Abstract

Infection by Schistosoma mansoni not only triggers the production of reactive oxygen species in liver but also leads to the alteration of antioxidant defences. To determine whether such events occur in school children, the absorbance of the liver organs of mice (as samples) were measured. Seven groups were used as controls of the parameters of seven different mice.

We found that, using a t-test, some group pair comparison had values higher than t-critical and some lower than t-critical. These findings indicate that oxidative stress might contribute to S. mansoni-associated pathology in school children.

The oxidative stress biomarker considered in this study, obtained by relatively simple techniques, may provide a useful biochemical index for the identification of almost asymptomatic patients who, however, are at risk of developing severe schistosomiasis.

CHAPTER ONE

INTRODUCTION

1.1 Introduction

Schistosomiasis is an acute and chronic disease caused by parasitic worms. People are infected during routine agricultural, domestic, occupational, and recreational activities, which expose them to infested water.

Lack of hygiene and certain play habits of school-aged children such as swimming or fishing in infested water make them especially vulnerable to infection. Schistosomiasis control focuses on reducing disease through periodic, large-scale population treatment with praziquantel; a more comprehensive approach including potable water, adequate sanitation, and snail control would also reduce transmission.

Estimates show that at least 290.8 million people required preventive treatment for schistosomiasis in 2018, out of which more than 97.2 million people were reported to have been treated. Schistosomiasis mostly affects poor and rural communities, particularly agricultural and fishing populations.

Women doing domestic chores in infested water, such as washing clothes, are also at risk and can develop female genital schistosomiasis. Inadequate hygiene and contact with infected water make children especially vulnerable to infection. (WHO, 2020).

Oxidative stress has generally been implicated as a cause or as a complication of various chronic diseases such as atherosclerosis, diabetes, HIV and Hepatitic infections. Intestinal helminths rank first among chronic infections in developing countries where several foci of Schistosoma mansoni and soil transmitted helminths (STH) exist. Studies have shown that oxidative stress is involved in tissue damage in intestinal helminthiasis.

Children of school age are usually the most infected with Schistosoma mansoni, Ascaris, and Trichuris. These insidious infections cause poor school performance, growth retardation, malnutrition and the loss of up to 4 Disability-Adjusted-Life-Years (DALY) amongst these children (Aloho et al., 2011).

Schistosomiasis continues to spread in many parts of the world, especially due to lack of sanitation and irrigation projects (Steinmann et al., 2006). Many works have been successful studying the life cycle of the parasite in its different hosts, aiming to understand the disease and to improve prevention programs and available treatments. Most clinical manifestations developed by infected individuals are related to complications derived from liver damage and a large number of studies focused on this organ. However, schistosomiasis is a complex syndrome affecting several organs and functions to different degrees (Carod-Artal, 2010).

The main organs affected in this pathology, such as liver, kidneys and spleen, are shifted to a pro-oxidant state in the course of the infection. Subsequently, oxidative stress parameters of liver, kidney and spleen with other organs affected by schistosomiasis in school children will be compared (de Oliveira et al. , 2013).

Proper check and safety of school children, who are most exposed to contracting the illness will be ensured following this project. Considering the recent WHO revision of the schistosomiasis treatment guidelines to include preschool aged children, and the recognition of gaps in our current knowledge on the disease and its treatment in this age group, there is now a concerted effort to address these shortcoming

1.2 Literature Review:

Schistosomiasis is a parasitic disease of significant medical and veterinary importance in many regions of the world. Recent shifts in global health policy have led towards the implementation of mass chemotherapeutic control programs at the national scale in previously ‘neglected’ countries such as those within sub-Saharan Africa.

Evolutionary theory has an important role to play in the design, application and interpretation of such programs. Whilst celebrating the rapid success achieved to date by such programs, in terms of reduced infection prevalence, intensity and associated human morbidity, evolutionary change in response to drug selection pressure may be predicted under certain circumstances, particularly in terms of the development of potential drug resistance, evolutionary changes in parasite virulence, transmission and host use, and/or competitive interactions with co-infecting pathogens. Theoretical and empirical data gained to date serve to highlight the importance of careful monitoring and evaluation of parasites and their hosts whenever and wherever chemotherapy is applied and where parasite transmission remains (Webster et al., 2008).

Reactive oxygen species and oxidation stress are the inevitable consequences of aerobic metabolism, with partially reduced and highly reactive metabolites of oxygen being formed in the mitochondria or as by-products of other cellular sources such as cytoplasm, the endoplasmic reticulum, the plasma membrane and peroxisomes (Sharma et al., 2012).

Superoxide radicals (O2•−), hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and singlet oxygen (1O2) are commonly defined reactive oxygen species (ROS); they are generated as metabolic by-products by biological systems.

Processes, like protein phosphorylation, activation of several transcriptional factors, apoptosis, immunity, and differentiation, are all dependent on a proper ROS production and presence inside cells that need to be kept at a low level. When ROS production increases, they start showing harmful effects on important cellular structures like proteins, lipids, and nucleic acids. A large body of evidences shows that oxidative stress can be responsible, with different degrees of importance, in the onset and/or progression of several diseases (i.e., cancer, diabetes, metabolic disorders, atherosclerosis, and cardiovascular diseases) (Pizzino et al. , 2017).

Schistosoma eggs are eliminated with feces or urine, depending on species. Under appropriate conditions the eggs hatch and release miracidia, which swim and penetrate specific snail intermediate hosts. The stages in the snail include two generations of sporocysts and the production of cercariae. Upon release from the snail, the infective cercariae swim, penetrate the skin of the human host, and shed their forked tails, becoming schistosomulae. A diagrammatical explanation of the life cycle of Schistosoma mansoni is represented below:

The schistosomulae migrate via venous circulation to lungs, then to the heart, and then develop in the liver, exiting the liver via the portal vein system when mature. Male and female adult worms copulate and reside in the mesenteric venules, the location of which varies by species (with some exceptions).

For instance, Schistosoma  japonicum is more frequently found in the superior mesenteric veins draining the small intestine, and Schistosoma mansoni occurs more often in the inferior mesenteric veins draining the large intestine. However, both species can occupy either location and are capable of moving between sites.

Schistosoma intercalatum and Schistosoma guineensis also inhabit the inferior mesenteric plexus but lower in the bowel than Schistosoma mansoni. Schistosoma haematobium most often inhabitsin the vesicular and pelvic venous plexus of the bladder , but it can also be found in the rectal venules. The females (size ranges from 7–28 mm, depending on species) deposit eggs in the small venules of the portal and perivesical systems.

The eggs are moved progressively toward the lumen of the intestine (Schistosoma mansoni, Schistosoma japonicum, Schistosoma mekongi, Schistosoma intercalatum/guineensis) and of the bladder and ureters (Schistosoma haematobium), and are eliminated with feces or urine, respectively (CDC, 2019)

Epidemiology:

Schistosomiasis is prevalent in tropical and subtropical areas, especially in poor communities without access to safe drinking water and adequate sanitation. It is estimated that at least 90% of those requiring treatment for schistosomiasis live in Africa. In Cameroon, four different types of schistosomiasis exist.

These are: Schistosoma mansoni, Schistosoma haematobium, Schistosoma intercalatum, and the hybrid resulting from the crossbreed of Schistosoma haematobium and Schistosoma intercalatum.  Schistosomiasis mostly affects poor and rural communities, particularly agricultural and fishing populations.

Women doing domestic chores in infested water, such as washing clothes, are also at risk and can develop female genital schistosomiasis. Inadequate hygiene and contact with infected water make children especially vulnerable to infection. Migration to urban areas and population movements are introducing the disease to new areas.

Increasing population size and the corresponding needs for power and water often result in development schemes, and environmental modifications facilitate transmission. With the rise in eco-tourism and travel “off the beaten track”, increasing numbers of tourists are contracting schistosomiasis. At times, tourists present severe acute infection and unusual problems including paralysis. Urogenital schistosomiasis is also considered to be a risk factor for HIV infection, especially in women (WHO, 2020).

Oxidative stress:

Oxidative stress is an imbalance between free radicals and antioxidants in your body. Free radicals are oxygen-containing molecules with an uneven number of electrons. The uneven number allows them to easily react with other molecules. Free radicals can cause large chain chemical reactions in your body because they react so easily with other molecules. These reactions are called oxidation.

They can be beneficial or harmful. Antioxidants are molecules that can donate an electron to a free radical without making themselves unstable. This causes the free radical to stabilize and become less reactive.

Oxidation is a normal and necessary process that takes place in your body. Oxidative stress, on the other hand, occurs when there’s an imbalance between free radical activity and antioxidant activity. When functioning properly, free radicals can help fight off pathogens. Pathogens lead to infections (healthline, 2020)

Methods used for the evaluation of oxidative stress in clinical samples:

Biomarkers of oxidative stress are therefore important tools in the assessment both of disease status and of the health-enhancing effects of antioxidants in humans. In this review, we aim to discuss different methods and approaches used for the evaluation of oxidative stress in clinical samples;

• Direct Measurement of Reactive Oxygen Species: Reactive oxygen species are the key molecules responsible for the deleterious effects of oxidative stress. Direct measurement of their cellular levels is therefore one approach to determine oxidative stress conditions. One way to estimate the cellular levels of reactive oxygen species is through the use of fluorogenic probes.
• Assessment of Oxidative Damage: Direct measurement of reactive oxygen species levels with high accuracy and precision is difficult due to their short lifespan and rapid reactivity with redox state regulating components. While peroxyl radicals and hydrogen peroxide are relatively stable molecules (with half-lives of seconds to minutes), hydroxyl radicals are very reactive (having a half-life of less than a nanosecond). Therefore, indirect measurement of reactive oxygen species by examining the oxidative damage these radicals cause to the lipids, proteins, and nucleic acids of the cells is a promising alternative approach to assess oxidative stress in clinical samples.
• Protein Damage: Protein carbonyl (PC) content is a commonly used marker of oxidative modification of proteins, providing significant evidence of oxidative stress in clinical samples. PCs are generated due to the oxidation of protein backbones and amino acid residues such as proline, arginine, lysine, and threonine by reactive oxygen species molecules. The oxidized proteins can be measured using the 2,4-dinitrophenylhydrazine (DNPH) method.
• Lipid Damage: Lipid peroxidation has been commonly used as an indicator of reactive oxygen species-mediated damage to cell membranes. Malondialdehyde (MDA) is one of the best studied end-products of peroxidation of polyunsaturated fatty acids in clinical samples and is frequently used to estimate oxidative stress conditions. The levels of MDA can be measured using thiobarbituric acid reactive substances (TBARS).
• DNA Damage: 8-Hydroxy-2-deoxyguanosine (8-OHdG) is one of the major oxidative modifications in DNA that is generated by hydroxylation of the deoxyguanosine residues. 8-OHdG residues can be excised from the DNA by enzymatic repair systems, leading to their circulation in the blood and subsequent excretion in the urine . Levels of 8-OHdG in blood and/or urine of patients can therefore be measured as a marker of oxidative DNA damage.
• Assessment of Antioxidant Status: The human body is equipped with an antioxidant system that serves to counterbalance the deleterious effects of oxidative free radicals. When the balance between antioxidants and reactive oxygen species, referred to as redox homeostasis, is disturbed, oxidative stress can occur. The disturbance of this prooxidant and antioxidant balance can be a result of increased free radical production, antioxidant enzyme inactivation, or excessive antioxidant consumption. Assessment of the antioxidant status can thereby be correlated to the extent of oxidative stress in clinical samples.

Redox homeostasis is regulated by two arms of antioxidant machineries: enzymatic components and non enzymatic, low molecular compounds. Several approaches have been developed to measure the different activities or levels of these antioxidants. Alternatively, the total antioxidant status can be evaluated to assess the oxidative state of clinical samples (Marrocco et al. , 2017)

Over the past decade, new opportunities for much broader schistosomiasis control have emerged (Fenwick, 2006). National efforts have delivered the antischistosomal drug praziquantel to school-age children across Uganda, Tanzania, Zambia, Burkina Faso, Mali, and Niger in collaboration with the Schistosomiasis Control Initiative of Imperial College, London (Garba et al. , 2009). Following on the heels of this program’s success, broader deworming initiatives are now being implemented to combine schistosomiasis treatment with ongoing regional or national programs for the control of onchocerciasis, lymphatic filariasis, and/ or the soil-transmitted helminths (STHs), hookworm, Ascaris, and Trichuris.

Expansion of such programs is being actively supported by governmental programs from donor nations and by many nongovernmental development organizations that are joining forces to develop national-level “deworming” projects in other sub-Saharan African countries (King, 2011). A research project like this will make some great contribution to all these efforts made by higher authorities.

1.2.5.   Relationship between schistosomiasis and oxidative stress:

Research on oxidative stress and related topics started out with chemistry and toxicology, then turned toward disease processes and in more recent times examined fundamental biological redox processes which underlie signaling in health and life processes in general.

The large body of knowledge on redox signaling fueled research activity on the role of oxidative stress under normal physiological conditions as opposed to exposure to supraphysiological oxidative challenge leading to damage of biomolecules and the subsequent consequences such as disturbed or disrupted redox signaling.

The Web of Science All Database Collection, at the beginning of 2018, shows about 360,000 entries under the topic “oxidative stress,” with over 30,000 new publications on this topic having appeared per year over the last 3 years. Obviously, a comprehensive analysis of current research development in this blossoming field is beyond the scope of this article. Oxidative stress pervades the chemical, biological, biochemical, and clinical-medical literature.

The apparently simple concept of an imbalance between oxidants and antioxidants, potentially leading to molecular damage, has evolved in recent years to focus on biological responses, including a disruption of redox signaling and control.

Related terms such as dietary oxidative stress, postprandial oxidative stress, physiological oxidative stress, photo oxidative stress, radiation-induced oxidative stress, oxidant stress, pro-oxidant stress, and oxidative stress status are presented. Also, reductive stress and nitrosative stress are addressed (Sies, 2019).

In some reports concerning Schistosoma mansoni, it was pointed out that levels of reduced glutathione and glutathione reductase were increased, while the activity of Glutathione-S-transferase was decreased in human and mice infected with Schistosoma mansoni. The same authors added that high levels of free radicals provide new evidence for organ damage since glutathione is decreased and lipid peroxides increased in different organs of hamsters infected with Schistosoma hematobium.

In this manner Schistosoma mansoni infection alters the hepatic levels of glutathione and activities of glutathione metabolizing enzymes and alterations may affect the capacity of the liver to detoxify or neutralize the toxic effects of endogenous and exogenous compounds (Sheweita et al.). Disruption of the glutathione system is known to trigger S. mansoni death. Indeed, the anthelmintic oltipraz causes oxidative stress, decreased GSH concentration and glutathione reductase activity, and increased GPx activity.

Oxidative stress is considered to be an important component of various diseases. A vast number of methods have been developed and used in virtually all diseases to measure the extent and nature of oxidative stress, ranging from oxidation of DNA to proteins, lipids, and free amino acids.

An increased understanding of the biology behind diseases and redox biology has led to more specific and sensitive tools to measure oxidative stress markers, which are very diverse and sometimes very low in abundance (Frijhoff et al., 2015).

During the past decade, research has revealed a widespread involvement of oxidative stress in a number of disease processes. Therefore, oxidative stress biomarkers are relevant in the evaluation of the disease status and the health-enhancing effects of antioxidants, although identifying markers of oxidative stress has been the focus of many studies, and several markers from various biomolecule sources have been proposed over the past decades (researchgate, May 2020). However, for some of them, there is a lack of consensus concerning validation, standardization, and reproducibility.

1.3 Rationale:

Since the focus of this study is on school children, it was designed to assess the relationship between schistosomiasis effect on the profile of oxidation stress biomarkers in school children. This is because from a wide range of statistics, school children have been found to be the most infected by schistosomiasis. Studying this can be used to understand the schistosomiasis infection, it’s positive or negative effects, as well as how it can be treated, since it is generally neglected in most places around the world.

1.4 Hypothesis:

The null hypothesis: Schistosomiasis infection has no impact on the profile of oxidation stress biomarkers in school children.

Alternative hypothesis: Schistosomiasis infection has an impact/effect on the profile of oxidation stress biomarkers in school children.

1.5 Objectives:

Main objective: To determine the impact of schistosomiasis on the profile of oxidative stress biomarkers in school children.

Specific objective:

1. Identifying school children infected by schistosomiasis.
2. Measuring the levels of their oxidation stress biomarkers.
3. Making comparisons with non-infected school children.