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JACOB NJERU NTHIGA TM 300-5262/2015 A research proposal submitted in partial fulfillment for the degree of Master of Medical Laboratory Science (Parasitology and Entomology option) in Jomo Kenyatta University of Agriculture and Technology 2018DECLARATION This research proposal is my original work and has not been presented in any institution leading to the award of a degree or any other award. Students Name JACOB NJERU NTHIGA Sign..Date Supervisors I confirm that this proposal was written by the above named student and has been submitted with our approval as supervisor. Name Dr. AMOS MBUGUA Jomo Kenyatta University of Agriculture and Technology Sign..Date Sign…………………………………….Date…………. Name DR CHRISTOPER ANJILI Centre for Biotechnology Research and Technology, Kenya Medical Research Institute SignDate ABBREVIATIONS S.G.specific gravity W.H.O…………………… HYPERLINK http//en.wikipedia.org/wiki/World_Health_Organization o World Health Organization World Health Organization R.P.M ..Revolution per minute K.N.H..Kenyatta national hospital D.E.A…Drug enforcement administration N.T.Ds..Neglected tropical diseases HYPERLINK http//en.wikipedia.org/wiki/Phenylpropanolamine o Phenylpropanolamine P.P.APhenylpropanolamine C.D.C.Center for Disease Control SSFS.sheather sugar floatation solution TABLE OF CONTENTS TOC o 1-3 h z u HYPERLINK l _Toc521575719 DECLARATION PAGEREF _Toc521575719 h ii HYPERLINK l _Toc521575720 ABBREVIATIONS PAGEREF _Toc521575720 h iii HYPERLINK l _Toc521575721 Table of Contents PAGEREF _Toc521575721 h iv HYPERLINK l _Toc521575722 ABSTRACT PAGEREF _Toc521575722 h v HYPERLINK l _Toc521575723 CHAPTER ONE PAGEREF _Toc521575723 h 1 HYPERLINK l _Toc521575724 INTRODUCTION PAGEREF _Toc521575724 h 1 HYPERLINK l _Toc521575725 1.1 Background Information PAGEREF _Toc521575725 h 1 HYPERLINK l _Toc521575726 1.2 Statement of the problem PAGEREF _Toc521575726 h 4 HYPERLINK l _Toc521575727 1.3 Justification of the study PAGEREF _Toc521575727 h 5 HYPERLINK l _Toc521575728 RESEARCH QUESTIONS PAGEREF _Toc521575728 h 6 HYPERLINK l _Toc521575729 HYPOTHESIS PAGEREF _Toc521575729 h 6 HYPERLINK l _Toc521575730 1.4. Objectives PAGEREF _Toc521575730 h 7 HYPERLINK l _Toc521575731 1.4.1 General objective PAGEREF _Toc521575731 h 7 HYPERLINK l _Toc521575732 1.4.2 Specific objectives PAGEREF _Toc521575732 h 7 HYPERLINK l _Toc521575733 CHAPTER TWO PAGEREF _Toc521575733 h 8 HYPERLINK l _Toc521575734 LITERATURE REVIEW PAGEREF _Toc521575734 h 8 HYPERLINK l _Toc521575735 2.1. Health effects of chewing C. edulis PAGEREF _Toc521575735 h 8 HYPERLINK l _Toc521575736 2.2 Chemistry and pharmacology of C. edulis PAGEREF _Toc521575736 h 9 HYPERLINK l _Toc521575737 2.3 Helminthes that may be associated with Khat consumption PAGEREF _Toc521575737 h 10 HYPERLINK l _Toc521575738 2.3.1.AscarislumbricoidesLinn.(Ascaridida Ascarididae) PAGEREF _Toc521575738 h 11 HYPERLINK l _Toc521575739 2.3.2 Trichuris trichiura Linn. (Trichocephalida Trichiuridae) PAGEREF _Toc521575739 h 11 HYPERLINK l _Toc521575740 2.3.3 Strongyloides stercoralis Bavay (Rhabditida Strongyloididae) PAGEREF _Toc521575740 h 12 HYPERLINK l _Toc521575741 2.3.4 Ancylostoma duodenale and Necator americana (Strongylia Ancyclostomatidae) PAGEREF _Toc521575741 h 13 HYPERLINK l _Toc521575742 2.4 Amoeba that may be associated with Khat consumption PAGEREF _Toc521575742 h 14 HYPERLINK l _Toc521575743 2.4.1 Genus Entamoeba PAGEREF _Toc521575743 h 14 HYPERLINK l _Toc521575744 2.4.2 Giardia lamblia PAGEREF _Toc521575744 h 15 HYPERLINK l _Toc521575745 CHAPTER THREE PAGEREF _Toc521575745 h 17 HYPERLINK l _Toc521575746 MATERIALS AND METHODS PAGEREF _Toc521575746 h 17 HYPERLINK l _Toc521575747 3.1. Study Site/Areas PAGEREF _Toc521575747 h 17 HYPERLINK l _Toc521575748 3.3 STUDY POPULATION PAGEREF _Toc521575748 h 17 HYPERLINK l _Toc521575749 3.4. METHODOLOGY FLOW CHART PAGEREF _Toc521575749 h 18 HYPERLINK l _Toc521575756 3.4 sample size determination PAGEREF _Toc521575756 h 19 HYPERLINK l _Toc521575757 3.5. COLLECTION OF C. edulis FROM STUDY SITE PAGEREF _Toc521575757 h 19 HYPERLINK l _Toc521575758 3.6.2 Parasite isolation using direct wet and iodine preparations PAGEREF _Toc521575758 h 21 HYPERLINK l _Toc521575759 3.6. 3 Determination of parasite distribution by vendor location PAGEREF _Toc521575759 h 22 HYPERLINK l _Toc521575760 3.6.4 Determination of hygiene standards among C. edulis vendors PAGEREF _Toc521575760 h 22 HYPERLINK l _Toc521575761 3.7 Data analysis PAGEREF _Toc521575761 h 22 HYPERLINK l _Toc521575762 3.8 Expected outcomes PAGEREF _Toc521575762 h 22 HYPERLINK l _Toc521575763 3.9 ETHICAL CONSIDERATION PAGEREF _Toc521575763 h 22 HYPERLINK l _Toc521575764 WORK PLAN PAGEREF _Toc521575764 h 23 HYPERLINK l _Toc521575765 BUDGET PAGEREF _Toc521575765 h 24 HYPERLINK l _Toc521575766 REFERENCE PAGEREF _Toc521575766 h 25 HYPERLINK l _Toc521575767 Appendix 1 PAGEREF _Toc521575767 h 28 ABSTRACT Catha edulis (Khat) is a stimulant containing the HYPERLINK https//en.wikipedia.org/wiki/Alkaloid o Alkaloid alkaloid HYPERLINK https//en.wikipedia.org/wiki/Cathinone o Cathinone cathinone, an HYPERLINK https//en.wikipedia.org/wiki/Amphetamine o Amphetamine amphetamine-like HYPERLINK https//en.wikipedia.org/wiki/Stimulant o Stimulant stimulant, responsible for excitement, HYPERLINK https//en.wikipedia.org/wiki/Anorectic o Anorectic loss of appetite, and HYPERLINK https//en.wikipedia.org/wiki/Euphoria o Euphoria euphoria following its consumption . It is a flowering plant shrub native to East Africa and the Arabian Peninsula, users simply chew the green C. edulis leaves keeping a ball of partially chewed leaves against the inside of the cheek. The most common form of C. edulis is as dried leaves which can be chewed, brewed into tea, sprinkled on food or smoked. Among communities from the areas where the plant is native, khat chewing has a history as a social custom dating back thousand of years. In Kenya its cultivated for economic purpose in Meru and and other areas near Mount Kenya region including Embu, Mbeere where it called muguka. Here its cultivated on irrigation using untreated water to keep the plant evergreen for marketing from various water tunnel and storage sources in urban and rural places. After harvesting C. edulis is packed in banana leaves enhance aeration and prevent drying. In street of Eastleigh, Kawangware, Kibra, Mathare, Dandora It is usually sold in open and in unsanitary places with unhygienic packaging and with exposure to dust. Furthermore the illiteracy of many of the people who sell it where and the fact that its chewed raw without sterilization expose users to health risk-one such potential hazards is expose and infection to parasite .The scope of research is to investigate whether human parasite are present on C.edulis leaves and may thus facilitate their transmission. The investigative study will aim at isolation of human parasite using floatation techniques and direct method of parasite identification associated with C. edulis from selected sites of Nairobi County, where C. edulis is consumed by most of the population. CHAPTER ONE INTRODUCTION 1.1 Background Information Catha edulis Forrsk (Celastrales Celastraceae) also known as khat is a HYPERLINK http//en.wikipedia.org/wiki/Flowering_plant o Flowering plant flowering plant (Curto et al., 2013).A native to the HYPERLINK http//en.wikipedia.org/wiki/Horn_of_Africa o Horn of Africa Horn of Africa and the HYPERLINK http//en.wikipedia.org/wiki/Arabian_Peninsula o Arabian Peninsula Arabian Peninsula (Saif-Ali et al.,2003). Among communities from these areas, C. edulis chewing has a history as social custom dating back thousands of year and it contains a HYPERLINK http//en.wikipedia.org/wiki/Monoamine o Monoamine monoamine HYPERLINK http//en.wikipedia.org/wiki/Alkaloid o Alkaloid alkaloid called HYPERLINK http//en.wikipedia.org/wiki/Cathinone o Cathinone cathinone,(kalix and braenden,1985) an HYPERLINK http//en.wikipedia.org/wiki/Amphetamine o Amphetamine amphetamine-like HYPERLINK http//en.wikipedia.org/wiki/Stimulant o Stimulant stimulant, which is said to cause excitement, HYPERLINK http//en.wikipedia.org/wiki/Anorectic o Anorectic loss of appetite and HYPERLINK http//en.wikipedia.org/wiki/Euphoria_(emotion) o Euphoria (emotion) euphoria (Hassan et al., 2000). In 1980, the HYPERLINK http//en.wikipedia.org/wiki/World_Health_Organization o World Health Organization World Health Organization (WHO) classified it as a HYPERLINK http//en.wikipedia.org/wiki/Drug_of_abuse o Drug of abuse drug of abuse that can produce mild-to-moderate HYPERLINK http//en.wikipedia.org/wiki/Substance_dependence o Substance dependence psychological dependence (less than tobacco or alcohol), although WHO does not consider C. edulis to be seriously addictive. The plant has been targeted by anti-drug organizations such as the drug enforcement administration ( HYPERLINK http//en.wikipedia.org/wiki/Drug_Enforcement_Administration o Drug Enforcement Administration DEA). It is a HYPERLINK http//en.wikipedia.org/wiki/Controlled_substance o Controlled substance controlled substance in some countries, such as HYPERLINK http//en.wikipedia.org/wiki/Canada o Canada Canada, HYPERLINK http//en.wikipedia.org/wiki/Germany o Germany Germany, the HYPERLINK http//en.wikipedia.org/wiki/United_Kingdom o United Kingdom United Kingdom, and the HYPERLINK http//en.wikipedia.org/wiki/United_States o United States United States, while its production, sale, and consumption are legal in other nations, including HYPERLINK http//en.wikipedia.org/wiki/Djibouti o Djibouti Djibouti, HYPERLINK http//en.wikipedia.org/wiki/Ethiopia o Ethiopia Ethiopia, HYPERLINK http//en.wikipedia.org/wiki/Somalia o Somalia Somalia, and HYPERLINK http//en.wikipedia.org/wiki/Yemen o Yemen Yemen. Khat is a slow-growing HYPERLINK http//en.wikipedia.org/wiki/Shrub o Shrub shrub that typically attains a height of between 1 and 5m (3.3 and 16.4ft) and in equatorial region, it can reach heights of up to 10 metres (33ft)(figure 1). The plant usually grows in HYPERLINK http//en.wikipedia.org/wiki/Desert_climate o Desert climate arid environments, at a temperature range of 5 to 35C (41 to 95F). It has HYPERLINK http//en.wikipedia.org/wiki/Evergreen o Evergreen evergreen HYPERLINK http//en.wikipedia.org/wiki/Leaf o Leaf leaves, which are 510cm long and 14cm broad. The shrubs flowers are produced on short axillary HYPERLINK http//en.wikipedia.org/wiki/Cyme_(botany) l Determinate_or_Cymose o Cyme (botany) cymes that are 48cm in length. Each flower is small, with five white petals. The HYPERLINK http//en.wikipedia.org/wiki/Samara_(fruit) o Samara (fruit) samara fruit is an oblong, three-valved capsule, which contains one to three seeds(figure2). (Robson et al., 1994) HYPERLINK http//upload.wikimedia.org/wikipedia/commons/5/56/Catha_edulis.jpg INCLUDEPICTURE http//upload.wikimedia.org/wikipedia/commons/5/56/Catha_edulis.jpg MERGEFORMATINET Figure 1 Catha edulis plant INCLUDEPICTURE http//www.plantsgalore.com/terms/images/images/PGC-G-Samara.jpg MERGEFORMATINET Figure 2 Samara fruit displaying paired seeds 1.2 Statement of the problem Human parasite infection are common in tropical and sub-tropical regions of the developing world especially in sub-Saharan Africa where poor domestic and environmental hygiene prevails, Poor quality source for drinking water ,inadequate sanitation facilities and overcrowding aid in human parasite transmission, which include amoeba and helminthes. Globally geohelminths impact the lives of millions of people. 819 million people are infected with roundworm (Ascariasis), while whipworm (Trichuriasis) are responsible for about 464.6 million infection while two hook worm species(Necator and Ancylostoma ) infect 438.0 million individual with Considering of 480 million people were infected with E. histolytica and 36 million developed disabling colitis with 40 thousand deaths attributed to amebiasis, and on a global scale, amebiasis likely ranks third among parasitic causes of death, behind only malaria and schistosomiasis. G.lamblia prevalence rate in temperate climates is 2-10 in adults whereas in tropical countries 50-80 of people are carriers G. lamblia is the most commonly identified intestinal parasite in the United state(US) as well as in Canada, Center for Disease Control( CDC) reports approximately 20,000 cases per year in US. According to one report from Ontario, Canada showed an incidence rate of 25.8 cases per 100,000 populations between 1990 and 1998. In developed countries, infection occurs most frequently among children care centers and congested population due to ingestion of mature cyst in drinking untreated water unhygienic food being common source of infection and can result in community wide epidemics. These group of intestinal parasite to are transmitted primarily through oral route. They have direct lifecycle and parasitic infection occurs through fecal contamination of foodstuffs and water supplies (Omitola et al.,2016). They are listed as part of neglected tropical diseases (NTDs) hence affecting more than one billion people worldwide mainly in Africa and mostly those living in remote rural, urban slum areas or conflict zones hence are obstacles to human settlement and social economic development of already impoverished communities, In 15 june 2016 Kenya launched the second NTD master plan2016-2020 to enhance elimination. In most places where it is grown, harvested and sold, there are usually no hygienic facilities such as well maintained sanitation and toilet facilities. And as Khat is usually chewed without cleaning the leaves it is sold in the open in areas where it is exposed to dust. All these factors can expose humans to a variety of pathogens ranging from bacteria, amoeba to intestinal geo helminthes. 1.3 Justification of the study An estimated 500,000 Kenyans depends on C. edulis cultivation(Nyongesa and Onyango (2010) and sale for their livelihood with Britains importing some 25million worth of the leaves annually before the 2014 ban .More than 15 cargo planes full of cargo planes full of khat also leave Kenya for Somalia dairy with retail value Of 400,000.Expert have also debated the impact of C. edulis on health and central nervous system which has led to banning in UK despite an official recommendation from regulatory agencies that it remains legal. The loss of British market which consumed between 2500 and 2800 tons a year has left many farmer and traders in Maua with huge loss. In consideration with mode of parasite transmission and in order to be able to control human pathogens that can be transmitted through the oral route, it is important to study the source of transmission. It is possible that because of the unhygienic manner C. edulis is handled chewing could be a source of transmission. Studies on the effects of the plant have only been based on other medical conditions ignoring parasite transmission. Through available parasitological techniques, attempts will be made to isolate and identify human parasites from C. edulis bought from different vendors in selected parts of Nairobi County. RESEARCH QUESTIONS Can human parasite cyst and ova be isolated from C. edulis leaves . What are the different species of human parasite associated with C. edulis. What are the predisposing factors on transmission of human parasite on( khat) C. edulis. Consumers HYPOTHESIS There are no human parasites contaminants associated with Catha edulis leaves from different vendors in Nairobi County. 1.4. Objectives 1.4.1 General objective To isolate and compare human parasite associated with C. edulis leaves in selected parts of Nairobi County (Eastleigh, Kawangware, Kibra, Mathare, Dandora ). 1.4.2 Specific objectives To isolate species of human parasite from C. edulis leaves sold by different vendors in Nairobi County. To compare the distribution of human parasite species associated with C. edulis leaves To study the hygiene associated with vendors of C. edulis in selected parts of Nairobi. CHAPTER TWO LITERATURE REVIEW 2.1. Health effects of chewing C. edulis Khat consumption induces mild HYPERLINK http//en.wikipedia.org/wiki/Euphoria_(emotion) o Euphoria (emotion) euphoria and excitement, similar to that conferred by strong coffee. Individuals become very talkative under the influence of the plant. The effects of oral administration of cathinone occur more rapidly than the effects of amphetamine pills roughly 15 minutes as compared to 30 minutes in amphetamine. Khat can induce HYPERLINK http//en.wikipedia.org/wiki/Mania o Mania manic behaviors and HYPERLINK http//en.wikipedia.org/wiki/Hyperactivity o Hyperactivity hyperactivity, similar in effects to those produced by amphetamine. The use of khat results in HYPERLINK http//en.wikipedia.org/wiki/Constipation o Constipation constipation. HYPERLINK http//en.wikipedia.org/wiki/Pupillary_response o Pupillary response Dilated pupils ( HYPERLINK http//en.wikipedia.org/wiki/Mydriasis o Mydriasis mydriasis) are prominent during khat consumption, reflecting the HYPERLINK http//en.wikipedia.org/wiki/Sympathomimetic o Sympathomimetic sympathomimetic effects of the drug, which are also reflected in increased heart rate and blood pressure. HYPERLINK http//en.wikipedia.org/wiki/Drug_withdrawal o Drug withdrawal Withdrawal HYPERLINK http//en.wikipedia.org/wiki/Symptom o Symptom symptoms that may follow occasional use include mild depression and irritability. Withdrawal symptoms that may follow prolonged khat use include HYPERLINK http//en.wikipedia.org/wiki/Lethargy o Lethargy lethargy, mild HYPERLINK http//en.wikipedia.org/wiki/Depression_(clinical) o Depression (clinical) depression, nightmares, and slight HYPERLINK http//en.wikipedia.org/wiki/Tremor o Tremor tremor. Khat is an effective HYPERLINK http//en.wikipedia.org/wiki/Anorectic o Anorectic anorectic (causes loss of appetite). Long-term use can precipitate negative impact on HYPERLINK http//en.wikipedia.org/wiki/Liver o Liver liver function, permanent HYPERLINK http//en.wikipedia.org/wiki/Tooth o Tooth tooth darkening (of a greenish tinge), susceptibility to HYPERLINK http//en.wikipedia.org/wiki/Peptic_ulcer o Peptic ulcer ulcers, and diminished drive. It is unclear if the consumption of khat directly affects the mental health of the user or not. Occasionally, a psychotic episode can result, resembling a HYPERLINK http//en.wikipedia.org/wiki/Hypomanic o Hypomanic hypo manic state in presentation. Immediate effects increased heart rate, blood pressure, HYPERLINK http//en.wikipedia.org/wiki/Euphoria o Euphoria euphoria and hyperactivity. Long-term effects HYPERLINK http//en.wikipedia.org/wiki/Depression_(clinical) o Depression (clinical) depression, infrequent, hallucinations, impaired inhibition (similar to alcohol), increased risk of HYPERLINK http//en.wikipedia.org/wiki/Myocardial_infarction o Myocardial infarction myocardial infarction (heart attack), HYPERLINK http//en.wikipedia.org/wiki/Psychosis o Psychosis psychosis in extreme cases in the genetically predisposed, oral cancer. Indeterminate effects include death and stroke following HYPERLINK http//en.wikipedia.org/wiki/Acute_coronary_syndrome o Acute coronary syndrome acute coronary syndrome (clogging of the artery) 2.2 Chemistry and pharmacology of C. edulis The stimulant effect of the plant was originally attributed to katin, HYPERLINK http//en.wikipedia.org/wiki/Cathine o Cathine cathine, a HYPERLINK http//en.wikipedia.org/wiki/Phenethylamine o Phenethylamine phenethylamine-type substance isolated from the plant (crombie 1980). However, the attribution was disputed by reports showing the plant extracts from fresh leaves contained another substance more behaviorally active than cathine(WHO,2006Balint,2012). In 1975, the related alkaloid HYPERLINK http//en.wikipedia.org/wiki/Cathinone o Cathinone cathinone was isolated, and its HYPERLINK http//en.wikipedia.org/wiki/Absolute_configuration o Absolute configuration absolute configuration was established in 1978. Cathinone is not very stable and breaks down to produce cathine and HYPERLINK http//en.wikipedia.org/wiki/Norephedrine o Norephedrine nor ephedrine. These chemicals belong to the HYPERLINK http//en.wikipedia.org/wiki/Phenylpropanolamine o Phenylpropanolamine PPA (phenylpropanolamine) family, a subset of the HYPERLINK http//en.wikipedia.org/wiki/Substituted_phenethylamine o Substituted phenethylamine phenethylamines related to HYPERLINK http//en.wikipedia.org/wiki/Amfetamine o Amfetamine amphetamines and the HYPERLINK http//en.wikipedia.org/wiki/Catecholamine o Catecholamine catecholamines HYPERLINK http//en.wikipedia.org/wiki/Epinephrine o Epinephrine epinephrine and HYPERLINK http//en.wikipedia.org/wiki/Norepinephrine o Norepinephrine norepinephrine. In fact, cathinone and cathine have a very similar molecular structure to amphetamine. Catha edulis is sometimes confused with HYPERLINK http//en.wikipedia.org/wiki/Methcathinone o Methcathinone methcathinone (also known as cat), a Schedule I substance that possesses a similar chemical structure to the khat plants cathinone active component. However, both the side effects and the addictive properties of methcathinone are much stronger than those associated with khat use.When khat leaves dry, the more potent chemical, cathinone, decomposes within 48 hours, leaving behind the milder chemical, cathine. Thus, harvesters transport khat by packaging the leaves and stems in plastic bags or wrapping them in banana leaves to preserve their moisture and keep the cathinone potent. It is also common for them to sprinkle the plant with water frequently or use refrigeration during transportation. HYPERLINK http//en.wikipedia.org/wiki/FileS-Cathinone.svg INCLUDEPICTURE http//upload.wikimedia.org/wikipedia/commons/thumb/c/ca/S-Cathinone.svg/220px-S-Cathinone.svg.png MERGEFORMATINET Figure1/ Cathinone structure When the C. edulis leaves are chewed, cathine and cathinone are released and absorbed through the mucous membranes of the mouth as well as the lining of the stomach. The action of cathine and cathinone on the HYPERLINK http//en.wikipedia.org/wiki/Reuptake o Reuptake reuptake of HYPERLINK http//en.wikipedia.org/wiki/Epinephrine o Epinephrine epinephrine and norepinephrine has been demonstrated in lab animals, showing that one or both of these chemicals cause(s) the body to recycle these neurotransmitters more slowly, resulting in the wakefulness and insomnia associated with khat use. Receptors for serotonin show a high affinity for cathinone, suggesting this chemical is responsible for feelings of euphoria associated with chewing khat. In mice, cathinone produces the same types of nervous pacing or repetitive scratching behaviors associated with amphetamines. The effects of cathinone peak after 15 to 30 minutes, with nearly 98 of the substance metabolised into norephedrine by the liver. Cathine is somewhat less understood, being believed to act upon the HYPERLINK http//en.wikipedia.org/wiki/Adrenergic_receptors o Adrenergic receptors adrenergic receptors causing the release of epinephrine and nor epinephrine. It has a HYPERLINK http//en.wikipedia.org/wiki/Half-life o Half-life half-life of about three hours in humans. The medication HYPERLINK http//en.wikipedia.org/wiki/Bromocriptine o Bromocriptine bromocriptine can reduce cravings and withdrawal symptoms within 24 hours. 2.3 Helminthes that may be associated with Khat consumption Helminthes transmitted through contamination can be strongly associated with khat consumption particularly those that spend part of their development in the soil 2.3.1.AscarislumbricoidesLinn.(Ascaridida Ascarididae) Human infections is acquired by ingesting fully embryonated eggs which have been accidentally picked up from polluted soil, from food or drink contaminated by viable embryonated eggs, or children who eat dirt. In the tropics, all ages are heavily parasitized, whereas in subtropical countries young children are more commonly infected than adults and more commonly pollute the soil. In countries where human feces are used as fertilizer for field crops or vegetable gardens, the adult population acquires infection from eating raw vegetables contaminated with fully embryonated eggs. Ascariasis is essentially a door yard and household infection, primarily propagated by the seeding of the soil immediately around the house with eggs present in promiscuous droppings of small children, who, in turn become reinfected from eggs which they pick up on their fingers and introduce into their mouth. Favorable seedbeds of Ascaris eggs may remain infective for many months. 2.3.2 Trichuris trichiura Linn. (Trichocephalida Trichiuridae) Conditions favorable for development of the unembryonated eggs of Trichiuris trichiura evacuated in human stool onto the ground consist of warm, shaded moist soil. Development to the infective stage requires about twenty-one days, after which an active first stage is coiled inside the shell. Infection results from the ingestion of these eggs obtained directly or indirectly from soil. The eggs are much resistant to temporary drought and heat than the Ascaris eggs, will not usually develop to the inactive stage on hard clay, ashes or cinders, and will not survive direct suns rays or intense cold, even in moist atmosphere, a moderately dry film of faeces will support survival for not more than two weeks. Areas of high incidence and heavy worm burden are usually those polluted by small children, who are often more commonly infected than are adults. In areas of high endemicity small children develop heavy infections yet the greatest prevalence occurs typically in children of primary school age who contaminate door yards soil with their feces and later pick up the fully embryonated eggs on their fingers and transfer eggs to their mouth. The exact mechanism by which T. trichiura affects the human host is not known but there are at least two important process at work, traumatic and allergy. Since the portal of entry into the human body is the mouth and the larva which escapes from the hatching e.g. requires no migration through the lungs, the first serious damage produced at the site of permanent attachment of the worm to the intestinal mucosa in the cecal area. If only few worms have become adult the traumatic damage is trivial, but many worms transmitted together may block the appendiceal lumen or cause marked irritation and inflammation of the epithelium of the caecum, appendix and ascending colon. Usually a few worms provoke very little allergic reactions, but a large number produces colitis and proctitis and frequently a secondary anaemia. 2.3.3 Strongyloides stercoralis Bavay (Rhabditida Strongyloididae) Man is the most important host of Strongyloides stercoralis. The infective stage larvae are developed in the soil, from which man becomes infected typically through skin contact. Strongyloidiasis tends to be particularly common in institutional groups, as mental hospitals and prisons in warm moist climates. It is more prevalent in adults than in children in the general population.The pathogenic effects of S. Stercoralis begin with the active entry of the parasite into the skin and continue long as it remains alive in the human body. The degree of systemic toxaemia and the hosts reaction are indicated by high eosinophilia, which characteristically develops during the acute stage of the infection. As well as by giant urticaria and other sensitisation reactions which frequently develop when a previously infected individual is subjected to re-exposure. 2.3.4 Ancylostoma duodenale and Necator americana (Strongylia Ancyclostomatidae) The propagation of human hookworm infection depends on 1.Adequate source of infection in the human population 2. Defecation habits ensuring that eggs of these hookworms will be deposited in favorable locations for extrinsic development of the parasite, 3.Appropriate conditions of environment (moisture and warmth) and of the soil (shade and sandy humus containing an abundant supply of nutritive material for developing larva 4.Opportunity for the infective stage to be exposed to the skin of man so that the parasitic phase may be re-established. In many tropical and subtropical countries, conditions are satisfactory for maintenance of the cycle through the greater part of the year. The infective stage larvae of the hookworm actively enter the skin with which they make contact usually they enter digital spaces between the toes of barefooted of persons who step on infested soils harboring the infective stage of the parasite. The most important manifestations of hookworm infections are caused by the adult worms in the intestine. The worms attach themselves to the gut mucosa by their buccal capsules. They suck in blood, which passes out undigested and not utilized through its intestines. A single Ancylostome adult can suck about 0.2ml blood a day, while the small Necator sucks in about 0.03ml per day. The worms frequently leave on site and attach themselves to another site. As the secretion of the worm contains anticoagulant activity, bleeding from the site continue for some time. This adds to the blood loss. This chronic blood loss over time leads to a microcytic hypochromic type of iron deficiency anemia 2.4 Amoeba that may be associated with Khat consumption 2.4.1 Genus Entamoeba Pathogenic HYPERLINK https//en.wikipedia.org/wiki/Entamoeba o Entamoeba Entamoeba histolitica infecting humans and other primates causes HYPERLINK https//en.wikipedia.org/wiki/Amoebiasis o Amoebiasis amoebiasis, E. histolytica is estimated to infect about 50 million people worldwide. Previously, it was thought that 10 of the world population was infected, but these figures predate the recognition that at least 90 of these infections were due to a second species, HYPERLINK https//en.wikipedia.org/wiki/E._dispar o E. dispar E. dispar. Mammals such as dogs and cats can become infected transiently, but are not thought to contribute significantly to transmission. When they cyst of E.histolytica reaches caecum or lower part of ileum encystations occursandan amoeba with four nuclei emerges and that divides by binary fission to form eight trophozoites. Trophozoites migrate to the large intestine and lodge in to the sub mucosal tissue where it grow and multiply by binary fission in large intestine (Trophozoite phase of life cycle is responsible for producing characteristics lesion of amoebiasis).hence number of trophozoites are discharged in to the lumen of the bowel and are transformed into cystic forms. However cysts thus formed are unable to develop in the same host and therefore necessitate transference to another susceptible host. The cysts are passed in the feces.E. histolytica can be distinguished from other amoebas by two major criteria.1)Nature of the nucleus of the trophozoite 2).Cyst size and number of its nuclei. Infections by Entamoeba histolytica occurs by ingestion of mature quadrinucleate cysts in fecally contaminated food, water, or hands. The quadrinucleate cyst is resistant to the gastric environment and passes unaltered through the stomach The active stage exists only in the host and in fresh loose feces HYPERLINK https//en.wikipedia.org/wiki/Microbial_cyst o Microbial cyst cysts survive outside the host in water, in soils, and on foods, especially under moist conditions on the latter. The infection can occur when a person puts anything into their mouth that has touched the feces of a person who is infected with quadrinucleate cyst of E.histolytica, swallows something, such as water or food that is contaminated, or swallows E. histolytica cysts picked up from contaminated surfaces or fingers. The cysts survive for only a few months outside of the host. When cysts are swallowed they cause infections by encysting in the digestive tract infection can be asymptomatic or can lead to HYPERLINK https//en.wikipedia.org/wiki/Dysentery o Dysentery amoebic dysentery or amoebic HYPERLINK https//en.wikipedia.org/wiki/Liver_abscess o Liver abscess liver abscess. Symptoms can include fulminating dysentery, bloody diarrhea, weight loss, fatigue, abdominal pain, and HYPERLINK https//en.wikipedia.org/wiki/Ameboma o Ameboma amoeboma. The amoeba can actually bore into the intestinal wall, causing HYPERLINK https//en.wikipedia.org/wiki/Lesion o Lesion lesions and intestinal symptoms, and it may reach the blood stream. From there, it can reach different vital organs of the human body, usually the liver, but sometimes the lungs, brain, spleen, etc. A common outcome of this invasion of tissues is a liver abscess, which can be fatal if untreated. 2.4.2 Giardia lamblia Infection occurs through ingestion of dormant HYPERLINK https//en.wikipedia.org/wiki/Microbial_cyst o Microbial cyst microbial cysts in contaminated water or food, or by the HYPERLINK https//en.wikipedia.org/wiki/FecalE28093oral_route o Fecaloral route fecaloral route (through poor hygiene practices). The cyst can survive for weeks to months in cold water, so can be present in contaminated wells and water systems, especially stagnant water sources, such as naturally occurring ponds, storm water storage systems, and even clean-looking mountain streams. Giardia lamblia is a microscopic parasite that causes the diarrheal illness known as giardiasis. Can also be found on surfaces, soil, food, or water that has been contaminated with feces from infected humans or animals .The may also occur in city reservoirs and persist after water treatment, as the cysts are resistant to conventional water treatment methods, such as HYPERLINK https//en.wikipedia.org/wiki/Water_chlorination o Water chlorination chlorination and HYPERLINK https//en.wikipedia.org/wiki/Ozonolysis o Ozonolysis ozonolysis. In addition to waterborne sources, fecaloral transmission can also occur, where community may have poor hygiene practices. However not all Giardia infections are symptomatic, and many people can unknowingly serve as carriers of the parasite. CHAPTER THREE MATERIALS AND METHODS 3.1. Study Site/Areas Catha edulis will be purchased from vendors within the Nairobi County. The selected areas for purchase where it is mostly chewed are Eastleigh, Kawangware, Kibra, Mathare, Dandora. There is absolute inaccessibility to the most basic amenities among them food, water, shelter and healthcare in part of above listed areas where khat is sold in street . Toilets are considered a luxury, like mathare Raw sewer openly drains from the nearby suburbs like Utalii Hotel, Muthaiga, and Survey of Kenya. The stench of raw sewer fills the air as human waste flows across the shanties, eventually draining into the nearby Nairobi River. Dandora is an eastern suburb in HYPERLINK https//en.wikipedia.org/wiki/Nairobi o Nairobi Nairobi, HYPERLINK https//en.wikipedia.org/wiki/Kenya o Kenya Kenya. It is part of the HYPERLINK https//en.wikipedia.org/wiki/Embakasi_division o Embakasi division Embakasi division. Surrounding neighbour hoods called estates include Kariobangi, Baba Dogo, Gitare Marigo and Korogocho .Dandora dumpsite has piled up waste uncontrollably leading to health, social, economic and political problems. This study will be significant because its findings provided insights on human parasite transmission on khat vendor and consume 3.2 Study Design A cross sectional study design will be conducted to assess human parasite ova and cyst on C. edulis leaves which will be purchased directly from vendors in selected part of Nairobi County 3.3 STUDY POPULATION This will consist of the collected samples of C. edulis leaves from vendors listed in the study areas 3.3.1 INCLUSION CRITERIA All khats vendors in the listed study site who will be selected 3.3.2 EXCLUSION CRITERIA 0.052 n125 n minimum sample size p prevalence rate 50 d2 absolute precision (5) 0.05 3.5. COLLECTION OF C. edulis FROM STUDY SITE Cluster sampling will be used in all the selected five study sites, An average of 300g of khat leaves from 25 vendors located in 5 study sites will be obtained and stored in a cool dry aerated bags during transportation. Samples not processed immediately will be refrigerated. A questionnaire having a number for confidentiality will be administered to each enrolled vendor by the principle investigator (PI). The participant may be assisted by the PI to understand the questionnaire where necessary (such as when participants are unable to understand the questions). The purpose of the questionnaire is to obtain bio-data (source of C.edulis leaves, site of vendor, public health consideration awareness on infection transmission, source of social amenities such as sewerage, toilet, food, water ), 3.5.1 Independent variables Catha edulis leaves 3.5.2 Dependent variables Catha edulis vendors and study sites 3.6.1 Parasite isolation using concentration method In order to isolate ova and cyst, Sheathers sugar floatation technique will be used (Shearer, 1923). The Sheathers sugar floatation solution (SSFS) will be prepared by mixing the following ingredients ComponentQuantityGranulated sugar Water 40 formaldehyde solution, U.S.P454 gm 355 ml 6 ml The mixture will be transferred into the upper half of a double boiler. The sugar will be dissolved by stirring up to 90oC and then cooled to room temperature. From the total 300 grams collected, 150 grams of C. edulis leaves and the branches that are usually chewed will be cut into small pieces and placed in 150 ml glass beaker containing the concentrated sugar solution (SG 1.300) and shaken for one hour. The leaves and stems will be removed using forceps, after which the sediment will be filtered through fabric screen into a different container to remove large particles. The filtrate will then be centrifuged at 3500, 2500, 1500 and 1000 revolutions per minute (rpm) for ten minutes. The resultant sediment will be transferred to slides and examined at x400 magnification for parasite cyst and ova. All ova and cysts will be identified using manuals available at Kenya Medical Research Institute and National Museums of Kenya. 3.6.2 Parasite isolation using direct wet and iodine preparations The remaining 150 grams of C. edulis leaves will be used for direct wet prep and iodine preparation for ova and cyst identification. Normal saline and lugols iodine5 solution will be prepared by mixing the following ingredients ComponentQuantityPotassium iodine(KI)10gIodine 5gDistilled water100ml Dissolve KI in about 20-30ml of distilled water .Add iodine and heat gently with constantly mixing until iodine is dissolved. Dilute upto 100ml with distilled water .store in a dark closed container. ComponentQuantitySodium chloride (NaCl)8.5gDistilled water100ml150 grams of C. edulis leaves and the branches that are usually chewed will be cut into small pieces and placed in 150 ml glass beaker containing normal saline and centrifuged for 10 minutes then sediment will be filtered through fabric screen into a different container to remove large particles. The filtrate will then be centrifuged at 3500, 2500, 1500 and 1000 revolutions per minute (rpm) for ten minutes. The resultant sediment will be transferred to slides and examined at x400 magnification for parasite cyst and ova. All ova and cysts will be identified using manuals available at Kenya Medical Research Institute and National Museums of Kenya. 3.6. 3 Determination of parasite distribution by vendor location Distribution of parasite in each site will be determined qualitatively whereby absence and presence of either or both of protozoa or helminthes will be recorded according to site sample will be collected. Samples will be designated a unique number according to site of collection which will facilitate easier recording of parasite presence. 3.6.4 Determination of hygiene standards among C. edulis vendors Hygiene in vendor site will be determined by use of questionnaire as in appendix 1 below. This will investigate the possible source of parasites which predispose the vendors and samples to contamination 3.7 Data analysis Comparison of parasites within and between vendors and sites will be done using NOVA Analysis of Variance. 3.8 Expected outcomes It is expected that information generated from the study will be useful in educating about the dangers of eating unclean C. edulis 3.9 ETHICAL CONSIDERATION Consent to participate in the study will be sought from each subject who will meet the criteria. The study purpose will be explained to each consenting participant in a language they are confer cent and they understand. Hence participant will be given opportunity to ask question if any. Study scientific and ethical approval will be sought from KNH review committee. WORK PLAN ACTIVITY2018—2019MARCH APRILMAYJUNE JULYAUG OCTNOV DECJAN 2019Feb 2019March April 2019Proposal DevelopmentOral presentation At JkuatProposal correctionKNH-UON Scientific review unit for approvalData collection Data entry and analysisThesis writing manuscript preppublication and thesis submission BUDGET Lab analysis and sample Parasitological Operational expenses(internet and consultation etc) Khat (8kg) 30000 16600 20000Equipment and Reagent Weigh machine Centrifuge tubes Reagents 5400 8000 10000Travelling and accommodations ( Stationary and data analysis15000 20000 Miscellaneous 25000 TOTAL 150000 REFERENCE Thamwiwat A, Mejia R, Nutman TB, Bates JT. Strongyloidiasis as a Cause of Chronic Diarrhea, Identified Using Next-Generation Strongyloides stercoralis-Specific Immunoassays. Curr Trop Med Reports. 20141(3)145-147. doi10.1007/s40475-014-0026-7. Skerratt LF. Strongyloides spearei n. sp. (Nematoda Strongyloididae) from the common wombatVombatus ursinus (Marsupialia Vombatidae). Syst Parasitol. 199532(2)81-89. doi10.1007/BF00009506. Bogitsh BJ (Burton J, Carter CE (Clint E, Oeltmann TN. Human Parasitology. UK Academic Press 2013. World Health Organization. Guidelines for the Safe Use of Wastewater, Excreta, and Greywater. third. Geneva World Health Organization 2006. Warfa, N., Klein, A., Bhui, K., Leavey, G., Craig, T., Alfred Stansfeld, S. (2007). Khat use and mental illness a critical review. Social Science Medicine (1982), 65(2), 30918. https//doi.org/10.1016/j.socscimed.2007.04.038 Abdulle, Sahal (2 January 2007). HYPERLINK https//www.reuters.com/article/2007/01/02/us-somalia-conflict-khat-idUSL0291421520070102 Somali Islamists are gone so khat is back. Reuters. Mogadishu. Retrieved 26 January 2014. HYPERLINK https//en.wikipedia.org/w/index.phptitleYousif_Al_Zarouniactioneditredlink1 o Yousif Al Zarouni (page does not exist) Al Zarouni, Yousif (2015). The Effects of Khat (Catha Edulis). London Yousif Al Zarouni. Anderson, David Beckerleg, Susan Hailu, Degol Klein, Axel (2007). HYPERLINK https//books.google.com/booksidGsavAwAAQBAJ The Khat Controversy Stimulating the Debate on Drugs. Berg. HYPERLINK https//en.wikipedia.org/wiki/International_Standard_Book_Number o International Standard Book Number ISBN HYPERLINK https//en.wikipedia.org/wiki/SpecialBookSources/978-1-84788-335-3 o SpecialBookSources/978-1-84788-335-3 978-1-84788-335-3. Beckerleg, Susan (2010). HYPERLINK https//books.google.com/booksidPVPFAAAAQBAJ Ethnic Identity and Development Khat and Social Change in Africa. New York Palgrave Macmillan US. HYPERLINK https//en.wikipedia.org/wiki/International_Standard_Book_Number o International Standard Book Number ISBN HYPERLINK https//en.wikipedia.org/wiki/SpecialBookSources/978-0-230-10778-6 o SpecialBookSources/978-0-230-10778-6 978-0-230-10778-6. Carrier, Neil C. M. (2007). HYPERLINK https//books.google.com/booksidHhtxBITJmtYC Kenyan Khat The Social Life of a Stimulant. Leiden BRILL. HYPERLINK https//en.wikipedia.org/wiki/International_Standard_Book_Number o International Standard Book Number ISBN HYPERLINK https//en.wikipedia.org/wiki/SpecialBookSources/90-04-15659-3 o SpecialBookSources/90-04-15659-3 90-04-15659-3. Gatter, Peer (2012). Politics of Qat The Role of a Drug in Ruling Yemen. Wiesbaden Ludwig Reichert Verlag. HYPERLINK https//en.wikipedia.org/wiki/International_Standard_Book_Number o International Standard Book Number ISBN HYPERLINK https//en.wikipedia.org/wiki/SpecialBookSources/978-3-89500-910-5 o SpecialBookSources/978-3-89500-910-5 978-3-89500-910-5. HYPERLINK http//www.qat-yemen.com/ Link to the table of contents and to selected chapters. Gebissa, Ezekiel (2004). HYPERLINK https//books.google.com/booksidga91oPVFb5MC Leaf of Allah Khat Agricultural Transformation in Harerge, Ethiopia 1875-1991. Athens, Ohio Ohio University Press. HYPERLINK https//en.wikipedia.org/wiki/International_Standard_Book_Number o International Standard Book Number ISBN HYPERLINK https//en.wikipedia.org/wiki/SpecialBookSources/978-0-85255-480-7 o SpecialBookSources/978-0-85255-480-7 978-0-85255-480-7. Retrieved 21 June 2016. Gebissa, Ezekiel (2010). HYPERLINK https//books.google.com/booksidj4EbAQAAMAAJ Taking the Place of Food Khat in Ethiopia. Trenton Red Sea Press. HYPERLINK https//en.wikipedia.org/wiki/International_Standard_Book_Number o International Standard Book Number ISBN HYPERLINK https//en.wikipedia.org/wiki/SpecialBookSources/978-1-56902-317-4 o SpecialBookSources/978-1-56902-317-4 978-1-56902-317-4. Gezon, Lisa (2012). HYPERLINK https//books.google.com/booksidHMhEdnT0BicC Drug Effects Khat in Biocultural and Socioeconomic Perspective. Walnut Creek Left Coast Press. HYPERLINK https//en.wikipedia.org/wiki/International_Standard_Book_Number o International Standard Book Number ISBN HYPERLINK https//en.wikipedia.org/wiki/SpecialBookSources/978-1-61132-788-5 o SpecialBookSources/978-1-61132-788-5 978-1-61132-788-5. Hilton-Taylor (1998). HYPERLINK http//www.iucnredlist.org/details/34617 Catha edulis. HYPERLINK https//en.wikipedia.org/wiki/IUCN_Red_List o IUCN Red List IUCN Red List of Threatened Species. Version 2006. HYPERLINK https//en.wikipedia.org/wiki/International_Union_for_Conservation_of_Nature o International Union for Conservation of Nature International Union for Conservation of Nature. Retrieved 12 May 2006. HYPERLINK https//en.wikipedia.org/wiki/Dale_Pendell o Dale Pendell Pendell, Dale (2002). Pharmakodynamis Stimulating Plants, Potions and Herbcraft Excitantia and Empathogenica. San Francisco Mercury House. Randrianame, Maurice Shahandeh, B. Szendrei, Kalman Tongue, Archer International Council on Alcohol and Addictions (1983). HYPERLINK https//books.google.com/booksidmSpOAQAAIAAJ The health and socio-economic aspects of khat use. Lausanne The Council. Shearer, A.L. (1923). The detection of intestinal protozoa and mange parasites by floatation technique. Flor-Parra, Ignacio Bernal, Manuel Zhurinsky, Jacob Daga, Rafael R. (2013-12-17). HYPERLINK https//www.ncbi.nlm.nih.gov/pmc/articles/PMC3892166 Cell migration and division in amoeboid-like fission yeast. Biology Open. 3 (1) 108-115. HYPERLINK https//en.wikipedia.org/wiki/Digital_object_identifier o Digital object identifier doi HYPERLINK https//doi.org/10.1242/bio.20136783 10.1242/bio.20136783. HYPERLINK https//en.wikipedia.org/wiki/International_Standard_Serial_Number o International Standard Serial Number ISSN HYPERLINK https//www.worldcat.org/issn/2046-6390 2046-6390. HYPERLINK https//en.wikipedia.org/wiki/PubMed_Central o PubMed Central PMC HYPERLINK https//www.ncbi.nlm.nih.gov/pmc/articles/PMC3892166 3892166 cdc.govPrevention. Center for Disease Control Prevention. Retrieved 24 October 2017 HYPERLINK https//www.cdc.gov/parasites/amebiasis/general-info.html Entamoeba histolytica. Appendix 1 ASSESSMENT OF HYGIENE CONDITIONS IN VENDORS AREA (QUESTIONAIRE FOR VENDORS) Information requested herein is for study purpose only and will be taken in confidence 1. Name of vendor. 2. Residence/location 3. Where is source of your supplies.. TICK WHERE APPLICABLE 5. Is their chances you can get contaminated miraa which can cause infection amongst youre consumer YES NO 6. Are there sewage leakage in these areas YES NO 7. Are latrines and toilet accessible YES NO 8. Is there caf around YES NO 9. Is there any source of water around within 30metres YES NO THANKS YOU VERY MUCH FOR YOUR COOPERATION PAGE MERGEFORMAT i PAGE MERGEFORMAT 24 -(0/,(,28H25D6,UDJLPQP0XWNHOPM M3,3MMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMM [email protected]/9cxp8U /[email protected]
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