Abstract

Schistosomiasis infection is an acute chronic and parasitic disease caused by blood flukes of the genus schistosoma. The work was interested in determining the effects of schistosomiasis infection on cytokine level in school children. Measurements of MDA concentration on liver homogenate and serum were conducted to determine these effects. Schistosomiasis infection will result to increase in the levels of pro-inflammatory cytokines as well as increase anxiety in Schistosomiasis infected subjects.

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1 INTRODUCTION

As stated by WHO (2020), schistosomiasis is an acute and chronic parasitic disease caused by blood flukes (trematode worms) of the genus schistosoma. Estimates show that at-least 299million people required preventive treatment in 2018. Preventive treatment which should be repeated over a number of years will reduce and prevent morbidity. Schistosomiasis infection have been reported from 78 countries. Schistosomiasis also called bilharzia is a disease causes by infection with fresh parasitic worms in certain tropical and subtropical counties (Gurarie et al., 2018) People become infected when larval forms of the parasite- released by fresh water snails penetrate the skin during contact with infested water. In the body, larvae develop into adult schistosomes (Roquis et al., 2018) There are two major forms of Schistosomiasis; intestinal and urogenital- caused by five main species of blood fluke; S. mansoni, S. japonicum, S. mekongi, S. intercalatum and Schistosoma haematobium. The WHO strategy for schistosomiasis control forcuses on reducing disease through periodic, targeted treatment with paraziquantael through the large scale treatment (preventive chemotherapy) of affected populations (Appiah et al., 2013) Groups targeted are school-aged children in endemic areas, adults considered to be at risk in endemic areas and people with occupations involving contact with infested water such as fisher men, farmers, irrigation workers and women whose domestic tasks bring them in contact with infested water. From Jun and Jianxian (2007), Cytokines are small secreted proteins released by cells that have a specific effect on the interactions and communications between cells. Cytokines may act on the cells that secrete them (autocrine action), on nearby cells (paracrine action) or in some instances on distant cells (endocrine action). There are both Pro-inflammatory and anti-inflammatory cytokines (Mutapi et al., 2007) Cytokines are made by many cell populations but the predominant producers are helper T cells (Th) and macrophages. Cytokines modulate the balance between humoral and cell based immune response, and they regulate the maturation, growth and responsiveness of particular cell populations. Cytokine are important in health and disease, specifically in host immune responses to infection, inflammation, trauma, sepsis, cancer and reproduction (Arunabha et al., 2007). From Meur et al (2017) Associations between Schistosoma antigen-induced cytokine profiles and infection intensity are significant. People with elevated schistosoma infection intensities are more likely to have a lower cytokine responses and vice versa. Infection intensity generally increase with IL-5 and decreases with Th1 cytokines TNF-α, IFN-ϒ and IL-2 (Miner et al., 2010).

People become infected when larval forms of the parasite released by fresh water snails penetrate the skin during contact with infested water. Infected individuals release Schistosoma eggs into water via their fecal material or urine. After larvae hatch from these eggs, the larvae infect a very specific type of freshwater snail. For example, in S. haematobium and S. intercalatum it is snails of the genus Bulinus, in S. mansoni it is Biomphalaria, and in S. japonicum it is Oncomelania. The Schistosoma larvae undergo the next phase of their lifecycles in these snails, spending their time reproducing and developing. Once this step has been completed, the parasite leaves the snail and enters the water column. The parasite can live in the water for only 48 hours without a mammalian host. Once a host has been found, the worm enters its blood vessels. For several weeks, the worm remains in the vessels, continuing its development into its adult phase. When maturity is reached, mating occurs and eggs are produced. Eggs enter the bladder/intestine and are excreted through urine and feces and the process repeats. If the eggs do not get excreted, they can become engrained in the body tissues and cause a variety of problems such as immune reactions and organ. Humans encounter larvae of the Schistosoma parasite when they enter contaminated water while bathing, playing, swimming, washing, fishing, or walking through the water (WHO 2020).

1.2.2 Diagnosis of Schistosomiasis infection.

Diagnosis of schistosoma infection can be done through a couple of methods.

1. Diagnosis through identification of eggs in stool: Microscopic identification of eggs in stool or urine is the most practical method for diagnosis. Stool S examination usually should be performed when infection with mansoni or S. japonicum is suspected, and urine examination is performed if S. haematobium is suspected. Eggs can be present in the stool in infections with all Schistosoma species. The examination can be performed on a simple smear (1 to 2 mg of fecal material). Because eggs may be passed intermittently or in small numbers, their detection is enhanced by repeated examinations or concentration procedures, or both. In addition, for field surveys and investigational purposes, the egg output can be quantified by using the Kato-Katz technique (20 to 50 mg of fecal material) or the Ritchie technique. Eggs can be found in the urine in infections with. S. haematobium (recommended time for collection: between noon and 3 PM) and with S. japonicum. Quantification is possible by using filtration through a nucleopore filter membrane of a standard volume of urine followed by egg counts on the membrane. Tissue biopsy (rectal biopsy for all species and biopsy of the bladder for S. haematobium) may demonstrate eggs when stool or urine examinations are negative. Identification of microhematuria in urine using urine reagent strips is more accurate than circulating antigen tests in the identification of active schistosomiasis in endemic areas. ( Ochodo et al 2015)
2. By Antibody detection

Antibody detection is use to indicate schistosome infection in people who have traveled to areas where schistosomiasis is common and in who eggs cannot be demonstrated in fecal or urine specimens. Test sensitivity and specificity vary widely among the many tests reported for the serologic diagnosis of schistosomiasis and are dependent on both the type of antigen preparations used (crude, purified, adult worm, egg, cercarial) and the test procedure. (Centers for Disease Control and Prevention) At the U.S. Centers for Disease Control and Prevention, a combination of tests with purified adult worm antigens is used for antibody detection. All serum specimens are tested by FAST-ELISA using S. mansoni adult microsomal antigen. A positive reaction (greater than 9 units/µl serum) indicates infection with Schistosoma species. Sensitivity for S. mansoni infection is 99%, 95% for S. haematobium infection, and less than 50% for S. japonicum infection. Specificity of this assay for detecting schistosome infection is 99%. Because test sensitivity with the FAST-ELISA is reduced for species other than S. mansoni, immunoblots of the species appropriate to the patient’s travel history are also tested to ensure detection of S. haematobium and S. japonicum infections. Immunoblots with adult worm microsomal antigens are species-specific, so a positive reaction indicates the infecting species. The presence of antibodies is indicative only of schistosome infection at some time and cannot be correlated with clinical status, worm burden, egg production, or prognosis. Where a person has traveled can help determine which Schistosoma species to test for by immunoblot. (Center for Disease Control and Treatment) In 2005, a field evaluation of a novel handheld microscope was undertaken in Uganda for the diagnosis of intestinal schistosomiasis by a team led by Russell Stothard from the Natural History Museum of London, working with the Schistosomiasis Control Initiative, London. (Ochodo et al 2015).

1. Molecular diagnostics:

Polymerase chain reaction (PCR) based testing is accurate and rapid. (Utzinger et al 2015) They, however, are not frequency used in countries were the disease is common due to the cost of the equipment and the technical experience required to run them (Utzinger et al 2015). Using a microscope to detect eggs costs less as compared to test with PCR. (Mutro et al 2019) Loop-mediated isothermal amplification are being studied as they are lower cost. LAMP testing is not commercially available as of 2019. ( Mutro et al 2019).

1.2.3 Treatment of schistosomiasis infection

Two drugs, Praziquantel and Oxamniquine, are available for the treatment of schistosomiasis. They are considered equivalent in relation to effcacy against S. mansoni and safety. (Appiah et al., 2013). Because of praziquantel’s lower cost per treatment, and oxaminiquine’s lack of efficacy against the urogenital form of the disease caused by S. haematobium, in general praziquantel is considered the first option for treatment (WHO). The treatment objective is to cure the disease and to prevent the evolution of the acute to the chronic form of the disease. All cases of suspected schistosomiasis should be treated regardless of presentation because the adult parasite can live in the host for years. According to (Brinkmann et al., 1988), Schistosomiasis is treatable by taking by mouth a single dose of the drug praziquantel annually. The WHO has developed guidelines for community treatment based on the impact the disease has on children in villages in which it is common: When a village reports more than 50 percent of children have blood in their urine, everyone in the village receives treatment. When 20 to 50 percent of children have bloody urine, only school-age children are treated. According to (Carter Center 2008), when fewer than 20 percent of children have symptoms, mass treatment is not implemented. viously been used to treat and prevent malaria, was recognised in 2008–2009 to be effective .Other possible treatments include a combination of praziquantel with metrifonate, artesunate, or mefloquine. From (Kramer et al., 2014), a Cochrane review found tentative evidence that when used alone, metrifonate was as effective as praziquantel. Another agent, mefloquine, which has pregainst Schistosoma .As stated in Xiao (2013) historically, antimony potassium tartrate remained the treatment of choice for schistosomiasis until the development of praziquantel in the 1980s.

1.2.4 Prevention of Schistosomiasis infection

Many countries are working towards eradicating the disease. The World Health Organization is promoting these efforts. In some cases, urbanization, pollution, and the consequent destruction of snail habitat have reduced exposure, with a subsequent decrease in new infections. The drug praziquantel is used for prevention in high-risk populations living in areas where the disease is common. The Centers for Disease Control and Prevention advises avoiding drinking or coming into contact with contaminated water in areas where schistosomiasis is common

1.2.5 Relationship between Schistosomiasis infection and cytokine levels

Experimental schistosome infections induce strong parasite-specific Th2 responses (Milner et al., 2010). According to Henri et al (2002), Hepatic fibrosis which affects 5-10% of subjects infected by Shistosoma mansoni, is caused by the T cell-dependent granuloma that develop around schistosome eggs. Experimental models of infection have shown that granuloma and fibrosis are tightly regulated by cytokines. From Njaanake et al (2014) pathological changes due to infection with Schistosoma haematobium include cytokine-mediated urinary tract inflammation. The involved cytokines may be excreted in urine and their presence in urine may therefore reflect S. haematobium related urinary tract pathology. The cytokine environment created during the development of helminth-specific immune response is thought to have effects on unrelated antigens by promoting regulatory effector responses. Systemic cytokines are promiscuous rather than antigen specific in their effects. Thus, cytokines stimulated by helminth antigens can modify the environment in which responses to other pathogens occur as illustrated by the systemic effects of gut-restricted helminths. Currently, this paradigm is thought to explain the observed negative associations between helminth infections and atopic⁄autoimmune diseases Milner et al (2010).

1.2 LITERATURE REVIEW

Brief overview of Schistosomiasis infection and Cytokines

Cytokines serve as molecular messengers between cells. Cytokines are proteins that are produced be cells. Cytokines regulate various inflammatory responses. Cytokines interact with cells of the immune system in order to regulate the body’s response to disease and infection, as well as mediate normal cellular processes in the body as stated in Eustice (2019). As stated by Arunabha et al (2016), about 200 cytokines are recognized to date. They have a high degree of helical structure and the molecules share a similar polypeptide fold with a four helical bundles. Cytokines serve various functions in the body such as; colony stimulating factors (stimulate the production of blood cells), growth and differentiation factors (function primarily in the development, tissue maintenance and repair), immune regulatory and pro-inflammatory cytokines (e.g interferons, interleukins, and TNF-alpha that function in the immune system) Eustice (2019). Pro-inflammatory cytokines play a role in the development of inflammatory and neuropathic pain. The anti-inflammatory are actually inflammatory cytokine antagonists. There equally exists evidence to suggest that chemokines are involved in initiating pain, as well as in the persistence of pain. Cytokines affect nearly every biological process viz. embryonic development, disease pathogenesis, non-specific response to infection, specific response to antigen, changes in cognitive functions and progression of the degenerative process of aging. They are also involve in stem cell differentiation, vaccine efficacy and allograft rejection. Such regulation of immune homeostasis is crutial for health and disease, and disruption of this balance results in many chronic pathophysiological states. Thus, it is imperative that therapeutic targeting of cytokine pathways holds great promises for patients suffering from serveral intractable chronic diseases. (Arunabha et al 2016).

Schistosoma haematobium and Schistosoma mansoni are endemic throughout Cameroon. S. haematobium is highly endemic in the regions of Far North and North (sahel area), and in the regions of Southwest and Littoral. S. mamsoni is highly endemic in the regions of Far North, North, Adamawa and Southwest. S. guineesis has been reported in the regions of Center and Littoral. Lack of hygiene and certain play habits of school-aged children such as swimming and fishing in infested water make them especially vulnerable to infection. Symptoms of schistosomiasis are caused the body’s reaction to the worms’ eggs. Intestinal schistosomiasis can result in abdominal pain, diarrhea and blood in stool. Liver enlargement is common in advanced cases, and it is frequently associated with accumulation of fluid in the peritoneal cavity and hypertension of the abdominal blood vessels. In such cases, there may also be enlargement of the spleen. The classic sign of urogenital schistosomiasis is hematuria (blood in urine), fibrosis of the bladder   and ureter and kidney damage is sometimes diagnosed in advanced cases. As stated by Milner et al (2010) of the three major human schistosome species, Schistosoma mansoni and S. japonicum adult parasites inhabit blood vessels of the intestine causing intestinal schistosomiasis, while S. haematobium adults are located in the bladder and pelvic plexuses causing urinary schistosomiasis. S. haematobium is the most prevalent species in sub-Saharan Africa, where it is responsible for a substantial amount of schistosome associated pathology.

The cytokine environment created during the development of helminth-specific immune responses is thought to have effects on unrelated antigens by promoting regulatory effector responses. Systemic cytokines are promiscuous rather than antigen specific in their effects. Thus, cytokines stimulated by helminth antigens can modify the environment in which responses to other pathogens occur as illustrated by the systemic effects of gut-restricted helminths. Currently, this paradigm is thought to explain the observed negative associations between helminth infections and atopic⁄autoimmune diseases Milner et al (2010).

1.3 Rationale

The rationale of the study is to better understand how schistosomiasis infection alter cytokine levels in school children. This can serve as a basis for researchers and drug producers to improve on the biochemistry of drugs and vaccines used against schistosomiasis infection. The study will as well increase an understanding of the role of cytokines in immunity and how their levels are the academic performance of school children in the locality.been regulated during an infection, in this case, Schistosomiasis infection. This study can also serve as a basis for further research on schistosomiasis prevalence and its consequent effects on the academic performance of school children in the locality.

1.4 Hypothesis

The null hypothesis: Schistosomiasis infection has no impact on the level of pro-inflammatory and anti-inflammatory cytokine in school children

Alternative hypothesis: schistosomiasis infection has an impact/effect on the levels of pro-inflammatory and anti-inflammatory cytokines in school children

1.5.0 General objective

Impact/effect of schistosomiasis infection on the levels of pro-inflammatory and anti-inflammatory cytokines in school children.

1.5.1 Specific objectives

1. Prevalence of schistosomaisis infection in children of Buea municipality
2. Pro-inflammatory (IL-1β, IL-6, TNF-α) and anti-inflammatory (IL-4, IL-10, IL-11) cytokine levels in both schistosomiases infected and uninfected children.
3. Schistosomiasis infection and anxiety in school children