ASSESSMENT OF PHYSICO CHEMICAL PARAMETERS OF WATER

CHAPTER ONE INTRODUCTION AND LITERATURE REVIEW 1.0 Introduction Malaria is a vector-borne infectious disease caused by an intracellular protozoan parasite of the genus ‘Plasmodium’ that infects multiple hosts such as humans and other mammalians (Latif et al., 2018). Commonly four main Plasmodium species: Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae and Plasmodium vivax have long been known to cause malaria in humans. Nowadays Plasmodium Knowlesi, previously confined to monkeys, is the fifth species that infect humans (Barber, 2017). However, P. falciparum and P. vivax are responsible for the majority of malaria cases and P. falciparum is mainly associated with deaths. Plasmodium is primarily transmitted by the bite of an infected female Anopheles mosquito. Infection can also occur through exposure to infected blood products and through congenital transmission. (Onyesom and Onyemakonor, 2011) The malaria parasite induces certain metabolic and biochemical changes within the host (Faucher et al., 2021). The disease's pathophysiology involves hemolysis and the release of parasitic waste products, which can lead to complications, including renal impairment. The disease's pathophysiology involves hemolysis and the release of parasitic waste products, which can lead to complications, including renal impairment. The administration of the right drug against the malaria parasite species to control morbidity and mortality is a very important demand. (WHO, 2015) Though the list of anti-malarial drugs is long, the most widely known are quinine, chloroquine (CQ), primaquine, and artemisinin and its derivatives. Due to the widespread resistance to monotherapies, the World Health Organization (WHO) recommended the use of Artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated falciparum malaria throughout malaria-endemic areas. Artemether-Lumefantrine (Art-L) having trade names Coartem®, is one of the ACTs. (Enato and Okhamafe, 2015) Urea and creatinine are vital markers for kidney function. Elevated levels of these markers can indicate renal dysfunction, which is a common complication in severe malaria cases (Augustine, 2018). Renal impairment is a known complication of severe malaria, often leading to acute kidney injury (AKI). The kidney damage in malaria can be attributed to several factors, including haemolysis, immune complex deposition, dehydration, and the direct effects of parasitic toxins. The timely and accurate assessment of renal function is crucial for managing malaria patients, especially those presenting with severe disease (Lamb et al.,2005). Urea is a by-product of protein catabolism, while creatinine is a waste product of muscle metabolism. Both are filtered by the kidneys, and their serum levels can indicate the glomerular filtration rate (GFR). Elevated levels of urea and creatinine suggest impaired renal function, which may necessitate immediate medical intervention. This study aims to investigate the serum levels of urea and creatinine in malaria patients and compare them with healthy individuals to assess the impact of malaria on renal function. Understanding these biochemical changes can help in the early diagnosis and treatment of renal complications in malaria. (Das, 2008) 1.1 Justification of the Study This study will provide statistically, medical information for public health professions, pharmacists, medical doctors, biochemists, researchers, community health planners and other health professionals. The information obtained provides a platform for further research, as it educates individuals on the relation of the serum levels of Urea as well as Creatinine in malaria subjects. 1.2 Aim of the study The aim of this study is to evaluate the biochemical estimation of Urea and Creatinine in malaria subjects. 1.3 Objectives of the study 1. To determine the biochemical estimation of Urea in malaria subjects. 2. To determine the biochemical estimation of Creatinine in malaria subjects. 1.4 Literature Review Malaria continues to be a great health problem in some of the most populated areas of the world. (Sharma and Awasthi, 2015) Globally, there were an estimated 229 million malaria cases in 2019 in 87 malaria endemic countries, declining from 238 million in 2000. The World Health Organization (WHO) African Region, with an estimated 215 million cases in 2019, accounted for about 94% of cases. (World Health Organization, 2020) Malaria is one of the leading public health problems in Ethiopia. The problem covers 75% of the landmass of the country and 68% of the populations are living in malaria-risk areas (Taffese et al., 2018). Plasmodium falciparum and P. vivax are the two dominant parasite species causing human malaria in Ethiopia, on average, with relative frequencies of about 62.8% and 37.2%, respectively. (Deress and Girma, 2019) In Ethiopia, malaria transmission is largely determined by altitude and climate. Most of the malaria transmission occurs between September and December, after the main rainy season June to August. In Yemen, the population at risk of malaria constituted 81% of the total population, with estimated one million cases in 2009. Hyperbilirubinemia, is a common feature of falciparum malaria and is attributed to hemolysis of both parasitized and non- parasitized erythrocytes and partly due to liver damage. Therefore, well-informed changes in blood parameters in malaria infection and hepatic function enable the clinician to establish reliable diagnosis and therapeutic interventions. (Alemu et al., 2011) 1.4.1 Discovery of Malaria Scientific studies on the discovery of malaria made their first advance in 1880, when Charles Louis Alphonse Laveran (1845-1922) – a French army doctor during the Franco-Prussian War, working in the military hospital of Constantine in Algeria observed parasites inside the red blood cells of infected people for the first time. He, therefore, proposed that malaria is caused by this organism, the first time a protest was identified as causing disease. (Jarcho, 1984) For this and later discoveries, he was awarded the 1907 Nobel Prize for discovering the single-celled protozoan that caused malaria (Laveran, 1978). A year later, Carlos Finlay, a Cuban doctor treating people with yellow fever in Havana, provided strong evidence that mosquitoes were transmitting disease to and from humans. (Tan and Sung, 2008) This work followed earlier suggestions by Josiah C. Nott, (Chernin, 1983) and work by Sir Patrick Manson, the father of tropical medicine”, on the transmission of filariasis. In April 1897, Sir Ronald Ross, a Scottish physician working in the Presidential General Hospital in Calcutta, proved the complete lifecycle of the malaria parasite in mosquitoes. He thus proved that the mosquito was the vector for malaria in humans by showing that certain mosquito species transmit malaria to birds. For this, he received the 1902 Nobel Prize in Medicine. (Harrison, 1978) The third piece of the human malaria puzzle—where sporozoites inoculated by mosquitoes undergo early development in the human host—was solved in 1948. Although previous researchers had found that bird malaria initially reproduced in tissues of the lymph system and the bone marrow, the sanctuary for primate and human malaria outside of red blood cells remained a mystery. Then H. E. Shortt, P. C. C. Garnham, and colleagues at the Ross Institute of the London School of Hygiene and Tropical Medicine detected malaria parasites in the livers of rhesus monkeys infected with a primate malaria species. They subsequently found similar stages in liver biopsy specimens from human volunteers experimentally infected with P. vivax, and confirmed the same early sanctuary for P. falciparum (Garnham, 1966). 1.4.2 Origin of Malaria Ancient writings and artifacts testify to malaria's long reign. Clay tablets with cuneiform script from Mesopotamia mention deadly periodic fevers suggestive of malaria. Malaria antigen was recently detected in Egyptian remains dating from 3200 and 1304 BC (Miller et al., 1994). Malaria's probable arrival in Rome in the first century AD was a turning point in European history. For the next 2,000 years, wherever Europe harboured crowded settlements and standing water, malaria flourished, rendering people seasonally ill, and chronically weak and apathetic. Many historians speculate that falciparum malaria (the deadliest form of malaria species in humans) contributed to the fall of Rome. The malaria epidemic of 79 AD devastated the fertile, marshy croplands surrounding the city, causing local farmers to abandon their fields and villages (Cartwright, 1991). But nowhere — past or present—has malaria exacted a greater toll than on Africa. It was a leading obstacle to Africa's colonization. Falciparum malaria was subsequently imported to the New World by African slaves initially protected by age-old genetic defences (sickle cell anemia) plus partial immunity gained through lifelong exposure. Their descendants were however more vulnerable. Deforestation and “wet” agriculture such as rice farming facilitated breeding of Anopheles mosquitoes. Portuguese traders who entered the African coastal plain in the late 1400s and early 1500s were the first foreigners to confront the killing fever. For the next 3 centuries, whenever European powers tried to establish outposts on the continent, they were repelled time and again by malaria, yellow fever, and other tropical scourges. By the 18th century, the dark specter of disease earned West and central Africa the famous epitaph, “the White Man's Grave.” In recent years, by virtue of climate, ecology, and poverty, sub-Saharan Africa has been home to 80 to 90 percent of the world's malaria cases and deaths. (Cartwright, 1991) 1.4.3 Mechanism of Action and Mode of Transfer Malaria is majorly caused by the transmission of the malaria parasite Plasmodium to humans by the bites of female Anopheles mosquitoes (only female mosquitoes feed on blood, thus males do not transmit the disease). Plasmodium parasites’ primary hosts and transmission vectors are the female Anopheles mosquitoes. Humans and other vertebrates are secondary hosts. (Nilsson et al., 2015) The mosquitoes first take in the parasite by feeding on the blood of an infected person. In the mosquitoes’ gut, the gametocytes (male and female) from the infected person fuse to form an ookinete that penetrates the gut lining and produces an oocyst in the gut wall. When the oocyst ruptures, it releases sporozoites that migrate through the mosquito's body to the salivary glands sporozoites. This is the phase of sexual reproduction. The mosquito then becomes ready to infect a new individual. The mosquitoes bite at night between dusk to dawn. Once within the humans, the malaria parasite undergoes two phases - an exoerythrocytic and an erythrocytic phase. i. Exoerythrocytic phase: The exoerythrocytic phase involves the maturation and development of the parasite in the liver. When an infected mosquito transmits the infection or sporizoites as it takes in a blood meal, the sporozoites in the mosquito's saliva enter the bloodstream and migrate to the liver of the human. The process of migration takes around 30 minutes after a bite. These sporozoites infect hepatocytes. This is followed by a multiplication of the sporozoites, known as asexual reproduction or multiplication. It takes around 6–15 days for this multiplication. The parasite then forms thousands of merozoites within the hepatocytes. The numerous merozoites lead to the rupture of their host cells and escape into the blood. Sometimes the sporozoites may instead, produce hypnozoites that lie dormant in the liver, usually seen in Plasmodium vivax and Plasmodium ovale. The periods of dormancy may range over several months (typically 6–12 months to around 3 years). Hypnozoites are responsible for long incubation and late relapses in these two species of malaria. (Nilsson et al., 2015) ii. Erythrocytic phase: The involvement of the red blood cells is called the erythrocytic phase. In the RBCs the merozoites multiply further asexually and burst the RBCs as they multiply releasing the merozoites in blood. The new merozoites then invade fresh red blood cells leading to further amplification. Each such amplification is thus characterized by a wave of fever. Some of the merozoites develop into male and female gametocytes that may be further transmitted to mosquitoes. Plasmodium parasites exist in various forms within the liver and blood but manage to escape the immune system. This is because in most of its forms it resides within the liver and blood cells and is relatively invisible to immune surveillance. Infected RBCs especially those with Plasmodium falciparum escape this destruction by developing adhesive proteins on the surface of the infected blood cells, causing the blood cells to stick to the walls of small blood vessels. This leads to sequestering the parasite from passage through the general circulation and the spleen. Transmission is also possible by blood transfusions from an infected person. (Nilsson et al., 2015) Figure 1.1: Life cycle of the malaria parasite Source: Antimalarial drug resistance, Klein E.Y. (2013) 1.4.4 Anti-malarial Medications Anti-malarial medications or simply anti-malarials are a type of antiparasitic chemical agent, often naturally derived, that can be used to treat or to prevent malaria specifically in three categories of individuals, (i) those with suspected or confirmed infection, (ii) those visiting malaria-endemic regions who have no immunity, to prevent infection via malaria prohylaxis, and (iii) in broader groups of individuals, in routine but intermittent preventive treatment in regions where malaria is endemic. (Ashley and Phyo, 2018) Currently, available anti-malarial drugs fall into three broad categories according to their chemical structure and mode of action. They include: i. Aryl aminoalcohol compounds: quinine, quinidine, chloroquine, amodiaquine, mefloquine, halofantrine, lumefantrine, piperaquine, tafenoquine. ii. Antifolate compounds (antifols): pyrimethamine, proguanil, chlorproguanil, trimethoprom iii. Artenisinin compounds: artemisinin, dihydroartemisinin, artemether, artesunate. (Ashley and Phyo, 2018) One important aspect in the mode of action of anti-malarial medications is interfering with the digestion of haemoglobin in the blood stages of malaria parasite life cycle in the liver and red blood cells; by: i. Binding to the haemoglobin and preventing it from being broken down by the parasites’ digestive enzymes. (e.g. chloroquine, quinine) ii. Undigested haemoglobin accumulates in the parasites’ food vacuole, leading to the formation of toxic fragments that can damage the parasites’ membranes. iii. By preventing the digestion of haemoglobin, anti-malarials starve the parasites of essential nutrients, thereby inhibiting its growth and development. iv. Ultimately, this interference with haemoglobin digestion leads to the death of the malaria parasite, which is the desired outcome of anti-malarial therapy. . (Ashley and Phyo, 2018) 1.4.5 Prevention and control of malaria Prevention of malaria includes medications, mosquito elimination and the prevention of bites. Prevention of malaria may be more cost-effective than treatment of the disease in the long run, but the initial costs required are out of reach of many of the world's poorest people. (Kajfasz, 2009) Vector control of malaria refers to methods used to decrease malaria by reducing the levels of transmission by mosquitoes. Some of the common effective methods for controlling malaria parasite transmission include: (Kajfasz, 2009) i. Anti-malarial medications – medications such as mefloquine, doxycycline, or the combination of atovaquone/proguanil (Malarone)—are frequently used for prevention. (Tickell-Painter et al., 2017) ii. Mosquito repellents iii. Insecticide-treated nets (ITNs) iv. Indoor residual spraying (IRS) – the spraying of insecticides on the walls inside a home. After feeding, many mosquitoes rest on a nearby surface while digesting the bloodmeal, so if the walls of houses have been coated with insecticides, the resting mosquitoes can be killed before they can bite another person and transfer the malaria parasite. (Enayati and Hemingway, 2010) v. Maintaining a clean environment vi. Malaria vaccines vii. Community participation and health education strategies promoting awareness of malaria and the importance of control measures have been successfully used to reduce the incidence of malaria in some areas of the developing world. (Kajfasz, 2009). 1.4.6 Urea Urea is a key biochemical marker used to assess kidney function and overall nitrogen balance in the body. It is the primary end product of protein metabolism, produced in the liver through the urea cycle. Urea is then transported in the blood to the kidneys, where it is filtered and excreted in urine. The measurement of urea levels in the blood (blood urea nitrogen, BUN) provides important information about renal function, liver function, and overall metabolic status. In the context of malaria, elevated urea levels can indicate renal impairment, which is a common complication of severe malaria (Owen et al., 1993) Urea is synthesized in the liver from ammonia, a toxic by-product of protein catabolism. The urea cycle involves several enzymatic steps that convert ammonia and carbon dioxide into urea and water. This cycle is crucial for detoxifying ammonia and maintaining nitrogen balance in the body. The normal concentration of urea in blood ranges from 7 to 20 mg/dL (2.5 to 7.1 mmol/L) in adults. Urea is freely filtered by the glomeruli in the kidneys and partially reabsorbed in the renal tubules. The reabsorption of urea is influenced by the body's hydration status and renal function. Elevated levels of urea in the blood can indicate decreased renal function or increased protein catabolism (Stevens et al., 2009). 1.4.7 Clinical Significance of Urea I. Kidney Function Assessment: - Blood urea nitrogen (BUN) levels are commonly measured to assess kidney function. Elevated BUN levels can indicate renal impairment, as seen in conditions such as acute kidney injury (AKI) and chronic kidney disease (CKD). Decreased kidney function leads to reduced urea excretion, resulting in higher blood levels. II. Liver Function and Metabolic Status: - Since urea is synthesized in the liver, abnormal urea levels can also indicate liver dysfunction. In liver disease, the conversion of ammonia to urea may be impaired, leading to decreased urea levels and elevated ammonia levels in the blood. III. Protein Metabolism: - Urea levels reflect the balance between protein intake and protein breakdown. High protein diets, excessive protein catabolism (as seen in febrile illnesses and hyper catabolic states), and gastrointestinal bleeding can lead to elevated urea levels. IV. Malaria and Kidney Function: - In malaria, particularly severe cases caused by Plasmodium falciparum, renal impairment is a frequent complication. The pathophysiology includes haemolysis, dehydration, and the release of inflammatory cytokines, which can all contribute to kidney damage. Monitoring urea levels in malaria patients is crucial for early detection and management of renal complications (Fossati et al., 1983). 1.4.8 Creatinine Creatinine is a crucial biochemical marker used to assess kidney function. It is a by-product of muscle metabolism, specifically from the breakdown of creatine phosphate, which is used by muscles for energy. The constant production of creatinine and its excretion through the kidneys make it a reliable indicator of renal function. Elevated levels of creatinine in the blood can indicate impaired kidney function or disease, necessitating its measurement in clinical settings, especially in conditions that can impact the kidneys, such as malaria (Stevens et al., 2009). Creatinine is formed from creatine, a molecule synthesized primarily in the liver and then transported to muscle tissues, where it is phosphorylated to create creatine phosphate. Creatine phosphate serves as a quick energy reserve in muscle cells. During muscle metabolism, creatine phosphate is converted into creatinine, which is then released into the bloodstream. The kidneys filter creatinine from the blood, and it is excreted in the urine. Because creatinine production is relatively constant and it is not significantly reabsorbed or secreted by the renal tubules, its serum concentration is a good indicator of the glomerular filtration rate (GFR) (Myers et al., 2006) 1.4.9 Clinical Significance of Creatinine I. Kidney Function Assessment: - Creatinine levels in the blood and urine are used to assess kidney function. Normal serum creatinine levels vary based on age, gender, and muscle mass but typically range from 0.6 to 1.2 mg/dL in adult males and 0.5 to 1.1 mg/dL in adult females. - Elevated serum creatinine levels can indicate reduced kidney function or kidney disease. Conditions such as acute kidney injury (AKI) or chronic kidney disease (CKD) often present with increased creatinine levels. II. Glomerular Filtration Rate (GFR): - The GFR is estimated using serum creatinine levels through equations like the Cockcroft-Gault formula, the Modification of Diet in Renal Disease (MDRD) study equation, and the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. These equations consider factors such as age, sex, and body size to provide an estimate of kidney function. III. Malaria and Kidney Function: - In malaria, particularly severe cases caused by Plasmodium falciparum, renal impairment is a common complication. The parasitic infection can lead to haemolysis, dehydration, and the release of inflammatory cytokines, all of which can contribute to kidney damage. - Monitoring creatinine levels in malaria patients is crucial for early detection and management of renal complications. Elevated creatinine levels in these patients can signal the onset of AKI, requiring prompt medical intervention (Fossati et al., 1983). CHAPTER TWO 2.0 MATERIALS AND METHOD 2.1 Materials 2.1.0 Study Centre The samples for the research work were drawn from malaria subjects/ patients who attended D-LIGHT medical laboratories Onitsha for their routine check-up. The Laboratory analysis was performed in the department of chemical pathology of the laboratory. 2.1.1 Subjects A total number of 100 malaria subjects were recruited into this study while another 20 apparently healthy subjects were also recruited into the study. These group of apparently recruited were referred to as Control subjects. These Control subjects were normal subjects. Blood samples were collected from all the subjects for laboratory evaluations of the immune system. The blood collected into the plain tubes were used for the analysis of the following; Urea and Creatinine Analysis 2.1.2 Oral Consent The oral consents of the subjects were obtained before embarking on this research work. 2.1.3 Exclusion Criteria Pregnant women and non-adults were excluded from the research work and also patients who were on herbal medicinal treatment. 2.1.4 Inclusion Criteria Only subjects who were suffering from malaria were recruited into the studies, while the apparently healthy subjects also recruited were not suffering from the disease entity. 2.1.5 Age Ranges The subjects recruited into the study were selected from different age ranges irrespective of their sexes. The classified age ranges were 21-30, 31-40, 41-50, 51- 60 and finally 61-70 years, while there was no indication of any age and sexes among the control subjects. 2.1.6 Equipments The following equipments were used in the course of embarking into this research work; 1. Water bottle 2. Centrifuge 3. Test tubes 4. Racks 5. Creatinine Reagent kit 6. Urea Reagent kit 7. Cuvette 8.Beaker 9. Micropipette (Micro flux, volume range:0-100microlitre) 10. Spectrophotometer 11. Stop watch 2.1.7 Determination of Urea by Berthelot method Principle Urea in serum is hydrolysed to ammonia in the presence of the urease. The ammonia reacts with phenol and hypochlorite in alkaline medium to form indophenol. The intensity of the colour indicates the concentration of urea in the sample. Nitroprusside is used to catalyse the reaction. This indophenol is then measured photometrically Procedure 0.01ml of serum was added to test, 0.01ml of urea standard was added to standard and 0.01ml of distilled water, added to blank. Following these additions, 0.1ml of urea reagent 1 was added to all the tubes, mixed thoroughly and incubated at 37OC for 10 minutes thereafter, 2.5mls of urea reagent 2 and 3 were each added to all the tubes, mixed thoroughly and incubated at 37OC for 15 mins and read at 540nmλ after blanking the spectrophotometer with the reagent blank. 2.1.8 Determination of Creatinine by Jaffe colorimetric method Principle Creatinine in alkaline solution reacts with picric acid to form a coloured complex. The amount of the complex formed is directly proportional to the creatinine concentration. Procedure A working reagent composed of equal volumes of Reagent A and B (Alkaline solution and Picric acid solution) was prepared. For each determination, 1ml of Reagent A was mixed with 1ml of Reagent B to give 2mls of working reagent for control and test. 0.1ml of standard serum was added to the tube marked standard, 0.1ml of the test serum was added to the tube marked test. 1ml of the working reagent was added to the tubes. The tubes were mixed and the absorbance of the tubes were read and noted within 30seconds. The absorbance of the tubes was read for the second time at exactly 2minutes. The tubes were read at a wavelength of 500nm. CHAPTER THREE RESULTS 3.0: Biochemical Estimation of urea and creatinine in malaria subjects. Table 3.1: The urea levels in malaria and control subjects Age Range Number of subjects n=100 Urea level in patient (mmol/L) Urea level in control subjects (mmol/L) P value 21-30 20 7.20 ± 1.44 6.30 ± 1.35 P>0.05 31-40 20 5.38 ± 1.36 6.30 ± 1.35 P0.05 41-50 20 87.60 ± 1.42 90.30 ± 1.40 P0.05 whereas other age range were statistically significant at P0.05). 4.1. 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