UNIVERSITY OF GONDAR

COLLEGE OF MEDICINE AND HEALTH SCIENCES

SCHOOL OF BIOMEDICAL AND LABORATORY SCIENCES

DEPARTMENT OF MEDICAL MICROBIOLOGY

PREVALENCE OF BACTERIAL ISOLATES, ANTIBIOTIC SUSCEPTIBILITY
PATTERNS AND RISK FACTORS AMONG PATIENTS WITH EAR INFECTION AT ENT
CLINIC OF , NORTHWEST ETHIOPIA

BY

A PROPOSAL SUBMITTED TO THE 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.

JANUARY, 2014

GONDAR, ETHIOPIA

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ACKNOWLEDGMENTS

I would like to acknowledge my advisors constructive comments for preparation of
this proposal.

My heartily gratitude also goes to who gave valuable comments during the proposal
development.

In addition, I want to acknowledge the Department of , School of Biomedical and
Laboratory Sciences, College of Medicine and Health Sciences, University of Gondar, for
giving me the chance on preparing this research proposal.

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TABLE OF CONTENTS

ACKNOWLEDGMENTS.............................................................................................. I
TABLE OF CONTENTS............................................................................................... II
LIST OF TABLES...................................................................................................... III
ABBREVIATIONS.................................................................................................... IV
SUMMARY.............................................................................................................. V
1. INTRODUCTION................................................................................................ 1
1.1. Background.................................................................................................. 1
1.2. Statement of the problem...................................................................................... 3
2. LITERATURE REVIEW........................................................................................ 5
3. SIGNIFICANCE OF THE STUDY........................................................................... 9
4. OBJECTIVES OF THE STUDY............................................................................. 10
4.1. General Objective............................................................................................. 10
4.2. Specific Objectives....................................................................................... 10
5. MATERIALS AND METHODS................................................................................ 11
5.1. Study area...................................................................................................... 11
5.2. Study design and period..................................................................................... 11
5.3. Population...................................................................................................... 11
5.3.1. Source population................................................................................ 11
5.3.2. Study population................................................................................. 11
5.3.3. Study subject....................................................................................... 11
5.4. Inclusion criteria.............................................................................................. 11
5.5. Exclusion criteria............................................................................................. 12
5.6. Variables........................................................................................................ 12
5.6.1. Dependent variables........................................................................... 12
5.6.2. Independent variables.........................................................................12

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5.7. Sample size and sampling technique......................................................................12
5.7. Data collection................................................................................................. 12
5.7.1. Socio-demographic and associated risk factor assessment.................12
5.7.2. Sample collection handling and transportation...................................13
5.7.3. Sample processing.............................................................................. 13
5.7.3. Quality control..................................................................................... 14
5.8. Data analysis................................................................................................... 15
5.9. Ethical considerations........................................................................................ 15
5.10. Result of dissemination plan.............................................................................. 15
6. WORK PLAN................................................................................................... 16
7. BUDGET......................................................................................................... 17
8. REFERENCES.................................................................................................. 20
9. ANNEX........................................................................................................... 24
Annex I-Dummy tables............................................................................................ 24
Annex II- Questionnaire.......................................................................................... 30
Annex III - Materials and procedures that will be used in the study......................................34
Annex IV- Information sheet and informed consent form..................................................36
Annex V- Questionnaire in Amharic............................................................................39
Annex VI-Information sheet and informed consent form in Amharic....................................41
Annex VII- Declaration........................................................................................... 43

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4
SUMMARY

Otitis media is an inflammation of the middle ear. It is one of the most common health
problems that affect children younger than 5 years. This is most commonly caused by the
buildup of fluid behind the ear drum, as a result of a blockage to the Eustachian tube.
Otitis media caused by the common respiratory pathogens Streptococcus such as
Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalisis often
preceded by a viral infection. Chronic supperative otitis media (CSOM) is caused by bacteria
found in the skin of the external ear when they enter into the middle ear through a chronic
perforation. Clinically otitis media presents with different signs, symptoms, and duration of
the exudates.
Acute otitis media (AOM) is acute inflammation the middle ear due to respiratory pathogens
and symptoms including otorrhoea occurring with less than two weeks of duration but in
CSOM more than two weeks.
Impaired hearing is the most frequent effect of otitis media and severe disability often
complicates this disease in developing countries. Hearing impairment impacts the ability to
work, to learn in school, or to develop basic language skills.
Objectives: To assess bacterial isolates, antibiotic susceptibility pattern and risk factors
among patients with ear infection at ENT clinic of Gondar University Hospital.
Methods: A cross sectional study will be conducted between January 2014 and June 2014
among patients with ear infection. From the eligible study population 166 study participants
will be selected by systematic random sampling. Data will be collected using a semi-
structured questionnaire for socio-demographic and ear discharge for isolation and antibiotic
susceptibility test and inoculated into appropriate media. The bacterial pathogens will be

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identified using standard microbiological methods. Antimicrobial susceptibility tests will be
performed using disk diffusion technique as per the standard Kirby-Bauer method. Finally
data will be entered and analyzed using SPSS version 20. Logistic regression will be used and
P values < 0.05 will be considered statistically significant.
Budget: This research will need a total cost of 23,694.00 Ethiopian Birr.

Key words: Otitis media, bacterial pathogens, antimicrobial resistance.

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1. INTRODUCTION

1.1. Background

Otitis media (OM) is an inflammatory disease of the mucosal lining of the middle ear by
pathogenic micro-organisms that are resident in the middle ear (1). It includes a variety of
clinical conditions with different signs and symptoms (2) Clinically OM presents has
different signs, symptoms, duration of the disease and type of exudates (3). Acute otitis
media (AOM) is acute inflammation the middle ear due to respiratory and other symptoms.
Otorrhoea occurring through tympanic perforation but with less than two weeks of duration
(4) and Chronic suppurative otitis media (CSOM) is the result acute otitis media and is
characterized by a persistent discharge from the middle ear (5) In developing countries the
natural course of the disease is different, leading to purulent otitis often with perforation and
further complications (6). In the developing countries, poverty, lack of specialists and limited
access to medical care amongst others increase complications of OM (7) and the global
burden of illness from CSOM involves 65-330 million individuals with draining ear 60% of
whom suffer from significant hearing impairment. Countries categorized by prevalence, as
proposed by WHO as CSOM prevalence rates of 1–2% were considered low and 3–6% high
and Ethiopia belongs to the latter category (8).
Otitis media is worldwide and a large part of the most common group of diseases in
childhood respiratory infectious diseases (9). Symptoms of upper respiratory infections are
often associated with otitis media in 94% cases (10). Otitis media is prevalent among children
than adults (11).
The pathogenesis of AOM is multi-factorial, both viruses and bacteria are implicated with the
common respiratory pathogens Streptococcus pneumoniae, Haemophilus influenzae, and
Moraxella catarrhalisis often preceded by a viral infection (12). These are respiratory
pathogens that may have been insufflated from the nasopharynx into the middle ear through
the Eustachian tube during upper respiratory infections (13). In CSOM the commonest
organisms isolated includes Pseudomonas aeruginosa, Escherichia coli, Staphylococcus
aureus, Streptococcus pyogenes, Proteus mirabilis, Klebsiella species) or anaerobic (e.g.
Bacteroides). These bacteria are infrequently found in the skin of the external canal, but may
proliferate in the presence of trauma, inflammation, lacerations or high humidity (14).

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Various antibiotics, including tetracycline, chloramphenicol and penicillin are effective in
treating OM. However, the recent emergence of penicillin-resistant Streptococcus pneumonia
and the increasing frequency of beta-lactamase producing strains of Moraxella catarrhalis
and Haemopihius influenzae are creating concern regarding the use of amoxicillin as
traditional first line empiric therapy for otitis media in younger children (15).
There are a few hospital-based studies documented in Ethiopia. A prospective study
conducted in Addis Ababa showed that out of 2, 334 patients with ear problem 69.8% had
otitis media of that the prevalence of COM with infected discharge was 52.8% and that of
AOM was 4.1% (16). Another prospective study findings within Addis Abeba showed that
chronic suppurative otitis media was 0.6% among the total hospital patient population and
22.3% in patients seen at the ear, nose and throat (ENT) clinic (17). Additionally a
retrospective study conducted on Gondar university hospital shows out of 228 ear discharge,
204 (89.5%) of microorganism isolates and of all microorganism isolates,192 (94.1%) had
multiple antibiotic resistant pattern (18). As it is indicated above, the morbidity due to ear
infection in Ethiopia is high and much attention is required in this regard. In Ethiopia
particularly in the study area, there is no prospective study which can fulfill the limitation of
retrospective studies. Therefore, the aim of this study is to assess prevalence of bacterial
isolates, antibiotic susceptibility pattern and risk factors among patients with ear infection at
the ENT Clinic of Gondar University Hospital, Gondar, North-west Ethiopia.

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1.2. Statement of the problem

Inflammation of the middle ear (OM) is one of the most common diseases, the leading cause
of hearing loss, and the most frequent indication for antimicrobial or surgical therapy in
children. The morbidity associated with OM is substantial in the aggregate, for example the
costs of therapy estimated over $5 billion each year for medical/surgical treatment and more
than 1 million operative procedures being performed annually in the USA(10).

Although impaired hearing is the most frequent effect of otitis media, or severe disability
often complicates this disease in developing countries. Hearing impairment impacts the
ability to work, to learn in school, or to develop basic language skills. Long term hearing loss
related to CSOM persisting throughout childhood and adolescence can be a significant
handicap(19). In Uganda community-based studies showed that perforation was present in
0.4% to 33.3% of children and youth; otorrhoea occurred in 0.4% to 6.1%; and mastoiditis
occurred in 0.19% to 0.74% also in a school survey, perforation was identified in 1.3% to
6.24% of students, and otorrhoea was found in 0.6% of patients who presented to ear, nose
and throat (ENT) clinics (20).

The above-mentioned reports from Ethiopia and elsewhere were tried to show that the burden
due to ear infection in terms of physician visits, medications, and absences from work and
school is very high, indicating that more attention needs to be given to this problem.
Therefore, this study will be undertaken to identify and isolate the bacterial etiological
agents, their antibiotic susceptibility pattern and risk factors among patients with ear
infections visiting ENT clinics of Gondar university hospitals Ethiopia.

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2. LITERATURE REVIEW

Otitis media is one of the most common and costly health problems that affect children
younger than 5 years with in the United States and the most common diagnosis associated
with the prescription of antibiotics (23). The cost of treatment was in excess of $US 5
billion(10). Similar estimates are most likely to describe OM disease burden in other
developed countries (24).
AOM remains a frequent global infection of childhood, with up to 80% of children having at
least one episode by three years of age. The peak incidence of the disease is between the ages
of 6 and 11 months. By one year of age, at least 60% of children have experienced 1 episode
and 17% have suffered from a minimum of 3 episodes of AOM (25).
A study was done in rural Bangladesh to assess the burden of AOM during the first two years
of life. Out of 252 newborns 0.9 episodes per child-year develop otitis media. 46% (n=115)
developed AOM: 36% (n=91) during the first year of life and 10% (n=24) during the second
year of life (p<0.001). AOM peaks occurred in the 6-12-month age-group and also the lowest
within the first three months of life (26).
Highest prevalence of CSOM in children is reported among the Inuits of Alaska, Canada and
Greenland, American Indians, and Australian Aborigines, and vary from7% to 46% and
Intermediate prevalence is reported in the South Pacific Islands, Africa, Korea, India, and
Saudi Arabia, ranging from 1% to 6%. The lowest prevalence is found in highly developed
industrial countries such as the UK and the US: <1% (27). The prevalence of chronic otitis
media within the African countries are markedly similar to one another, ranging from 0.4% to
4.2% (8).
In specific high-risk populations the Inuits of Alaska (30–40%), Australian Aborigines (12–
33%), and a few Native Americans (4–8%) and Prevalence of OME was most strongly
associated with age (P-value < 0.001). Other factors significantly associated are gestational
age, birth weight, breastfeeding, day-care attendance, number of siblings, season, and parent-
reported ear infection, hearing loss, mouth breathing and common cold with (P-value < 0.05)
(28).

Study on 300 patients with OM age ranging from 3 months -12 years were investigated for
possible risk factors of OM. Results: Residence in rural areas, artificial feeding, low
socioeconomic standard (SES), parental smoking, allergic rhinitis, adenoid hypertrophy,
chronic tonsillitis, URTI, LRTI and introduction of foreign body (FB) were significant

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(P<0.01). Bacteriological examination showed that the prevailing bacteria in cases of AOM
were Streptococcus pneumoniae, (24.8%), Staphylococcus aureus (24.8%), Pseudomonas
aeruginosa(15.2%), Haemophilus influenzae (9.5%) and Streptococcus pyogenes (7.6%). On
the other hand, the prevailing bacteria in CSOM were Proteus mirabilis (31%), Pseudomonas
aeruginosa(26.8%), Klebsiella species (14.1%), Staphylococcus aureus (8.5%) and E. coli
(5.6%) (29).

Another cross sectional study conducted on 191 otitis media patients seen in the Dessie
referral hospital, age range was 3/12–70years. 83.2% of them were identified as chronic otitis
media. The frequency of otitis media below 15years was 45.0%. Of 207 isolates, Proteus spp.
was the leading one, 48 (23.2%) followed by Staphylococcus aureus 44 (21.3%). Escherichia
coli, Enterobacter, Citrobacter, and Klebsiella spp. were 100% resistant to Amoxicillin. Age
less than 15years were the most affected groups. Proteus spp. S. aureus, and Pseudomonas
spp. were the major isolated microorganism. Gentamycin and Ciprofloxacin were relatively
effective antibiotics. However, all isolates showed multi drug resistance (40).

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3. SIGNIFICANCE OF THE STUDY

Inflammation of the middle ear is a major health problem, and the most frequent cause of
impaired hearing, and indication for antimicrobial or surgical therapy in children. Death or
severe disability often complicates this disease especially in developing countries. Therefore,
it needs epidemiological studies in Ethiopia.

It is known that the health policy of our country is a prevention strategy so, in order to
implement appropriate prevention measures, it is important to identify specific risk factors for
otitis media infection among individual who has ear infection based on epidemiological data.
Moreover, this study is highly significant on preparing preventing measures based on the
identified most significantly associated risk factors and it gives a clue for policy makers to
put their strategic plan on the risk groups. The present study will be providing knowledge of
definitive diagnosis of ear infection and update information for appropriate management of
ear infections, prevention and control of morbidity, disability and mortality of individual and
communities as a whole. It also serves as base line data for health facilities, for rational use of
antibiotics for prophylaxis, treatment and other researchers who further study on ear
infection. Finally, the results will be compared with the previous studies done in Ethiopia and
elsewhere in the world.

4. OBJECTIVES OF THE STUDY

4.1. General Objective
 To assess the prevalence, risk factors and antimicrobial susceptibility pattern of
bacterial isolates among patients with ear infection attending at ENT clinic of Gondar
University Hospitals.

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4.2. Specific Objectives
 To determine bacterial isolates from patients with ear infections at the ENT clinic of
Gondar University Hospital.
 To identify possible risk factors for ear infection.

5. MATERIALS AND METHODS

5.1. Study area

The study will be conducted in Gondar University Hospital, which is 727 km far from Addis
Ababa, located in North Gondar Zone of the Amhara Region. It is 65 kilometer north of Lake
Tana .

5.2. Study design and period

A cross-sectional study will be conducted from January, 2014 to June, 2014.

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5.3. Population

5.3.1. Source population

The source populations of the study will be all patients attending at the ENT clinic of Gondar
University Hospital during the study period.
5.3.2. Study population

The study populations will be all patients with ear discharge attending at the ENT clinic of
Gondar University Hospital during the study period.
5.3.3. Study subject

The study subject will be individual patient who are selected with ear discharge attending at
the ENT clinic of Gondar University Hospital during the study period

5.4. Inclusion criteria

All patients with ear discharge, who visited ENT clinic during the study period, will be
included in the study.

5.5. Exclusion criteria
All patients with ear discharge, who take antibiotic within 2 weeks during the study period,
will be excluded in the study.

5.7. Sample size and sampling technique

The sampling technique will be systematic random sampling and the sample size (n) will be
calculated by taking highest prevalence of ear infection in previous study in Gondar
University Hospital 92%(40).The expected margin of error (d) will be 0.05 and the
confidence interval (Zα/2) will be 95%. Contingency for the unknown circumstance will be
10%.
(1.96)2 × 0.92× o .8
n= (0.05)2 =

Antimicrobial susceptibility testing:

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Antimicrobial susceptibility testing will be performed for all isolates according to the criteria
of the National Committee for Clinical Laboratory Standards (NCCLs) by disk diffusion
method (42).
5.7.3. Quality control

Prior to the beginning of any data collection, all data collectors will be trained by the
Principal Investigator on an overview of the assessment and its objectives. A pre-test will be
given to standardize the questionnaire. Then, each questionnaire will be checked at the site of
collection before the study participant leaves to complete if there is any unfilled question. All
specimens will be collected according to the standard operating procedure of specimen
collection(43). To ensure the accuracy of data, double data entry method will be used. The
sterility of culture media will be ensured by incubating 5% of each batch of the prepared
media at 37oc for 24 hours. Performance of all prepared media will be also checked by
inoculating international standard-strains such as Escherichia coli (ATCC 25922),
Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853). To
standardize the inoculums density of bacterial suspension for the susceptibility test, 0.5
McFarland standard will be used (43).

5.8. Data analysis

Data will be entered and analyzed using SPSS version 20.0. Simple frequency will be applied
to see the distribution of socio-demographic variables. Logistic regressions will be used to
study the effect of many independent variables on the dependent variable. P -values ≤ 0.05
will be considered statistically significant.

5.9. Ethical considerations

Ethical clearance will be obtained from the ethical review committee of School of
Biomedical and Laboratory Sciences, College of Medicine and Health Sciences, Bahir Dar
University. The laboratory result of the study participant will be contacted to their doctors for
appropriate treatment.

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6. WORK PLAN

Activities Responsi Nov Dec Jan Feb Mar Apr May Jun
No bility

1 Topic selection PI

Prepare proposal
submit to Advisors,
donor

2 Obtain fund & ethical PI
approval from UOG

3 Preparation of study PI
tool

4 Prepare for study work PI

5 Data collection and PI+ DC
processing

6 Data entry PI

7 Data analyses & report PI
writing

8 Thesis defense PI

DC= Data collector

PI=Principal investigator

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7. BUDGET

Laboratory EQUIPMENT – SUPPLIES COST

Items Unit Number/amount Unit Price in Total Price in
Birr Birr

Nutrient agar(500gm) Bottle 2 750 1500

Mac Conkey agar (500gm) Bottle 1 650 650

Trypticase soy broth(500gm) Bottle 1 750 750

Mueller Hinton agar(500gm) Bottle 1 750 750

Triple sugar iron agar(500gm) Bottle 1 550 550

Urea broth base(500gm) Bottle 1 550 550

Lysine iron agar(500gm) Bottle 1 550 550

Motility testing medium(500gm) Bottle 1 550 550

Citrate agar(500gm) Bottle 1 550 550

Antibiotic disks Pack 13 100 1300

Kovac's reagent Bottle 1 500 500

3% hydrogen per oxide Bottle 1 250 250

Wire loop piece 4 20 80

McFarland standard Bottle 1 100 100

Gauze Roll 1 200 200

TOTAL Laboratory EQUIPMENT 17,784.
SUPPLIES COST 00

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