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Evaluation of the Relationship between Malaria Infection and Signicant Spread of Electrolyte Variation in Patients: Within and Outside Limit Analytic Model.

Background: Malaria is one of the most common diseased conditions across most developing countries caused by one of four species of Plasmodium. Electrolyte imbalance and mineral disturbances are majorly identied clinical symptoms in various infectious diseases including malaria. Malaria infection has also been shown to be associated with abnormalities in uids, electrolytes and acid base balances which are capable of enhancing disease severity. The aim of this study was to investigate the effects of malaria infection on electrolytes parameters. Methods: Finger prick blood samples were collected from Two-Hundred (200) malaria-suspected subjects representing all age groups and gender. The Giemsa-stained blood smears were carefully examined according to standard procedure. Demographic information was obtained using structured questionnaires. Results: Pearson's Correlation Coecient technique was used to investigate the relationship, and the strength of association between the variables. The mean bound of patients’ sodium level was observed to fall within the specied normal limits of 125mmol/L – 145mmol/L; except for positive malaria patients belonging to the MP-(++) which will fall below the 125mmol/L (i.e. 126.25mmol/L – 1.77mmol/L = 124.48mmol/L). Conclusion: In our study we found that Plasmodium falciparum showed more alteration in electrolytes parameters than Plasmodium vivax. This study discovered a signicant linear relationship based on the Pearson product-moment correlation between creatinine and urea, potassium and chloride, potassium and creatinine, potassium and urea. The mean sodium and chloride level of positive malaria [MP-(++)] patients were observed to fall outside the normal limit.


Introduction
Malaria is one of the most widespread infectious diseases among humans and endemic in tropical and subtropical regions with highest prevalence in Africa and Southeast Asia, It is also one of the major public health problems in Nigeria, contributing a quarter of the malaria burden in Africa. (1). Humans are infected by ve Plasmodium species which are P. falciparum, P. malariae, P. ovale, P. vivax and P. knowlesi. although malaria infection is mainly caused by P. vivax and P. falciparum (2). Electrolytes are de ned as minerals found in blood and other body uids. For appropriate physiological processes to take place hence, vital for the essential life processes. These are the ionized salts (minerals) found in humans' body uids and the blood stream. for this reason it is vital to determine the optimum range mostly during infection (3). In various infections including malaria, electrolyte imbalances and mineral disturbances were identi ed as the most indicative clinical symptoms. Hyponatraemia, hyperkalaemia, hypocalcaemia and hypomagnesaemia usually develops as a result of infection with Plasmodium (4). The major cationic extracellular uid is known as Sodium (Na), which aids the regulation of normal water distribution and osmotic pressure in several body uids. Na + ion disturbance is a cause of major health problems (5). Hyponatraemia is referred to as decline in the amount of Na concentration and considered as an important clinical manifestation of malaria.
Coincidentally, decreased levels of Na exaggerates the disease symptoms and results in severe malaria condition in most patients (6). For all body cells, tissues and organs to accurately function, Potassium (K) is identi ed as a crucial electrolyte which is vital for the proper function of all body cells, tissues and organs. It further enhances the maintenance of blood pH and water levels in the body. A major importance of Potassium is its role in skeletal and smooth muscle contraction. Hypokalaemia, most times is a common complication of severe malaria. A noticeable treatment for acidosis in malaria is a decline in the level of Potassium (7). Calcium (Ca) on the other hand is considered an essential nutrient for human body whereby bones and teeth in the body are strengthened as a result of the presence of calcium in the body due to its relevance in maintaining health and nutrition of the body (8).
During a malaria infection, most noticeable symptom is reduction in calcium level. Clinical symptoms associated with malaria like sweating, shivering, fever, high pulse rate are most times causes of reduction in calcium levels (9). Magnesium (Mg) is an important element which is a cofactor of more than 300 enzymes. It is involved in the regulation of blood glucose, protein synthesis, neuromuscular function, blood pressure and various biochemical processes. (10). In most cases of malaria caused by P.
falciparum, there is an observed decline in the levels of Magnesium due to the illness (11). Electrolyte imbalance appears because of malaria and can likely lead to a virulent onset of the disease. The present study aimed to nd out the levels of Na, K, Ca and Mg in malarial patients suffering from both P. vivax and P. falciparum in our study location.

Sample collection
Study area and population The study was conducted at the Landmark University Medical Center Omuaran -Nigeria, which is a tertiary health institution of learning. The study population comprised of randomly selected patients attending the outpatient department of the Medical facility.

Ethical permit and consent
All procedures complied with the declaration of Helsinki A proposal of the project was submitted to the Ethical Review Committee of the Landmark University Ethical Committee approved the protocol for this study.

Questionnaire and sample size
The sample size used in this research work was obtained from Two-Hundred (200) volunteers. Subjects recruited were informed about the study and their consents obtained. Well-structured questionnaires were used to collect demographic data and other pertinent information.

Sample collection and processing
A total of Two-Hundred (200) samples were included in this study consisting of 80 samples with peripheral blood lm evidence of falciparum malaria, 60 samples of vivax malaria and 60 samples were taken from healthy subjects as control. The electrolytes levels (Na, K, Cl-and HCO3-) in malaria patients were analyzed using standard procedures according to manufacturer's guidelines. Estimation of renal function and liver function tests were done using the standard method. Three-Five (3-5) ml of blood was collected from each patient aseptically. Standard procedures were strictly adhered during the assay process. Blood samples aseptically collected were stored at 4-8 °C prior use. Data related to subjects such as age, gender and Plasmodium species responsible for malaria were also collected. The determination of electrolytes in whole blood was performed using wet acid digestion method followed by atomic absorption spectrophotometry. Whole blood was used for electrolyte determination in the present study Estimation of electrolytes From each sample, 1 ml of whole blood was shifted into beaker and 0.5 ml of distilled water was added in it followed by the addition of 1 ml of Hydrogen peroxide (H 2 O 2 ) and 4 ml of Nitric acid (HNO 3 ) for wet acid digestion. The beaker was covered and left overnight. Next day, samples were heated on hot plate and H 2 O 2 was added drop wise until solution became clear. After ltration, de-ionized water was added to make volume up to50ml and was stored in Te on tubes (12).After wet acid digestion, the blood samples were analyzed for determination of Na, K, Ca and Mg through Atomic absorption spectrophotometer (AA 6600 Shimadzu). Standards were used for the standard curve formation and estimation of electrolyte levels in the samples.

Data analysis
Questionnaires which were handed out were manually rechecked for accurate entry of data. The data were analyzed using Pearson's correlation was used to measure linear relationship between two continuous random variables., Chi-square test was used to compare several Variables while the critical level for statistical signi cance was set at p = 5% (0.05). ANOVA test was used to observe a signi cant difference between the four categories of malaria patients.

Result
The Pearson product moment correlation usually denoted as 'r' between the electrolytes levels are presented in the upper triangle of Table 1 while the lower triangle shows the probability value for determining signi cance. A strong positive and signi cant correlation was observed between creatinine and urea (r = 0.916 and p-value = 0.000) at p-value < 0.01. Potassium was observed to have a weak positive and signi cant correlation with chloride (r = 0.244 and p-value = 0.029) at p-value < 0.05, creatinine (r = 0.292 and p-value = 0.009) and urea (r = 0.327 and p-value = 0.003) at p-value < 0.01 The mean bound distribution of the electrolyte levels across all the four categories of malaria patients is presented in Table 2. Key: Mean ± Standard Deviation The mean bound of patients' sodium level was observed to fall within the speci ed normal limits of 125 mmol/L -145 mmol/L (Table 2), except for positive malaria patients belonging to the MP-(++) which will fall below the 125 mmol/L (i.e. 126.25 mmol/L -1.77 mmol/L = 124.48 mmol/L). The spread of the sodium data taken from the patients were observed to fall within the normal limits leaving only the boxplot's lower whisker out, that is, observations falling within the rst quartile, except for MP-(++) patients with observations in the rst to second quartile spreading outside the normal lower limit (Fig. 2).
The mean bound of patients' potassium level was observed to fall within the speci ed normal limits of 2.8 mmol/L -4.8 mmol/L ( Table 2). Almost all the box spread of the potassium data taken from the patients were observed to fall within the normal limits leaving only the lower whisker out, that is, the observations in the rst quartile and half of it for the MP-(+++). The case is different for the negative tested patients, the box and its whiskers were almost engulfed within the normal limits (Fig. 3).
The mean bound of the chloride level of MP-(+) and MP-(+++) malaria (positive category) patients' was observed to fall within the speci ed normal limits of 90 mmol/L -115 mmol/L ( Table 2). The lower bound of the mean for negatively tested and MP-(++) malaria (positive) patients were observed to fall below the lower limit of 90 mmol/L (i.e. 90.85 mmol/L -1.14 mmol/L = 89.71 mmol/L and 89.20 mmol/L -0.98 mmol/L = 88.22 mmol/L) respectively. However, the box spread of the chloride data taken from the patients were observed to fall slightly outside the normal lower limit for negatively tested and positively MP-(+++) tested patients, and about half of the observations falling outside the normal lower limit for positively MP-(+) tested patients. The case is entirely different for positively MP-(++) tested patients, all the observations in the rst to the third quartile spreads below the normal lower limit (Fig. 4).
The mean bound of patients' carbonate level was observed to fall completely within the speci ed normal limits of 20 mmol/L -30 mmol/L ( Table 2). The spread of the carbonate data taken from the patients were observed to fall within the normal limits except for MP-(+) patients with observations in the fourth quartile region spreading outside the normal upper limit (Fig. 5).
The mean bound of patients' creatinine level was observed to fall within the speci ed normal limits of  Table 2). The high variation in the mean bound of the negatively tested patients can be attributed to the greatly distant outliers in the observations (Fig. 5). The spread of the creatinine data taken from the patients were observed to fall within the normal limits except for the MP-(++) patients with observations in the fourth quartile region spreading outside the normal upper limit (Fig. 6).
The mean bound of patients' urea level was observed to fall within the speci ed normal limits of 2.0 mmol/L -8.0 mmol/L (Table 2), except for the negatively tested malaria patients which have the upper bound of their average distribution to be above the 8.0 mmol/L (i.e. 7.57 mmol/L + 2.36 mmol/L = 9.93 mmol/L). The spread of the urea data taken from the patients were observed to fall within the normal limits with each of the categories having at least one outlier; two females tested MP-nil, a male and a female tested MP-(+), a male tested MP-(++) and two males and one female tested MP-(+++) (Fig. 7).  (Table 3). Although this was expected as the boxplots in Fig. 2-7 overlaps, Fig. 8 further shows the similar variation possessed by the four categories of malaria patients across the six considered electrolyte measures. The distance noticed in the creatinine level for the malaria patients can be attributed to the value of the outliers observed.

Discussion
Malaria is a common parasitic disease of tropical and subtropical regions of the world (13).It is a highly devastating parasitic disease caused by intra-erythrocytic protozoa of genus Plasmodium. The major species responsible for deaths and that are widely spread are Plasmodium, P. falciparum and P. vivax Plasmodium parasite resides in the red blood cells resulting in their lysis. When variation in the electrolyte level occurs due to the presence of malaria parasite, it may be detected through the comparison of whole blood electrolyte levels in blood of patients and healthy subjects (14).
The human body acts as a bioelectric process while electrolytes are both the switch and the energy source for the body (15). Electrolyte imbalance has been severally observed as the commonly identi ed complication in severe cases of malaria. Electrolyte imbalance acts as an indicator for the level of disease virulence due to its close association with the severe P. falciparum and P. vivax malaria (16). In this study, blood samples were used for the evaluation of electrolytes in malaria patients and healthy individuals.
Findings from this study indicate that malarial infection led to the observed reduction in the levels of Na referred to as hyponatraemia condition. Alterations in Na level can result in several health conditions (17).Various literature shows reports of increased vasopressin(ADH) secretion which either appropriately or inappropriately is a key factor in the reduced level of sodium in malaria, as sodium is able to gain entry into the infected cells and cause loss of blood (18). Hyponatraemia has been identi ed as a common outcome of malaria (16). Ikekpeazu et al. also reported a signi cant lowering of sodium and Potassium levels in malaria infection (20). Hyponatraemia has been reported to occur frequently in patients suffering from P. falciparum malaria than in P.vivax malaria (16,21). Minor changes in potassium level can cause weakness, fatigue and rapid heartbeat. Therefore, its balance is very important for the normal physiology of human body (22).
The present ndings showed a decline in K level due to Plasmodium infection,This nding is similar to the report of Ikekpeazu (20). Enhanced urinary removal of K and hypokalemia has been reported as common outcome of malaria infection (12). Plasmodium presence may lower the K levels and aggravates the complications associated with malaria disease. P. falciparum infected individuals were frequently observed with hypokalaemia as compared to P. vivax infected individuals (16).
However Maitland et al. observed no change in the level of Na and K in patients suffering from malaria as compared to healthy individuals (23).Alterations in the levels of Ca during malaria infection can cause several changes in the body such as muscle cramps, osteoporosis, etc. (24). It was found that Hypocalcaemia in the blood samples of malaria patients was observed compared to healthy subjects. Previous studies carried out revealed a reduction in the level of Ca in malaria patients (25).Trophozoites concentrate calcium in their internal compartment for metabolism (26). Our nding emphasizes a need to manage the electrolyte level of malaria patients by estimating the serum sodium and potassium ratio, while estimation of the Hyponatraemia and hypokalaemia levels could be a good marker for the severity of the disease in patients.

Conclusion
Levels of Na, K, Ca and Mg are in uenced by the presence of both P. falcipaum and P. vivax malaria. In order to curb the spread of malaria, there is an urgent need to also manage the electrolyte derangements for overall management of malaria. It can be concluded that mineral supplementation can aid the prevention of disease severity. In this study the mean bicarbonate level of the negative [MP-nil] and positive [MP-(+++)] malaria patients were observed to fall outside the normal limit. The out of limit measurement in the mean of the negative malaria patients was traced to the presence of the outliers. Boxplots which includes the normal limits were presented for better visualization of the data spread across each category. However, an ANOVA test was used to observe a signi cant difference between the four categories of malaria patients.