDIGRAVIDS

6^th 0(0.0) 0(0.0)

7^th 0(0.0) 2(33.3)

8^th 0(0.0) 1(16.7)

9^th 0(0.0) 3(50.0)

TOTAL 0(0.0) 6(100)

MULTIGRAVIDS

6^th 0(0.0) 2(28.6)

7^th 0(0.0) 2(28.6)

8^th 0(0.0) 0(0.0)

9^th 0(0.0) 3(42.8)

TOTAL 0(0.0) 7(100)

As with malaria- associated rapid breathing, there were also no cases of malaria- associated convulsion among infants across all the months and gravidities in the trial group.

However, episodes with frequencies of 1(11.1), 3(33.3), 5(55.6) and 0(0.0) of malaria- associated convulsion were observed among infants in the overall, whose mothers started taking the supplement at the 6^th, 7^th, 8^th and 9^th months respectively.

Within the primigravids , 0(0.0), 1(20.0), 4(80.0),and 0(0.0) episodes were observed for the 6^th, 7^th, 8^th and 9^th month groups respectively. Infants of the secundigravids whose mothers started taking the placebo at the 6^th, 7^th, 8^th and 9^th months had episodes with the following frequencies; 1(50.0), 1(50.0), 0(0.0) and 0(0.0) respectively.

Among the multigravids, 0(0.0), 1(50.0), 1(50.0) and 0(0.0) frequencies of episodes of convulsion were observed for the infants of mothers that started receiving the placebo at the 6^th, 7^th, 8^th and 9^th months respectively. Infants of the primigravids had the highest episodes of the morbid condition. (Table 11)

Table 11: Frequency (%) of malaria- associated convulsion among infants

OVERALL

Months Trial Placebo

6^th 0(0.0) 1(11.1)

7^th 0(0.0) 3(33.3)

8^th 0(0.0) 5(55.6)

9^th 0(0.0) 0(0.0)

TOTAL 0(0.0) 9(100)

PRIMIGRAVIDS

6^th 0(0.0) 0(0.0)

7^th 0(0.0) 1(20.0)

8^th 0(0.0) 4(80.0)

9^th 0(0.0) 0(0.0)

TOTAL 0(0.0) 5(100)

SECUNDIGRAVIDS

6^th 0(0.0) 1(50.0)

7^th 0(0.0) 1(50.0)

8^th 0(0.0) 0(0.0)

9^th 0(0.0) 0(0.0)

TOTAL 0(0.0) 2(100)

MULTIGRAVIDS

6^th 0(0.0) 0(0.0)

7^th 0(0.0) 1(50.0)

8^th 0(0.0) 1(50.0)

9^th 0(0.0) 0(0.0)

TOTAL 0(0.0) 2(100)

Key: Values in parenthesis indicate percentages.

In the trial group of the overall birth weights of the infants, the weight of infants whose mothers received the vitamin A supplement at the 7^th month was significantly lower (p˂0.05) than those of other infants with different supplementation periods. In the placebo group, no significant difference (p˃0.05) was observed when the birth weight of infants whose mothers received vitamin A placebo at different months was compared. When the trial and placebo group birth weights were compared, a significant difference (p˂0.05) was found only in the weights of the infants whose mothers received the supplement at the 8^th and 9^th months, with the placebo group being significantly lower (p˂0.05) than the trial group.

In both trial and placebo groups of the primigravids, there was no significant difference in the birthweights of the infants whose mothers started receiving the vitamin A supplement at different months (p˃0.05). But when the weights of the infants in the trial and placebo groups were compared, a significant difference (p˂0.05) was observed in the infants whose mothers received the supplement at the 8^th and 9^th months. Generally, the birth weights of the placebo group were observed to be smaller than those of the trial group.

Within the trial group of the secundigravids, a significant difference (p˂0.05) was observed only in the birth weights of the infants whose mothers took the supplement at the 7^th month. In the placebo group, there was no significant difference in the birth weights of the infants whose mothers took the supplement from the 6^th month through the 9^th month. When the birth weights of the trial group were compared with those of the placebo group, those of the placebo group were generally smaller than those in the trial group, though no significant difference was observed (p˃0.05).

In the trial group of the multigravids, there was no significant difference in the birth weights of the infants whose mothers received the vitamin A supplement in all the months. Within the placebo group, there was also no significant difference (p˃0.05) in the birth weights across the months. When the trial group was compared with the placebo group, a significant difference (p˂0.05) was observed only in the birth weights of the infants whose mothers took the supplement at the 8^th month.

Table 12 Effects of maternal Vitamin A supplementation on infant birth weights ( kg)

OVERALL

Trial Placebo

6^th month 2.94±0.39^b 2.68±0.39^{a ns}

7^th month 2.64±0.19^a 2.58±0.13^{a ns}

8^th month 2.93±0.32^b 2.61±0.21^{a *}

9^th month 2.87±0.31^b 2.64±0.28^{a *}

PRIMIGRAVIDS

Trial Placebo

6^th 2.98±0.46^a 2.47±0.06^{a ns}

7^th 2.65±0.21^a 2.62±0.11^{a ns}

8^th 2.82±0.13^a 2.50±0.12^{a *}

9^th 2.95±0.33^a 2.60±0.18^{a *}

SECUNDIGRAVIDS

6^th 3.00±0.00^b 2.70±0.30^{a ns}

7^th 2.63±0.21^a 2.64±0.13^{a ns}

8^th 3.03±0.27^b 2.73±0.31^{a ns}

9^th 2.80±0.33^b 2.58±0.08^{a ns}

MULTIGRAVIDS

6^th 2.85±0.50^a 2.95±0.78^{a ns}

7^th 2.79±0.41^a 2.53±0.12^{a ns}

8^th 2.93±0.37^a 2.61±0.15^{a *}

9^th 2.93±0.38^a 2.69±0.40^{a ns}

Key: values in a column with different alphabets as superscripts are significantly different. (p˂0.05)

Ns—no significant difference (p˃0.05) , determined by t- test

*--- significant difference (p˂0.05), determined by t- test.

The overall mean malaria parasite density did not differ statistically in infants whose mothers received the supplement at the 6^th month,7^th month, and 9^th month (p˃0.05). However, the mean parasite density of infants whose mothers received the supplement at the 6^th month differed from that of the infants whose mothers received the supplement at the 8^th month. Among the placebo group, there was no difference in mean parasite density among the infants whose mothers received the placebo at the 6^th and 7^th months. There was also no difference among those whose mothers took the supplement at the 8^th and 9^th months. When the t- test was used to compare the difference in mean parasite densities, among the trial and placebo groups, it was observed that there waere significant differences across the months, with the placebo group having higher mean parasite densities than the trial group.

In the trial group of the primigravids, there was no difference in mean parasite densities of infants whose mothers took the supplement from the 6^th month through the 9^th month, though mean parasite density was lowest among infants in the 8^th month. Among infants from mothers in the placebo group, there was no difference in mean parasite densities of infants whose mothers took the supplement at the 6^th and 7^th months. There was also no difference in mean parasite density of infants of mothers from the 8^th and 9^th month. However, those in the 6^th and 7^th months differed from those in the 8^th and 9^th months. Malaria parasite density in the trial and placebo group had no statistical difference in the 6^th and 7^th months (p>0.05) while within the 8^th, and 9^th month, there was a statistical significant difference between the trial and placebo groups (p˂0.05). There were generally higher mean parasite densities in the placebo group when compared with the trial group.

Among the trial group of the secundigravids, there was no significant difference in mean parasite densities of the infants whose mothers received the supplement at the 6^th month, 8^th month and 9^th month. The mean parasite densities differed among infants whose mothers started receiving the Vitamin A supplement at the 7^th and 8^th months (p˂0.05). In the placebo group, there was no significant difference (p˃0.05) in the parasite density of infants whose mothers started taking the vitamin A placebo from the 6^th month through the 9^th month. However, when the trial and placebo groups were compared, significant statistical differences were observed in all the months, with the mean parasite densities being higher among the placebo groups.

Within the trial group of the multigravids, difference in mean malaria parasite densities was only observed among infants whose mothers started receiving the supplement at the 6^th month. Infants in that group also had the least malaria parasite densities.

Among the placebo group, the mean malaria parasite density of infants whose mothers received the placebo supplement at the 6^th month differed from those at the 7^th, 8^th and 9^th months (p˂0.05). Also, the mean parasite density of those whose mothers received the supplement at the 7^th month also differed from that of other months. However, there was no significant difference in the mean parasite density of infants whose mothers received the supplement at the 8^th and 9^th months (p˃0.05). When the trial and placebo groups were compared, a statistical significant difference (p˂0.05) was observed in all the months with mean parasite densities being higher in the placebo group.

Table13: Effects of maternal vitamin A supplementation on infant malaria parasite densities.

Malaria parasite densities (μL)

OVERALL

Months Trial Placebo

6^th 910.00±118.93^a 1645.60±114.14^a*

7^th 1351.70±90.19^ab 1837.58±76.04^a*

8^th 1447.38±196.91^b 2301.13±125.28^b*

9^th 1154.86±72.30^ab 2350.54±75.03^b*

PRIMIGRAVIDS

6^th 1293.33±293.63^a 1830.00±205.03^{a ns}

7^th 1660.00±217.39^a 1892.00±171.95^{a ns}

8^th 1271.30±111.33^a 2415.00±125.62^b*

9^th 1308±152.74^a 2476±147.31^b*

SECUNDIGRAVIDS

6^th 900.00±140.00^ab 1996.67±224.42^a*

7^th 1328±147.73^b 1936.92±139.42^a*

8^th 897.14±135.79^a 2093.85±120.92^a*

9^th 1104.00±104.51^ab 2296.67±129.50^a*

MULTIGRAVIDS

6^th 708.24±94.54^a 1250.53±112.51^a*

7^th 1232.31±122.84^b 1749.43±110.18^b*

8^th 1237.14±91.34^b 2131.43±93.10^c*

9^th 1065.26±123.54^b 2320.00±119.34^c*

Key:

Values in a column with different alphabets as superscripts are significantly different (p˂0.05).

Ns--- No significant difference (p˃0.05), determined by T- test

*-- Significant difference (p˂0.05) ,determined by T- test.

There was no significant difference in the overall mean blood glucose concentration in both the trial and placebo groups of infants whose mothers received the vitamin A supplement from the 6^th month through the 9^th month (p˃0.05). But when the trial group was compared with the placebo group, a significant difference (p˂0.05) was observed in all the months.

Also, in both the trial and placebo groups, no significant difference in the mean blood glucose levels of the infants of the primigravids who started receiving the supplement from the 6^th month to the 9^th month (p˃0.05). But when the trial group was compared with the placebo group, a statistically significant difference (p˂0.05) was observed; with the mean blood glucose levels being higher in the trial group than in the placebo group.

Among the secundigravids, there was also no significant difference (p˃0.05) in both the trial and placebo groups in mean blood glucose concentrations of infants of mothers who took the Vitamin A supplement from the 6^th month through the 9^th month. But when the trial and placebo groups were compared, statistically significant difference (p˂0.05) was observed in all the months with values in the trial group being higher than those in the placebo group.

Within the trial group of the multigravids, no significant difference (p˃0.05) was obtained in the blood glucose concentrations of infants whose mothers received the supplement from the 6^th month to the 9^th month.

Among the placebo group, the mean blood glucose concentrations of infants whose mothers started the placebo at the 6^th month differed from those whose mothers started it at the 8^th, and 9^th months. But when the trial and placebo groups were compared, a statistically significant difference (p˂0.05) was observed across all the months, with the mean blood glucose levels of infants in the trial group also being higher than those in the placebo group.

Table 14 Effects of maternal vitamin A supplementation on mean blood glucose concentrations (Mg/dl) in the infants.

Blood glucose concentrations (mg/dl)

OVERALL

Months Trial Placebo

6^th 58.72±4.80^a 51.81±6.37^a*

7^th 59.54±5.53^a52.40±5.10^a*

8^th 59.79±5.02^a 52.64±5.70^a*

9^th 59.28±3.97^a 53.51±5.19^a*

PRIMIGRAVIDS

6^th 59.12±4.39^a 53.00±5.34^a*

7^th 60.00±4.67^a 52.09±3.74^a*

8^th 60.05±5.43^a 50.77±5.58^a*

9^th 58.71±3.60^a 51.15±4.66^a*

SECUNDIGRAVIDS

6^th 52.21±8.63^a

7^th 59.75±4.50^a 55.31±4.79^a*

8^th 60.97±5.40^a 53.21±5.79^a*

9^th 60.15±4.03^a 53.71±4.72^a*

MULTIGRAVIDS

6^th 58.44±3.90^a 50.26±5.40^a*

7^th 59.29±6.48^a 51.14±5.22^ab*

8^th 58.66±4.27^a 53.22±5.28^bc*

9^th 58.61±3.84^a 54.80±5.24^c*

Key:

Values in a column with different alphabets as superscripts are significantly different (p˂0.05).

Ns--- No significant difference (p ˃ 0.05) as determined by t- test

*---- Significant difference (p ˂ 0.05) as determined by t- test.

In both the trial and placebo groups, there were no significant differences in the overall mean haemoglobin concentrations of infants whose mothers took the vitamin A supplement from the 6^th month through the 9^th month. But when the mean haemoglobin concentrations were compared between the trial and placebo groups, significant differences (p ˂ 0.05) were observed across the months; with the mean haemoglobin concentrations of infants of the placebo group being smaller than those of the trial group.

Among the trial group of the primigravids there was no significant difference in the mean haemoglobin concentrations of infants of the primigravids that received the supplement from the 6^th month through the 9^th month. Within the placebo group, there was no significant difference in the mean haemoglobin concentration of the infants of mothers that received the placebo at the 6^th, 8^th, and 9th months of pregnancy ( p˃ 0.05), but a significant difference (p˂0.05) was observed in the mean haemoglobin concentrations of infants of mothers that took the supplement at the 7^th month and 9^th months; with that of the 9^th month being the least. When the trial group was compared with the placebo group, significant differences, (p˂0.05) were observed in the mean haemoglobin concentrations of those of the 6^th, 8^th and 9^th months. No significant difference was observed in those at the 7^th month (p˃0.05).The values of the placebo group were lower in all the months when compared with the trial group.

There was no significant difference in the mean haemoglobin concentration of infants whose mothers took the supplement in the secundigravids group from the 6^th month through the 9^th month in both the trial and placebo groups (p˃0.05). When the trial and placebo groups were compared, no significant difference was observed in the mean haemoglobin concentration of infants whose mothers took the supplement at the 6^th month. But significant differences were observed in the mean haemoglobin concentrations of infants whose mothers took the supplement at the 7^th, 8^th and 9^th months.

In the trial group of the multigravids, there was also no significant difference in the mean haemoglobin concentrations of infants of mothers who received the vitamin A supplement in all the months. But in the placebo group, the mean haemoglobin concentrations of infants whose mothers started receiving the supplement at the 7^th month differed only from those whose mothers started the supplement at the 8^th month. However, when the trial and placebo groups were compared, no significant difference (p˃0.05) was observed in the mean haemoglobin values of infants whose mothers received the supplement at the 6^th and 8^th months, but there was a significant difference in the haemoglobin concentrations of infants whose mothers started the supplement at the 7^th and 9^th months (p˂0.05). Generally, the haemoglobin concentrations of the placebo group were smaller than those of the trial group.

Table15. Effects of maternal vitamin A supplementation on mean haemoglobin concentrations (g/dl) of infants.

OVERALL

Months Trial Placebo

6^th 11.04±1.02^a 10.23±0.97^a*

7^th 11.11±0.90^a 10.38±1.02^a*

8^th 11.07±0.91^a 10.49±0.88^a*

9^th 10.95±0.96^a 10.31±0.99^a*

PRIMIGRAVIDS

6^th 11.06±1.16^a 9.93±0.49^ab*

7^th 11.03±0.97^a 10.35±0.90^{b ns}

8^th 11.01±0.88^a 10.09±0.92^ab*

9^th 10.63±1.16^a 9.85±0.75^a*

SECUNDIGRAVIDS

6^th 11.08±0.73^a 10.16±1.31^{a ns}

7^th 10.99±0.88^a 10.46±0.97^a

8^th 11.19±0.90^a 10.75±0.74^a*

9^th 11.05±0.87^a 10.30±0.96^a*

MULTIGRAVIDS

6^th 11.01±1.00^a 10.59±0.98^{ab ns}

7^th 11.22±0.93^a 10.33±1.10^a*

8^th 11.04±0.95^a 10.81±0.78^{b ns}

9^th 11.07±0.81^a 10.54±1.03^{ab *}

Within the trial group of the overall, there was no significant difference in the effects of the supplement on the mean malaria- associated fever episodes among infants whose mothers started receiving the vitamin A supplement at the 6^th, 7^th, 8^th and 9^th months respectively. Although at the 7^th month, there seems to be a relatively higher mean episode of the morbidity. Also, within the placebo group, there was no significant difference. But when the trial group was compared with the placebo group, significant differences (p˂0.05) were observed in all the months except in the 7^th month.

In the trial group of the primigravids, the effects of the vitamin A supplement on mean malaria- associated fever episodes among infants was different in infants whose mothers started receiving the supplement at the 7^th month when compared with those whose mothers started it at the 6^th and 8^th months but it did not differ significantly from the infants whose mothers started receiving the supplement at the 9^th month. Within the placebo group, the differences in the effects of the supplement were observed among infants whose mothers commenced the supplement at the 7^th and 9^th months.

When the trial group was compared with the placebo group, significant differences (p˂0.05) were observed among the 7^th and 8^th month groups, while no significant difference (p˃0.05) was observed in the 6^th and 9^th month groups. The mean malaria fever episodes were in most cases higher in the placebo group than in the trial group.

Among the secundigravids, there was no significant difference in the mean malaria- associated fever episodes from the 6^th month through the 9^th month in both the trial and placebo groups. When the trial group was compared with the placebo group, no significant difference (p˃0.05) was also observed in all the months at which the regimen commenced, although there seems to be higher mean episodes among the placebo than in the trial group.

There was no significant difference in the mean malaria fever episodes among infants whose mothers started receiving the supplement at their 6^th ,7^th, 8^th and 9^th months into pregnancy in both the trial and placebo groups. But when the trial and placebo groups were compared, statistical significant differences (p˂0.05) were observed in all the months except in the 9^th month which had no significant difference (p˃0.05). There were generally higher mean malaria fever episodes among infants from the placebo group in comparison with those of the trial group (table 16).

Table 16: Effects of maternal Vitamin A supplementation on malaria-associated fever episodes among infants.

OVERALL

Months Trial Placebo

6^th 1.33±0.71^a 2.55±0.70^a*

7^th 1.95±0.97^a 2.16±0.70^{a ns}

8^th 1.52±0.60^a 2.36±0.80^a*

9^th 1.71±0.85^a 2.28±0.75^a*

PRIMIGRAVIDS

6^th 1.50±1.00^a 1.50±0.58^{ab ns}

7^th 3.00±0.00^b 1.00±0.00^a*

8^th 1.33±0.50^a 1.83±0.75^ab*

9^th 1.83±0.75^ab 2.25±0.50^{b ns}

SECUNDIGRAVIDS

6^th 1.00±0.00^a 2.33±0.58^{a ns}

7^th 2.14±1.06^a 2.17±0.98^{a ns}

8^th 1.50±0.55^a 2.00±0.63^{a ns}

9^th 1.67±0.71^a 2.60±0.89^{a ns}

MULTIGRAVIDS

6^th 1.25±0.50^a 2.00±0.00^{a *}

7^th 1.50±0.80^a 2.25±0.62^{a *}

8^th 1.70±0.67^a 2.56±0.73^{a *}

9^th 1.71±0.42^a 2.11±0.78^{a ns}

Key: Values in a column with different alphabets as superscripts are significantly different. (p˂0.05)

Ns—no significant difference (p˃0.05) , determined by t- test

*--- significant difference (p˂0.05), determined by t- test.

There was no significant difference in the overall effect of the supplement on mean malaria- associated cough among infants whose mothers started receiving the vitamin A from the 6^th month in pregnancy through the 9^th month in both the trial and placebo groups. Also, when the trial group was compared with the placebo group, no statistical significant difference was observed in the mean episodes of the morbid condition among infants whose mothers commenced the supplements across the months.

In the trial group of the primigravids, there was no significant difference in the mean episode of cough from the 6^th month group through the 9^th month group. Among the placebo group, significant difference was observed between infants of the 8^th and 9^th month groups. However, when the trial group was compared with the placebo group, significant difference (p˂0.05) was statistically observed among the infants whose mothers started receiving the supplement at the 8^th month.

No significant mean episode of malaria cough difference was observed across the months in both the trial and placebo groups of the secundigravids. When the trial and placebo groups were compared, there was also no statistical significant difference (p˃0.05) in mean malaria cough among infants of mothers from all the groups.

Among the multigravids, significant differences in malaria coughs were also not observed across the months in both the trial and placebo groups. And when the trial and placebo groups were compared, no significant difference was observed also across the months (p˃0.05). (Table17).

Table 17: Effects of maternal vitamin A supplementation on malaria- associated cough in infants.

OVERALL

Months Trial Placebo

6^th 1.43±0.53^a 1.38±0.52^{a ns}

7^th 1.20±0.42^a 1.46±0.69^{a ns}

8^th 1.38±0.51^a 1.41±0.62^{a ns}

9^th 1.27±0.60^a 1.17±0.39^{a ns}

PRIMIGRAVIDS

6^th 1.00±0.00^a 1.50±0.58^{ab ns}

7^th 1.00±0.00^a 1.00±0.00^ab

8^th 1.00±0.00^a 1.83±0.75^b*

9^th 1.75±0.48^a 1.00±0.00^{a ns}

SECUNDIGRAVIDS

6^th 1.00±0.00^a 1.50±0.71^{a ns}

7^th 1.75±0.50^a 1.67±0.67^{a ns}

8^th 1.25±0.50^a 1.20±0.45^{a ns}

9^th 1.17±0.41^a 1.25±0.50^{a ns}

MULTIGRAVIDS

6^th 1.50±0.58^a 1.00±0.00^{a ns}

7^th 1.00±0.00^a 1.38±0.52^{a ns}

8^th 1.43±0.53^a 1.17±0.41^{a ns}

9^th 1.00±0.00^a 1.00±0.00^a

Key: Values in a column with different alphabets as superscripts are significantly different. (p˂0.05)

Ns—no significant difference (p˃0.05) , determined by t- test

*--- significant difference (p˂0.05), determined by t- test.

There was no significant difference in the malaria- related diarrhea episodes among the trial group of the overall. And within the placebo group, there was no significant difference in the mean diarrhea episodes of infants whose mothers started taking the supplement at the 8^th and 9^th month. There was also no difference among those whose mothers started the supplement at both the 6^th and 7^th months. However, those in the 8^th and 9^th months differed from

those in the 7^th month. When the trial group was compared with the placebo group, a statistically significant difference (p˂0.05) was only observed in the mean malaria- associated diarrhea of infants whose mothers started taking the supplement at the 7^th month.

Within the trial group of the primigravids, there were no significant differences in the mean malaria- associated diarrhea among infants whose mothers started receiving the supplement from the 7^th month through the 9^th month. However, the mean malaria diarrheoa episodes among infants of mothers of the 6^th month group differed from those of other months. Within the placebo group, there were no differences in the mean of the episodes among infants of mothers from both the 6^th and 7^th months. There were also no significant differences among those from the 8^th and 9^th months. However, the 6^th and 7^th month groups differed from those of 8^th and 9^th months. When the trial group was compared with the placebo group, a statistically significant difference (p˂0.05) was observed in the 7^th month group, while the 8^th month group had no statistical significant difference (p˂0.05).

Among the secundigravids, no significant difference was observed within the trial group from the 6^th month through the 9^th month. Also, no significant difference was observed across the months among the placebo group. However, when the trial group was compared with the placebo group, a significant difference (p˂0.05) was observed only among infants from the 8^th month group.

Across the months in both the trial and placebo groups of the multigravids, there was no significant difference in mean malaria- related diarrhea episodes among the infants of mothers of the group. When the trial group was compared with the placebo group, no significant differences (p˃0.05) were observed in the mean episodes among infants of mothers of the 7^th and 8^th month groups (table 18).

Table 18: Effects of maternal Vitamin A supplementation on malaria- associated diarrhea episodes in infants

OVERALL

Months Trial Placebo

6^th 1.00±0.00^a 1.50±0.54^{ab ns}

7^th 1.00±0.00^a 1.67±0.80^{b *}

8^th 1.14±0.38^a 1.19±0.40^{a ns}

9^th 1.00±0.00^a 1.00±0.00^a

PRIMIGRAVIDS

6^th 2.00±0.00^a 2.00±0.00^b

7^th 1.00±0.00^b 2.50±0.71^b*

8^th 1.50±0.71^b 1.00±0.00^{a ns}

9^th 1.00±0.00^b 1.00±0.00^a

SECUNDIGRAVIDS

6^th 0.00±0.00^a 1.00±0.00^a

7^th 1.00±0.00^a 1.00±0.00^a

8^th 0.00±0.00^a 1.20±0.45^{a *}

9^th 1.00±0.00^a 1.00±0.00^a

MULTIGRAVIDS

6^th 1.00±0.00^a 1.00±0.00^a

7^th 1.00±0.00^a 1.33±0.58^{a ns}

8^th 1.00±0.00^a 1.29±0.49^{a ns}

9^th 0.00±0.00^a 1.00±0.00^a

Key: Values in a column with different alphabets as superscripts are significantly different. (p˂0.05)

Ns—no significant difference (p˃0.05), determined by t- test

*--- significant difference (p˂0.05), determined by t- test.

In the trial group of the overall, there was no significant difference in the mean malaria- associated vomiting among infants of mothers who started taking the vitamin A supplement from the 6^th month through the 9^th month of pregnancy. Within the placebo group, no significant was observed in the mean malaria- associated vomiting among infants in the 8^th and 9^th months. But significant difference was observed between infants of mothers from the 6^th month and those of the 8^th and 9^th month groups. When the trial group was compared with the placebo group, no significant difference (p˃0.05) was observed in the mean malaria- associated vomiting episodes of infants from mothers of the 6^th and 7^th month groups.

Among the trial group of the primigravids, there was no significant difference in the mean malaria related vomiting episodes among infants whose mothers started the supplement from the 6^th, 7^th, 8^th and 9^th months. Within the placebo group, infants of mothers who started receiving the placebo at the 7^th and 8^th months of pregnancy did not differ in their mean vomiting episodes. They did not significantly differ from the infants of the 6^th month group. However, they significantly differed from infants of mothers of the 9^th month group.

Among the secundigravid trials, the mean malaria- associated vomiting episodes did not differ across the months. In the placebo group, the mean vomiting episodes differed only among infants of mothers of the 6^th month group. When the trial group was compared with the placebo group, no significant difference (p˃0.05) was observed in the 8^th month group.

In both the trial and placebo groups of the multigravids, there was no significant difference in the vomiting episodes among the infants across the months. Also, when the trial and placebo groups were compared, no significant difference (p˃0.05) were observed among infants of the 7^th and 9^th month groups, although the mean vomiting episodes were generally relatively higher among the placebo groups than in the trial group (table 19).

Table 19: Effects of maternal Vitamin A supplementation on malaria- associated vomiting episodes in infants.

OVERALL

Months Trial Placebo

6^th 1.00±0.00^a 1.60±0.89^{b ns}

7^th 1.00±0.00^a 1.18±0.40^{ab ns}

8^th 0.00±0.00^a 1.00±0.00^a

9^th 1.00±0.00^a 1.00±0.00^a

PRIMIGRAVIDS

6^th 1.00±0.00^a 1.50±0.71^ab

7^th 1.00±0.00^a 1.00±0.00^a

8^th 0.00±0.00^a 1.00±0.00^a

9^th 0.00±0.00^a 2.00±0.00^b

SECUNDIGRAVIDS

6^th 1.00±0.00^a 3.00±0.00^b

7^th 0.00±0.00^a 1.00±0.00^a

8^th 0.00±0.00^a 1.00±0.00^a

9^th 1.00±0.00^a 1.25±0.50^{a ns}

MULTIGRAVIDS

6^th 1.00±0.00^a 1.00±0.00^a

7^th 1.00±0.00^a 1.50±0.58^{a ns}

8^th 0.00±0.00^a 1.00±0.00^a

9^th 1.00±0.00^a 1.50±0.58^{a ns}

Key: Values in a column with different alphabets as superscripts are significantly different. (p˂0.05)

Ns—no significant difference (p˃0.05), determined by t- test

*--- significant difference (p˂0.05), determined by t- test.

In the trial group, the overall, all the gravidities, and across the months, no cases of rapid breathing were observed among the infants. In the placebo group of the overall, a significant difference was observed only among infants of mothers of the 8^th month group.

Among the primigravids, malaria- associated rapid breathing among infants of the 6^th month group differed from those of other months. There was no significant difference between those in the 8^th and 9^th month groups. However, those in the 7^th month group differed from those in the 9^th month.

Within the secundigravids, no significant difference was observed in the rapid breathing of the infants of mothers across the months.

In the multigravids, there were also no differences in the rapid breathing of the infants across all the months (table 20)

Table 20: Effects of maternal vitamin A supplementation on malaria- associated rapid breathing in infants.

OVERALL

Months Trial Placebo

6^th 0.00±0.00^a 1.00±0.00^a

7^th 0.00±0.00^a 1.00±0.00^a

8^th 0.00±0.00^a 1.33±0.58^b

9^th 0.00±0.00^a 1.00±0.00^a

PRIMIGRAVIDS

6^th 0.00±0.00^a 0.00±0.00^a

7^th 0.00±0.00^a 1.00±0.00^b

8^th 0.00±0.00^a 1.50±0.71^bc

9^th 0.00±0.00^a 2.00±0.00^c

SECUNDIGRAVIDS

6^th 0.00±0.00^a 0.00±0.00^a

7^th 0.00±0.00^a 1.00±0.00^a

8^th 0.00±0.00^a 1.00±0.00^a

9^th 0.00±0.00^a 1.00±0.00^a

MULTIGRAVIDS

6^th 0.00±0.00^a 1.00±0.00^a

7^th 0.00±0.00^a 1.00±0.00^a

8^th 0.00±0.00^a 0.00±0.00^a

9^th 0.00±0.00^a 1.00±0.00^a

Key: Values in a column with different alphabets as superscripts are significantly different. (p˂0.05)

Ns—no significant difference (p˃0.05) , determined by t- test

*--- significant difference (p˂0.05), determined by t- test.

As with rapid breathing, the entire trial group had no cases of convulsion. Within the placebo group of the overall, the absence of episodes of convulsion in infants whose mothers belong to the 9^th month group made them differ from the rest of the groups.

In the primigravids, there were no cases of malaria-related convulsion episodes in infants of the 6^th and 9^th month groups. There were no differences in malaria-related convulsion episodes among infants of mothers from the 7^th and 8^th month group. Among the secundigravids, there were no statistical differences in convulsion episodes among infants from the groups across the months. Although infants from mothers of the 6^th and 7^th month groups appear to differ, the differences were non- significant.

There were also non- significant differences in the cases of the morbid condition across the months among infants from the multigravids (table 21)

Table 21: Effects of maternal Vitamin A supplementation on malaria associated convulsion

OVERALL

Months Trial Placebo

6^th 0.00±0.00^a 1.00±0.00^b

7^th 0.00±0.00^a 1.00±0.00^b

8^th 0.00±0.00^a 1.25±0.50^b

9^th 0.00±0.00^a 0.00±0.00^a

PRIMIGRAVIDS

6^th 0.00±0.00^a 0.00±0.00^a

7^th 0.00±0.00^a 1.00±0.00^b

8^th 0.00±0.00^a 1.33±0.58^b

9^th 0.00±0.00^a 0.00±0.00^a

SECUNDIGRAVIDS

6^th 0.00±0.00^a1.00±0.00^a

7^th 0.00±0.00^a 1.00±0.00^a

8^th 0.00±0.00^a 0.00±0.00^a

9^th 0.00±0.00^a 0.00±0.00^a

MULTIGRAVIDS

6^th 0.00±0.00^a0.00±0.00^a

7^th 0.00±0.00^a 1.00±0.00^a

8^th 0.00±0.00^a 1.00±0.00^a

9^th 0.00±0.00^a 0.00±0.00^a

Within the trial group of the overall, there were no significant differences in the effects of the supplement on infantile age at first malaria parasitaemia. Although infants born by mothers who started taking the supplement at the 6^th month seem to harbor the parasites earlier in life in comparison to others, the differences were insignificant. Among the placebo group, the age (in months) at which the infants developed malaria parasitaemia for the first time differed among infants of mothers in the 6^th and 7^th month groups. It also differed between the 6^th and 9^th month groups. However, there were no significant differences in age at first malaria- parasitaemia onset among infants of mothers of the 6^th and 8^th month, 7^th and 8^th month and 8^th and 9^th month groups. When the trial group was compared with the placebo group, significant differences (p˂0.05) were observed in the 7^th and 8^th month groups, while non- significant difference (p˃0.05) were observed in the 6^th and 9^th month.

There were no significant differences in age at first malaria parasitaemia across all the months among infants of the primigravids mothers of the trial group. There were also no significant differences among infants across the months in the placebo group. However, when the trial and placebo groups were compared, a statistical significant difference (p˂0.05) was only observed among infants of mothers of the 8^th month group.

Within the secundigravids, there was no significant difference in the effect of the vitamin A supplement on age at first malaria parasitaemia of the infants across all the months among the trial group. There were also no significant differences across the months in the placebo group. But when the trial and placebo groups were compared, significant differences (p˂0.05) in age at first malaria parasitaemia of the infants were observed among infants born by mothers in the 6^th and 7^th month groups. Conversely, non-significant differences (p˃0.05) were observed in the 8^th and 9^th month groups.

Among the trial group of the multigravids, the infantile age at first malaria parasitaemia differed only in the 6^th and 8^th month groups. In the placebo group, the age at first malaria parasitaemia significantly differed among infants of mothers in the 6^th month group and those of the 8^th and 9^th months, but does not differ significantly when compared with infants of mothers of the 7^th month group. Also, those in the 8^th and 9^th month groups do not differ significantly from those of the 7^th month.

When the trial group was compared with the placebo group, there were non- significant differences (p˃0.05) across the months. Although there were certain cases of non significant differences across the gravidities, the age (in months) at which the infants become infected with the malaria parasites were generally lower (younger age) in the placebo group than in the trial group.

Table 22: Effects of maternal Vitamin A supplementation on infantile age (in months) at first malaria parasitaemia

OVERALL

Months Trial Placebo

6^th 4.56±2.24^a 3.46±1.04^{a ns}

7^th 6.20±1.94^a 4.61±1.41^{b *}

8^th 6.32±2.60^a 4.44±1.70^{ab *}

9^th 5.05±2.42^a 4.79±1.78^{b ns}

PRIMIGRAVIDS

6^th 4.75±1.44^a 3.75±1.50^{a ns}

7^th 5.50±2.50^a 4.60±1.52^{a ns}

8^th 6.00±1.01^a 3.38±1.06^{a *}

9^th 5.00±1.00^a 4.50±1.73^{a ns}

SECUNDIGRAVIDS

6^th 7.00±0.00^a 3.67±1.15^{a *}

7^th 6.71±1.70^a 4.50±1.38^{a *}

8^th 6.29±1.89^a 4.83±2.40^{a ns}

9^th 4.67±2.40^a 4.00±1.55^{a ns}

MULTIGRAVIDS

6^th 3.00±0.00^a 3.00±0.00^a

7^th 6.00±2.52^ab 4.69±1.44^{ab ns}

8^th 6.60±2.63^b 5.11±1.26^{b ns}

9^th 5.57±2.70^ab 5.63±1.92^{b ns}

Key: Values in a column with different alphabets as superscripts are significantly different. (p˂0.05)

Ns—no significant difference (p˃0.05) , determined by t- test

*--- significant difference (p˂0.05), determined by t- test.

In the trial group, the correlations between malaria parasitaemia and glucose (r=0.018), and malaria parasitaemia and infantile age at first malaria parasitaemia (r=0.091) were positive, even though the associations were insignificant (p˃0.05). However, negative correlations that were also not significant (p˃0.05) were found between malaria parasitaemia and haemoglobin (r= -0.026) and malaria parasitaemia and fever (r= -0.155). Correlations between the infants blood glucose and haemoglobin concentrations and blood glucose concentrations and age at first malaria parasitaemia were positive (r= 0.074 and r= 0.228) respectively.

The correlation between glucose and haemoglobin was however not significant (p˃0.05) while that between glucose and age was significant (p˂0.05). However, the correlation between fever and blood glucose concentrations was negatively and non- significantly correlated (r= -0.174; p˃0.05).The haemoglobin concentrations (Hb) correlated positively but non- significantly with fever episodes (r= 0.100; p˃0.05) while it correlated negatively and also non- significantly with age at first malaria parasitaemia (r= -0.093; p˃0.05). Malaria fever episodes correlated negatively but significantly with age at first malaria parasitaemia (r= -0.267; p˂0.05).

Within the placebo group, malaria parasitaemia correlated positively though non- significantly with blood glucose concentrations, haemoglobin concentrations, and with age at first malaria parasitaemia (r=0.090, p˃0.05; r= 0.088, p˃0.05; r= 0.009, p˃0.05) respectively. However, it correlated negatively with fever (r= -0.220), though not significantly (p˃0.05). The blood glucose concentration correlated positively and significantly with haemoglobin (r= 0.176; p˂0.05), but correlated negatively and non- significantly with malaria fever episodes, and age at first malaria parasitaemia respectively (r= -0.183, p˃0.05; r= -0.020, p˃0.05). The haemoglobin concentrations (Hb), correlated negatively and non- significantly with malaria fever (r= -0.221, p˃0.05). However, it correlated positively but also non- significantly with age at first malaria parasitaemia (r=0.041; p˃0.05). Fever episodes correlated negatively and non-significantly with age at first malaria parasitaemia (r= -0.042, p˃0.05)

Table : Correlations of parameters

Trial group

Para Glucose Hb Fever Infantile Age

Para 1 0.018 -0.026 -0.155 0.091

Glucose 0.018 1 0.074 -0.174 0.228^*

Hb -0.026 0.074 1 0.100 -0.093

Fever -0.155 -0.174 0.100 1 -0.267^*

Infantile age 0.091 0.228^* -0.093 -0.267 1

Placebo group

Para 1 0.090 0.088 -0.220 0.009

Glucose 0.090 1 0.176^** -0.183 -0.020

Hb 0.088 0.176^** 1 -0.221 0.041

Fever -0.220 -0.183 -0.221 1 -0.042

Infantile age 0.009 -0.020 0.041 -0.042 1

Key:

Para—parasitaemia

Glucose—blood glucose concentration

Hb—haemoglobin concentration

Fever—fever episodes

Infantile age in months

*----- correlation is significant at 0.05 level (2-tailed).

**---- correlation is significant at the 0.01 level

Within the trial group, the mean age of the multigravids differed significantly from the rest of the gravidities. The primigravids had the least mean age while the multigravids had the highest mean age.

In the placebo group, the mean age of mothers differed across the gravidities, with the primigravids having the least mean age.

When the trial group was compared with the placebo group, a statistically significant difference (p<0.05) was obtained in the mean age of the primigravids. The mean age of both the secundigravids and the multigravids differed non- significantly (p>0.05).

Table : Mean age of mothers

Trial Placebo

Overall 26.93±5.37 26.56±5.50^ns

Primigravids 23.24±4.43^a 20.00±2.59^{a *}

Secundigravids 25.63±3.37^a 27.58±4.20^{b ns}

Multigravids 30.20±4.85^b 29.93±3.83^{c ns}

Key: Values in a column with different alphabets as superscripts are significantly different. (p˂0.05)

Ns—no significant difference (p˃0.05) , determined by t- test

*--- significant difference (p˂0.05), determined by t- test.

There was no significant effect of parasitaemia on any of the parameters- birth weight, blood glucose concentration, haemoglobin concentration and fever in both the trial and placebo groups. However, parasitaemia had a relatively higher effect though non-significant on fever, among the placebo group (r=0.220, r²=0.048, p=0.06).

Table : Regression: Association of parasitaemia with other parameters

Parasitaemia

Trial Group R R² P value

Birth weight 0.099 0.001 0.436

Glucose 0.018 0.000 0.789

Haemoglobin 0.026 0.001 0.699

Fever 0.155 0.024 0.171

Placebo Group

Birth weight 0.018 0.000 0.886

Glucose 0.090 0.008 0.102

Haemoglobin 0.088 0.008 0.110

Fever 0.220 0.048 0.061

DISCUSSION

The study is a placebo-controlled mother-infant dyad field trial conducted in Ebonyi state, Nigeria. A total of 76 pregnant women who received 10,000 I.U of Vitamin A soft gels three times a week and 76 who received placebo in equal strength were followed up until they delivered their babies. On delivery, 200,000 I.U of the Vitamin A were administered to the supplemented group within 8 weeks post partum and the regimen was continued every three months until the end of the study after the 12^th month. Vitamin A devoid of its active ingredients was administered to the placebo group at the same periods in equal strengths and frequencies. The infants were breastfed by their mothers and were monitored for malaria parasites and for malaria – associated symptoms and morbidities.

Among the 64 available birth weight records from each of the supplemented and placebo groups, a total of 3(4.7%) low birth weights were observed in the trial group while 13(20.3%) low birth weights were recorded from the placebo groups (Table 4). The mean birth weights that were generally higher in the supplemented group than in the placebo group (table 12) is in consonance with the findings of Kumwenda et al., 2002 who reported an increase in birth weights of infants of human immunodeficient virus (HIV) – infected women who received Vitamin A with iron and folate when compared with the placebo group that received daily doses of iron and folate alone. The findings of this study also conforms with those of Jaya and Shatrugna, 1976; and Pauth iet al., 1991 who found higher birth weights among the Vitamin A supplemented group, when compared compared with the placebo group, #though the result was not significant# the reported insignificant differences, according to the authors could be attributed to the smallness of the sample sizes. Kaestel et al., (2005) also gave a conforming report to this finding, although they used multimicronutrient supplementation.

However, some findings disagree with this work. A trial from England conducted among South Asian immigrants with higher risks of lower birth weights than their British counterparts, reported no differences in birth weights (Howell et al., 1986). In addition, a randomized blinded trial conducted among HIV-positive women in Tanzania found no significant effect in mean birth weight (Fawzi et al., 1998).

The total frequencies of malaria fever episodes and the mean malaria fever episodes were higher in the placebo group (table 5 and 16 respectively). These higher frequencies in febrile episodes are in consonance with the findings of Shankar et al., (1991), who reported a 30% lower P. falciparum febrile episodes in the Vitamin A group, than in the placebo group, on their work on effect of Vitamin A supplementation on morbidity due to Plasmodium falciparum in young children in Papua, New Guinea. The findings of this work also agrees with that of Zeba et al. (2008), who reported 22% fewer episodes in the supplemented group than in the placebo group, when they administered combined Vitamin A with Zinc to Burkina Faso children.

However, these findings do not agree with that of Idindili et al. (2007), who reported no significant difference in serum retinol and incidence of illness in the infants of two groups in their work on randomized controlled safety and efficacy trial of 2 Vitamin A supplementation schedules in Ifakara in Southern Tanzania. The work is also not in consonance with that of Fernandes et al. (2012), who reported that postpartum maternal supplementation with 400,000 I.U of Vitamin A does not provide any additional benefits in the reduction of illness in children. Binka et al. (1995) on Vitamin A supplementation and childhood malaria in northern Ghana found no effect of the supplementation on death from malaria, fever episodes, malaria parasitaemia or probable malaria illness. Vitamin A supplementation for breast feeding mothers was reportedly reviewed to offer limited benefits in the improvement of infant morbidity (Oliveira-Menegozzo et al., 2010)

In the mean malaria – associated fever episodes (table 16), differences in significant effects in both the supplemented and placebo groups were observed across the gravidities, with the secundigravids having no significant difference at all across the months while the multigravids had significant differences in almost all the months. These differences explain the gravidity-dependent pattern of infant susceptibility to malaria and its morbidities as explained by Mutabingwa et al. (2005), who reported that placental malaria (PM) decreases susceptibility in the offspring of primigravids and increases the susceptibility in the offspring of the multigravids. Based on previous findings, expectations were that the infants of the primigravids should be more susceptible to malaria infection and the associated morbidities than infants of the secundigravids and multigravids.

The relative greater episodes of malaria cough (table 7) and the greater though non-significant mean malaria associated cough (table 17) observed among the placebo group in comparison with the supplemented counterparts is in line with Gebremedhin et al. (2009), who found no significant association between Vitamin A supplementation and occurrence of diarrhoea and symptoms of acute respiratory infections including cough, rapid breathing/difficulty in breathing. However, the findings of this study is not in consonance with that carried out in Ghana, that reported no effect at all of Vitamin A supplementation on probable malaria illness (Binka et al., 1995). The greater frequencies observed in the primigravidae over other gravidities in both the trial and placebo groups is in line with the gravidity-dependent explanation of Mutabingwa et al. (2005).

The greater frequencies of diarrhoea episodes among infants of the placebo group when compared with the supplemented group infants (table 8) and the greater relative though mostly non-significant (p>0.05) mean malaria- associated diarrhoea episodes among the placebo group over the supplemented group (table 18) could indicate the potency, though non-significant effect of Vitamin A supplementation in the prevention of diarrhoea in malaria infected infants. This finding is in line with the reports of (Lie et al., 1993; Barreto et al., 1994), who reported 60% and 6% reductions respectively in their works in China and Brazil. This finding is also in agreement with Beaton et al., (1993), who reported reduction in diarrhoea disease with Vitamin A supplementation. Rahman et al., (2001), also reported a decrease in the severity of some episodes of diarrhoea in children especially when Vitamin A is supplemented with Zinc.

However, some findings disagree with this work, Bloem et al., (1990), Ghana VAST study team (1993), and Dibley et al., (1996), all reported no effect of the supplement in the control of malaria diarrhoea while Stansfield et al., (1992) in his work in Haiti even reported 11% increase in diarrhoea episodes, following Vitamin A supplementation. The significant differences (p<0.05) observed in the 7^th month group of the overall, 7^th month group of the primigravids and 8^th month group of the secundigravids could be attributable to gravidity-dependent absorption mechanisms, which the affected months could be the months with most efficient absorption potentials and utilization of the Vitamin A supplement by the bodies of the pregnant women. Although this generalization is subject to further research and needs to be elucidated more, the respective affected months all fall at the middle of the third trimester of pregnancy, the period when the foetus starts to absorb the Vitamin A across the maternal placenta (Stoltzfus, 1994; Ortega et al., 1997; Azais-Braesco and Pascal, 1998).

The lower frequencies of vomiting episodes 8(100%) vs 40(100%), 1(100%) vs 10(100%), 2(100%) vs 14(100%), and 5(100%) vs 16(100%) for Vitamin A supplemented group against placebo group from the overall down to the multigravidae respectively (table 9), is similar to the findings of Arthur et al., (1992) who reported 13% reduction in the mean daily prevalence of vomiting in their work when they supplemented high dose Vitamin A in a 4- monthly schedule in Ghana. The mean vomitting episodes were also relatively lower in the supplemented group in comparison with the placebo group, though no significant difference was observed (p>0.05).

There was totally no observed case of frequencies of rapid breathing among infants of Vitamin A supplemented women. This could be a clear indication of Vitamin A’s potency in the clearance of respiratory associated infections as has been documented by some studies.

Significant reductions in the incidence of respiratory infections were observed in children under 3years of age who received 200,000 I.U of Vitamin A at 6 months interval (Lie et al., 1993). Tielsch et al., (2008) in their work in India where they dosed live born infants of night blind women with either Vitamin A or placebo, reported that infants of night blind women were at approximately 30% excess risk for respiratory infection after adjusting for potentially confounding factors. Since night blindness is an indication of Vitamin A deficiency, the reverse of the report by Tielsch et al., (2008) is that Vitamin A deficiency could potentially reduce respiratory disease by approximately some 30%.

However, a number of studies report that Vitamin A supplementation has no effect on respiratory disease. Ramakrishnan et al., (1995) reported that supplementin children under 5years old with a single, double or triple dose of 200,000I.U of Vitamin A did not affect the severity or incidence of respiratory illness. In addition, Coutsoudis et al., (2000) reported no evidence of improvement in neonatal or post neonatal respiratory problem associated with Vitamin A supplementation on morbidity of low birth weight neonates. Both the supplemented and placebo group neonates did not differ in the occurrence of the respiratory distress.

The mean rapid breathing differed most though non-significantly within the primigravidae. This is in line with normal expectations since the primigravids are known to be more prone to plasmodium infections than the other gravidities.

The overall total number of episodes of convulsion among infants of the placebo group is 9 (table 11). This figure could be considered insignificant when compared to with the total number of 76 infants in that group and with the episodes of other morbidities. There was almost no significant value observed in the mean convulsion episodes among the infants (table 21). This insignificant association of Vitamin A supplementation with convulsion episodes among the infants is similar to the findings of Mwanga-Amumpaire et al., (2012), who reported no difference in the resolution of convulsion (p=0.37) among the two treatment arms of Vitamin A and placebo in their work on the effect of Vitamin A adjuvant therapy for cerebral malaria in children in Mulago.

Infants of mothers of the placebo group were infected at early months of their lives during the follow-up period. While those from mothers that received the Vitamin A supplement were infected at more later months. The overall range in mean ages of the infants at first malaria parasite infection (malaria parasitaemia) expressed in months were 4.56±2.24 – 6.32±2.60 and 3.46±1.04 – 4.79±1.78 in both the Vitamin A supplemented group and the placebo respectively (table 22).

This finding agrees with (Zeba et al., 2008), who reported a longer time to first malaria fever episodes (p=0.015) among infants of the supplemented group in their work when they administered a single dose of 200,000I.U of Vitamin Ainc to a group and placebo to another group of children for six months in Burkina Faso. The reported differences in time to first malaria infection in this study were significant at the 7^th and 8^th months in the overall, and at 7^th month in the primigravidae, 6^th and 7^th months of the secundigravidae. It is an established fact that at early post natal months of life, infants immunity are generally low. This low immunity therefore renders them more susceptible to infections including that of malaria parasites, especially in a malaria hyperendemic and stable transmission areas. Vitamin A supplementation must have been responsible for prolonging time to first malaria infection observed in the supplemented group. Reports of work on infection in children within their 12months of life abound. One of such reports is that of Mabunda et al., (2009), who reported peak malaria infections that was associated with fever in children less than 12months of age in Mozambique.

The range of the overall mean malaria parasite densities per microlitre ( µL) of blood were 910.00±118.93 – 1447.38±196.91 and 1645.60±114.14 – 2350.54±75.03 in the supplemented and placebo groups respectively (table 13).

Except in the 7^th month of the primigravidae, the mean parasite densities were statistically significant (p<0.05) in all the months across the gravidities. The findings of this study disagrees with that carried out in 1995 in Ghana, where Vitamin A was administered to pre-school children but no statistically significant effect was observed in P. falciparum infection morbidity or mortality (Binka et al., 1995).

No evidence of a morbidity or mortality benefit to the infants was reported by Gogia and Sachder (2010) in their review on maternal postpartum Vitamin A supplementation. However, the basis of their review might be centred more on the curative rather than the prophylactic potentials of Vitamin A. this is because they stated that the utilization of the supplementation programme as an intervention in public health programmes be made only in the prevention of infant morbidity or mortality.

However, the findings of this study is in line with Shankar et al., (1999) in a double blind placebo – controlled trial in Papua, New Guinea, who reported a 36% lower geometric mean parasite density with a 30% reduction (p=0.0013) in the frequency of P. falciparum episodes in the Vitamin A group than in the placebo group of pre-school children. Children aged 12-36 months were reported to have benefited more by having a 35%(p=0.0023) fewer malaria attacks and a 68% reduction in parasite density. The parasite clearance efficacy of Vitamin A has been reported in in vitro studies. Davies et al., (1998) reported that in vitro addition of free retinol to P. falciparum cultures resulted in the reduction of parasite replication. Quarterly Vitamin A supplementation to pre-school children in Tanzania also gave rise to a decreased risk of malaria mortality (Fawzi et al., 1999).

In the overall level, in all the months, and across the gravidities, statistical significant differences (p<0.05) were observed in the mean blood glucose concentrations of the infants (table 14). The mean blood glucose concentrations of the infants in the placebo group women were significantly lower (p<0.05) than those in the Vitamin A supplement group. This lower blood glucose concentration in the placebo group could be as a result of the metabolic effect of Plasmodium falciparum parasites and their disruptive activities on the blood glucose concentrations and homeostasis of the hosts.

The Plasmodium parasites have been reported to increase glucose consumption by 50-100 folds in infected red blood cells as compared to uninfected cells and that most of the glucose is metabolized to lactic acid (Roth, 1990). The parasites are highly dependent on glucose and very sensitive to oxidative stress (Preuss et al., 2012). The mechanism of glucose metabolism by the P. falciparum in the infected red blood cells (placebo group) may have been blocked, deleted or reversed in the erythrocytes of the uninfected cells (Vitamin A supplemented group) by the Vitamin A supplement.

However, it is interesting to note that even though the blood glucose concentration observed among the placebo group in this study did not reach hypoglycaemic levels (<40mg/dl), the reduced levels might have accounted for the few rapid breathing and convulsion cases; symptoms of hypoglyceamia observed in this work.

The range of the overall mean haemoglobin concentrations (mg/dl) of the infants of the supplemented group was 10.95±0.96 – 11.11±0.90 while that of the placebo group infants was 10.23±0.97 – 10.49±0.88 (table 15) at the overall level, significant differences (p<0.05) were observed in all the months while across the gravidities, some but few non-significant cases were also observed. Also at the overall, all of the infants born by the mothers of the placebo group had mild anaemia (Hb<11g/dl). But in the primigravids, infants of women who fall within the 6^th and 9^th month groups had morderate anaemia (Hb <10g/dl). Infants of the secundigravidae and multigravidae all had mild anaemia. No cases of severe anaemia (Hb<7g/dl) was observed in any level of gravidity. The 100%mild anaemia observed in the placebo group of this study could be attributed to Vitamin A deficiency at the sub-clinical level. The subclinical stage of the micronutrient deficiency could have been the reason why no clinical manifestation of signs of the deficiency like Bitot’s spots, Xerolphthalmia and keratomalacia was observed among the studied infants but Vitamin A deficiency at the clinical stage is known to cause some of its associated morbid conditions

The 100% mild anaemia observed in this study is similar to the findings of Nabakwe et al.,(2005) who reported 92% moderate anaemia in their work on young children in Western Kenya. It is also similar to that of Nkwo – Akereji et al (2008) who reported microcytic anaemia cases in 81.4% of the studied children in Cameroon. Vitamin A supplementation has always been known to boost levels of haemoglobin and reduce anaemia when right doses are supplemented. The greater haemoglobin levels observed in this study in infants born by the Vitamin A supplemented group in comparison to those born by the women in the placebo group is in line with the findings of Kumwenda et al., (2002) who reported a decrease in anaemia in the Vitamin A supplemented group of antenatal women in Malawi. However, the findings of this work do not agree with that of Milller et al., (2006), who reported that Vitamin A supplementation had no effect on haemoglobin or anaemia on infants in their work where they supplemented women and their neonates with Vitamin A in Zimbabwe.

The age at which the infants of supplemented mothers became infected with malaria parasites correlated negatively and significantly with malaria fever episodes but positively and significantly with glucose concentrations (r= -0.267, p<0.05 and r= 0.228, p< 0.05). but in the placebo group, the age at which the infants became infected with the malaria parasites correlated negatively but insignificantly with the febrile episodes and blood glucose concentrations (r= -0.042, p>0.05 anr r= -0.020, p>0.05) respectively (table )

The implication of the finding among infants of the supplemented women is that with increasing age in months of the infants, the malaria fever episodes decreasessignificantly (p<0.05) irrespective of parasite density. This could be attributed to the development of immunity as the infants become exposed to more infective mosquito bite.

A similar observation with respect to decline in malaria fever episodes with advancement in age has been made by Beadle et al., (1995), who reported that in areas of high malaria endemicity, the incidence of malaria associated fevers increases with age for the first 6 months of life and then gradually declines. Cox et al., (1994) also reported a decline in fever threshold.

In the placebo group, the age at which the infants became infected with malaria parasites also correlated negatively but insignificantly (p>0.05) with malaria fever episodes. The significant decline in malaria fever episodes observed in the supplemented group but not in the placebo group could be attributed to the Vitamin A potential in the reduction of fever incidence in infants with age in self reported febrile cases.

A significant positive correlation (r= 0.228, p<0.05) was observed between age of the infants and blood glucose levels in the supplemented group while a non-significant negative correlation of the two parameters respectively were observed in the placebo group (r= -0.020, p>0.05).

The negative correlation, though non- significant observed in the placebo group could be explained by the fact that with increase in age of the infants, they become more exposed to more infective mosquito bites which could lead to increase in the parasite density in their blood. High malaria parasite density is known to result to hypoglycaemia, with its associated symptoms including rapid breathing, convulsion and coma. The high parasite density could also make them not to feed well, thereby reducing blood sugar level. Akpede et al., (1993) reported coma, higher prevalence of repeated convulsion and hypoglyceamia in older children than in the younger ones.

The significant positive correlation observed in the supplemented group indicates an increase in the blood sugar level. With age, the Vitamin A supplement may have mitigated by reducing the parasite density and increasing the feeding appetite of the infants, thereby maintaining high levels of blood sugar with increasing age.

None of the parameters – birth weight, glucose, haemoglobin and fever (table ) had significant regression coefficient. However, fever had stronger associateion with the highest coefficient of determination (R² =0.042), the findings of this work is in line with that of Orogade (2004) who reported fever as the most strongly associated clinical feature (OR= 8.12, p= 0.002) in his work on neonatal malaria.

CONCLUSION

The increasing resistance of Plasmodium species to a vast array of antimalarials jeopardizes the global human health and is of great concern to health care givers. There is therefore a need for adjuvants to the antimalarials.

The malaria parasitaemia and morbidity prophylactic capacity of Vitamin A has been ascertained even though not significant at some statistical levels. Since Vitamin A supplementation administered according to the current WHO supplementation guidelines does not pose any health hazard to the recipients, it could be recommended at prepartum and postpartum levels, and/or administered directly to individuals ≥6 months of age as adjuvant regimen in the routine malaria prevention and control measures in areas where malaria is a public health problem and Vitamin A is deficient even at subclinical levels.

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EFFECT OF MATERNAL VITAMIN A SUPPLEMENTATION ON INFANT MALARIA PARASITAEMIA AND MORBIDITY IN EBONYI STATE, NIGERIA.

PG/Ph.D/13/xxxxx

DEPARTMENT OF ZOOLOGY,

UNIVERSITY OF NIGERIA, NSUKKA

SUPERVISORS: PROFESSORS

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