UNIVERSITY OF GONDAR

COLLEGE OF MEDICINE AND HEALTH SCIENCES

SCHOOL OF BIOMEDICAL AND LABORATORY SCIENCES
DEPARTMENT OF MEDICAL MICROBIOLOGY

Prevalence, Associated Risk Factors and Antimicrobial Resistance Patterns of

Salmonella and Shigella Species among Diarrheic Pediatric Population
Attending at Gondar City Healthcare Institutions, Northwest Ethiopia.

By: - Amare Alemu (Bsc)

Advisors: - Setegn Eshetie (Bsc, Msc)

Tigist Engda (Bsc, Msc)

A PROPOSAL SUBMITTED TO THE DEPARTMENT OF MEDICAL MICROBIOLOGY

SCHOOL OF BIOMEDICAL AND LABORATORY SCIENCES, COLLEGE OF MEDICINE
AND HEALTH SCIENCES, UNIVERSITY OF GONDAR FOR PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN MEDICAL
MICROBIOLOGY
DECEMBER, 2017

GONDAR, ETHIOPIA

I
ACKNOWLEDGEMENT
I would like to thank Setegn Eshetie and Tigist Engida to let me come up with this valuable research topic
and offering for their tremendous technical support. I would like to extend my gratitude to School of
Biomedical and Laboratory Sciences and Department of Medical Microbiology for their technical support
and enrolling me as MSc student.

I
Table of Contents
ACKNOWLEDGEMENT ............................................................................................................................. I

ABBREVIATIONS ........................................................................................................................................... IV

ABSTRACT...................................................................................................................................................... V

1. INTRODUCTION ......................................................................................................................................... 1

1.1. Background ........................................................................................................................................ 1

1.2. Statement of the problem ................................................................................................................... 3

1.3. LITERATURE REVIEW .............................................................................................................................. 4

1.3.1. Prevalence of Salmonella and Shigella in Diarrheic Pediatric Patients .......................................... 4

1.3.2. Antimicrobial Susceptibility Patterns of Salmonella and Shigella ................................................. 6

1.3.3. Associated Risk Factors for Salmonella and Shigella in Diarrheic Pediatric Patients .................... 9

2. JUSTIFICATION OF THE PROPOSED STUDY ................................................................................. 11

3. OBJECTIVES ......................................................................................................................................... 12

3.1. General objectives ............................................................................................................................ 12

3.2. Specific objectives ........................................................................................................................... 12

4. METHODS AND MATERIALS ............................................................................................................ 13

4.1. Study area......................................................................................................................................... 13

4.2. Study design and study period ......................................................................................................... 13

4.3. study populations ............................................................................................................................. 13

4.3.1. Source populations .................................................................................................................... 13

4.3.2. Study population ....................................................................................................................... 13

4.4. Inclusion and exclusion criterion ..................................................................................................... 13

4.4.1. Inclusion criteria .......................................................................................................................... 13

4.4.2. Exclusion criteria ......................................................................................................................... 13

4.5. Sample size determination and sampling techniques ........................................................................... 14

4.6. Study variables ..................................................................................................................................... 15

II
 4.6.1. Dependent variables: - .................................................................................................................. 15

4.6.2. Inpendent variables: - .................................................................................................................... 15

4.7. Definition of terms ............................................................................................................................... 15

4.8. Sample processing and Laboratory analysis ..................................................................................... 16

4.9. Data management and quality assurance ........................................................................................ 16

4.10. Quality control ............................................................................................................................... 17

4.11. Data analysis .................................................................................................................................. 17

4.12. Ethical consideration ...................................................................................................................... 18

4.13. Dissemination of results................................................................................................................ 18

5. WORK PLAN ......................................................................................................................................... 19

6. BUDGET PROPOSAL................................................................................................................................. 20

7.REFERENCE ............................................................................................................................................... 23

ANNEXES ..................................................................................................................................................... 28

Annex 1: ENGLISH VERSION OF THE INFORMATION SHEET ............................................................... 28

Patient information Sheet form (አአአአ) ........................................................................................... 30

ANNEX 2: ASSENT FORM ......................................................................................................................... 31

INFORMED ASSSENT (አአአአ)................................................................................................................ 36

ANNEX 3: QUESTIONNAIRES ................................................................................................................... 40

ANNEX 4: DUMMY TABLE ....................................................................................................................... 42

III
ABBREVIATIONS
AIDS---------------------Acquired Immuno Deficiency Syndrome
CDC--------------------Centers for Disease Control
C. jejuni----------------Campylobacter jejuni
CLSI--------------------Clinical Laboratory Standards Institute
HCL----------------------Hydrochloric acid
HIV------------------------Human Immune Virus
MLST-------------------- Multiple Loci Sequencing Typing
MUAC---------------------Middle Upper Arm Circumference
NTS----------------------Non-Typhoid Salmonellosis
PFGE--------------------Pulse-Field Gel Electrophoresis
PI------------------------Principal Investigator
PMNS-------------------Polymorphonuclear Cells
SBMS------------------School of Biomedical and Laboratory Sciences
SD1---------------------ShigellaDysentery Type 1
SPP.--------------------Species
SPSS--------------------Statistical Package for Social Sciences
UOG---------------------University of Gondar
USA---------------------United States of America
WHO--------------------World Health Organization

IV
ABSTRACT
Introduction: - Globally, Salmonellae and Shigella infections remain a major public health threat
and the significant cause of morbidity and mortality especially in the pediatric population. A
regular surveillance system needs to be in place, in order to explore the burden, antimicrobial
resistance pattern and associated risk factors for Salmonella and Shigella infections.

Objectives: - To assess the prevalence, antimicrobial susceptibility patterns and associated risk
factors of Shigella and Salmonella infections among diarrheic pediatric population attending at
Gondar city healthcare institutions.

Methods: - A cross-sectional study will be conducted in Gondar city healthcare institutions from
at the beginning of Jan 2018 to July 2018. A systematic random sampling technique will be used
to enroll 272 study subjects. Structured questionnaires will be used to gather socio-demographic
data and environmental factors. A stool sample will be collected from diarrheic pediatric patients
and inoculated onto MacConkey media, salmonella and shigella agar and xylose-lysine
deoxycholate agar. Further identification of the species will be carried out by sub-culturing the
organisms in biochemical tests. The disc diffusion method will be used to determine the
antimicrobial susceptibility of the isolates. A 0.5 McFarland solution will be used to standardize
the suspensions. The SPSS version 20 statistical package will be used for data entry and analysis.
Multivariable logistic regression will be carried out to point out associated risk factors. In all cases
P-value <0.05 will be considered as statistical significant.

Work plan: - This study will be carried out at the beginning of December 2017to July 2018.

Budget: - To conduct this study a total of 88,843 Ethiopian birrs will be required.

Keywords: - Salmonella, Shigella, Prevalence, risk factors, Pediatric population

V
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. This is mainly attributed to Salmonella,
and Shigella species (1). The Salmonella and Shigella are members of the Enterobacteriaceae
characterized by non-lactose fermenters, gram-negative rods, non-spore formers and facultative
anaerobes (2). The Shigella species are non-motile, and non-gas producers’,(2, 3) and Salmonella
species are motile, produces acid and gas from glucose, normally inhabit the intestines of animals
and humans(2, 4).
A remarkable characteristic of 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, 3). 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(2, 5).
The virulence factors associated with Salmonella species such as adhesion, invasion, and toxin
genes are clustered in certain areas of the chromosome known as “Salmonella pathogenicity
islands (2, 6). Shigellosis is only a human disease caused by the four species of genus Shigella and
is characterized by the increase in the frequency of stool motion and the presence of blood, mucous
and pus in the stool (2, 7). The Shigella species are limited to the intestinal tract of humans and
cause bacillary dysentery leading to watery or bloody diarrhea (8). To initiate infection, as few as
100 ingested Shigella microorganisms are enough to cause acute diarrhea after 4-7 days (7). 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 a
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 (3,
9). 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 (2, 5). The Shigella species have a lot of virulence factors that allow it to adhere
to the epithelium of the intestine, survive stomach acid, invade host cells, evade immune responses,
and introduce toxins into the body. Invasion of plasmid antigen B (IpaB) initiates binding to the

1
host cell and initiating pathways that kill macrophages upon infection, IpaC activates proteins to
form the actin-polymerizing complex that allows Shigella to move and spread within host cells (2,
6).
The Salmonella and Shigella are transmitted from person to person usually by asymptomatic
carriers and via contaminated food, flies, feces, fingers, and water (2, 3, 10). A severe infection of
diarrhea in children is highly associated with risk factors such as poor environmental sanitation
and hygiene, poverty and malnutrition (7, 11).

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. Blood culture and bone marrow aspirate may be used if the source and trained
personnel are available (4). 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, 4).

Drug-resistant Salmonella and Shigella infections are largely encountered and indiscriminate use
of antimicrobials has contributed to the development of resistance in gastrointestinal pathogens,
including misuse, overuse, quality and potency of the antimicrobial agents (1). A research which
were conducted in gondar before 10 years ago showed that 58.5% of patients were 0-5 years old
which is relatively high and needs further research.

2
1.2. Statement of the problem
Shigellosis and salmonellosis are still accounting for a significant proportion of morbidity and
mortality cases, especially in children with diarrhea in developing countries. Shigellosis which is
recognized by the WHO as the main cause of death among pediatric patients in developing
countries (3). Salmonellosis is also major bacterial enteric illness in human and animal which is a
public health burden and results in an economic loss in the society. Globally, non-typhoidal (NTS)
illness is estimated to be responsible for 93.8 million cases of gastroenteritis and 155,000 deaths
annually (12).
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 are attributable to
shigellosis including children under 5 years of age (13). Two hundred million to more than one
billion cases of diarrhea result worldwide due to Salmonella infections every year, leading to 3
million deaths (14). 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 (15).
In Africa, an estimate of 115 people dies of diarrheal diseases every hour, mostly of shigellosis
and salmonellosis (3, 16).
Over the past decades, Shigellae and Salmonellae show a persistent increase in antimicrobial
resistance to routinely prescribed antimicrobials (17-19). In Ethiopia, Salmonella and Shigella
have been reported to be resistant to first-line antibiotics such as ampicillin, tetracycline, and
amoxicillin (7, 8).
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 explore the prevalence, drug resistance and risk factors of Salmonella
and Shigella infections among diarrheic pediatric patients in Gondar city, Northwest Ethiopia.

3
1.3. LITERATURE REVIEW
1.3.1. Prevalence of Salmonella and Shigella in Diarrheic Pediatric Patients
Shigellosis is an important cause of acute diarrhea all over the world. It has high prevalence and it
is severed in children especially 5 years of a child. Shigella was responsible for more than 10% of
the cases of acute diarrhea in Teresina, Brazil (20). In a study carried out on the prevalence of
serogroups in Southern Trinidad, reported that within 0-10 years of age, the prevalence of the total
shigella species was 16% (21). A study from Henan, China reported 47% of salmonella species
were isolated from children less than 14 years of age (22).

In a study conducted in Iran, the prevalence of Shigella in pediatric patients was 3.8% in between
two and five years of age, there is the highest prevalence of Shigella which was 55.6% (23).
Another study from Iran also reported that the prevalence of Shigella species was 46.5% (24). As
a study from Shigella species over five years at a tertiary-care teaching hospital in northern India
indicates that the most commonly affected age-group was 1-5 years (39.6%), followed by the age-
group of less than one year (27.3%) (25). As noted by a study conducted at Amirkola children's
hospital, North of Iran, the prevalence of shigellosis among diarrheal patients was 14.05% (26). A
research conducted by Saudi Arabia on Salmonella serogroup reported that a higher percentage of
Salmonella species (61.3%) was isolated from children less than 15 years old. The highest number
(46.9%) was isolated from children of the age group 1- 3 years old (27).

According to a research carried out on prevalence and antimicrobial susceptibility of Salmonella

species associated with childhood acute gastroenteritis in federal capital territory Abuja, Nigeria,
2.3% of them were positive for Salmonella isolates and 3 of them were in the age group 6-12
months and children aged 0-5 months had the highest Salmonellae infections (4.1%), followed by
13-24 months with (3.5%) (28). In a study from Dare Salam, Tanzania shows the prevalence of
40% in children with bloody diarrhea and Salmonella has a prevalence of 5.7% (29). A research
from southern Libya points out a higher percentage of infection (36%) by Salmonella species
below 15 years of child age (30).

According to a study conducted among diarrheal patients at some selected health facilities in Addis
Ababa Ethiopia, the prevalence of Shigella in stool samples was found to be 9.1%, and (3.9%) of
children were found to be infected with Salmonella species (11). Similarly, in a study from Addis

4
Ababa, Ethiopia, the prevalence of Salmonella from gastroenteritis patients within 0-5 years of the
child was 6.3% (31). According to a study conducted in the isolation rate of Shigella species among
diarrheal patients attending at Hiwot Fana Hospital, Harar, Ethiopia, the prevalence of Shigella
within 0-5 years of age was 17.7% (19).

As indicated in a study carried out on the prevalence of Salmonella and Shigella species isolated
from outpatients, Jimma University specialized hospital, southwest Ethiopia, only two Shigella
isolates (1.1%) were encountered among children <4 years old and the prevalence of Salmonella
has relatively higher in children which was 31.58% (17). A research conducted in Jimma, Ethiopia,
from pediatric diarrheic outpatients, the prevalence of Salmonella species was 15.4% (32). In
another study conducted on the prevalence of intestinal parasite, Shigella and Salmonella species
among diarrheal children in Jimma health center, Jimma, southwest Ethiopia, the prevalence of
Salmonella and Shigella was 6.2% and 2.3% respectively (16). In a study carried out on the
serogroups of Shigella species among pediatric outpatients in Southwest, Jimma, Ethiopia, showed
that the prevalence of Shigella isolates was 20.1% (33).

Another a study carried out on the prevalence of Shigella, Salmonella and Campylobacter species
and their antimicrobial susceptibility patterns among under-five children with diarrhea in Hawassa
town, south Ethiopia, the prevalence of both Shigella and Salmonella was 3.3%(34) . In a research
conducted on the prevalence of Shigella among diarrheic children under-5 years of age attending
at Mekelle health center, northern Ethiopia, showed a prevalence of 13.3 %. The lowest (3.9 %)
and highest (22.6 %) Shigella isolates in this study was revealed from the age group of 6–11 and
12–23 months respectively (35).

As noted by the study on the prevalence of Shigella species at felege hiwot referral hospital, Bahir
Dar, Ethiopia, 2009, the prevalence of Shigella species for patients under 5 years of age was 5.1%
and 6-14 years of age was 3.3% (18). A study from Koren, Ethiopia, indicates that the prevalence
of Shigella in children was 2.6% (36).

According to a research conducted on biodiversity of Shigella isolates at Gondar university

hospital, northwest Ethiopia, reported that 58.5% of patients were 0-5 years old (37). In another
research from a five-year antimicrobial resistance pattern observed in shigella species isolated
from stool samples in Gondar University Hospital,northwest Ethiopia, report that children
accounted for more than a third (36.4%) of all Shigella positive patients(38).

5
1.3.2. Antimicrobial Susceptibility Patterns of Salmonella and Shigella
Increasing antimicrobial resistance has complicated the selection of empirical antibiotics for the
treatment of shigellosis and salmonellosis. A study from United States (USA) reports that 64% of
the Shigella isolates were resistant to two or more antimicrobial agents including the combination
of ampicillin, streptomycin, sulphamethoxazole (9). A study of Salmonella and Shigellainfections
in children, Yucatan, Mexico shows that the increase in shigellosis in the pediatric population is
a major public health concern (39).Similarly, the study in Brazil point out, more than 50% of
Shigella isolates from pediatric patients were resistant to ampicillin and sulfamethoxazole-
trimethoprim (20). Feeding of stock animals with food containing antibiotics plays a significant
role in the development of multidrug-resistant Salmonella. Studies in the 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 (4).

A study done on antimicrobial resistance pattern of Shigella species over five years at a tertiary-
care teaching hospital in north India, indicates that a high degree of resistance to most commonly-
used drugs, such as nalidixic acid (94.3%), ampicillin (95.8%), co-trimoxazole (94.5%),
tetracycline (79.4%), ciprofloxacin (60.2%), and chloramphenicol (63.3%) (25).

A surveillance of antimicrobial susceptibility patterns among Shigella species isolated in China

during the 7-year period of 2005-2011, shows 91.0% were multidrug resistant (resistant to 2 or
more agents). The highest resistance rate was found for nalidixic acid (96.4%), followed by
ampicillin (93.2%), tetracycline (90.9%), and trimethoprim/sulfamethoxazole (80.8%) (40).

As a study conducted from stool samples among hospitalized children in Abadan, Iran, shows
47.2% of Shigellaisolates were resistant to two or more antibiotics including trimethoprim-
sulphamethoxazole, ampicillin, and tetracycline (41).

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 (25). A
research from Abuja, Nigeria indicates the resistance pattern to antibiotics used was highest for
Amoxicillin, Cephalexin and Cefuroxime 55.6% (28) .

6
A study conducted among diarrheal patients at some selected health facilities in Addis Ababa
Ethiopia, showed that the resistance rates of Shigella species were high for ampicillin (95.7%),
Augmentin (91.4%), trimethoprim/sulphamethoxazole (52.2%) and resistances for one or more
antibiotics were observed among 91.3% of the isolates of Shigella species. But 70.0% of isolated
Salmonella species were resistance to one or more antibacterial antibiotics (11).

According to a study conducted on the prevalence and antimicrobial resistance in Salmonella and
Shigella species isolated from outpatients, Jimma University specialized hospital, Southwest
Ethiopia, the highest multidrug resistance(MDR)Salmonella species (42.1%) was observed for
combinations of two antibiotics such as resistance to tetracycline and ampicillin (17). In another
research done in Jimma, indicate, the most common resistance among Salmonella isolate was those
combinations containing ampicillin, tetracycline, cephalothin, and chloramphenicol (32). A
research on the antimicrobial and serogroups of Shigella species among pediatric outpatients in
the southwest, Jimma, Ethiopia, showed more than 53% of Shigella isolates in each serogroup
were resistant to tetracycline, ampicillin, cephalothin (33).

As indicated by the study on the prevalence and antimicrobial susceptibility patterns of Shigella
species at Felege hiwot referral hospital, Bahir Dar, Ethiopia, 2009; the highest prevalence of
antibiotic resistance was documented against streptomycin (96.9%) followed by ampicillin and
tetracycline (93.8%), cephalothin (90.7%), amoxicillin, (75%), and co-trimoxazole (62.5%). In
this study, 93.8% isolates of Shigella species were resistant to ampicillin, streptomycin, and
tetracycline, 78.1% of the isolates were resistant to ampicillin, tetracycline, and amoxicillin. 84.4%
of Shigella isolates tested were resistant to ampicillin, tetracycline, streptomycin, and cephalothin
(18).

Salmonella species shows the highest resistance to ampicillin (81.2%), cephalothin (86.4%),
chloramphenicol (83.7%), erythromycin (100.0%), gentamicin (75.6%), sulfonamide (81.1%),
tetracycline (94.5%) and trimethoprim/sulfamethoxazole (75.7%) (7).

As the research from serodiversity and antimicrobial resistance pattern of Shigella isolates at
Gondar University Teaching Hospital, Northwest Ethiopia, indicated that all the Shigella isolates
in the study showed the highest resistance rates to tetracycline (90%), co-trimoxazole (84.6%),
ampicillin (78.9%) and chloramphenicol (67.8%), and lowest resistance rates to gentamicin
(12.2%), ciprofloxacin (2.2%), and norfloxacin (1.1%) (37). In a study conducted on high level of

7
antimicrobial resistance in Shigella Species isolated from diarrheal patients in University of
Gondar Teaching Hospital, Gondar, Ethiopia reported that 90.8% of the Shigella species were
resistant to one or more antibiotics and 81.5% of the Shigella species tested exhibited multiple
resistances to up to six antimicrobials agents (42).

8
1.3.3. Associated Risk Factors for Salmonella and Shigella in Diarrheic Pediatric Patients
In the endemic regions, the presence of several serotypes of Salmonella and Shigella cause several
episodes of the disease that occurs in childhood. In the first 6 months of life, children usually live
in a more protected environment and receive protective factors conferred by breastfeeding (20,43).
After this age, although still immunologically naïve, they come into contact with the
microorganisms more frequently. Consequently, they are more susceptible to the infection and
develop the disease and progressive protection against the types of the microorganism circulating
in that region (10, 20). Thus, it is possible to explain the low frequency of shigellosis up to 6
months of age, its increase between 6 months and 2 years old and the drop after this age (43).The
hot and humid climate facilitates microbial breeding, and the food is more vulnerable to bacterial
contaminations, which may influence the survival and transmission of pathogens (10).Literacy
status of the mothers or guard of the children, the source of drinking water, latrine usage and
regular hand washing habit of the study subjects were among the characteristics ofthe patients (18).

Some risk factors vary with age and the weaning practices of the children; bottle-feeding is highly
associated with diarrhea in children with age between 1 to 6 months. Feeding of a child with a
bottle may be contaminated 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 (5).

Feeding type also influenced the rate of infection; children on solid food recorded the highest
Salmonellae infection of 3.2%, followed by those on breast milk (2.5%), while those on a
combination of breast milk and formula milk had no detectable level of Salmonellae infection (28).

The risk to salmonellosis is increased due to the following factors; absence of effective vaccines,
modifying handwashing behavior after defecating to control prolonged community outbreaks and
identifying high-risk groups and targeting prevention measures (43). The widespread occurrences
of Salmonella and Shigella are attributed to several factors including malnutrition and
undernutrition, 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 (44).

9
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 (43).

The risk of death due to shigellosis may be severe in infants and adults older than 50 years, children
not breastfed and malnutrition. 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 (3). Other risk factors predisposing
to non-typhoidal salmonellosis (NTS) infection include immunosuppression, decreased gastric
acidity, recent use of antibiotics, changes in the intestinal flora, hemoglobinopathies, and extremes
of age (4).

10
2. JUSTIFICATION OF THE PROPOSED STUDY
Salmonellosis and Shigellosis are endemic in most developing countries and is the most important
cause of bloody diarrhea worldwide. Though Salmonellosis and Shigellosis are a public threat in
Africa especially in the pediatric population, little is known about the burden, antibiotic resistance
and risk factors of the infections in the pediatric population in Ethiopia especially in Gondar.
Knowledge on Salmonella and Shigella pathogens are essential and a study that is designed to
assess its prevalence in diarrheic pediatric patients in the area remains poorly understood.

understanding the prevalence, antimicrobial susceptibility and associated risk factors in diarrheal
infections particularly due to Salmonella and Shigella is essential to design effective control and
effective preventive strategies. 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. The previous studies report the prevalence of salmonella and shigella
without specifying the age groups from 0-14 years which is the most susceptible age group.

This study will also show the burden of Salmonella and Shigella infections among diarrheic
pediatric patients in Gondar city, Northwest Ethiopia. This study also gives up-to-date information
on the cases for policy makers and health managers.

Therefore, this study is proposed to feel this gap and contribute to producing evidence on the
prevalence, antimicrobial susceptibility patterns and associated risk factors for Salmonella and
Shigella infections in pediatric populations to help the health service to have knowledge based
medical decisions in the prevention and treatment of such diseases.

11
3. OBJECTIVES
3.1. General objectives
 To assess the prevalence, antimicrobial susceptibility pattern and associated risk
factors of Shigella and Salmonella infections among diarrheic pediatric population
attending at 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 identify the associated factors for shigellosis and salmonellosis in diarrheic

pediatric populations attending at Gondar town health institutions.

12
4. METHODS AND MATERIALS
4.1. Study area
The study will be conducted at Gondar town health institutions. Gondar is found 747 KM far from
the capital city of Ethiopia in the Northwest part of the country. Based on the 2007 census
conducted by the Central Statistical Agency of Ethiopia (CSA), it covers an area of 29280 square
kilometers and the city has an estimate of >300,000 total populations (45) with one referral hospital
and 8 health centers and two private specialized pediatric clinics which includes, Gondar, Gabriel,
Woleka, Ginbot 20, Blajig, Maraki, Azezo,and Tseda Health Centers, Enat and Dr mihretie
specialized pediatric clinics, which are currently giving health service to the community. Among
these by convenience, we randomly select five of health centers and two specialized pediatric
clinics including Gondar, Gebriel, Ginbot 20, Maraki, and Azezo health centers. From private
specialized pediatric clinics Enat and Dr Mihretie specialized pediatric clinics are selected as well.
4.2. Study design and study period
A cross-sectional study will be conducted in Gondar city healthcare institutions to assess the
prevalence, antimicrobial susceptibility pattern and associated factors in diarrheic pediatric
populations from Jan 2018 to March 2018 in Gondar town health institutions.
4.3. study populations
4.3.1. Source populations
All pediatric population who live in Gondar town are the source of population.
4.3.2. Study population
All diarrheic pediatric populations seeking medical service in Gondar city health care facilities
will be a study population.
4.4. Inclusion and exclusion criterion
4.4.1. Inclusion criteria
All diarrheic pediatric patients from age 0-14 years old attending at selected Gondar city health
institutions will be included in the study.
4.4.2. Exclusion criteria
Patients, who have a history of antibiotic treatments for the past two weeks from the day of data
collection, will be excluded from the study. Unable to give required sample and those who are
above 14 years old will be also excluded.

13
4.5. Sample size determination and sampling techniques
In study carried out on the serogroups of Shigella species among pediatric outpatients (age in
between 0 and 14) in southwest, Jimma, Ethiopia, showed that the prevalence of Shigella
isolates was 20.1% (33). By taking prevalence that is 20.1%, and using single population
proportion formula and 95 % of confidence interval with 5% of margin of error sample size
calculated as follow

n= (1.96)2*0.201(1-0.201) = 247
(0.05)2
n=247. When 10 % non-response rate added, it will be 272. So, the sample size will be 272.
Systematic random sampling technique will be used to select the study participants. In the study
area, all the patients who were attending before one month of the study were 321. The estimated
number of patients in the data collection time from ( Jan 2018-MAR 2018 E.C) will be 963. To
determine the k value. Population size (N)=963and sample size(n)=272. Therefore, k= N/n=
963/272=3.5=4 Then every 4th cases of diarrheal pediatric patients will be selected as study
subjects. The total sample size will be allocated proportionally to Gondar comprehensive
referral hospital and to the five health centers and two private specialized pediatric clinics based
on the size of patients with diarrhea. In gondar comprehensive referral hospital around 423

14
diarrheic pediatric patients while there will be 90 diarrheic pediatric patients in Gondar health
center, 62 in Maraki health center, 28 in Gebriel health center, 14 in Ginbot 20 health cente, 91
in Azezo health center, 120 in Enat specialized pediatric clinic, and 135 in Dr Mihretie
specialized pediatric cilinic.

4.6. Study variables

4.6.1. Dependent variables: -
Salmonella and Shigella infections
Antimicrobial susceptibility patterns
4.6.2. Inpendent variables: -
Socio-demographic features (age, sex, educational status of the mothers or guard of the
children and residence), Toilet usage habit, Water supply, Nutritional status, Family
income and Hygiene practice

4.7. Definition of terms

Multidrug resistance: resistance to two or more antibiotics in different classes (11,40)

Pediatric population: the segment of the population with the age group ranging from 0 to 14
years (32,33).

Diarrhea: passing loose, watery, muciod and bloody stools three or more times a day (7).

4.8. Data Collection and Processing

4.8.1. Data collection

Relivant informations and clinical data related to the study will be collected using structured and
pretested questionnaires. The questionnaires will be translated from English to Amharic and then
back to English by another person for a cross check and used to obtain information of the
diarrheic pediatric patients.

4.8.2. Sample collection and transportation

For under 5-the children or the mother/guardian will be given a clean plastic stool container and
oriented about sample collection after interviewed with some pretested structured questionnaires.
For adult children, they will be given a clean, dry, disinfectant free container and instructed to
15
collect diarrheic stool sample as soon as they come for laboratory examination. By using selenite
F broth as a transport media, it will be transported to department of medical microbiology
laboratory of gondar university for microbial identification.

4.8. Sample processing and Laboratory analysis

Inoculation: Stool samples from diarrheic pediatric patients will be immediately transferred to a
transport medium (caryblair transport medium if available or selenite F broth will be used as a
transport medium) and transported to department of medical microbiology of gondar university
for further antimicrobial invetigations. Inoculation onto Salmonella-Shigella agar and xylose-
lysine-deoxycholate agar (XLD) and MacConkey, selenite F broth, and incubated at 370C for 24
hours will be performed.

Identification: 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: Biochemical tests performed will be triple sugar iron agar, indole, urea,
Simon’s citrate agar, and motility tests.

Antimicrobial susceptibility 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 selected antimicrobials. 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 will be measured in millimeters, using a metal caliper, and interpreted as
sensitive; intermediate and resistance following the method of CLSI 2016.

4.9. Data management and quality assurance

To generate quality and reliable data all questions in the 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

16
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 stool samples will be tested according to the manufactures instruction. And all quality issue
will be maintained by using standard operating procedures in the 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 the post-analytical stage.

4.10. Quality control

The standard operating procedures will be strictly followed. The culture media will be checked
for sterility and performance and American Type Culture Collection (ATCC) strains (E. coli
ATCC 25922, S. Typhi ATCC 13311, S.entritidisATCC 13076, S. sonni ATCC 25331) will be
used as controls for culture and sensitivity testing (20).

4.11. Data analysis

After data collection, the corresponding code number will be written carefully. The data generated
will be entered into EPI Info version 7(CDC, USA). The data will be imported from EPI Info and
will be analyzed by the 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. A 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.

17
4.12. Ethical consideration
The study will be conducted after obtaining ethical clearance from school of biomedical and
laboratory sciences and from ethical review committe. The study participants will be told that they
have full right to participate or not to participate. The assent (for 0-6 years old children) and
consent (for 7-14 years old children) will be taken from children's parents or guardians after they
understood the purpose of the study. All the subjects' data will be kept in full confidentiality and
will not be disclosed to an 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.

4.13. Dissemination of results

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 the manuscript will be prepared and submitted for publication.

18
 5. 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 city healthcare 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

Ethical clearance RERC

Data collection PI

Data entry and analysis PI

Final thesis write-up PI

Final submission of thesis PI

PI= Principal investigator RERC = Research Ethics Review Committee

19
6. BUDGET PROPOSAL
Table 2: personnel costs

No Title Qualification Rate Duration of 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 stationary materials, 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 8 1.5 10

Pen Pcs 20 5 100

Labeling tape Role 2 20 40

Media preparation manual Each 8 1 birr/page 8

(SOP)

20
 Registration book for result Each 1 100 100
documentation

Writing pad (small size) for Piece 8 15 120

training

Plaster Role 8 20 1600

Sub-total 3978

Table 4: Laboratory media, reagents and materials

Description Unit Quanti Quantity/unit Unit Price Total price

ty Birr Cent Birr Cent
Disposable glove Box 10 100X10 100 00 1000 00
Applicator stick Box 01 75*1 75 00 75 00
Cotton 100gm Roll 01 1X1 60 00 60 00
Petri dish box 100 100X12 100 00 1000 00
Salmonella-shigella agar 500 gram 01 1*2000 2000 00 2000 00
Xylose-lysine agar 500 gram 01 1*2000 2000 00 2000 00
Selenite F broth 500 gram 01 1*1500 1500 00 1500 00
MacConkey Agar 500gram 01 1X1500 1500 00 1500 00
Simmons Citrate 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) Medium 500gram 01 1X1500 2000 00 2000 00
Lysine decarboxylase 500gram 01 1X1500 2000 00 2000 00
Tryptone Soya Broth 500gram 01 1X1500 2000 00 2000 00
Absolut Alcohol 99.8% Litre 3 1x3 100 00 300 00
solution
Microscope slides size box 10 10x50 100 00 1000 00
27x75 mm thickness
1.2mm frosted
Immersion oil (Must litre 1 100ml 100 00 100 00
have proper refractive
index and density for
microscopy)

21
 Sub total 23835 00

Table 5: Proposed antimicrobials

Name of drugs Disk Quantity Unit price Birr Cent Total

content
Ampicillin 02 200 400 00
Ciprofloxaciline 5ug 02 200 400 00 400
Cotrimoxazole 30ug 02 200 400 000 400
Amoxacillin- 20/10ug 02 200 400 00 400
clavulanate
Chloramphenicol 30ug 02 200 400 00 400
Tetracycline 30ug 02 200 400 00 400
Cefuroxime 30ug 01 500 500 00 500
Tobramycin 10ug 01 500 500 00 500
Gentamycin 10ug 01 500 500 00 500
Amikacin 30ug 01 500 500 00 500
Sub-total 4000

Table 6: Budget summary

Type of cost Birr Cent

Personnel costs 52800 00
Stationary materials, reagents and supply 3978 00
Laboratory media, reagents and materials 23835 00
Proposed antimicrobials 400 00
Total 84,613 00
Contingency (5%) 88613 00
Grand Total 88,843 00

22
7.REFERENCE
1.Asrat D. Shigella and Salmonella serogroups and their antibiotic susceptibility patterns in
Ethiopia. 2008.

2.Greenwood D, Slack RC, Barer MR, Irving WL. Medical Microbiology E-Book: A Guide to
Microbial Infections: Pathogenesis, Immunity, Laboratory Diagnosis and Control. With
STUDENT CONSULT Online Access: Elsevier Health Sciences; 2012.

3.Organization WH. Guidelines for the control of shigellosis, including epidemics due to Shigella
dysenteriae type 1. 2005.

4.Sánchez-Vargas FM, Abu-El-Haija MA, Gómez-Duarte OG. Salmonella infections: an update

on epidemiology, management, and prevention. Travel medicine and infectious disease.
2011;9(6):263-77.

5.Nesa M, Khan M, Alam M. Isolation, identification and characterization of salmonella serovars

from diarrhoeic stool samples of human. Bangladesh Journal of Veterinary Medicine.
2012;9(1):85-93.

6.Coburn B, Grassl GA, Finlay B. Salmonella, the host and disease: a brief review. Immunology
and cell biology. 2007;85(2):112-8.

23
7.Demissie A, Wubie T, Yehuala FM, Fetene M, Gudeta A. Prevalence and antimicrobial
susceptibility patterns of Shigella and Salmonella species among patients with diarrhea attending
Gondar Town Health Institutions, Northwest Ethiopia. Sci J Pub Health. 2014;2(5):469-75.

8.Reda AA, Seyoum B, Yimam J, Fiseha S, Jean-Michel V. Antibiotic susceptibility patterns of

Salmonella and Shigella isolates in Harar, Eastern Ethiopia. Journal of Infectious Diseases and
Immunity. 2011;3(8):134-9.

9.Sivapalasingam S, Nelson JM, Joyce K, Hoekstra M, Angulo FJ, Mintz ED. High prevalence of
antimicrobial resistance among Shigella isolates in the United States tested by the National
Antimicrobial Resistance Monitoring System from 1999 to 2002. Antimicrobial agents and
chemotherapy. 2006;50(1):49-54.

10.Qu M, Lv B, Zhang X, Yan H, Huang Y, Qian H, et al. Prevalence and antibiotic resistance of
bacterial pathogens isolated from childhood diarrhea in Beijing, China (2010–2014). Gut
pathogens. 2016;8(1):31.

11.Mamuye Y, Metaferia G, Birhanu A, Desta K, Fantaw S. Isolation and antibiotic susceptibility

patterns of Shigella and Salmonella among under 5 children with acute diarrhoea: A cross-
sectional study at selected public health facilities in Addis Ababa, Ethiopia. Clinical Microbiology:
Open Access. 2015.

12.Crump JA, Luby SP, Mintz ED. The global burden of typhoid fever. Bulletin of the World
Health Organization. 2004;82(5):346-53.

13.Kotloff KL, Winickoff JP, Ivanoff B, Clemens JD, Swerdlow DL, Sansonetti PJ, et al. Global
burden of Shigella infections: implications for vaccine development and implementation of control
strategies. Bulletin of the World Health Organization. 1999;77(8):651.

14.Kumar Y, Sharma A, Sehgal R, Kumar S. Distribution trends of Salmonella serovars in India

(2001–2005). Transactions of the Royal Society of Tropical Medicine and Hygiene.
2009;103(4):390-4.

15.Zhang J, Jin H, Hu J, Yuan Z, Shi W, Yang X, et al. Antimicrobial resistance of Shigella spp.
from humans in Shanghai, China, 2004–2011. Diagnostic microbiology and infectious disease.
2014;78(3):282-6.

24
16.Beyene G, Tasew H. Prevalence of intestinal parasite, Shigella and Salmonella species among
diarrheal children in Jimma health center, Jimma southwest Ethiopia: a cross sectional study.
Annals of clinical microbiology and antimicrobials. 2014;13(1):10.

17.Lamboro T, Ketema T, Bacha K. Prevalence and Antimicrobial Resistance in Salmonella and

Shigella Species Isolated from Outpatients, Jimma University Specialized Hospital, Southwest
Ethiopia. Canadian Journal of Infectious Diseases and Medical Microbiology. 2016;2016.

18.Debas G, Kibret M, Biadglegne F, Abera B. Prevalence and antimicrobial susceptibility

patterns of shigella species at Felege Hiwot Referral Hospital, Northwest Ethiopia. Ethiop Med J.
2011;49(3):249-56.

19.Mekonnen H, Kebede A, Menkir S. Isolation rate and drug resistance patterns of Shigella
species among diarrheal patients attending at Hiwot Fana Hospital, Harar, Ethiopia. Ethiopian
Journal of Science and Technology. 2014;7(1):15-25.

20.Nunes MdR, Magalhães PP, Penna FJ, Nunes JM, Mendes EN. Diarrhea associated with
Shigella in children and susceptibility to antimicrobials. Jornal de pediatria. 2012;88(2):125-8.

21.Orrett FA. Prevalence of Shigella serogroups and their antimicrobial resistance patterns in
southern Trinidad. Journal of health, population, and nutrition. 2008;26(4):456.

22.Xia S, Hendriksen RS, Xie Z, Huang L, Zhang J, Guo W, et al. Molecular characterization and
antimicrobial susceptibility of Salmonella isolates from infections in humans in Henan Province,
China. Journal of clinical microbiology. 2009;47(2):401-9.

23.Talebreza A, Memariani M, Memariani H, Shirazi MH, Shamsabad PE, Bakhtiari M.

Prevalence and Antibiotic Susceptibility of Shigella Species Isolated From Pediatric Patients in
Tehran. Archives of Pediatric Infectious Diseases. 2016;4(1).

24.Rahbar M, Deldari M, Hajia M. Changing prevalence and antibiotic susceptibility patterns of

different Shigella species in Tehran, Iran. The Internet J Microbiol. 2007;3(2).

25.Urvashi SS, Dutta R. Antimicrobial resistance pattern of Shigella species over five years at a
tertiary-care teaching hospital in north India. Journal of health, population, and nutrition.
2011;29(3):292.

25
26.Savadkoohi RB, Ahmadpour-Kacho M. Prevalence of Shigella species and their antimicrobial
resistance patterns at Amirkola children hospital, North of Iran. Iranian Journal of Pediatrics.
2007;17(2):118-22.

27.Altayyar IA, Abdalla AM. Antibiotic Susceptibility Patterns and Sero-grouping of Salmonella
Isolated from Clinical Specimens in Eastern Province, Saudi Arabia. Emer Life Sci Res.
2015;1(2):8-12.

28.Cajetan ICI, Bassey BE, Florence IN, Nnennaya IR, Casmir AA. Prevalence and Antimicrobial
Susceptibility of Salmonella Species Associated with Childhood Acute Gastroenteritis in Federal
Capital Territory Abuja, Nigeria. 2013.

29.Moyo SJ, Gro N, Matee MI, Kitundu J, Myrmel H, Mylvaganam H, et al. Age specific
aetiological agents of diarrhoea in hospitalized children aged less than five years in Dar es Salaam,
Tanzania. BMC pediatrics. 2011;11(1):19.

30.Altayyar IA, Elbreki MF, Ali MO, Ali AA. Prevalence and Antimicrobial Susceptibility
Patterns of Salmonella spp Isolated from Gastroenteritis Patients, Southwestern, Libya. Appl. Med.
and Bio. Res. Vol 1 (1), 2016: p 2-6

31.Eguale T, Gebreyes WA, Asrat D, Alemayehu H, Gunn JS, Engidawork E. Non-typhoidal

Salmonella serotypes, antimicrobial resistance and co-infection with parasites among patients with
diarrhea and other gastrointestinal complaints in Addis Ababa, Ethiopia. BMC infectious diseases.
2015;15(1):497.

32.Mache A. Salmonella serogroups and their antibiotic resistance patterns isolated from
diarrhoeal stools of pediatric out-patients in Jimma Hospital and Jimma Health Center, South West
Ethiopia. Ethiopian Journal of Health Sciences. 2002;12(1).

33.Mache A. Antibiotic resistance and sero-groups of Shigella among paediatric out-patients in

southern Ethiopia. East African medical journal. 2001;78(6):296-9.

34.Getamesay M, Getenet B, Ahmed Z. Prevalence of shigella, salmonella and cmpylobacter

species and their susceptibility patters among under five children with diarrhea in hawassa town,
south Ethiopia. Ethiopian Journal of Health Sciences. 2014;24(2):101-8.

26
35.Kahsay AG, Teklemariam Z. Prevalence of Shigella among diarrheic children under-5 years of
age attending at Mekelle health center, north Ethiopia. BMC research notes. 2015;8(1):788.

36.Desenclos J, Zergabachew A, Desmoulins B, Chouteau L, Desve G, Admassu M. Clinical,

microbiological and antibiotic susceptibility patterns of diarrhoea in Korem, Ethiopia. The Journal
of tropical medicine and hygiene. 1988.

37.Tiruneh M. Serodiversity and antimicrobial resistance pattern of Shigella isolates at Gondar

University teaching hospital, Northwest Ethiopia. Jpn J Infect Dis. 2009;62(2):93-7.

38.Yismaw O, Negeri C, Kassu A. A five-year antimicrobial resistance pattern observed in

Shigella species isolated from stool samples in Gondar University Hospital, northwest Ethiopia.
Ethiopian Journal of Health Development. 2006;20(3).

39.Zaidi MB, Estrada-García T, Campos FD, Chim R, Arjona F, Leon M, et al. Incidence, clinical
presentation, and antimicrobial resistance trends in Salmonella and Shigella infections from
children in Yucatan, Mexico. Frontiers in microbiology. 2013;4.

40.Yang H, Chen G, Zhu Y, Liu Y, Cheng J, Hu L, et al. Surveillance of antimicrobial

susceptibility patterns among Shigella species isolated in China during the 7-year period of 2005-
2011. Annals of laboratory medicine. 2013;33(2):111-5.

41.Jomezadeh N, Babamoradi S, Kalantar E, Javaherizadeh H. Isolation and antibiotic

susceptibility of Shigella species from stool samples among hospitalized children in Abadan, Iran.
Gastroenterology and Hepatology from bed to bench. 2014;7(4):218.

42.Huruy K, Kassu A, Mulu A, Gebretsadik S, Andargie G, Tadesse T, et al. High level of

antimicrobial resistance in Shigella species isolated from diarrhoeal patients in University of
Gondar Teaching Hospital, Gondar, Ethiopia. Pharmacology Online. 2008;2:328-40.

43. 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 Sciences. 2010;3(1).

44. Tadesse G. Prevalence of human Salmonellosis in Ethiopia: a systematic review and meta-
analysis. BMC infectious diseases. 2014;14(1):88.

27
45.Commission FDRoEPC. Summary and statistical report of the 2007 population and housing
census–population size by age and sex. Addis Ababa D.

ANNEXES
Annex 1: ENGLISH VERSION OF THE INFORMATION SHEET
My name is Amare Alemu and I am Msc student in medical microbiology at Gondar University
College of Health science, school of biomedical and laboratory 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

28
The purpose of this study is to assess the Prevalence, antimicrobial susceptibility and associated
risk factors of Shigella and Salmonella infection among diarrheic pediatric populations attending
at Gondar city 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.

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 and medicine in Gondar University)

Cell phone: +251924466550

29
E-mail: amare.vip@gmail.com

Ethical Review Board –address

Po.box.196

Patient information Sheet form (አአአአ)

አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ2አአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ15አአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ

የየየየየየየየየየየየየየየየ

የ.የየየየየየየየ-
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ15
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአ

የ.የየየየየየየ-
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ

የ.የየየየየየየየየየ-
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአ

የ.የየየየየየየየየየየየ-አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ

የ.የየየየየየየየ-አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ

የ.የየየየየየየየየየየ-
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ

30
የየየየየ-አአአአአአአአአአአአአአአአአአአአአአአአአአአአአ::

የየየየየየየየየየየአ-

አአአአአአ

አአአአአአአአአአአአአአአአ

አአአአአአአአአአ

አአአአ-0924466550

E-mail: amare.vip@gmail.com

ANNEX 2: ASSENT FORM

Written Assent English:

Name of researching organization(s): - Gondar University

31
Title of the project: Prevalence, Associated Risk Factors and Antimicrobial Resistance
Patterns of Salmonella and Shigella Species among Diarrheic Pediatric Population Attending at
Gondar City Healthcare Institutions, Northwest Ethiopia.

Serial no _________________________
Card no _________________________
Name of study participant child: ___________

Investigator's Statement

My name is Amare Alemu and I am Msc student in medical microbiology at Gondar University
College of Health science, school of biomedical and laboratory sciences, and department of
medical microbiology. I am here to study the problem of childhood salmonellosis and shigellosis.I
will ask you/your child to be in this research study. The purpose of this assent form is to give you
the information you will need to help you decide whether or not to be in the study. Please read the
form carefully. You may ask questions about the purpose of the research, what we would ask you
to do, the possible risks and benefits, your rights as a volunteer, and anything else about the
research or this form that is not clear. When all of your questions have been answered, you can
decide if you want to be in the study or not.

Purpose: We are asking you to participate in this registry to learn more about the prevalence,
antimicrobial susceptibility patterns and associated risk factors of Salmonella and Shigella
infections on pediatrics. Salmonella and Shigella are well known causative agents of diarrhea in
children. The objective of the registry is to collect information on the prevalence and antimicrobial
susceptibility patterns in pediatric population. In addition, the registry is studying the risk of
developing of salmonellosis and shigellosis in pediatric populations.

Procedures: I will request information from your child medical records: diagnosis, medical
history including his/her history of infections, related medical problems, types oftreatment you
have received for diarrheic salmonellosis and shigellosis, copies of reports that describe results
ofprevious treatment. I will also ask for information about the health of your immediate family
members (children, sisters, brothers, parents and grandparents).Examination will involve only
laboratory examinations of stool samples. I will givecup to your child to collect diarrheic stool

32
samples. Then, I will examine the stool by using standard microbial cultures and biochemical tests
to detect the presence or absence of Salmonella and Shigella infections in your child stool.

Potential risks and discomfort: I am asking for your medical information, so the primary risk is
to your privacy. I will store your information in locked file cabinets which are located in an office
space that is locked at all times. Only the researchers will have routine access to your information.
While I will make every effort to maintain the confidentiality of your information is completely
secure. Researchers at other Universities working with the registry study may have access to
information about you that is coded with a study number instead of your name. Officials at the
University of Gondar and government offices sometimes review studies, such as this one, to make
sure they are being done safely and according to local, state and federal regulations. The study
records will not be used to put you at legal risk of harm. Your name will not be used in any
published reports about this registry study.

Direct benefits: There is no direct benefit to you from participating in this registry study but the
information learned through this research is valuable in understanding the condition in your child
and in the development of new treatments. If there is any positive finding in laboratory examination
the result will be reported to your physician for appropriate treatment and management.

Right to refuse or withdraw:The following information should be read to each study participant:
Participation in this study is voluntary. If you do not want to be in this research, no one will be
angry or disappointed with you. It’s your choice. You can think about it and tell us later if you
change your mind. On the other hand, you can say "yes" now and decline later on without any
negative consequences to you.

Who to contact:You can ask me questions now or later. I have written a cell phone number and
address where you can reach us or, if you are nearby; you can come and see us. If you want to talk
to someone else that you know like your doctor or family friend, etc., that's okay too.).

You are kindly request to approve your participationand I________________ hereby to approve
my agreement with my signature.
Participant’s signature: __________________________Date__________________
Principal Investigator s signature: __________________Date___________________

Thank you for your participation.

33
Certificate of assent (for children ≤6 years) and consent for 7-14 years old child
I understand the research is about investigation of in 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.I understand
that I will be inquired to give stool sample. In addition, I will be inquired to provide data on the
risk factors of Salmonella and Shigella infections
All the information related to this study has been given in the language I understand. I have been
informed that all the information I shall provide to the interviewer will be kept confidential. I also
know that I have the right to withhold information, skip questions to answer or to withdraw from
the study any time. I have acquainted nobody will impose me to explain the reason of withdrawal.
It is also enlightened there would have no effect at all in my health benefit or other administrative
effect that I get from all sectors. I have assured the right to ask information that is not clear about
the study before and/or during the study by contacting.
For families or attendants of patients unable to respond
I______________________________________ parent/guardian/attendant, after being fully
Informed about the purpose of this study, hereby give my consent on the patient’s Participation
in this study. I understand that my child free to withdraw at any time without penalty or loss of
benefits.
1.Institute:
a. Name: University of Gondar
b. E-mail:
c. Website: www.uog.edu.et
d. Tel:-
e. Fax:
f. P.O.Box: 296,Gondar, Ethiopia
2.Principal investigator:
a. Name: Amare Alemu Melese (Msc candidate)
b. Mob: +251 (0)924466550
c. E-mail: amare.vip@gmail.com
3.Supervisor:
a. Name: ________________________
b. Mob: __________________________
c. E-mail: ________________________
4.Data collector
a. Name: ________________________

34
 b. Mob: __________________________
c. E-mail: ________________________
I have read this information (or had the information read to me). I have had my questions answered
and know that I can ask questions later if I have them.
I agree to take part in the research.
OR
I do not wish to take part in the research and I have not signed the assent below.
___________ (initialed by child/minor)
Only if child assents:
Print name of child: ___________________
Signature of child: ____________________
Date: ________________
Day/month/year
If unable to read and write:
A literate witness will sign (if possible, this person should be selected by the participant, not be a
parent, and should have no connection to the research team). Participants who are unable to read
and write should include their thumb print as well.
I have witnessed the accurate reading of the assent form to the child, and the individual has had
the opportunity to ask questions. I confirm that the individual has given consent freely.
Print name of witness (not a parent): _________________ AND Thumb print of participant
Signature of witness: ______________________
Date: ________________________
Day/month/year
I have accurately read or witnessed the accurate reading of the assent form to the potential
participant, and the individual has had the opportunity to ask questions. I confirm that the
individual has given assent freely.
Print name of data collector: _________________
Signature of data collector: ___________________
Date: __________________
Day/month/year
Statement by the data collector
I have accurately read out the information sheet to the potential participant, and to the best of my
ability.I confirm that the child/child’s mother or guard was given an opportunity to ask questions

35
about the study, and all the questions asked by himself/herself or by their mother/guard have been
answered correctly and to the best of my ability. I confirm that the individual has not been coerced
into giving consent, and the consent has been given freely and voluntarily.
A copy of this assent form has been provided to the participant.
Print name of data collector: ________________________
Signature of data collector: __________________________
Date ___________________________
Day/month/year
Copy provided to the participant ________ (initialed by data collector/supervisor)
Parent/Guardian has signed an informed consent
i) Yes___________
ii) No___________ (initialed by data collector/supervisor)

INFORMED ASSSENT (አአአአ)

የ/ የየየየየየየየየየየየየየ (14)

የየየየየየየየየየየየየየየአ-አአአአአአአአአአ

36
የየየየአአአአ-
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
14አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአ

የየየየየየየየ-
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ14
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ

የየየየ የየየየ የየ የየየየአ አአአአአአአአአአአአአአአአአአአአአአ 14

አአአአአአአአአአአአአአአአአአአአአአአአአአአ/አአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአ/አአአአአአአአአአአአአአአአአአአአአአአአአአአአ/አአአአአአአአ
አአአአአአአአአአአ/አአአአአአአአ45
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአ/አአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአ

የየየየየየየአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአ

የየየአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ

37
የየየአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ:
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ

የየየየየየየየየየየአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአ

የየየየየየየየየየየአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአ

የየየየየየየየየየየየየአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ

የየየየየየየየየየየየየየአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአ

 አአአአአአአአአአ

 አአአአአአአአአአአአአአአአአአአአአአአአአአአአ 0924466550

 አአአአአአአአአአአአአአአአአአአአአአአአአ____________________________

የ.የየየየየየየየየየየየ

የየየየ አአአአ- አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ14አአአአአአአአአ

አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
2አአአአ(አአአአ)አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ14አአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ

38
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ

1. አአአአአአአአአአአ

2. አአአአአአአአአአአአአአአአአአአአአአአአአአአአ +251 924466550

3. አአአአአአአአአአአአአአአአአአ_________________

አአአአአአአአአአአአ________________________

አአአአአ……………………………

አአአአአአአ…………………

አአ
………………አአአአአአአአአአአአአ/አአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአ15
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ
አአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአአ

አአአአአአአ……………………አአአ……………………አአ……………

አአአአአአአአአ…………………አአአ……………………አአ……………

39
ANNEX 3: QUESTIONNAIRES
Table 6: ENGLISH VERSION QUESTIONNAIRE

Part one: questionnaire on socio-demographic characteristics

Identification code ----------------------------------

Questionnaire on identification of the Alternative choice for Skip Code

respondents Responses
No.

1 Sex 1. Male

2. Female

2 Age _______years

3 Residence 1. urban

40
 2. rural

4 What is your educational statusof the 1.Illitrate

child or parent?
2. Only read & write

3. Primary completed

4.Secondary completed

5. College and university

6 Drinking water sourceof the child 1. Unprotected

2. surface water

3.Piped tap water

7 Toilet usage of the family 1. open

2. simple pit latrine

3.-------

9 Handwashing of parents 1.before preparing food

2. after defecating child

3. after toilet use

4…………

10 Nutritional status of the child as 1.normal (MUAC greater than

measured by the clinicians 11 cm)

2.undernutritioned (MUAC
less than 11cm)

3. Weight

4. lateral edema

41
 ANNEX 4: DUMMY TABLE
Table 7: prevalence of salmonella and shigella infections based on socio-demographic
characteristics in diarrheic padiatric patients in gondar city healthcare institutions from
Jan 2018-March 2018.
Variables Parameters Frequency Percent
Age __________years
Sex Male
Female
Educational status of the 1.illitrate
child or parents 2.only read and write
3.primary school
4.secondary school
5.college and university

Resisdence Urban
Rural

Table 8: prevalence of salmonella and shigella infection with respect to environmental

sanitation in gondar city healthcare institutions from Jan 2018-Mar 2018
42
 Variables Parameter Total subjects tested p-
value
Salmonella Shigella
Positive Negative Positive Negative
Drinking water Unprotected
source Piped
Tap sources
Toilet usage Open
Simple pit latrin

Flushed toilet
Hand washing after Yes
toilet visiting No

Hand washing Yes

before meal No

Table 9: Some of the antimicrobials proposed to be done

Types of Disk Zone diameter break points nearest to whole mm
antibiotics content
Susceptibility Intermediate Resistance Zone Result
reading (S,I,R)
(mm)
Ampicillin

43
Ciprofloxaciline 5ug
Cotrimoxazole 30ug
Amoxacillin- 20/10ug
clavulanate
Chloramphenicol 30ug
Tetracycline 30ug
Cefuroxime 30ug
torbamycin 10ug
Gentamycin 10ug
Amikacin 30ug

44