University of Gondar

College of Medicine and Health sciences

School of Biomedical and Laboratory Sciences
Department of Medical Microbiology

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

Advisers: - Setegn Eshetie ( BSC, MSC)
Tigist Engida ( BSC, MSC)

October, 2017
Gondar, Ethiopia

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 ACKNOWLEDGEMENT
I would like to thank SETEGN ESHETIE AND TIGIST ENGIDA 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 assistance in to propose this title.

Table of Contents

ACKNOWLEDGEMENT-------------------------------------2
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

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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
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

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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

LIST OF TABLES AND FIGURES

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
4
AIDS---------------------acquire immunodeficiency syndrome
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

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SUMMARY
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]. NTS illnesses continue to impose a significant burden on the population’s
health in industrialized and underdeveloped countries. The burden of enteric fever is
poorly characterized in many developing countries due to a scarcity of resources for
diagnosis [2].
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].
Shigella is one of the most important causes of gastroenteritis and death of 3-5 million
of children under the age of 5 years in developing countries [49].
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.

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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 the sample is 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 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 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
1.1. BACKGROUND
Diarrhea causing pathogens are the second leading cause of morbidity and mortality
worldwide; 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
enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni and Clostridium difficile,
Shigella 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

8
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 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.

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 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].
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; a study that is designed to assess its prevalence in diarrheic
pediatric patients in the area remains poorly understood.

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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.

1.3. LITERATURE REVIEW

1.3.1. Epidemiology and Historic background.
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 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

11
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 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].

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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 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

13
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 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

14
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

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.

15
2. Significance 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.

3. Objectives
3.1. General objectives
 To determine the prevalence, antimicrobial susceptibility and associated risk
factors of shigella and salmonella species among diarrheic pediatric children
attending at selected Gondar town health institutions.
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.

16
4. METHODS AND MATERIALS
4.1. Study area: - Study was conducted at Gondar town health institutions, which is
located in Gondar city administration, Amhara Region, Ethiopia. Gondar is found in the
Northwest part of Ethiopia, 747KM northwest of the capital city, Addis Ababa. 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 centers and two
private specialized pediatric clinics that include, Gondar, Gebrieal, Ginbot 20, Maraki,
Health Centers, Enat and Dr mihrete specialized pediatric clinics, which are currently
giving health service to the community.
4.2. Study design and study period
Across sectional study will be conducted in Gondar town health institutions to evaluate
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
Source populations: - All the populations who lives in and around selected Gondar
town were the source of population.
Study population: -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

There are studies which were conducted in different regions related with this study.
Taking prevalence from the previous study which was conducted in Addis Ababa,
Ethiopia, in under 5-children shigella were 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.

17
N= (1.96)2*0.13*(1-0.13)
(0.04)2

n=272. When 10 % contingency added, it will be 300. So, the 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 number of patients in the data collection time from (DEC 2017-MAR
2017 E.C) will be 1284. To determine the k value. Population size (N)=1284 and sample
size(n)=300. Therefore, k= N/n= 1284/300=4.28. Then every 4th cases of diarrheal
pediatric patients 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.

18
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: -
socio-demographic variables
 Age
 Sex
 Residence
 Toilet usage habit
 Water supply
 Nutritional status
 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 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

19
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 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 incubation, the plates will
be examined for characteristic colony growth and gram stain will be done and further
bacterial species will be identified following standard biochemical test procedure.
Biochemical tests performed will be triple sugar iron agar, indole, urea, Simon’s citrate
agar, lysine iron agar, and motility tests. Suspension of test organisms will be prepared
by picking pure 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].

20
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.

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.

21
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 Responsible Time in months

performed Person
Oct Nov Dec Jan Feb Mar Apr May Jun

Finalizing proposal PI + advisor

Ethical clearance RERC

Data collection PI

Data entry and analysis PI

Final thesis write-up PI

Final submission of PI
thesis
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
2 Data entry & statistician 300ETB 30 9000
analysis
3 Cleaning and Sanitary 150ETB 120 18,000
washing
4 Transport 15ETB 120 1800
Total 52,800ETB

Table 3: Budget for laboratory materials, reagents and supplies

Items Mx. Unit Quantity Unit price Total price
A4 paper Rim 10 100 1, 000
Permanent markers Pack 10 100 1, 000
Pencil Pack 1 10 10
Pen Pcs 20 5 100
Labeling tape Role 2 20 40
Media preparation manual Each 1 1 birr/page 50
(SOP)
Registration book for result Each 2 100 200
documentation
Writing pad (small size) for Piece 4 15 60
training
Flip chart for training Piece 03 100 300
Plaster Role 5 20 100
Sub-total 2, 800

Table 4: Laboratory equipment and reagents

Description unit Quant Quantity/unit Unit Price Total price
ity Birr Cent Birr Cent
McCartney tubes Pack 5 5x12 1000 00 5000 00

24
Disposable glove Box 10 100X10 100 00 1000 00
Applicator stick Box 01 75*1 75 00 75 00
Bleach Bottle 5 1X5 20 00 100 00
Acetone Alcohol 500ml 01 1 100 00 100 00
decolorizer
Cotton 100gm Roll 01 1X1 60 00 60 00
Petri dish box 100 100X12 100 00 1000 00
Blood Agar Base 500gram 01 1X1500 1500 00 1500 00
MacConkey Agar 500gram 01 1X1500 1500 00 1500 00
Simmons Citrate Agar 500gram 01 1X1500 1500 00 1500 00
Manitol salt agar 500gram 01 1X1500 1500 00 1500 00
Triple sugar iron agar 500gram 01 1X1500 1500 00 1500 00
Kovas Reagent 100ml 01 1X1500 1500 00 1500 00
Muller Hinton agar 500gram 01 1X1500 2000 00 2000 00
Urea Agar Base 500gram 01 1X1500 800 00 800 00
Motility (S.I.M) 500gram 01 1X1500 2000 00 2000 00
Medium
Lysine decarboxilase 500gram 01 1X1500 2000 00 2000 00
Tryptone Soya Broth 500gram 01 1X1500 2000 00 2000 00
Ceftazidime pack 2 5x50disks 450 00 900 00
Cefuroxime packs 2 5x50discs 450 00 900 00
Cephalexine packs 2 5x50discs 450 00 900 00
Cefixime of 5µg packs 2 5x50discs 450 00 900 00
Ceftriaxone of 5µg packs 2 5x50discs 450 00 900 00
Ceftazidime of 30µg packs 2 5x50discs 450 00 900 00
Cefoxitin of 30µg packs 2 5x50discs 450 00 900 00
chloramphenicol packs 2 5x50discs 450 00 900 00
Cefuroxime of 4µg packs 2 5x50discs 450 00 900 00
Tetracycline packs 2 5x50discs 450 00 900 00
Penicillin G packs 2 5x50discs 450 00 900 00
Gentamycin packs 2 5x50discs 450 00 900 00
Clindamycin packs 2 5x50discs 450 00 900 00
nitrofurantoine packs 2 5x50discs 450 00 900 00
Grams iodine 500gram 1 1 1500 00 1500 00
Safranine 500gram 1 1 1500 00 1500 00
Cristal violet 500gram 1 1 1500 00 1500 00
Absolut Alcohol Litre 3 1x3 100 00 300 00
99.8% solution
Hydrogen Peroxide litre 1 100ml 100 00 100 00
3% Solution
Microscope slides box 10 10x50 100 00 1000 00
size 27x75 mm
thickness 1.2mm
frosted

25
 Immersion oil (Must litre 1 100ml 100 00 100 00
have proper refractive
index and density for
microscopy)
Sub total 48, 135 00

Table 5: Budget summary

Type of cost Birr Cent
Stationary 2, 800 00
Laboratory equipments and reagents 48, 135 00

Personnel costs 52,800 00

Total 103735 00
Contingency (5%) 5,186.75 00
Grand Total 1080921.75 00
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51. Mohammedaman Mama and Getaneh Alemu, Prevalence, antimicrobial
susceptibility patterns and associated risk factors of Shigella and Salmonella among
food handlers in Arba Minch University, South Ethiopia, Mama and Alemu BMC
Infectious Diseases (2016) 16:686
52. Abdullahi, M. INCIDENCE AND ANTIMICROBIAL SUSCEPTIBILITY PATTERN OF
SALMONELLA SPECIES IN CHILDREN ATTENDING SOME HOSPITALS IN KANO
METROPOLIS, KANO STATE –NIGERIA, Bayero Journal of Pure and Applied
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53. INA´ CIO M. MANDOMANDO, EUSE´ BIO V. MACETE, JOAQUIM RUIZ, SERGI
SANZ, FATIMA ABACASSAMO, XAVIER VALLÈS, et al ETIOLOGY OF DIARRHEA IN
CHILDREN YOUNGER THAN 5 YEARS OF AGE ADMITTED IN A RURAL HOSPITAL
OF SOUTHERN MOZAMBIQUE, Am. J. Trop. Med. Hyg., 76(3), 2007, pp. 522–527

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54. Haifei Yang, M.D. Guosheng Chen, M.D., Yulin Zhu, M.D., Yanyan Liu, M.D., Jun
Cheng, M.D., Lifen Hu, M.D,et al, Surveillance of Antimicrobial Susceptibility Patterns
among Shigella Species Isolated in China during the 7-Year Period of 2005-2011
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S. Gunn and Ephrem Engidawork, Non-typhoidal Salmonella serotypes, antimicrobial
resistance and co-infection with parasites among patients with diarrhea and other
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110

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.

Name: Amare Alemu

Signature: ______________

31
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
The undersigned agrees 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: ____________________

32
Approval of the advisor (s)
Advisors

Name Signature Date

1. ________________ ________________ _______________
2. ________________ ________________ _______________

ANNEXES
Annex 1: ENGLISH VERSION OF THE INFORMATION SHEET
My name is Amare Alemu and I am Msc student in microbiology at Gondar University
College of 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

Incentives: there is no special incentive that you will be given for participating in this
research.

33
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)

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
የታይፎይድ እና የደም ተቅማጥ አምጭ ተህዋሲያን በህጻናቱ ላይ ያለበትን ደረጃ፣ ለመዳህኒቱ
የሚሰጡትን ምላሽ እና ለነዚህ ተህዋሲያን የሚያጋልጡ ሁኔታዎችን ለማዎቅ የሚካሄድ ጥናት ነው።

የጥናቱ ተሳታፊዎች የመረጃ ቅጽ

ሀ.የጠናቱ አላማ፡-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።

ለ. ፈቃደኝነት፡-እርስዎ በጠናቱ ላይ በሙሉ ፈቃደኝነት እንዲሳተፉ እየጠየቅን በጥናቱ ላይ ለመሳተፍ

ፈቃደኛ ከሆኑ ለሚቀርብልዎት መጠይቅ ምላሽ ከሰጡ በኋላ የሰገራ ናሙና እንዲሰጡ ይጠየቃሉ፡፡

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፡፡

34
መ.የሚያስከትለው ጉዳት፡-በዚህ ጥናት በመሳተፍዎ በእርስዎ ላይ የሚያስከትለው ችግር የለም፡፡
ሠ.ሚስጥራዊነት፡-የእርስዎ የግል መረጃ በሙሉ ሚስጥራዊነት የተጠበቀ ይሆናል፡፡
ረ.ውጤቱን ስለመጠየቅ፡-ከእዚህ ጥናተ በኋላ በሽታውን በተመለከተ ሪፖርት ይፃፋል፡፡ሆኖም
የእርስዎ ማንነት የሚገልፅ መረጃ የማይካተት መሆኑን እና ችግሩን ለማሳዎቅ ብቻ የሚውል ይሆናል፡፡

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 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

35
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።

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።

የታካሚው ስም……………………ፊርማ……………………ቀን……………

36
የተመራማሪው ስም…………………ፊርማ……………………ቀን……………

ANNEX 3: QUESTIONNAIRES
Table 6: ENGLISH VERSION QUESTIONNAIRE

Part one: questionnaire on socio-demographic characteristics

Identification code ----------------------------------
Questionnaire on identification of Alternative choice for Skip Code
No. the respondents Responses
1 Sex 1. Male
2. Female

2 Age _______years
3 Residence 1. urban
2. rural
1. Housewife
4 What is your occupation? 2. Farmer
3. Merchant
4. Civil servant
5. Student
6. Other
5 What is your educational status? 1.Illitrate
2. Only read & write
3. Primary completed
4.Secondary completed

37
 5. College and university

6 Religion 1. Orthodox
2. Muslim
3. Protestant
4. Others
7 What is your ethnic group? 1. Amhara
2. Tigray
3. Oromo
4. Kimant
5. Others

8 Family income/month ETB 1. <776

2. >776

9 Drinking water source 1. Unprotected

2. surface water
3.Piped tap water
10 Toilet usage 1. open
2. simple pit latrine
3.-------
11 Hygiene practice 1. daily
2. within 3 days
3. within 7 days

12 Handwashing 1. before preparing

food
2. after defecating
child
3. after toilet use
4…………

13 Nutritional status 1.norman

2.undernutritioned
3.--------------------

38
39