Mekuanint G. Comments

University of Gondar Formatted: Font: (Default) Times New Roman
College of Medicine and Health sciences

School of Biomedical and Laboratory Sciences
Department of Medical Microbiology
Formatted: Font: (Default) Times New Roman
Prevalence, Antimicrobial Susceptibility and Associated Risk Factors of

Shigella And Salmonella Infection among Diarrheic Pediatric Populations
Attending at Gondar Town Health Institution, Northwest Ethiopia.
By: - Amare Alemu(BSc, MSc candidate)

Advisers: - Setegn E ()
Tigist E ()
October, 2017
Gondar, Ethiopia
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ACKNOWLEDGEMENT
I would like to thank Setegn Eshetie and Tigist E. to let me come up with this valuable research
topic and offering and for his tremendous technical support. I would like to extend my gratitude to
SBLS and department of medical microbiology for their technical support and enrolling me as
MSc student.
Table of Contents Formatted: Font: (Default) Times New Roman
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ACKNOWLEDGEMENT-------------------------------------2 Formatted: Font: (Default) Times New Roman
LIST OF TABLES AND FIGURES------------------------4

ABREVIATIONS----------------------------------------------5
SUMMARY ----------------------------------------------------6-7
1. INTRODUCTION-----------------------------------------8-9
1.1 BACKGROUND----------------------------------------8-9
1.2. STATEMENT OF THE PROBLEM-------------10-11
1.3 LITERATURE REVIEW----------------------------11-12
1.3.1 EPIDEMIOLOGY--------------------------------11-12
1.3.2 TRANSMISSION--------------------------------------12
1.3.3 RISK FACTOR ASSOCIATION-------------------13
1.3.4 PATHOGENESIS-------------------------------------13
1.3.5 DIAGNOSIS--------------------------------------------14
1.3.6 TREATMENT, PREVENTION AND CONTROL-14-15
1.3.7. ANTIMICROBIAL SUSCEPTIBILITY PATTERN--------15
2. SIGNIFICANCE OF THE STUDY-----------------------------16
3. OBJECTIVES-------------------------------------------------------16
3.1 GENERAL OBJECTIVE----------------------------------------16
3.2 SPECIFIC OBJECTIVES--------------------------------------16
4. MATERIALS AND METHODS -------------------------------17
4.1 STUDY AREA----------------------------------------------------17
4.2 STUDY DESIGN AND STUDY PERIOD------------------17
4.3 STUDY POPULATIONS--------------------------------------17
4.3.1. Source population-------------------------------------------17
4.3.2. Study population---------------------------------------------17
4.4. INCLUSION CRITERIA---------------------------------------17
4.5 EXCLUSION CRITERIA--------------------------------------17
4.6 SAMPLE SIZE DETERMINATIO N AND SAMPLING TECHNIQUES-----17
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4.7 STUDY VARIABLES---------------------------------------------18
4.7.1 DEPENDENT VARIABLES----------------------------------18
4.7.2 INDEPENDENT VARIABLES-------------------------------18
4.8. OPERATIONAL DEFINITIONS------------------------------------18
4.9. SAMPLE COLLECTION AND MICROBIAL IDENTIFICATION PROCEDURES--18
4.9.1. Structured questioners---------------------------------------18
4.9.2. Laboratory analysis-------------------------------------------19
4.9.3 DATA MANAGEMENT AND QUALITY ASSURANCE--20
4.9.4.DATA ANALYSIS-------------------------------------------------20
5. Ethical Considerations-----------------------------------------------21
6.DISSEMINATION OF RESULTS----------------------------------21
7. WORK PLAN------------------------------------------------------21-22
8. BUDGET PROPOSAL-----------------------------------------------22
BUDGET FOR LABORATORY REAGENT AND SUPPLIES-------22-23
LABORATORY EQUIPMENT AND REAGENT----------------24
BUDGET SUMMARY-------------------------------------------------24
REFERENCE------------------------------------------------------25-30
ANNEXES---------------------------------------------------------------30
Annex 1: ENGLISH VERSION OF THE INFORMATION SHEET------31
Patient information Sheet form (አማርኛ) ------------------------------------32

ANNEX 2: CONSENT FORM ---------------------------------------------33
INFORMED CONSENT (አማርኛ) ---------------------------------------------34

ANNEX 3: QUESTIONNAIRES ---------------------------------------------36
ENGLISH VERSION QUESTIONNAIRE ------------------------------------36
ANNEEX 4: DUMMY TABLES ---------------------------------------------36 Formatted: Font: (Default) Times New Roman
LIST OF TABLES AND FIGURES

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Table 1: A time schedule for the study of prevalence, antimicrobial susceptibility and associated
risk factors of salmonellosis and shigellosis in diarrheic pediatric patients attending at Gondar
town health institutions, northwest Ethiopia, from December 2017 to July 2017
Table 2: Manpower costs
Table 3: Budget for laboratory materials, reagents and supplies
Table 4: Laboratory equipment and reagents
Table 5: Budget summary
Table 6: ENGLISH VERSION QUESTIONNAIRE
Abbreviation
AIDS---------------------acquire immunodeficiency syndrome
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CLSI--------------------Clinical Laboratory Standards Institute
CDC--------------------centers for disease control
C. jejuni----------------campylobacter jejuni
E. coli-------------------Escherichia coli
ETEC--------------------Enterotoxigenic Escherichia coli
HCL----------------------hydrochloric acid
HIV------------------------human immune virus
MLST------------------- multiple loci sequencing typing
NTS----------------------non-typhoid salmonellosis
PI------------------------Principal investigator
PMNS-------------------polymorphonuclear cells
PFGE------------------ pulse-field gel electrophoresis
RERC------------------Research Ethics Review Committee
SD1---------------------shigellae dysentery type 1
SBMS------------------school of biomedical and laboratory sciences
SPP.--------------------species
WHO--------------------world health organization
SPSS--------------------statistical package for Social Sciences
USA---------------------united states of America
UOG---------------------university of Gondar
Commented [M1]: Since this is the summery, Please

merge the general characteristics of shigella and salmonella.
Summery must be on one page. So come with very general
things
SUMMARY Formatted: Font: Times New Roman
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Introduction: The Shigellae are members of the enterobacteriaceae, non-lactose fermenters,
non-motile, and non-gas producers’ gram-negative rods. The genus Shigella includes four
species: S. dysenteriae, S. flexneri, S. boydii and S. sonnei, also designated groups A, B, C and
D, respectively [31]. Genus Salmonella are heterogeneous group, gram negative rods, non-
lactose fermenter, facultative anaerobic, non-spore forming, motile, produces acid and gas from
glucose, normally inhabit the intestines of animals and humans [10,2].
Salmonella species causes typhoidal and non-typhoidal fevers. S.typhoid and S.paratyphoid
cause typhoid and other salmonella isolates causes non-typhoidal enteric fever [2,10]. Shigellosis
is endemic in many developing countries and it occurs as epidemics causing considerable
morbidity and mortality [53]. Shigella dysenteriae type 1 (Sd1) is especially important because it
causes the most severe disease and may occur in large regional epidemics [31,36].
Salmonella and shigella infections can be transmitted by direct and indirect feco-orally.
Contaminated materials and fecal matters from animals can transmit the diseases [49,32,2].
The relative antimicrobial susceptibility of different Shigella species may vary geographically. It
may be due to pattern of antibiotic using for treatment of shigellosis [44].
Treatment and control measures must consider on the control of the spread of the pathogens
within the community and from person to person [31]. Salmonellosis and shigellosis can be
treated with antibiotics even though there is the increase in antibiotic resistance from time to
time [34,38,54].
This study which is conducted on “prevalence, antimicrobial susceptibility and associated risk
factors of salmonella and shigella infections among diarrheic pediatric populations in Gondar
town health institutions, northwest Ethiopia. It may have a lot of importance out of which it will
indicate the burden of the bacteria in the region and also it will serve as a base line for other
researchers who need to work on salmonella and shigella.
Objectives: To determine the prevalence, antimicrobial susceptibility and associated risk

factors of shigella and salmonella species among diarrheic pediatric children attending at Gondar
town health institutions.
Methods: Across sectional study will be conducted in Gondar town health institutions. Once Commented [M2]: In methods section in addition to
study design, area and period you should put what sampling
the sample is collected, protozoa parasites will be identified through direct microscopy using technique to be applied to select HC and study participants,
saline wet mount at each study sites and part of the stool will be kept in transport media, and sample size determination and sample size, general
transported using ice box to microbiology department of Gondar university for further laboratory principles. No need of detail description in this
section
microbiological investigations. One-gram of stool sample will be collected from each diarrheic
pediatric patient using sterile screw capped tubes containing transport media (Cary Blaire or Formatted: Font: (Default) Times New Roman
peptone water media) and transported to microbiology department of Gondar university for Formatted: Font: (Default) Times New Roman
further microbiological investigations. Inoculation and incubation of the specimens on the
standard culture media performed. The primary isolates will be sub cultured on the available
biochemical tests for further identification. Antimicrobial susceptibility tests will be carried out
by using disc diffusion method using Mueller-Hinton agar. A standardized suspension of the
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bacterial isolate will be prepared and turbidity of the inoculum will be compared with 0.5
McFarland turbidity standard.
Work plan: -. A time schedule for the study of prevalence, antimicrobial susceptibility and
associated risk factors of salmonellosis and shigellosis in diarrheic pediatric patients attending at
Gondar town health institutions, northwest Ethiopia, from December 2017 to July 2017.
Budget: - To conduct this study a total of 107091 birr will be required.
Expected outcome: This study will indicate Prevalence, antimicrobial susceptibility and
associated risk factors of shigella and salmonella infection among diarrheic pediatric populations
attending at Gondar town health institution, Northwest Ethiopia.
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1. INTRODUCTION Commented [M3]: Throughout your document use
Justified
1.1. BACKGROUND New times roman style
Heading bold with 14’
Sub heading bold with 12’
Diarrhea causing pathogens are the second leading cause of morbidity and mortality worldwide; Body 12 and 1.5 space
mainly children under the age of 5 years are at high risk. The organisms responsible are some
viruses (E.g. rotaviruses, Norwalk-like viruses), bacteria such as Formatted: Font: (Default) Times New Roman
enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni and Clostridium difficile, Shigella Formatted: Font: (Default) Times New Roman
spp., Salmonella spp., Cryptosporidium spp. and Giardia lamblia [1].
The Shigellae are members of the enterobacteriaceae, non-lactose fermenters, non-motile, and
non-gas producers’ gram-negative rods. The genus Shigella includes four species: S. dysenteriae,
S. flexneri, S. boydii and S. sonnei, also designated groups A, B, C and D, respectively [31]. Genus
Salmonella are heterogeneous group, gram negative rods, non- lactose fermenter, facultative
anaerobic, non-spore forming, motile, produces acid and gas from glucose, normally inhabit the
intestines of animals and humans [10,2].
A remarkable characteristic in Salmonella pathogenesis is the invasion of non-phagocytic cells.
Salmonella will penetrate into the intestinal epithelial cells by inducing their own uptake, in a
complex and active process that morphologically resembles phagocytosis [2]. They invade the
mucosa of the small and large intestines and produce inflammation. Invasion of intestinal epithelial
cells induces an inflammatory reaction which causes diarrhea due to salmonella infections [10,15].
The inoculum of bacteria that must be swallowed in order to cause infection is uncertain and varies
with the serotype [10]. People who have compromised immune systems, older adults, pregnant
women, infants and children are at high risk for a serious complication due to Salmonella food
poisoning [15]. Salmonella causes self-limited gastro-enteritis and the more severe forms of
systemic typhoid fever.
Shigellosis is only a human disease caused by the four species of genus Shigella and is
characterized by the increase in frequency of stool motion and the presence of blood, mucous and
pus in the stool [10]. Shigella species are limited to the intestinal tract of humans and cause
bacillary dysentery leading to watery or bloody diarrhea [11]. Symptoms of shigellosis may
include acute abdominal pain, fever and bloody stools. S. flexneri is predominant causative agent
of shigellosis in developing countries and S. sonnei in industrialized countries [14]. Shigellosis is
basically a disease of poor, crowded communities that do not have adequate sanitation or clean
water [8 ,9]. Shigellae are transmitted from person to person usually by asymptomatic carriers and
via contaminated food, flies, faeces, fingers, and water. To initiate infection, as few as 100 ingested
Shigella microorganisms are enough to cause an acute diarrhea after 4-7 days. After the organisms
enter the human body, they remain in the cytoplasm of the epithelial cells and spread laterally to
invade adjacent cells which result in the formation of abscesses and ulcerations with high
concentration of neutrophils in the stools. Because of delay in humoral responses, complication
and mortality rate due to shigellosis in children is higher than in other age groups [31,33].
salmonellosis is the most common food borne disease in both developing and developed countries
although incidence rates vary according to the country in both developing and developed countries
[4]. The highest incidence of infection is among the very young and elderly. Mortality is highest
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in children less than one year old. The highest susceptibility of this age group may be due to the
fact that children less than 2 months old produce little hydrochloric acid (gastric HCL), a natural
barrier to many microorganisms [10,31,15]
A severe infection of diarrhea in children is highly associated with risk factors such as poor
environmental sanitation and hygiene, poverty and malnutrition. Some risk factors vary with age
and the weaning practices of the children; bottle-feeding is highly associated with diarrhea to
children with age between 1 to 6 months. Feeding of a child with bottle may be contaminate and
cause diarrheal diseases. Unable to adapt to bottle feeding affects the nutritional status of the child.
Malnutrition lowers the immunity of the child and exposes them to diarrheal diseases [15].
Drug resistance is the decreased sensitivity or the complete insensitivity of microbes to
antimicrobial drugs [11]. Antimicrobial resistance of Salmonella and Shigella are emerging global
challenges. Many studies showed that, in most endemic countries, especially in Asia and sub-
Saharan Africa, there was an emergence of multidrug resistance to frequently prescribed
antimicrobials [43]. In Ethiopia, Salmonella and Shigella have been reported to be resistant to first
line antibiotics such as ampicillin, tetracycline and amoxicillin [3,11,]. The resistance of
salmonella and shigella complicated the selection of antimicrobials and Many factors have
contributed to the development of resistance in gastrointestinal pathogens, including misuse,
overuse, quality and potency of the antimicrobial agents [1].
In Ethiopia particularly in Gondar, there is no currently study conducted about the prevalence,
antimicrobial susceptibility and associated risk factors that contributes to shigellosis and
salmonellosis in diarrheic pediatric patients. As the antimicrobial susceptibility varies from time
to time, there is a need for updating the empirical antimicrobial susceptibility data periodically to
adopt some new clinical treatments. Therefore, this study will show the burden of the bacteria
among diarrheic pediatric patients in Gondar town, Northwest Ethiopia. This study also gives up-
to-dated information on the cases for policy makers and health managers.
1.2. Statement of the problem

Shigellosis and salmonellosis are still accounts for a significant proportion of morbidity and
mortality cases, especially in children with diarrhea in developing countries. Shigellosis and
salmonellosis becomes a major global public health problem. Shigellosis which is recognized by
the WHO as the main cause of death among pediatric patients in developing countries [31].
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Salmonellosis is major bacterial enteric illness in human and animal which is a public health
burden and results an economic loss in the society. It can be typhoidal or non-typhoidal. Typhoid
fever is a global health problem. According to the WHO 1996 annual report, the global burden
of typhoid fever is 16 million illness and 600,000 deaths [32]. Globally, non-typhoidal (NTS)
illness is estimated to be responsible for 93.8 million cases of gastroenteritis and 155,000 deaths
annually. Even though NTS occurs worldwide, mortality due to NTS infection primarily occurs
in the developing world [55].
The annual number of Shigella burden throughout the world was estimated to be 164.7
million, of which 163.2 million were in developing countries (with 1.1 million deaths) and 1.5
million in industrialized countries. A total of 69% of all episodes and 61% of all deaths
attributable to shigellosis involve children under 5 years of age [4]. Two hundred million to more
than one billion cases of diarrhea result worldwide due to Salmonella infections every year,
leading to 3 million deaths [48. In Africa, an estimate of 115 people dies of diarrheal diseases
every hour, mostly of shigellosis and salmonellosis which may be due to contaminated food and
water due to poor sanitation and hygiene practices [25,31].
Antibiotic resistance is a drug where a microorganism has developed the ability to survive
exposure to an antibiotic [5]. Over the past decades shigellae and salmonellae shows a persistent
increase in antimicrobial resistance to routinely prescribed antimicrobials [12,16,17]. Over the
last 50 years, Shigella shows extraordinary progression in acquiring plasmid-encoded resistance
to first line antimicrobial drugs [4]. Feeding of stock animals with food containing antibiotics
plays a significant role in the development of multidrug-resistant Salmonella. Studies in USA on
cattle and Denmark on pigs have shown that concerning spread of multidrug-resistant Salmonella
in association with the use of antibiotics in the animals’ food [2].
There are studies in Ethiopia that shows the prevalence of shigellae in all age groups in
Jimma, Gondar, Hawasa, Harar, Bahirdar and Mekelle with high prevalence and antibiotic
resistance [26,29,17,16,19] respectively. The positivity rate of Shigella, carried out in Gondar,
among children under-5 years of age was 31.1 % [17].
In order to design preventive strategies, the explanation of the mode of spread of the pathogen
is essential; designed to assess its prevalence in diarrheic pediatric patients in the area remains
poorly understood.
Therefore, this study is proposed to feel this gap and contribute to produce evidence on the
prevalence, antimicrobial susceptibility patterns and associated risk factors of salmonella and
shigella infections in diarrheic pediatric populations in Gondar town health institutions,
northwest Ethiopia. Commented [M4]: In this section you should talk about
Prevalence of Shigella and salmonella
1.3. LITERATURE REVIEW Associated risk factors of shigella and salmonella
Antimicrobial susceptibility of shigella spp and salmonella
1.3.1. Epidemiology and Historic background. spp of previous study conducted in different areas
Transmission, Dx, Rx and others must be stated in
introduction section in 1 paragraph each
Evolutionary evidence shows that Salmonella species (S.Typhi) exists with mankind since
ancient times. It was spread across the planet when human beings were hunters. Typhi was not Formatted: Font: (Default) Times New Roman
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recognized as etiologic agent of enteric fever until 1880 BC. Its epidemiology varies according
to the species [2]. Salmonella was discovered by Arl Joseph Eberth, in the abdominal lymph
nodes and the spleen in 1879. The genus “Salmonella” was named after Daniel Elmer Salmon
(veterinary pathologist) [2,41].
Shigellosis is one of the most common diarrheal diseases in humans worldwide. The
epidemiology of salmonella and Shigella species depends on the country: S. flexneri is
predominant in developing countries, whilst S. sonnei is most reported in developed countries
[23]. S. sonnei has become dominant in some Asian countries. Due to international travel and
trade of animals and food products, there is a shift in the prevalence of specific shigellae strain
types and serovars in different places [6,23,2].
Salmonella which has 2500 different serotypes, is a leading cause of foodborne infections
worldwide [20]. Based on a research conducted for 3-year period, in a web-based surveillance,
Salmonella enterica serovar Enteritidis was by far the most common serotype reported from
human isolates globally. In 2002, it accounted for 65% of all isolates, followed by S.
Typhimurium at 12% and S. Newport at 4% [63]. S. Enteritidis represented 85% of isolates in
Europe but only 9% in Oceania. In Latin America and the Caribbean, S. Typhi accounted for the
greatest proportion of salmonellae (13%) [2,33,63]. In Asia, from 2000 through 2002, Japan,
Korea, and Thailand together reported S. Enteritidis as the most common human serotype [2].
A study that is conducted on the sero-grouping of salmonella and shigella in Ethiopia shows that
in shigella: Serogroup B (S. flexneri) was the most commonly isolated species (54.0%), followed
by group A (S. dysenteriae) (22.4%), group D (S. sonnei) (15.8%) and group C (S. boydii)
(7.8%) [1]. Among salmonella, the most commonly isolated serogroup was group B (81.1%),
followed by group D (S.typhi) (10.8%) and group C (8.1%) [1].
Many studies show that in 2000, outbreaks of bloody diarrhea due to Sd1 that were resistant
to fluoroquinolones occurred in India and Bangladesh. In Central America, the most recent large
epidemic lasted from 1969 to 1973 and was responsible for more than 500,000 cases and 20,000
deaths [31,64]. In recent years, according to the research conducted in china (2004-20011)
approximately 125 million cases of Shigella infections occur annually in Asia, of which 14,000
are fatal. In China, shigellosis is one of the top four notifiable infectious diseases, with 1.7 million
episodes of bacillary dysentery, and 200,000 patients admitted to hospitals each year [23]. 1.4
million cases of food-borne salmonella disease have been reported in USA alone [33]. There is a
slight increase (4.2%) compared with 1996 and a large increase compared with 2005 (12.3%); this
could be attributed to increased reports from several states, including Texas and California [33,64].
In the same time in USA, the national incidence of laboratory confirmed shigella was 3.5 per
100,000 population. This was isolated frequently from children < 5 years of age, who accounted
for 31.1% of all isolates [33].
In developing countries, a number of studies report the high prevalence of salmonellosis and
shigellosis. These diseases are important cause of morbidity and mortality especially in children
[39]. Widespread out breaks of shigellosis due to multiple antibiotic resistant Shigellae has been
documented in Central America, Asia, and Africa [31]. In developing countries salmonellosis and
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shigellosis account for a significant proportion of morbidity and mortality in diarrheic pediatric
patients. According to the research conducted in 1994, an explosive outbreak among Rwandan
refugees in Zaïre caused approximately 20,000 deaths during the first month alone. Between 1999
and 2003, outbreaks were reported in Sierra Leone, Liberia, Guinea, Senegal, Angola, the Central
African Republic and the Democratic Republic of Congo [31].
In Ethiopia, a limited number of studies on the prevalence of shigella, salmonella and associated
drug resistance have been carried out mainly in Addis Ababa [40,47,55], Gondar [3,26,30,50],
Mekelle [19], Hawassa [29], Harar [11,17], Bahirdar [16], Arba Minchi [51], Jimma [9,12],
southwest [39] and west shoa gedo zone [5].
1.3.2. Transmission
Typhoid (enteric fever) and Non-typhoidal Salmonella (NTS, e.g. food poisoning) is an
important public health problem worldwide. Shigellosis (bacillary dysentery), the result of
infection with Shigella, is one of the most common diarrhea-related causes of morbidity and
mortality in children under 5 years in developing countries [36].
Primarily salmonella and shigella transmitted through ingestion of contaminated food and
water [11]. Salmonella and shigella are known food-borne diseases [40]. Direct or indirect
contact with infected animals and/or persons or from contact with pets such as cats, dogs,
rodents, reptiles, or amphibians can transmit the disease. Several recent outbreaks have also been
associated with consumption of contaminated plant products such as sprouts, tomatoes, fruits,
peanuts, and spinach [55].
1.3.3. ASSOCIATED RISK FACTORS

The risk to salmonellosis is increased due to the following factors; absence of effective vaccines,
modifying hand washing behavior after defecating to control prolonged community out breaks
and identifying high risk groups and targeting prevention measures [52]. The widespread
occurrences of salmonella and shigella are attributed to several factors including malnutrition
and under nutrition, HIV-AIDS, the close relationship between man and animals, the widespread
field slaughtering practices, the raw meat consumption habits in some societies, the unhygienic
food handling practices and the water sources in the population are suggestive evidences of their
higher occurrence than is estimated in several studies [28].
The change in feeding conditions of a child may expose the child to food- borne and water-borne
infections. A research that is carried out in Nigeria shows that mothers who are advised to feed
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their babies with breast milk only until the age of 6 months could not have salmonellosis in the
age group of 0-4 months [52].
The risk of death due to shigellosis may be severe in infants and adults older than 50 years,
children not breastfed and malnutritional. Refugees and internally displaced persons who live in
common overcrowded, impoverished areas with poor sanitation, inadequate hygiene practices,
and unsafe water supplies are at higher risk factors in getting of shigellosis [31]. Other risk
factors predisposing to NTS infection include immunosuppression, decreased gastric acidity,
recent use of antibiotics, changes in the intestinal flora, hemoglobinopathies, and extremes of age
[2]. Humans and a few primates are the only reservoir of Shigella [31,63].
1.3.4. PATHOGENESIS
As of other enteric bacteria salmonella and shigella species require a mechanism to survive
through the digestive tract and colonize a host and cause disease. Salmonella spp. can infect both
warm and cold-blooded hosts. This wide range reflects the ability of this pathogen to sense and
adapt to a range of different environments, including the interior of macrophages [10]. This
ability of the organism to avoid fusion of Salmonella containing vacuoles with dendritic cell
lysosomes in the intestine is the mechanism by which it can escape of killing. By surviving
within macrophages, Salmonella species will be carried to the spleen, lymph nodes and
throughout the reticuloendothelial system [2,10]. The availability of gene sequence allows
researchers to understand the pathogenicity of salmonella [2]. Infection is initiated when a
sufficient number of microorganisms are ingested that can survive the acidity of the digestive
tract and the effect of digestive bile salts before intestinal colonization. In shigella the infective
dose is small and causes bacillary dysentery [3,10]. It infects the M cells in the Peyer’s patches
of the large intestine [10,31]. Salmonella causes mainly Typhoidal salmonellosis (S.typhi and
S.paratyphi) and non-typhoidal salmonellosis (all salmonella serovars) [20]. In some cases,
salmonella causes bacteremia. Shigella causes bloody dysentery or watery diarrhea. In
developing countries salmonella and shigella causes childhood gastroenteritis [61,30,28].
Shigellosis typically present with diarrhea characterized by the frequent flow of bloody stool
with or without mucus, abdominal cramp and tenesmus are common [31,1,2]. Salmonellosis
shows symptoms of like diarrhea, vomiting fever, and abdominal pain these occur 12-36 hours
after eating infected food, chills, fever, and prostration [12, 51].
1.3.5. DIAGNOSIS
Salmonellosis and shigellosis cannot be distinguished reliably from other causes of bloody
diarrhea on the basis of clinical features alone. Routine microscopy must be performed and the
presence of PMNs suggests a bacterial etiology but does not necessarily indicate salmonellosis or
shigellosis; it may be C. jejuni or diarrheogenic E. coli. To identify accurately culture and
biochemical tests must be performed. Blood culture and bone marrow aspirate may be used if the
source and trained personnel are available [2]. Molecular techniques are also more necessary to
identify them correctly. The most common methods currently in use are the pulse-field gel
electrophoresis (PFGE) and multiple loci sequencing typing (MLST) [2,10, 31]. The Widal test
developed in 1896, utilizes a suspension of killed S. typhi as antigen to detect serum antibodies
14
against the flagellar and somatic antigens. In developing countries, the Widal test could be the only
laboratory tool available for diagnosis [2].
1.3.6. TREATMENT, PREVENTION AND CONTROL

Prevention of salmonellosis and shigellosis can be primarily on measures to the spread the
organism within the community.
1. Health education: - Teaching the child bearing mothers and school children about these
diseases and spreading the information in the local communities via health and religious
institutions, mass media, schools, and markets by using posters, drama etc.
2. Hand washing: - Hand-washing using soap is important after defecation, after cleaning a child
who has defecated, after disposing of a child’s stool, before preparing or handling food, and
before eating.
3. pure water supply: - The use of surface water for drinking, like water from a river, pond, or
open well, should be discouraged. To be used for drinking, it must be disinfected with chlorine
or it must be boiled.
4. breastfeeding: - breast feeding until 6 months must be promoted and continue breast feeding
with other nutrients for about 3 years are advisable.
5. Other prevention methods: -Other prevention methods should be promoted in the general
communities. In this regard health education must stress on the preparation and consumption of
safe food supply and on the disposal of environmental wastes. Vaccine trials should be carried
out to prevent it. But still there is no WHO recommended vaccine that is effective in preventing
shigella infections. Currently there is a trial against S.flexneri but still it is under development
[31]. In other countries, heat-killed, phenol preserved whole cell salmonella vaccines containing
a mixture of culture of S.typhi and S.paratyphi have been used. But these were not effective.
Capsular (vi) polysaccharide replaces the existing vaccine. Now oral live-attenuated salmonella
vaccine is used [2,10].
Another essential measure to prevent the increased number of antibiotic resistant NTS strains is
restricting the use of antimicrobials with animal food and vaccinations [2].
Salmonellosis and shigellosis can be treated with antimicrobials that is known to be effective.
Supportive measures such as rehydration, feeding and zinc supplementation should be provided
to treat shigellosis.
Shigellosis, which continues to have an important global impact, cannot be adequately
controlled with the existing prevention and treatment measures. Innovative strategies, including
development of vaccines against the most common serotypes, may provide substantial benefits
[4,63].
1.3.7. Antimicrobial susceptibility patterns
15
Effective antibiotic treatment reduces the progression of the diseases, the duration of fecal
shedding of the pathogen, the risk of lethal complications and interrupts further transmission of
the diseases [8]. Mobile genetic elements such as resistance plasmid, transposons, and genomic
islands on the bacterial genome are responsible for multidrug resistant isolates [18,64]. Various
salmonella and shigella species become resistant for frequently prescribed antibiotics [7]. A high
resistance rates for ampicillin, amoxicillin and tetracycline has been reported from many studies
worldwide. In developing countries, the real resistance of salmonella among diarrheic children is
not well known due to variation in population characteristics, under reporting and poor laboratory
techniques [9]. Several studies worldwide have reported increase in resistance of antimicrobials.
According to a research conducted in Iran, 47.2% of Shigella, isolates was resistant to two or more
antibiotic [35]. A research carried out in Harar, Eastern Ethiopia, salmonella and shigella isolates
were 100% resistant to both amoxicillin and ampicillin [11]. A study that is conducted in Gondar,
Northwest Ethiopia, shows that from stool samples,90.8% of the shigella isolates were resistant to
one or more antibiotic agent(s) and 87.8% were multi-drug resistant [30]. This shows the increase
in the resistance of frequently prescribed antimicrobial.
2. Significance Justification of the study

Salmonellosis and Shigellosis is endemic in most developing countries and is the most important
cause of bloody diarrhea worldwide. Shigellosis is estimated to cause at least 80 million cases of
bloody diarrhea and 700,000 deaths each year. Ninety-nine percent of infections caused by
Shigella occur in developing countries, and the majority of cases (~70%), and of deaths (~60%),
occur among children less than five years of age [31]. Salmonella species are the most common
cause of bacterial food-borne infections. A study conducted in Gedo hospital, West Shoa zone,
Oromia state, Ethiopia reveals the prevalence of 33.3% from mucoid stool sample [26]. In Gondar,
northwest Ethiopia, it has 1.08% prevalence among diarrheic patients [3]. But most of the studies
carried out in Ethiopia focuses on patients who complains gastroenteritis without concerning the
age groups. Most of the studies that was done here in Gondar town targets all patients who
complains gastroenteritis. Still there is no a research that is done on the pediatric patients
specifically and associated risk factors that predisposes children to salmonellosis and shigellosis.
Therefore, this study is proposed to feel this gap and contribute to produce evidence on the
prevalence, antimicrobial susceptibility patterns and associated risk factors for salmonellosis and
shigellosis to help the health service to have knowledge based medical decisions in the prevention
and treatment of such diseases.
16
3. Objectives Commented [M5]: Main section of your document should
be in separate page
3.1. General objectives Formatted: Font: (Default) Times New Roman
 To determine the prevalence, antimicrobial susceptibility and associated risk factors Formatted: Font: (Default) Times New Roman
of shigella and salmonella species among diarrheic pediatric children attending at
selected Gondar town health institutions, Northwest Ethiopia.
3.2. specific objectives
 To determine the prevalence of shigella and salmonella species among diarrheic
pediatric populations
 To determine the antimicrobial susceptibility patterns of shigella and salmonella
species among diarrheic pediatric populations.
 To assess the associated factors for shigellosis and salmonellosis in diarrheic
pediatric populations attending at Gondar town health institutions.
4. METHODS AND MATERIALS

4.1. Study area: - Study waswill be conducted at Gondar town health institutions., which is
located in Gondar city administration, Amhara Region, Ethiopia. Gondar is found 747 KM far
from the capital city of Ethiopia in the Northwest part of the countryEthiopia, 747KM northwest
of the capital city, Addis Ababa. It The city is one of the fastest growing urban areas in Ethiopia
and covers an area of 29280 square kilometers Based on the 2007 census conducted by the
Central Statistical Agency of Ethiopia (CSA). The town has an estimate of >300,000 total
populations [13] with one referral hospital (University of Gondar Referral Hospital) and 4 health Commented [M6]: Does Gondar contains only 4 HCs
centers and two private specialized pediatric clinics that include, Gondar, Gebrieal, Ginbot 20, Or are they the only HC which give Child health service
Maraki, Health Centers, Enat and Dr mihrete specialized pediatric clinics, which are currently Formatted: Font: (Default) Times New Roman
giving health service to the community. Formatted: Font: (Default) Times New Roman
4.2. Study design and study period

A cross sectional study will be conducted in Gondar town health institutions to evaluate explore
the prevalence, antimicrobial susceptibility and associated factors in diarrheic pediatric
populations from December 2017 to June 2017 in Gondar town health institutions.
4.3. STUDY POPULATIONS
17
Source populations: - All children attending pediatrics clinic in Gondar town health institutions.
the populations who lives in and around selected Gondar town were the source of population. Formatted: Font: (Default) Times New Roman, Font
color: Red
Study population: - All children attending pediatrics clinic in selected health institutions in the
study period. all diarrheic pediatric populations attending in selected Gondar town health
institutions during the study period are study populations.
Inclusion and exclusion criterion: -

4.5. Inclusion: - All diarrheic pediatric patients from 0-14 years old attending at selected Gondar
town health institutions.
4.6. Exclusion: - All patients out the target age group and diarrheic pediatric patients who had
been taking antibiotic treatment in the last 14 days.
4.6. Sample size determination and sampling techniques Commented [M7]: Sample size should calculated
separately for prevalence of salmonella and prevalence of
The sample size will be determined with single population proportion formula. There are studies shigella, and Associated risk factors then take the largest
sample size from the calculated
which were conducted in different regions related with this study. Taking prevalence from the Formatted: Font: (Default) Times New Roman
previous study which was conducted in Addis Ababa, Ethiopia, in under 5-children shigella were Formatted: Font: (Default) Times New Roman
found to be 9.1% and salmonella were found to be 3.9% [47]. By taking the combination of the
two prevalence’s, we found 13%. Using 95 % of confidence interval with 4% of margin of error
sample size will be calculated as follows.
18
N= (1.96)2*0.13*(1-0.13)
(0.04)2 Commented [M8]: Why you use 4% margin of error
n=272. When 10 % contingency non-response rate added, it will be 300. So, the final sample
size will be 300.
Systematic random sampling technique will be used to select the study participants. In the study
area all the patients who was attending before one month of the study was 321. The estimated Commented [M9]: Is this from one institution or from all
selected health institutions?
number of patients in the data collection time from (DEC 2017-MAR 2017 E.C) will be 1284. To I think it is better to use simple random sampling technique
or convenience sampling technique.
determine the k value. Population size (N)=1284 and sample size(n)=300. Therefore, k= N/n= When you allocate proportionally, you should show the
allocated sample size to each health institution in flow chart
1284/300=4.28. Then every 4th cases of diarrheal pediatric pa tients will be selected as study
subjects.
The total sample size will be allocated proportionally to the one hospital (University of Gondar
Hospital), to five health centers and two private specialized pediatric clinics based on the size of
the patients with diarrhea.
4.7. STUDY VARIABLES

4.7.1. Dependent variables: -
 prevalence of salmonella and shigella
 antimicrobial susceptibility of salmonella and shigella
 associated risk factors for salmonellosis and shigellosis
4.7.2. Independent variables: - Commented [M10]: List factors to be increase resistance
socio-demographic variables to Abcs and Factors to be increase prevalence of shigella
and salmonella
 Age Formatted: Font: (Default) Times New Roman
 Sex Formatted: Font: (Default) Times New Roman
 Residence
 Toilet usage habit
 Water supply
 Nutritional status
19
 Educational status of the mothers or the guard of the children
 Family income
 Hygiene practice
 Occupation

5.8. Operational definitions
 Bloody diarrhea: Refers to any diarrheal episode in which the loose or watery stools
contain visible red blood. But it does not include blood present in streaks on the surface of
formed stool that can only detected in microscopical examination or biochemical tests in
which stools are black due to the presence of digested blood cells (melena) [3,31].
 Bacillary dysentery. This is dysentery caused by Shigella. The term is often used to
distinguish shigellosis from amoebic dysentery, caused by Entamoeba histolytica [10,31].
 Invasive diarrhea: Refers to diarrhea caused by bacterial pathogens including Shigella,
and some Salmonella, E. coli and Campylobacter jejuni, that invade the bowel mucosa,
causing inflammation and tissue damage [10,31].
 Typhoid: an enteric fever which is caused by S.typhi and S. paratyphi [10,2]
 Non-typhoid: a disease caused by salmonella species other than S.typhi and S.paratyphi
[55]
5.9. Sample collection and microbial identification procedures
Structured questionnaires : will be used to collect sociodemographic factors and associated risk
factors. The questioner will be translated from English to Amharic and then back to English by
another person for cross check and used to obtain information of the diarrheic pediatric patients.
For under 5- children the mother or guardian will be given a clean plastic stool container and
oriented about sample collection after interviewed with some pretested structured questionnaires.
Once collected, protozoa parasites will be identified through direct microscopy using saline wet
mount at each study sites and part of the stool will be kept in transport media, and transported
using ice box to microbiology department of Gondar university for further microbiological
investigations. One-gram of stool sample will be collected from each diarrheic pediatric patient
using sterile screw capped tubes containing transport media (Cary Blaire or peptone water media)
and transported to microbiology department of Gondar university for further microbiological
investigations. Inoculation and incubation of the specimens on the standard culture media
performed. The primary isolates will be subculture on the available biochemical tests for further
identification. Antimicrobial susceptibility tests will be carried out by using disc diffusion method
using Mueller-Hinton agar. A standardized suspension of the bacterial isolate will be prepared and
turbidity of the inoculum will be compared with 0.5 McFarland turbidity standard.
4.9.1. Laboratory analysis:
Stool samples from diarrheic pediatric patients will be collected in a clean, dry, disinfectant-free
suitable wide-necked container and immediately transferred to a transport medium and inoculate Commented [M11]: Why you will use these all media?
onto Salmonella-Shigella agar (Oxoid) and MacConkey (Oxoid), selenite F broth (Oxoid,UK),
xylose-lysine-deoxycholate agar (XLD) (Oxoid, UK) and incubated at 370C for 24 hours. After
20
incubation, the plates will be examined for characteristic colony growth and gram stain will be Commented [M12]: Why you will examine with this
done and further bacterial species will be identified following standard biochemical test procedure. staining? I think you will use selective media.
Biochemical tests performed will be triple sugar iron agar, indole, urea, Simon’s citrate agar, lysine Formatted: Font: (Default) Times New Roman
iron agar, and motility tests. Suspension of test organisms will be prepared by picking pure Formatted: Font: (Default) Times New Roman
colonies with a sterile wire loop suspended in sterile nutrient broth and incubated for 2 hrs. The
density of suspensions to be inoculated will be determined by comparing with 0.5 McFarland
standards. A sterile cotton swab will be used and the excess suspension will be removed by gentle
rotation of the swab against the surface of the tube and then spread evenly over the Muller Hinton
agar plate. Susceptibility testing will be performed on isolates using agar disc diffusion technique
against ampicillin (10 μg), amoxicillin (10μg), tetracycline (30 μg), trimethoprime-
sulphamethoxazole (30μg), gentamicin (10μg), kanamycin (25μg), nalidixic acid (30 μg),
chloramphenicol (30μg), norfloxacin (10μg), ciprofloxacin (5μg), etc. The plates will be left at
room temperature for 30 minutes for diffusion then incubated for 18-24 hours at 370C. After 18-
24 hrs, the zone of growth of inhibition around each disc was measured in millimeters, using a
metal caliper, and interpreted as sensitive; intermediate and resistance following the method of
CLSI [3,5,11,12].
4.9.2. DATA MANAGEMENT AND QUALITY ASSURANCE

To generate quality and reliable data all questions in structured questionnaire will be prepared in
a clear and precise way and translated into local language (Amharic). Data collectors will be
trained; the entire questionnaire will be checked for completeness, during and after data
collection by the data collectors. Moreover, all laboratory procedures will be done by
maintaining the quality control procedures. All the necessary media will be checked by known
positive and negative samples before sample preparation and examination.
The raw data (the laboratory, clinical and demographic data) will be checked for completeness
and representativeness prior entry to the database. The questionnaires will also be pre-tested in
similar patients which are not part of the study and then the necessary adjustments will be made.
The stool samples will be tested according to the manufactures instruction. And all quality issue
will be maintained by using standard operating procedures in detection of salmonella and
shigella in stool sample during pre-analytical, analytical and post analytical stages.
For the reliability of the results good laboratory practices will be performed starting from the
pre-analytical stage (sample collection transportation) analytical stage (sample processing or
analyzing) up to post analytical stage.
21
4.9.3. DATA ANALYSIS
After data collection, the corresponding code number will be written carefully. The data generated
will be entered in to the Microsoft-Excel spreadsheet 2016 (Microsoft Cop., USA) and EPI Info
version 7(CDC, USA). The data will be imported from EPI Info and will be analyzed by statistical
package for Social Sciences (SPSS) software version 20.0(IBM, USA). Descriptive statistics will
be computed and data will be presented using figures and tables. Binary logistic regression will be
used to show associations of different variables with the dependent variable. Moreover, a
multivariate analysis will be done to identify factors that are independently associated. P-value
less than 0.05 will be considered statistically significant. In addition to this multivariate analysis
using logistic regression model will be computed to know factors which independently influence
the occurrence of dependent variables.
5. Ethical consideration
The study will be conducted after obtaining institutional ethical clearance from Research and
Publication Office of the University of Gondar. Official cooperation letters will be obtained from
Gondar University, Amhara Regional Health Bureau and zonal health offices, from Gondar town
municipal administration health office to do this research in the available health centers in their
catchment area. The study participants will be told that they have full right to participate or not to
participate in the study. Written informed consent was obtained from voluntary participants and
parents or guardians for children during data collection. All the subjects’ data will be kept with
full confidentiality and will not be disclosed to unauthorized person. Results of the laboratory
examinations that have a direct benefit in the health of the study participants will be informed to
physicians and the participants. Individuals who were found positive for bacterial and parasite
were treated as per the national guidelines.
6. Dissemination of result
Findings of this study will be disseminated to study health facilities, Woreda and zonal health
administrations, Amhara regional Health office, SBMS, UOG and other concerned bodies.
Moreover, the results will be presented to the scientific community in uog, national and
international conferences and manuscript will be prepared and submitted for publication.
22
7. Work plan
Table 1: A time schedule for the study of prevalence, antimicrobial susceptibility and associated
risk factors of salmonellosis and shigellosis in diarrheic pediatric patients attending at selected
Gondar town health institutions, northwest Ethiopia, from December 2017 to July 2017.
Tasks to be performed Responsible Time in months

Person Oct Nov Dec Jan Feb Mar Apr May Jun
Finalizing proposal PI + advisor Formatted: Font: (Default) Times New Roman
Ethical clearance RERC Formatted: Font: (Default) Times New Roman
Data collection PI Formatted: Font: (Default) Times New Roman
Data entry and analysis PI Formatted: Font: (Default) Times New Roman
Final thesis write-up PI Formatted: Font: (Default) Times New Roman
Final submission of thesis PI Formatted: Font: (Default) Times New Roman
PI= Principal investigator RERC = Research Ethics Review Committee
23
8. BUDGET PROPOSAL
Table 2: personnel costs
No Title Qualification Rate Duration on Total
work in days
1 Data collectors Lab.tech. 200ETB 120 24000 Formatted: Font: (Default) Times New Roman
2 Data entry & statistician 300ETB 30 9000 Formatted: Font: (Default) Times New Roman
analysis
3 Cleaning and Sanitary 150ETB 120 18,000 Formatted: Font: (Default) Times New Roman
washing
4 Transport 15ETB 120 1800 Formatted: Font: (Default) Times New Roman
Total 52,800ETB Formatted: Font: (Default) Times New Roman
Table 3: Budget for laboratory materials, reagents and supplies

Items Mx. Unit Quantity Unit price Total price
A4 paper Rim 10 100 1, 000 Formatted: Font: (Default) Times New Roman
24
Permanent markers Pack 10 100 1, 000 Formatted: Font: (Default) Times New Roman
Pencil Pack 1 10 10 Formatted: Font: (Default) Times New Roman
Pen Pcs 20 5 100 Formatted: Font: (Default) Times New Roman
Labeling tape Role 2 20 40 Formatted: Font: (Default) Times New Roman
Media preparation manual Each 1 1 birr/page 50 Formatted: Font: (Default) Times New Roman
(SOP)
Registration book for result Each 2 100 200 Formatted: Font: (Default) Times New Roman
documentation
Writing pad (small size) for Piece 4 15 60 Formatted: Font: (Default) Times New Roman
training
Flip chart for training Piece 03 100 300 Formatted: Font: (Default) Times New Roman
Plaster Role 5 20 100 Formatted: Font: (Default) Times New Roman
Sub-total 2, 800 Formatted: Font: (Default) Times New Roman
Table 4: Laboratory equipment and reagents Formatted: Font: (Default) Times New Roman
Description unit Quanti Quantity/unit Unit Price Total price

ty
Birr Cent Birr Cent
McCartney tubes Pack 5 5x12 1000 00 5000 00 Formatted: Font: (Default) Times New Roman
Disposable glove Box 10 100X10 100 00 1000 00 Formatted: Font: (Default) Times New Roman
Applicator stick Box 01 75*1 75 00 75 00 Formatted: Font: (Default) Times New Roman
Bleach Bottle 5 1X5 20 00 100 00 Formatted: Font: (Default) Times New Roman
Acetone Alcohol 500ml 01 1 100 00 100 00 Formatted: Font: (Default) Times New Roman
decolorizer
Cotton 100gm Roll 01 1X1 60 00 60 00 Formatted: Font: (Default) Times New Roman
Petri dish box 100 100X12 100 00 1000 00 Formatted: Font: (Default) Times New Roman
Blood Agar Base 500gram 01 1X1500 1500 00 1500 00 Formatted: Font: (Default) Times New Roman
MacConkey Agar 500gram 01 1X1500 1500 00 1500 00 Formatted: Font: (Default) Times New Roman
Simmons Citrate Agar 500gram 01 1X1500 1500 00 1500 00 Formatted: Font: (Default) Times New Roman
Manitol salt agar 500gram 01 1X1500 1500 00 1500 00 Formatted: Font: (Default) Times New Roman
Triple sugar iron agar 500gram 01 1X1500 1500 00 1500 00 Formatted: Font: (Default) Times New Roman
25
Kovas Reagent 100ml 01 1X1500 1500 00 1500 00 Formatted: Font: (Default) Times New Roman
Muller Hinton agar 500gram 01 1X1500 2000 00 2000 00 Formatted: Font: (Default) Times New Roman
Urea Agar Base 500gram 01 1X1500 800 00 800 00 Formatted: Font: (Default) Times New Roman
Motility (S.I.M) 500gram 01 1X1500 2000 00 2000 00 Formatted: Font: (Default) Times New Roman
Medium
Lysine decarboxilase 500gram 01 1X1500 2000 00 2000 00 Formatted: Font: (Default) Times New Roman
Tryptone Soya Broth 500gram 01 1X1500 2000 00 2000 00 Formatted: Font: (Default) Times New Roman
Ceftazidime pack 2 5x50disks 450 00 900 00 Formatted: Font: (Default) Times New Roman
Cefuroxime packs 2 5x50discs 450 00 900 00 Formatted: Font: (Default) Times New Roman
Cephalexine packs 2 5x50discs 450 00 900 00 Formatted: Font: (Default) Times New Roman
Cefixime of 5µg packs 2 5x50discs 450 00 900 00 Formatted: Font: (Default) Times New Roman
Ceftriaxone of 5µg packs 2 5x50discs 450 00 900 00 Formatted: Font: (Default) Times New Roman
Ceftazidime of 30µg packs 2 5x50discs 450 00 900 00 Formatted: Font: (Default) Times New Roman
Cefoxitin of 30µg packs 2 5x50discs 450 00 900 00 Formatted: Font: (Default) Times New Roman
chloramphenicol packs 2 5x50discs 450 00 900 00 Formatted: Font: (Default) Times New Roman
Cefuroxime of 4µg packs 2 5x50discs 450 00 900 00 Formatted: Font: (Default) Times New Roman
Tetracycline packs 2 5x50discs 450 00 900 00 Formatted: Font: (Default) Times New Roman
Penicillin G packs 2 5x50discs 450 00 900 00 Formatted: Font: (Default) Times New Roman
Gentamycin packs 2 5x50discs 450 00 900 00 Formatted: Font: (Default) Times New Roman
Clindamycin packs 2 5x50discs 450 00 900 00 Formatted: Font: (Default) Times New Roman
nitrofurantoine packs 2 5x50discs 450 00 900 00 Formatted: Font: (Default) Times New Roman
Grams iodine 500gram 1 1 1500 00 1500 00 Formatted: Font: (Default) Times New Roman
Safranine 500gram 1 1 1500 00 1500 00 Formatted: Font: (Default) Times New Roman
Cristal violet 500gram 1 1 1500 00 1500 00 Formatted: Font: (Default) Times New Roman
Absolut Alcohol 99.8% Litre 3 1x3 100 00 300 00 Formatted: Font: (Default) Times New Roman
solution
Hydrogen Peroxide 3% litre 1 100ml 100 00 100 00 Formatted: Font: (Default) Times New Roman
Solution
26
Microscope slides size box 10 10x50 100 00 1000 00 Formatted: Font: (Default) Times New Roman
27x75 mm thickness
1.2mm frosted
Immersion oil (Must litre 1 100ml 100 00 100 00 Formatted: Font: (Default) Times New Roman
have proper refractive
index and density for
microscopy)
Sub total 48, 135 00 Formatted: Font: (Default) Times New Roman
Table 5: Budget summary Formatted: Font: (Default) Times New Roman
Type of cost Birr Cent

Stationary 2, 800 00 Formatted: Font: (Default) Times New Roman
Laboratory equipments and reagents 48, 135 00 Formatted: Font: (Default) Times New Roman
Personnel costs 52,800 00 Formatted: Font: (Default) Times New Roman
Total 103735 00 Formatted: Font: (Default) Times New Roman
Contingency (5%) 5,186.75 00 Formatted: Font: (Default) Times New Roman
Grand Total 1080921.75 00 Formatted: Font: (Default) Times New Roman
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and Ephrem Engidawork, Non-typhoidal Salmonella serotypes, antimicrobial resistance and co-
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Comparison of the antibiotic resistance patterns among Shigella species isolated from pediatric
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International Journal of Biomedical and Advance Research 2015; 6(05): 413-417
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Declaration
I, the undersigned, senior medical microbiology student declare that this thesis is my original
work in partial fulfillment of the requirement for the degree of Master of Science in advanced
medical microbiology.
32
Name: Amare Alemu
Signature: ______________
Place of submission: School of biomedical and laboratory science, College of Medicine and
Health Sciences, University of Gondar.
Date of Submission: ____________________________
This thesis work has been submitted for examination with my/ our approval as university
advisor(s).
Advisors
Name Signature
1. ________________________ ______________________
2. ________________________ ______________________
ASSURANCE OF INVESTIGATOR
33
The undersigned agree s to accept responsibility for the scientific, ethical and technical conduct
of the research project and for provision of required progress reports as pre-terms and conditions
of the research and publications office of the University of Gondar.
Name of the student: Amare Alemu
Date: ___________________ Signature: ____________________
Approval of the advisor (s)
Advisors
Name Signature Date

1. ________________ ________________ _______________
2. ________________ ________________ _______________
ANNEXES Commented [M14]: All laboratory principles and

procedures should be annexed
Annex 1: ENGLISH VERSION OF THE INFORMATION SHEET Formatted: Font: (Default) Times New Roman
My name is Amare Alemu and I am Msc student in microbiology at Gondar University College of Formatted: Font: (Default) Times New Roman
Health science, school of biomedical sciences, and department of medical microbiology. I am
doing a research on the Prevalence, antimicrobial susceptibility and associated risk factors of
shigella and salmonella infection among diarrheic pediatric populations attending at selected
Gondar town health institution, Northwest Ethiopia.
Purpose of the study
The purpose of this study is to determine the Prevalence, antimicrobial susceptibility and
associated risk factors of shigella and salmonella infection among diarrheic pediatric populations
attending at Gondar town health institution, Northwest Ethiopia. In order to design preventive
strategies, the explanation of the mode of spread of these potentially fatal pathogens is crucial;
particularly since its prevalence in the study area is still remain poorly understood, therefore this
study will assess the prevalence, antimicrobial susceptibility and associated risk factors of
salmonella and shigella infection.
Participation: For this study to be successful we need your participation. And I am asking you
to participate voluntarily in this study. If you are voluntary to participate in this study, you are
expected to understand and sign the informed consent. Then Socio demographic and clinical
information related to salmonella and shigella infection will be filled on the questionnaire. Stool
sample will be collected for laboratory analysis at the time of the encounter, the end of the day,
or the following morning by attending laboratory technicians.
Expected benefits: your participation in this study will benefit for the region and the nation
as a whole. If there is any positive finding in laboratory examination the result will be reported to
your physician for appropriate treatment and management
34
Incentives: there is no special incentive that you will be given for participating in this research.
Confidentiality: All personal information you give and data obtained from laboratory
analysis will be kept confidential. Formats containing data will be kept locked.
Sharing the result: results will be written about the finding of the study, either through
publication or any other means. The result will not bear any information relevant to your
personality in any way.
Contact address
If the study subjects have question or problem related with the present study, you can contact the
principal investigator at any time using the following address.
Principal Investigator: Mr. AMARE ALEMU (candidate of MSc Microbiology

Department College of Health Sciences Gondar University
Cell phone: +251924466550
E-mail: amare.vip@gmail.com
Ethical Review Board –address
Po.box.196
Patient information Sheet form (አማርኛ) Formatted: Font: (Default) Times New Roman
አማረ አለሙ እባላለሁ ፡፡የጎንደር ዩኒቨርሲቲ የህክምና ማይክሮባዮሎጅ የ2ኛ ዲግሪ ተማሪ ነኝ፡፡ በጎንደር
ከተማ እና በአካባቢዋ በሚገኙ ጤና ተቋማት ለመታከም ከሚመጡ ከ15 በታች በሆኑ ህጻናት ላይ Formatted: Font: (Default) Times New Roman
የታይፎይድ እና የደም ተቅማጥ አምጭ ተህዋሲያን በህጻናቱ ላይ ያለበትን ደረጃ፣ ለመዳህኒቱ የሚሰጡትን Formatted: Font: (Default) Times New Roman
ምላሽ እና ለነዚህ ተህዋሲያን የሚያጋልጡ ሁኔታዎችን ለማዎቅ የሚካሄድ ጥናት ነው። Formatted: Font: (Default) Times New Roman
የጥናቱ ተሳታፊዎች የመረጃ ቅጽ Formatted: Font: (Default) Times New Roman
ሀ.የጠናቱ አላማ፡-የዚህ ጥናት አላማ በጎንደር ከተማ እና አካባቢዋ በሚገኙ ጤና ተቋማት ለህክምና Formatted: Font: (Default) Times New Roman
ከሚመጡ ከ15 በታች በሆኑ ህጻናት ላይ የታይፎይድ እና የደም ተቅማጥ አምጭ ተህዋሲያን በህጻናቱ ላይ Formatted: Font: (Default) Times New Roman
ያለበትን ደረጃ፣ ለመዳህኒቱ የሚሰጡትን ምላሽ እና ለነዚህ ተህዋሲያን መያዝ ምክንያት የሚሆኑ ነገሮችን Formatted: Font: (Default) Times New Roman
ለማዎቅ ነው።
ለ. ፈቃደኝነት፡-እርስዎ በጠናቱ ላይ በሙሉ ፈቃደኝነት እንዲሳተፉ እየጠየቅን በጥናቱ ላይ ለመሳተፍ Formatted: Font: (Default) Times New Roman
ፈቃደኛ ከሆኑ ለሚቀርብልዎት መጠይቅ ምላሽ ከሰጡ በኋላ የሰገራ ናሙና እንዲሰጡ ይጠየቃሉ፡፡
35
ሐ.የሚያገኙት ጥቅም፡-ባክቴሪያው መኖሩ በላቦራቶሪ ከተረጋገጠ በኋላ ተገቢውን መዳህኒት
እንዲዎስዱ ውጤቱ ለሃኪም ተልኮ በሃኪሙ ትዕዛዝ መዳህኒቱን እንዲዎስዱ ይደረጋል፡፡የእርስዎ በዚህ
ጥናት ተሳታፊ መሆን ለክልሉ እነዲሁም ለሃገር ጠቀሜታ አለው፡፡ Formatted: Font: (Default) Times New Roman
መ.የሚያስከትለው ጉዳት፡-በዚህ ጥናት በመሳተፍዎ በእርስዎ ላይ የሚያስከትለው ችግር የለም፡፡
ሠ.ሚስጥራዊነት፡-የእርስዎ የግል መረጃ በሙሉ ሚስጥራዊነት የተጠበቀ ይሆናል፡፡ Formatted: Font: (Default) Times New Roman
ረ.ውጤቱን ስለመጠየቅ፡-ከእዚህ ጥናተ በኋላ በሽታውን በተመለከተ ሪፖርት ይፃፋል፡፡ሆኖም Formatted: Font: (Default) Times New Roman
የእርስዎ ማንነት የሚገልፅ መረጃ የማይካተት መሆኑን እና ችግሩን ለማሳዎቅ ብቻ የሚውል ይሆናል፡፡ Formatted: Font: (Default) Times New Roman
አድራሻ፡-ማንኛውንም ወይም ጥርጣሬ ካለዎት ይህንን አድራሻ ይጠቀሙ::
የተመራማሪው አድራሻ፡-አማረ አለሙ

ማይክሮባዮሎጅ ትምህርት ክፍል
ጎንደር ዩኒቨርሲቲ
ሞባይል-0924466550
E-mail: amare.vip@gmail.com
ANNEX 2: CONSENT FORM
Written consent English:
Name of researching organization(s): - Gondar University
Title of the project: Prevalence, antimicrobial susceptibility and associated risk factors of
shigella and salmonella infection among diarrheic pediatric populations attending at Gondar
town health institution, Northwest Ethiopia.
Serial no _________________________
Card no _________________________
Name of study participant child: ___________________________
I have been requested to participate about this study, which plans to determine. Prevalence,
antimicrobial susceptibility and associated risk factors of shigella and salmonella infection
among diarrheic pediatric populations attending at Gondar town health institution, Northwest
Ethiopia. I have been informed this study which involves collecting of stool specimen. During
collection of the specimen I have been told that there is no harm and I have also read the
information sheet or it has been read to me. I have been also informed that all information
contained within the questionnaire is to be kept confidential. Moreover, I have also been well
informed of my right to keep hold of information, decline to cooperate and drop out of the study
if I want and that none of my actions will have any bearing at all on my overall health care and
36
clinic access. It is therefore with full understanding of the situations that I agreed to give the
informed consent voluntarily to the researcher to use my child stool specimen for the
investigation. I agree that I am contributing to the treatment of my fellows by participating in this
project. I have asked some questions and clarification has been given to me. I have given my
consent freely to participate in
the study, and I________________ hereby to approve my agreement with my signature.
Participant’s signature: __________________________Date_____________________
Principal Investigator s signature: __________________Date_____________________
Thank you for your participation.
INFORMED CONSENT (አማርኛ)

የፈቃደኝነት መጠየቂያ ቅጽ
ጥናቱን የሚያካሂደው ድርጅት፡-ጎንደር ዩኒቨርሲቲ

የጥናቱ ርዕስ፡-የዚህ ጥናት አላማ በጎንደር ከተማ እና አካባቢዋ በሚገኙ ጤና ተቋማት ለህክምና
ከሚመጡ ከ15 በታች በሆኑ ህጻናት ላይ የታይፎይድ እና የደም ተቅማጥ አምጭ ተህዋሲያን በህጻናቱ ላይ
ያለበትን ደረጃ፣ ለመዳህኒቱ የሚሰጡትን ምላሽ እና ለነዚህ ተህዋሲያን መያዝ ምክንያት የሚሆኑ ነገሮችን
ለማዎቅ ነው። Formatted: Font: (Default) Times New Roman
ተራ ቁጥር……………………………
የካርድ ቁጥር…………………
እኔ ………………የተባለኩት የህጻኑ ወላጅ/አሳዳጊ በጎንደር ከተማ እና አካባቢዋ በሚገኙ

ጤና ተቋማት ለህክምና ከሚመጡ ከ15 በታች በሆኑ ህጻናት ላይ የታይፎይድ እና የደም
ተቅማጥ አምጭ ተህዋሲያን በህጻናቱ ላይ ያለበትን ደረጃ፣ ለመዳህኒቱ የሚሰጡትን ምላሽ
እና ለነዚህ ተህዋሲያን መያዝ ምክንያት የሚሆኑ ነገሮችን ለማዎቅ በሚደረገው ጥናት
ለምርምሩ የሚያስፈልጉ መጥይቆች እና የልጄን የሰገራ ናሙና ለመስጥት በሚገኝ ቋንቋ
የተብራራልኝ ስለሆነ በጥናቱ ለመሳተፍ ሙሉ ፈቃደኛ መሆኔን በፊርማየ አረጋግጣለሁ።
37
የታካሚው ስም……………………ፊርማ……………………ቀን……………
የተመራማሪው ስም…………………ፊርማ……………………ቀን……………
ANNEX 3: QUESTIONNAIRES
Table 6: ENGLISH VERSION QUESTIONNAIRE
Part one: questionnaire on socio-demographic characteristics

Identification code ---------------------------------- Formatted: Font: (Default) Times New Roman
Questionnaire on identification of Alternative choice for Skip Code Formatted: Font: (Default) Times New Roman
the respondents Responses
No.
1 Sex 1. Male Formatted: Font: (Default) Times New Roman
2. Female
2 Age _______years
3 Residence 1. urban Formatted: Font: (Default) Times New Roman
2. rural
1. Housewife Formatted: Font: (Default) Times New Roman
4 What is your occupation? 2. Farmer
38
3. Merchant
4. Civil servant
5. Student
6. Other
5 What is your educational status? 1.Illitrate Formatted: Font: (Default) Times New Roman
2. Only read & write

3. Primary completed
4.Secondary completed
5. College and university
6 Religion 1. Orthodox Formatted: Font: (Default) Times New Roman
2. Muslim
3. Protestant
4. Others
7 What is your ethnic group? 1. Amhara Formatted: Font: (Default) Times New Roman
2. Tigray
3. Oromo
4. Kimant
5. Others
8 Family income/month ETB 1. <776 Formatted: Font: (Default) Times New Roman
2. >776
9 Drinking water source 1. Unprotected Formatted: Font: (Default) Times New Roman
2. surface water
3.Piped tap water
10 Toilet usage 1. open Formatted: Font: (Default) Times New Roman
2. simple pit latrine
39
3.-------
11 Hygiene practice 1. daily Formatted: Font: (Default) Times New Roman
2. within 3 days
3. within 7 days
12 Handwashing 1. before preparing Formatted: Font: (Default) Times New Roman

food
2. after defecating
child
3. after toilet use
4…………
13 Nutritional status 1.norman Formatted: Font: (Default) Times New Roman
2.undernutritioned
3.--------------------
40

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University of Gondar Formatted: Font: (Default) Times New Roman

College of Medicine and Health sciences

Prevalence, Antimicrobial Susceptibility and Associated Risk Factors of

By: - Amare Alemu(BSc, MSc candidate)

Table of Contents Formatted: Font: (Default) Times New Roman

LIST OF TABLES AND FIGURES------------------------4

Patient information Sheet form (አማርኛ) ------------------------------------32

INFORMED CONSENT (አማርኛ) ---------------------------------------------34

LIST OF TABLES AND FIGURES

Commented [M1]: Since this is the summery, Please

Objectives: To determine the prevalence, antimicrobial susceptibility and associated risk

1.2. Statement of the problem

1.3.3. ASSOCIATED RISK FACTORS

1.3.6. TREATMENT, PREVENTION AND CONTROL

1.3.7. Antimicrobial susceptibility patterns

2. Significance Justification of the study

4. METHODS AND MATERIALS

4.2. Study design and study period

Inclusion and exclusion criterion: -

4.7. STUDY VARIABLES

4.9.2. DATA MANAGEMENT AND QUALITY ASSURANCE

Tasks to be performed Responsible Time in months

Finalizing proposal PI + advisor Formatted: Font: (Default) Times New Roman

Ethical clearance RERC Formatted: Font: (Default) Times New Roman

Data collection PI Formatted: Font: (Default) Times New Roman

Final thesis write-up PI Formatted: Font: (Default) Times New Roman

Final submission of thesis PI Formatted: Font: (Default) Times New Roman

PI= Principal investigator RERC = Research Ethics Review Committee

Total 52,800ETB Formatted: Font: (Default) Times New Roman

Table 3: Budget for laboratory materials, reagents and supplies

Pencil Pack 1 10 10 Formatted: Font: (Default) Times New Roman

Pen Pcs 20 5 100 Formatted: Font: (Default) Times New Roman

Labeling tape Role 2 20 40 Formatted: Font: (Default) Times New Roman

Plaster Role 5 20 100 Formatted: Font: (Default) Times New Roman

Sub-total 2, 800 Formatted: Font: (Default) Times New Roman

Description unit Quanti Quantity/unit Unit Price Total price

Table 5: Budget summary Formatted: Font: (Default) Times New Roman

Type of cost Birr Cent

Personnel costs 52,800 00 Formatted: Font: (Default) Times New Roman

Total 103735 00 Formatted: Font: (Default) Times New Roman

Contingency (5%) 5,186.75 00 Formatted: Font: (Default) Times New Roman

Grand Total 1080921.75 00 Formatted: Font: (Default) Times New Roman

REFERENCE Commented [M13]: In this section

14. Maria do Rosário C. M. Nunes,1 Paula P. Magalhães, Francisco J. Penna,

50. Huruy K, Kassu A, Mulu A, Gebretsadik S, Andargie G, Tadesse T, Birhan W, Worku N,

Date of Submission: ____________________________

Name Signature Date

ANNEXES Commented [M14]: All laboratory principles and

Principal Investigator: Mr. AMARE ALEMU (candidate of MSc Microbiology

የተመራማሪው አድራሻ፡-አማረ አለሙ

Thank you for your participation.

INFORMED CONSENT (አማርኛ)

ጥናቱን የሚያካሂደው ድርጅት፡-ጎንደር ዩኒቨርሲቲ

እኔ ………………የተባለኩት የህጻኑ ወላጅ/አሳዳጊ በጎንደር ከተማ እና አካባቢዋ በሚገኙ

Part one: questionnaire on socio-demographic characteristics

Formatted: Font: (Default) Times New Roman

4 What is your occupation? 2. Farmer

2. Only read & write

Formatted: Font: (Default) Times New Roman

6 Religion 1. Orthodox Formatted: Font: (Default) Times New Roman

Formatted: Font: (Default) Times New Roman

2. simple pit latrine

12 Handwashing 1. before preparing Formatted: Font: (Default) Times New Roman