Prevalence of helicobacter pylori and intestinal parasite and their associated risk factors among school children at selam fire elementary school in akaki kality, addis ababa, ethiopia

ADDIS ABABA UNIVERSITY COLLEGE OF HEALTH SCIENCE SCHOOL OF ALLIED HEALTH SCIENCE DEPARTMENT OF MEDICAL LABORATORY SCIENCE Prevalence of Helicobacter pylori and intestinal parasite and their associated risk factors among school children at Selam Fire Elementary School in Akaki Kality, Addis Ababa, Ethiopia By: Abebe Worku (BSc) Advisors: Kassu Desta (MSc, PhD fellow) Mistire Wolde (MSc, PhD) A thesis submitted to Addis Ababa University, College of Health Sciences, Department of Medical Laboratory Science, in partial fulfillment of the requirements for the degree of master in Clinical Laboratory Science (Diagnostic and public health microbiology) June /2017/ Addis Ababa, Ethiopia I ADDIS ABABA UNIVERSITY COLLEGE OF HEALTH SCIENCES SCHOOL OF ALLIED HEALTH SCIENCES DEPARTMENT OF MEDICAL LABORATORY SCIENCES This is to certify that the thesis prepared by Abebe Worku, entitled: Prevalence of Helicobacter pylori and intestinal parasite and their associated risk factors among school children at Selam Fire Elementary School in Akaki Kality, Addis Ababa, Ethiopia and submitted in partial fulfillment of the requirements for the degree of Master of Science in Clinical Laboratory Sciences (Diagnostic and Public Health Microbiology) complies with the regulations of the University and meets the accepted standards with respect to originality and quality BY: ABEBE WORKU (BSc) Approved by the Examining Board Chairman, Dep Graduate Committee Signature _ Advisors Signatures _ _ Internal Examiner Signature Date External Examiner Signature _ Date _ Acknowledgment Primarily my heartfelt thanks go to the Almighty God, And my all Families Then my thanks go to Addis Ababa University, Department of Medical Laboratory Sciences for arranging a program to conduct my MSc Thesis work I would like to express my sincere gratitude and deep appreciation to my advisors, Kassu Desta (MSc, PhD fellow) and Dr Mistre Wolde (MSc, PhD) whose advice and support made this work fruitful Their guidance was very clear since the beginning of the process and their input always valuable in term of giving direction and helps to solve problems in this thesis Finally, my special thanks also go staffs working in Selam Fire Health Center Laboratory; and Akaki kality Sub City education Bureau and to Selam Fire Elementary School director, and all staffs, in which the research has been conducted, also the school children who participated in this study Table of Contents Pages Acknowledgment……………………………………………………………………… II Table of contents ………………………………………………………………………… III List of Tables………………………………………………………………………… …….VI List of Figures……………………………………………………………………………….VII List of Abbreviation……………………………………………………………………… VIII Operational definition……………………………………………………………………… IX Abstract……………………………………………………………………………………….X 1.Introduction………………………………………………………………… .1 1.1 Background…………………… ……………………………………………………… 1.2 Statement of the problem…………………………………………………………………3 1.3 Significance of the study………………………………………………………………….4 2.Literature review………….…………………………………………………………………5 2.1 Prevalence of H Pylori………… .5 2.2 Risk factor for H pylori……………………………………………………………….….6 2.3 Prevalence of intestinal parasite … 2.4 Risk factors associated for intestinal parasite … .9 Objectives……………………………………………………………………… .10 3.1 General objective……………………………………………………………………… 10 3.2 Specific objectives……………………………………………………………………….10 Materials and methods…………………………………………………………………….11 4.1 Study Design…………………………………………………………………………….11 4.2 Study Area……………………………………………………………………………….11 4.3 Study Duration………………………………………………………………………… 11 4.4 Population……………………………………………………………………………… 11 4.4.1 Source population……………………………… ……….………………………… 11 4.4.2 Study population……………………………………………………………………….12 4.5 Inclusion and Exclusion criteria…………………………………………………………12 4.5.1 Inclusion criteria……………………………………………………………………….12 4.5.2 Exclusion criteria………………………………………………………………………12 4.6 Variables of the Study………………………………………………………………… 12 4.6.1 Independent variables………………………………………………………… …… 12 4.6.2 Dependent variable .12 4.7 Sample size determination and sampling……………………………………………… 12 4.7.1 Sample size determination…………………………………………………………… 12 4.7.2 Sampling procedures………………………………………………………………… 13 4.8 Data collection tools and procedures…………………………………………………….15 4.8.1 Demographic characteristics and exposure to risk factors…………………………….15 4.8.2 Specimen collection and transportaion ……………………………………………….15 4.8.3 Laboratory tecquniqies ……………………………………………………………… 15 4.8.3.1 Direct wet mount… 15 4.8.3.2 Formal Ether concentration techniques……….…………………………………….15 4.8.3.3 H Pylori stool antigen test…….…………………………………………………….16 4.9 Data management and Quality control………………………………………………… 16 4.9.1 Pre-analytical phase……………………………………………………………………17 4.9.2 Analytical phase……………………………………………………………………….17 4.9.3 Post-analytical phase………………………………………………………………… 17 4.10 Data Processing and Analysis………………………………………………………….19 4.11 Ethical consideratios……………………………………………………………………19 Result………………………………………………………………………………………20 Discussion…………………………………………………………………………………27 7.Limitaion of the study………………………….………………………………………… 30 8.Conculusion and Recommendation ……………………………………………………… 31 References…………………………………………………………………………………32 10 List of annexes……………………………………………………………………………37 Annex I: English version of participant information sheet ………………………………….37 Annex II: Amharic version of participant information sheet……………………………… 40 Annex III: English version informed consent form………………………………………….42 Annex IV: Amharic version informed consent form…………………………………………43 Annex V: English version informed assent form…………………………………………….44 Annex VI: Amharic version informed assent form………………………………………… 45 Annex VII: English version questionnaires………………………………………………… 46 Annex VIII : Amharic version questionnaires……………………………………………….49 Annex IX: laboratory standard operating producers…………………………………………52 For helicobacter pylori stool antigen test…………………………………………………52 For direct stool examination……… …………………………………………………… 55 For stool sedimentation concentration technique…………………………………………56 Annex X: Data entry work sheet for participants’ laboratory test results………………… 58 Annex XI: Selection participants from various arms /grade…………………………… ….59 Annex XII: Declaration……… …………………………………………………………….60 List of tables Page Table5.1 Socio demographic characteristic of Selam Fire Elementary School children …20 Table5.2 Shows the distribution of the study population by household population characteristics, and student’s behavioral characteristic in Selam Fire Elementary School children…………………………………….……………………………………………… 21 Table 5.3 Association between risk factors and H Pylori infection at Selam Fire Elementary School children … 22 Table5.4 Association between risk factors and Intestinal parasites infection at Selam Fire Elementary School children ……….……………………………………………………… 23 Table 5.5 The Co-infection of IPI with H pylori at Selam Fire Elementary School children…………………………………………… .26 List of figures Page Fig.1 Conceptual framework………………………………………………………………….9 Fig.2 A diagrammatic representation of sampling procedures………………………………14 Fig.3 Work flow of the study……………………………………………………………… 18 Fig.4.The distribution of intestinal parasite among study subjects………………………… 25 List of abbreviations Ag: Antigen ENAO: Ethiopian National Accreditation Office ELISA: Enzyme linked immune sorbent assay FECT: Formal ether concentration technique HP: Helicobacter pylori HpSA: Helicobacter pylori stool antigen ICT: Immune chromatographic test IgG: immunoglobulin G IPI: Intestinal parasitic infection KG: Kindergarten MALT: Mucosa-associated lymphoid tissue MUAC: Mean Upper Arm Circumference NaCl: Sodium Chloride PTA: Parents Teachers Association SOP: Standard operating producers SPSS: Statistical package for social study STH: Soil transmitted helminthes UK: United Kingdom WGO: World Gastroenterology Organization WHO: World health organization Operational definitions Helicobacter pylori stool Antigen test (HPSA): is a lateral flow chromatographic immunoassay for the qualitative detection of H pylori antigen in human faecal specimen Prevalence: is a measurement of all individuals affected by the disease at a particular time Co-infection: the simultaneous presence of two or more infections, which may increase the severity and duration of one or both Children: A person between birth and puberty School children: a child attending school A Primary school: is a school for children between the ages of and 18 Annex VII English version of Questionnaire Addis Ababa University Collage of Health Sciences, School of Allied Health Science Department of Medical Laboratory Science Questionnaires: for the demographic characteristics, assessment of risk factors for H pylori, intestinal parasitic for students, who were learning at Elementary School: Participant Identification School name Year Participant code no: _ Participants address (Sub city) Telephone signature Grade _Block _ Data collector name _date signature Demography of the child Part one: - For Children from KG level to grade eight Age in yr Weight in kg. Height _ MUAC _ Sex? Male Female Residence of the children? Rural Urban Shoe wearing habit? Sometimes Always Not at all Does the child have a habit of washing hands after using toilet? Sometimes Always Not at all 56 Part two: - For Family/Guardian Age in yr Sex? Male Female Residence of family /guardians? Rural Urban Does the child report abdominal pain more than times per week? Yes No Does the students have taken / given a de-worming before the last six weeks? Yes No How much family /guardian monthly income? 4000 How many bedrooms you have in the house? 1-2 3-5 ≥ above How many persons live in the house? < 4-6 >6 57 What is family or guardian level of education? Illiterate Read and write Primary school Secondary school above secondary school 10 What is your family/home drinking water Source? Tap/ bono water Bottled water Boiled tap water Mineral water 11 Do you have a Toilet in your house? Yes No 12 What type of toilet used in your house? Pit latrine Flush toilet Open field 58 Annex VIII: Amharic version of Questionnaire የጨጓራ ህመምች ምክንያት የሆነውን የኤች ፓይልሪ ባክቴሪያ እና የሆድ ውስጥ ትሊትልች በአንዯኛ ዯርጃ ት/ቤት በሌጆች ሊይ ስሊሇው ስርጭትና፡ አጋሊጭ ምክንየትና መንሴዎችን ሇይቶ ሇማወቅ ሇማጥናት የተዘጋጀ ቃሇ መጠየቅ ፡፡ የተሳትፎመሇያ የት/ቤቱ ስም ዓ/ም መሊያ ቁጥር : _ አድራሻ (ክፊሇ ከተማ ) ስሌክ ቁጥር ፊርማ የት/ ዯርጃ _ህንፃ ቁጥር _ የመርጃ ስብሳቢ ስም ቀን ፊርማ ክፍሌ አንድ ፡- ከ KG – ኛ ክፇሌ ሊለ ተማሪዎች መሠረታዊመረጃ እድሜ ክብዯት -3 ቁመት የክንድ መጠን ሌኬት ፆታ ወንድ ሴት መኖሪያቦታ? ገጠር ከተማ ጫማየማድረግሌምድአሇው/ አሊት? አንዳንዴ ሁሌጊዜ አይ የሊትም/ውም መጸዳጃ ቤት ከተጠቀመ/ቸ በዋሊእጅየመታጠብሌምድአሇው/አሊት? አንዳንዴ ሁሌጊዜ አይ የሊትም/ውም 59 ክፍሌ ሁሇት፡- ሇቤተሠብ/ ሇአሳዳጊዎች ; እድሜ ፆታ ወንድ ሴት መኖሪያቦታ? ገጠር ከተማ ሌጆት በሳምንት ከ ሶስት ጊዜ በሊይ የሆድ ህመም ስሜት ይስማዋሌ? አዎ ይስማዋሌ አይ አይስማውም በት/ቤቱ የፀር ሆድ ትሊትሌመድሀኒትተስጥቶየውቃሌ? አዎ ያውቃሌ አይ አያውቅም የቤተሠብ/ የአሳዳጊ ወርሀዊ ገቢ? 4000 ስንት መኝታ ክፍሌ ቤት አሇዎት ? 1-2 3-5 ከ በሊይ ምን ያህሌ ስው በቤት ውስጥ ይኖራሌ? < 4-6 > 60 የአሳዳጊው /የወሊጅ የትምህርት ዯረጃ? ያሌተማረ ማንበብና መጻፍ አንዯኛ ዯርጃ ትምህርት ሁሇተኛ ዯርጃ ትምህርት ከሁሇተኛ ዯርጃ ትምህረት በሊይ 10 ቤተሠቡ ሇመጠጥ የሚጠቀመው ውሃ አይነት? የቧንቧውሃ የታሸገ የፕሊስቲ የፇሊ የቧንቧ ውሃ የጉድጋድ /የከርስ ምድር ውሃ 11 በቤት ውስጥ የመጸዳጃ ቤት አልት? አዎ አሇኝ አይ የሇኝ 12 ቤተሠብ የሚጠቀመው የመጸዳጃ ቤት አይነት? ባሇ ጉድጎድ ሽንት ቤት ውሃ መሌቀቂያ ያሇው ሽንት ቤት ሜዳ ሊይ መጸዳዳት 61 Annex IX: Standard operational Procedure for Laboratory investigation Well-trained laboratory technologist/technician was collected stool in order to ensure that appropriate stool specimen is obtained and quality control for H pylori stool antigen test and intestinal parasite Standard operational procedures (SOP) for Helicobacter pylori Stool Antigen test 1.1 Purpose The H pylori Ag Rapid test is a lateral flow chromatographic immunoassay for the qualitative detection of H pylori antigen in human faecal specimen It is intended to be used by professionals as a screening test and as an aid in the diagnosis of infection with H pylori Any reactive specimen with the H pylori Ag Rapid test must be confirmed with alternative testing method(s) The H pylori Ag Rapid test uses a colloid gold conjugated monoclonal anti-H.Pylori antibody and another monoclonal anti-H pylori antibody to specifically detect H pylori antigen present in the faecal specimen of an infected patient The test is user friendly, accurate, and the result is available within 15 minutes 1.2 Test Principle The H pylori Ag Rapid test is a sandwich lateral flow chromatographic immunoassay The test strip consists of: a burgundy colored conjugate pad containing monoclonal anti- H pylori antibody conjugated with colloid gold (anti-H.P conjugates) and a nitrocellulose membrane strip containing a test band (T band) and a control band (C band) The T band is pre-coated with another monoclonal anti-H.P antibody, and the C band is pre-coated with goat anti-mouse IgG antibody When an adequate volume of extracted faecal specimen is dispensed into the sample well of the test cassette, the specimen migrates by capillary action across the cassette H pylori antigens if present in the specimen was bind to the anti-H Pylori conjugate The immune complex is then captured on the membrane by the pre-coated antibody, forming a burgundy colored T band, indicating an H pylori positive test result Absence of the T band suggests that the concentration of H pylori antigens in the specimen is below the detectable level, indicating an H pylori negative test result 62 Reagents and Materials Provided Individually sealed foil pouches containing:  One cassette test device  One desiccant Sample extraction tubes, each containing 2ml of extraction buffer Plastic droppers for transferring watery stool One package inserts (instruction for use) 1.3 Test Procedure Bring the specimen and test components to room temperature if refrigerated or frozen Mix the specimen well prior to assay once thawed When ready to test, open the pouch at the notch and remove the test strip Place the strip on a clean, flat surface Fill the plastic dropper with the specimen Holding the dropper vertically, dispense drop (about 30-45 µL) of specimen into the sample pad making sure that there are no air bubbles Then add drop (about 35 – 50 µL) of Sample Diluents immediately and wait for 15 minutes Set up timer Results read within 15 minutes 1.4 Test Quality Control The test contains an internal control (C band) which should exhibit a burgundy colored band of the immune complex of goat anti-mouse IgG/mouse IgG-gold conjugate regardless of the color development on the T band If the C band does not develop, the test result is invalid and the specimen must be retested with another device 63 Interpretation of Assay Result of H pylori test Negative Result: If only the C band is developed, the test indicates that no detectable H pylori antigen is present in the specimen The result is negative Positive Result: If both C and T bands are developed, the test indicates the presence of H pylori antigen in the specimen The result is positive Invalid: If no C band is developed, the assay is invalid regardless of any color development on the T band as indicated below Repeat the assay with a new test device Excess faecal specimen can lead to invalid test results; if this is the cause, re-sample and re-test (see instructions for collection of specimen) Safety precaution Consider any materials of human origin as infectious and handle them using standard bio-safety procedures 64 SOP for Direct stool examination 2.1 Purpose of the test For detection and identification of parasites in wet mount preparation of stool 2.2 Principle of the test The value of wet preparations lies in the fact that certain protozoa trophozoites retain their motility which may aid in their identification Definitive identification however may not be possible, especially for amoeba, since the nuclei of trophozoites and cysts are often not clearly visible Wet preparations on fresh unpreserved liquid stool should be performed and examined as soon as possible (within 30 minutes of passage) and on soft/formed stool within 60 minutes of passage provided that prior arrangements have been made with the lab 2.3 Test Procedure Place a drop of fresh physiological saline on one a slide, to avoid contaminating the fingers and stage of the microscope, not use too large a drop of saline Using a wire loop or piece of stick, mix a small amount of specimen, about mg, (matchstick head amount) with the saline Make smooth thin preparations Cover preparation with a cover glass Sample from different areas in and on the specimen or preferably mix the faeces before sampling to distribute evenly any parasites in the specimen Do not use too much specimen otherwise the preparations will be too thick, making it difficult to detect and identify parasites Examine systematically the entire saline preparation for larvae, ciliates, helminthes eggs, cysts, and oocysts Use the 10x objective with the condenser iris closed sufficiently to give good contrast Use the 40x objective to assist in the detection and identification of eggs, cysts, and oocysts Always examine several microscope fields with this objective before reporting ‘No parasites found’ Report the number of larvae and each species of egg found in the entire saline preparation 65 SOP for Stool Sedimentation Concentration technique 3.1 Purpose of the test Sedimentation methods (using centrifugation) lead to the recovery of all protozoa, oocysts, spores, eggs, and larvae present; however, the preparation contains more debris If one technique is selected for routine use, the sedimentation procedure is recommended as being the easiest to perform and least subject to technical error 3.2 Principle By centrifugation, this concentration procedure leads to the recovery of all protozoa, eggs, and larvae present; however, the preparation contains more debris than is found with the flotation procedure Ethyl acetate is used as an extractor of debris and fat from the feces and leaves the parasites at the bottom of the suspension The formol ether sedimentation concentration is recommended as being the easiest to perform, allows recovery of the broadest range of organisms, and is least subject to technical error 3.3 Test Procedures Using a rod or stick, emulsify an estimated 1g (pea size) of faeces in about ml of 10% formol water contained in a screw cap bottle or tube from the surface and several places in the specimen Add a further 3–4 ml of 10% v/v formol water, cap the bottle, and mix well by shaking Sieve the emulsified faeces, collecting the sieved suspension in a beaker Transfer the suspension to a conical (centrifuge) tube made of strong glass, copolymer, or polypropylene Add 3–4 ml of diethyl ether or ethyl acetate Stopper the tube and mix for minute If using a Vortex mixer leave the tube unstoppered and mix for about 15 seconds (it is best to use a boiling tube) * Do not use a rubber bung or a cap with a rubber liner because ether attacks rubber 66 With a tissue or piece of cloth wrapped around the top of the tube, loosen the stopper (considerable pressure will have built up inside the tube) Centrifuge immediately at 750–1 000 g (approx 3000 rpm) for minute After centrifuging, the parasites will have sedimented to the bottom of the tube and the faecal debris will have collected in a layer between the ether and formol water Using a stick or the stem of a plastic bulb pipette, loosen the layer of faecal debris from the side of the tube and invert the tube to discard the ether, faecal debris, and formol water Return the tube to its upright position and allow the fluid from the side of the tube to drain to the bottom Tap the bottom of the tube to re-suspend and mix the sediment Transfer the sediment to a slide, and cover with a cover glass 10 Examine the preparation microscopically using the 10objective with the condenser iris closed sufficiently to give good contrast Use the 40 objective to examine small cysts and eggs To assist in the identification of cysts, run a small drop of iodine under the cover glass 3.4 Microscopic result interpretation No ova of parasite seen, if there is no finding Examine systematically the entire saline preparation for larvae, ciliates, helminthes eggs, cysts, and oocysts 67 Annex X: Data entry work sheet for participants’ laboratory test results 001 002 003 004 005 006 007 008 009 010 011 012 013 014 68 Negative Positive MUAC Height Weight Sex Age Grade Ro: no Helicobacter stool Ag test Intestinal parasites Formol-ether concentration methods Direct microscopy (wet method) Annex XI: Selection participants from various arms /grade would made by balloting/lottery method as described in the above As summarized as follow: Level of School Total number of Chance of students students enrolled to be selected is 35% /students from each grade Male Female Male Female KG -1 30 36 10 12 22 KG -2 24 20 8 16 KG -3 38 36 13 13 26 Total 92 92 31 32 64 pary school Male Female Male Female Grade 1A 23 26 10 19 Grade 1B 15 36 13 21 Grade 2A 20 32 11 19 Grade 2B 21 28 10 18 Grade 3A 24 34 12 20 Grade 3B 24 31 12 20 Grade 4A 24 35 12 21 Grade 4B 33 25 12 21 Grade 5A 35 45 12 16 28 Grade 5B 37 43 13 15 28 Grade 6A 26 37 13 22 Grade 6B 30 34 10 12 22 Grade 7A 30 42 10 16 26 Grade 7B 30 45 10 16 26 Grade 8A 17 25 11 17 Grade 8B 17 25 10 16 Grade 8C 18 23 14 516 658 186 289 Pre-school Sum Total 69 Total number of participant 422 Annex XII: Declaration As thesis advisor, I hereby certify that I have read and evaluated this thesis prepared under my guidance, by Abebe Worku ; entitled: ‘Prevalence of Helicobacter pylori and intestinal parasite and their associated risk factors among school children at Selam Fire Elementary School in Akaki Kality, March to June 2017, Addis Ababa, Ethiopia Abebe Worku (BSc) _ Principal investigator Signature Date I recommend it to be submitted as fulfilling the thesis requirement Advisor Signature Kassu Desta (MSc, PhD fellow Mistire Wolde (MSc PhD) _ 70 Date ... entitled: Prevalence of Helicobacter pylori and intestinal parasite and their associated risk factors among school children at Selam Fire Elementary School in Akaki Kality, Addis Ababa, Ethiopia and. .. between risk factors and H Pylori infection at Selam Fire Elementary School children … 22 Table5.4 Association between risk factors and Intestinal parasites infection at Selam Fire Elementary. .. determine the prevalence intestinal parasite among school children  To assess the risk factors associated with H pylori and intestinal parasite among school children 20 MATERIALS AND METHODS 4.1