Received on: March 08, 2018
Accepted on: March 18, 2018
Published on: March 25, 2018
*Corresponding author: Erhabor Osaro, Department of Haematology and Blood Transfusion Science, Usmanu Danfodiyo University, Sokoto, Nigeria. E-mail: firstname.lastname@example.org; Tel: 234-813-962-5990
Malaria is one of the most important tropical infectious diseases. Despite the fact that it is a completely preventable disease, a staggering 3.4 billion people continues to be at risk of the disease globally with 1.2 billion people at high risk particularly in high endemic and predominantly developing countries. In 2012, malaria was responsible for the death of approximately 482,000 under-five children. In Nigeria, Plasmodium falciparum malaria is responsible for around 60% of the out-patient visits to health facilities, 30% of childhood death, 25% of death in children under one year and 11% of maternal deaths. This study was carried out to determine the effect of Plasmodium falciparum parasitaemia on the serum ascorbic acid level of 60 malaria parasitized children of African descent aged 6 month-5 years visiting the children emergency unit and paediatric unit of Sokoto State Specialist Hospital in Sokoto, North Western, Nigeria. Controls included 30 non-parasitized children. Serum vitamin C (ascorbic acid) was assayed using a standard chemical method. Data were analyzed using SPSS 22.0 statistical package. A p-value ≤0.05 was considered significant in all statistical comparisons. The mean ascorbic acid level of plasmodium- parasitized children (0.22 ± 0.00 mg/dL) was significantly lower compared to non -infected controls (0.55 ± 0.34 mg/dL), p=0.000. Plasmodium parasitaemia was more prevalent among children in the 6 months-30 months age group (61.67%) compared to children in the 31 months -60 months age group (38.33%) (p<0.05). Male children were more predisposed to malaria (71.67%) compared to female children (28.330%) (p<0.05). Plasmodium parasitaemia was more prevalent among children whose mothers with no formal education. Plasmodium falciparum was the predominant malaria specie identified. There is need for routine monitoring of ascorbic acid levels in plasmodium falciparum parasitized children as well as educational enlightenment on need to seek medical attention following potential malaria infection, keep their environment clean to eradicate the vector and use of insecticide-treated nets.
L-Ascorbic Acid; Children; African; Plasmodium Falciparum Malaria; Nigeria
In Nigeria, malaria is endemic and stable, being a major cause of morbidity and mortality, resulting in 25% of infant and 30% of childhood mortality . Tragically, the health status of children under the age of fve and women has remained a major barrier to Nigeria's development. About 30% -40% of out-patient consultations and paediatric admission are due to malaria with mortality rate for children admitted to hospital with severe malaria as high as 15-30% [2, 3]. Clinical presentation of malaria overlaps with other common illnesses and attempts to develop clinical scoring system and predictive value have proved unsuccessful .
Children under 5 years of age are one of most vulnerable group affected by malaria. There were an estimated 438,000 malaria deaths around the world in 2015, of which approximately 69% were among children under 5 years of age. In high transmission areas, partial immunity to disease is acquired during childhood. In such settings, the majority of malarial disease, and particularly severe disease with rapid progression to death, occur in young children without acquired immunity .
Malaria is caused by protozoan (Plasmodium), transmitted by female anopheles mosquitoes, which typically bite between dust and dawn . The parasite then travels to the liver where they mature and reproduce. Five species of plasmodium can infect and be spread by humans (P. falciparum, P. vivax, P. malariae P. ovale and P. knowlesi) . But among the fve, P. falciparum is the main cause of malaria and death in sub Saharan Africa. Malaria is not a uniform disease, it encompasses many manifestations and it is impact varies on epidemiological setting. In children symptoms are varied and often mimic other common childhood illness particularly gastroenteritis, meningitis, pneumonia, fever and headache. However, children are more likely to have high fever (>400c), which may also lead to febrile convulsion. Nausea and vomiting are also common especially for P. falciparum .
Vitamin C, also known as L-ascorbic acid, is a water'soluble vitamin that is naturally present in some foods, added to others, and available as a dietary supplement . Vitamin C serves as an antioxidant to protect intracellular and extracellular component from free radical damage. It mops up free radicals and forms the less reactive ascorbyl radical. The ascorbyl radical can then be either reduced to ascorbic acid or oxidized to dehydroascorbic acid .
It is a universally accepted fact that malaria is a disease of public health importance globally. The disease causes the greatest suffering among poor people, particularly in Africa. In 2001, the World Health Organization (WHO) ranked malaria as the eighth-highest contributor to the global disease burden as reﬂected in disability-adjusted life years (DALYs) and the second highest in Africa . Despite the availability of the malaria control measure and intervention, the morbidity and mortality in under-fives is still unacceptably high particularly in developing countries .
Vitamin C functions as an antioxidant to protect intracellular and extracellular and extra cellular component from free radical damage . It appears to reduce oxidation mediated damage to DNA in lymphocytes and may enhance production of interleukin-1 and tumor necrosis factor-a. Vitamin C also reduces T - cell death and increase NK activity. It has a higher concentration in neutrophils and appears to be used during infection to prevent oxidative damage .
Ascorbic acid has antioxidant properties and is reported to mop up free radicals. Since malaria infection imposes tremendous oxidative stress on the host, the antimalarials are often prescribed with vitamin C or similar antioxidant supplements. The antioxidant effect in erythrocytes has been reported to depend upon the presence or absence of glutathione. In the presence of glutathione, ascorbic acid has synergistic antioxidant activity against haem-mediated cell toxicity . In glutathione defcient red cells, as often happens in parasitized RBCs due to oxidative stress, ascorbic acid can react with iron or iron containing compounds to generate hydrogen peroxide or hydroxyl radical and heighten the hemolytic mechanisms in malaria . Vitamin C may have additional negative effects in malaria. Results from an experimental study have shown that concomitant administration of artemether and ascorbic acid compromised the rates of parasite clearance in P. berghei malaria infection in mice. This effect was more pronounced at higher doses of ascorbic acid. The high doses of vitamin C by itself could inhibit growth of malaria parasite to some extent . There is paucity of data on the ascorbic acid level among children with acute Plasmodium falciparum malaria particularly in Nigeria. This aim of this study was to determine the ascorbic acid levels among children (6 months-5 years) with Plasmodium falciparum malaria in Specialist Hospital, Sokoto. It is envisaged that result from this study apart from adding to scientifc knowledge, will provide valuable information that will optimize the care offered to children with malaria in the area.
Materials and Methods
The study was conducted in Specialist Hospital, Sokoto State, Nigeria. The hospital serves as a referral center for people from Sokoto, Kebbi and Zamfara and the neighboring Niger and Benin Republic, in the West African sub region. The study area is located in Sokoto State, which is in the extreme North-Western part of Nigeria between longitude 05ο 111 to 13o 031 East latitudes 13o 001 to 13o 061 North. The State share borders with the Republic of Niger to the North, Kebbi State to the West and South East and Zamfara to the East. The State covers a land area of about 32,000 square kilometers and with a population of 4.602298 million based on the United Nation Population Fund projection . Sokoto is, on a whole, a very hot area. However, maximum daytime temperatures are most of the year generally under 40oC (104oF). The warmest months are February to April. The raining season is from June to October during which showers are a daily occurrence, although rarely last long compared to that of the wet tropical regions. The indigenous inhabitants of the area are the Hausas and Fulani. Other ethnic group resident in the State includes; Igbo, Ebira, Yoruba, Igala as well as Buzus from the neighboring Niger Republic. Farming and crop production are the major occupation of the people living in the study area. The major crops grown in the area includes millet, sorghum, ground nuts, cowpea and tobacco. Livestock reared includes cattle, sheep, goat, donkey, camel, horses and poultry .
The subjects for this study included 60 consecutively recruited malaria parasitized children aged 6 month-5 years. Controls included 30 non-parasitized children. Subjects were recruited from among the children visiting the emergency unit and Paediatric Department while controls were recruited from among children visiting the immunization unit of Specialist Hospital, Sokoto North-Western Nigeria.
Sample Size Determination
The sample size was determined according to Cochran (1999) using the formula:
n = minimum sample size
z = standard normal deviation and probability.
p = prevalence or proportion of value to be estimated from previous studies.
q = Proportion of failure (= 1 - P)
d = precision, tolerance limit, the minimum is 0.05.
Therefore n = z2 pq/d2
Where Z = 95% (1.96)
P = 27.29% 
q = 1 - P = (1-0.27)
d = 5% (0.05)
Therefore n = (1.96)2 (0.27) (1-0.27) / (0.05)2 = 3.8416 x (0.27) (0.73)/0.0025
n = 303
The following children that met the inclusion criteria (children aged between 6 months - 5 years with confrmed Plasmodium parasitaemia visiting the Paediatric Outpatient Department of specialist Hospital Sokoto, Nigeria and children whose parents and guardian give a written informed consent for their ward to be included in the study were recruited into the study. Exclusion Criteria
The following children who did not meet the inclusion criteria (children aged < 6 months and > 5 years and children who parents or guardian refused to give a written informed consent for their ward to participate in the study were excluded from the study.
The research was a case-control study involving Plasmodium falciparum malaria parasitized children (subjects) and non-malaria parasitized controls. The aim of the study was to assess the level of ascorbic acid among 60 children aged (6month-5 years) with malaria. Thirty (30) age and gender- matched non-parasitized children will be monitored as controls. Sample ﬂow chat is shown in figure 1.
Figure 1: Sample Flow Chat
Ethical approval for this study will be obtained from the Ethics Committee of Specialist Hospital, Sokoto.
Diagnosis and speciation of malaria was made by demonstration of parasite in Giemsa-stained thin and thick blood smear . Serum vitamin C (ascorbic acid) was assayed using chemical method described by Natelson .
The data analysis was performed using statistical package of social sciences (SPSS) version 22.0. Data was presented as mean ± standard error of mean (SEM) and percentage. Student t-test was used for mean comparison between the subjects and controls. A p-value of less than or equal to 0.05 (p≤0.05) was considered as statistically signifcant in all statistical analysis.
The result of 60 malaria subjects recruited from the Paediatric units of Specialist Hospitals and 30 apparently healthy children as controls recruited from the immunization clinics of the same hospitals were analyzed. The malaria subjects presented with fever with chills and rigors, prostration, headache, abdominal pain, splenomegaly, nausea and vomiting. The entire subject was found positive for Plasmodium falciparum only. A structured interviewer administered questionnaire was used to obtain socio-demographic information of the subjects. Blood samples were analyzed for malaria testing and ascorbic acid levels in the laboratory.
Table 1 shows the socio-demographic characteristics of the malaria subjects and controls and reveals that majority of the subjects 37 (61.67%) and control group 17 (56.7%) were in the age range of 31-60 months. The distribution of patients and control group based on ethnicity shows that majority of the subjects and control participants were Hausa, 59 (98.3) and 27 (90%) respectively. The distribution of the patients based on maternal education status shows that none of the mothers were educated to tertiary level, 5 (8.33%) had secondary education, 16 (26.67%) had primary education and 39 (65%) had informal education as compared with control where none of the mothers were educated to tertiary education, 11 (36.67%) had secondary education, 9 (30%) had primary education and 10 (33.3%) had informal education. The distribution of patient and control based on gender shows that majority were male 43 (71.67%) and 22 (73.33%) respectively.
Table 2 revealed that ascorbic acid levels was signifcantly lower among patients subjects as compared to controls (p<0.05).
Table 1: The Socio-Demographic Characteristics of Subjects and Controls
Key: Group1=Control group and Group 11= Subjects
Table 2: The Level of Vitamin C and Vitamin B5 in Children with Malaria and Controls
Key: Data are presented as mean ± SEM. Group1 = Control Group, Group 11= Subjects, Vitamin C (Asorbic acid), (s) =statistically significant
Malaria is endemic throughout most of the tropics. Ninety-fve countries and territories have ongoing transmission . Of the approximately 3.2 billion people living in malarious countries, 1.2 billion are at high risk. The World Health Organization (WHO) states that there were 214 million (range 149 to 303 million) cases of symptomatic malaria in 2015. Between 2000 and 2015, the incidence of malaria decreased by 32 percent, and the number of annual malaria cases estimated by the WHO decreased by 18% .
The children with malaria included in this study were aged 6 months- 5 years. This conforms with previous reports that children under 5 years of age are one of most vulnerable groups affected by malaria. There were an estimated 438, 000 malaria deaths around the world in 2015, of which approximately 69% were children under 5 years of age. In high transmission areas, partial immunity to disease is acquired during childhood. In such settings, the majority of malarial disease, and particularly severe disease with rapid progression to death, occur in young children without acquired immunity .
In this present study, we observed a higher male gender predisposition to malaria infection. Our fnding is consistent with previous reports which indicated that Falciparum malaria was profound in males than female [22, 23]. Our fnding is also consistent with the report of a previous study in Nigeria which indicated that the prevalence of malaria was found to be higher among male children less than fve years than female counterpart . Our fnding is also consistent with previous reports which showed that male children generally suffered more malaria compared to their female counterpart [25, 26].
It was observed in this study that the majority of the mothers of the plasmodium parasitized subjects (65%) had no formal education; the remaining (26.6%) had primary education and the other (8.33%) had secondary education. Our fnding is consistent with previous studies which found a strong statistical association between child health, survivorship, improved nutritional status and increased immunization uptake with maternal education levels [27, 28]. Other studies in African setting have also yielded similar conclusions. A study in Kenya found that women reporting some higher level of education were more likely to own and use insecticide- treated bed net obtained from retail sector outlets than those without any formal education .
In this study, serum ascorbate levels were signifcantly decreased among plasmodium parasitized subjects when compared to control. This fnding is consistent with several studies which showed a decrease in the vitamin-C levels in malarial patients [30, 31]. This observation is also in agreement with a previous investigation performed in Pakistan, which showed that a lower concentration of vitamin A and vitamin C is common among 1 - 5-year-old children correlated with higher malarial parasitaemia . Similarly, our fnding is consistent with a previous report that investigated the serum ascorbic acid concentration in 150 acute falciparummalaria patients aged two to ten years and observed that the mean serum ascorbic acid concentration of healthy controls was signifcantly higher compared to malaria parasitized subjects .
Vitamin-C is known as an antioxidant because by donating its electrons, it can prevent other compounds from getting oxidized . Malarial infection activates the immune system of the body and thereby leads to release of reactive oxygen species (ROS). Ascorbic acid has antioxidant properties and is reported to mop up free radicals. Since malaria infection imposes tremendous oxidative stress on the host, the antimalarials are often prescribed with vitamin C or similar antioxidant supplements . Plasmodia cells accumulate protective enzymes (catalase, glutathione peroxidase and superoxiade dimutase) that are depleted in the red blood cells of the host. Enhanced production of hydrogen peroxide (H2O2) and free oxygen radicals and a decrease in antioxidant enzymes have been observed in parasitized erythrocytes [35, 36]. Moreover, reduced antioxidant enzymes defense in the RBCs of P. falciparum infected patients may be responsible for higher levels of lipid peroxidation and oxidative stress in children with moderate and high parasitaemia. Thus, the enhanced oxidative stress to erythrocytes in children with moderate and high parasiteamia may be endogenously stimulated by malaria parasites during their consumption of hemoglobin and further leading to anaemia . Ascorbic acid plays an important role in conserving plasma lipids from ROS. However, it is rapidly oxidized when challenged by oxidants released from activated polymorphonuclear neutrophils .
In this study, we observed that pplasmodium falciparum was found to be the predominant specie.
Our fnding is consistent with previous reports [37, 38, 39, 40, 41] in different parts of Nigeria which found that Plasmodium falciparum was the predominate specie responsible for malaria in Nigeria.
This study indicates that Plasmodium parasitaemia has a signifcant impact on the ascorbic acid level of children with Plasmodium falciparum malaria. The ascorbic acid was signifcantly lower in patient when compared with control. Malaria is more prevalent among the lower socioeconomic class among children whose mothers have no formal education. This study recommends that; there is need to routinely monitor ascorbic acid levels among plasmodium parasitized children in Sokoto, Nigeria. There is need to promote the use of insecticides- treated bed net and the use of mosquito repellent to reduce the incidence of malaria among children in Sokoto, Nigeria. Preventive treatment with antimalarial drugs and effective follow up to prevent the development of anemia and thrombocytopenia and to prevent the development of drug resistant strains. There is need for the enrollment of more females in school to improve access to formal education. There is need for community awareness program to strengthen the malaria prevention program by educating parents on the benefts of effective environmental sanitation aimed at destroying the breeding sites of Anopheles mosquito -the vector of malaria. More large-scale population-based studies should be carried out to assess the serum Ascorbic acid level in malaria patient in Nigeria. Vitamin C supplementation may be of therapeutic advantage in clinical outcome of the patients and thus recommend Vitamin C supplementation in malaria therapy.
Subjects for this case - control study were consecutively recruited from among children presenting to the Paediatric Outpatient Department of Specialist Hospital Sokoto, Nigeria with confrmed Plasmodium falciparum malaria. The limitation of this study is that an opportunistic sample size of 60 subjects was studied instead of the calculated sample size of 303 subjects because of the challenge associated with recruitment of subjects. There is the possibility that the fndings may be different if the ideal sample size was used.
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