Bertha Ochuma

 CHAPTER ONE INTRODUCTION 1.1 BACKGROUND OF STUDY Otitis Media is the most common disease of childhood, with about 65 –330 million people suffering from ear infection worldwide and 60% of them have significant hearing impairment (Woodfield et al. 2008). Otitis media is the inflammation of the middle ear cleft and the tympanum with otorrhoea lasting from 2  weeks to more than 3  months, with permanent perforation mainly caused by bacteria (Adoga et  al. 2010; Mesfin and Muluken 2014). It is estimated that 20,000 people die each year from otitis media and the overall burden of these diseases is higher in the poorest countries (Appiah-Korang et al. 2014). The health-economic burden of ear infection is also severe especially in Africa and other developing nations where the disease prevalence is estimated as high as 11% (Akinpelu et al. 2008, (WHO, 2004). According to World Health Organization (WHO) report, Ethiopia belongs to the high ear infection burden countries (WHO 2004). Complications of Otitis Media (OM) such as perforation of the tympanic membrane, otitis externa and mastoiditis affect balance, motor control and hearing. Even though ear infection is primarily a disease of infants and young children, it can also affect adults (Farhan et al. 2011). The disease may begin in childhood or as a complication of untreated or inadequately treated acute suppurative otitis media or may be chronic from onset (Afolabi et al. 2012). The microorganisms may gain entry to the middle ear through a chronic perforation. Children tend to have higher predisposition to ear infection than adults because anatomy of the Eustachian tube in children permits easier access of organism through the nasopharynx. Moreover, the incidence is higher in males than females (Afolabi et al. 2012; Seid et al. 2013). The prevalence of otitis media varies from place to place. In the developed world like United States of America (USA) and Europe, it is declining because of awareness; but in developing countries, it is on the rise (Adoga et  al. 2010). In developing countries untreated Otitis Media leads to purulent otitis often with perforation and further complications including recurrent acute otitis media, persistence of middle ear effusion which requires the insertion of drainage tube and often leads to hearing impairment, mastoiditis, meningitis, chronic otitis media, brain abscess and sepsis (Seid et al. 2013). The hearing impairment produced by otitis media also affect intellectual performance and language development (Ilechukwu et al. 2017, Roberts et al. 2008) The etiology, frequency and antimicrobial resistance patterns of ear infection is different in different geographical area and climate conditions (Abera and Kibret 2011; Wasihun and Zemene 2015). According to reports of many studies, Pseudomonas aeruginosa, Staphylococcus aureus,, Klebsiella pneumoniae and Escherichia coli are the common organisms isolated from cases of ear infection (Abera and Kibret 2011; Muluye et al. 2013; Seid et al. 2013). 1.2 STATEMENT OF PROBLEM While muchis known about thefactors contributingOME,thereremain a number ofkey questions yet to beanswered.Both host and environmental factors contribute to the development of OME. Host factors include age, sex, inherited predisposition to OME and anatomical factors. In addition, environmental factors such as pathogen exposure, tobacco smoke exposure, allergy, population density, seasonal variation and breastfeeding also play a role. Based on our current knowledge,these factors combine to contribute to Eustachian tube dysfunction. However, Eustachian tubedysfunction in itself is usually not sufficient to cause OME, but in the presence of apathogen, whether throughviralorbacterialinfection,OM candevelop, which subsequentlyresults in OME in 40%ofcases. As a result,thereisampleresearchontherolethat viruses and bacteriaplayinthepathogenesis of OME, their identification and characterization 1.3 JUSTIFICATION OF STUDY The most frequently detected bacteria are Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. (4-7) Recently, polymerase chain reaction (PCR) techniques were adapted to detect bacterial DNA in middle ear effusions, and their use increased the frequency of positive results for these bacteria in the examined effusions by nearly 80% . (8-10) The acquired knowledge about the prevalence of microorganisms responsible for or involved in OME cases may help select the most appropriate antimicrobials and minimize complications that might require surgery. 1.4 AIM AND OBJECTIVE OF STUDY To isolate bacterial pathogens responsible for Otitis Media Characterizethe bacterial pathogens present in patients with OME using culture independent techniques CHAPTER TWO LITERATURE REVIEW 2.1 Otitis Media It is likely that humans have always suffered from infections of the middle ear and its associated sequelae. Evidence of perforations of thetympanic membrane and destruction of the mastoid were identified in 2600 year old Egyptian mummies (Hoffman and Hudgins, 2002, Chan otitis) Today, otitis media continues to be a worldwide problem for children. Otitis media (OM) refers to a spectrum of disease involving the Middle-ear however, its nomenclature and classification have changed over the years. [3] Otitis media is a generic term defined as an inflammation of the middle ear without reference to a specific etiology or pathogenesis. Because all pneumatized spaces of the temporal bone are contiguous, inflammation of the middle ear may involve inflammation in the other 3 spaces: the mastoid, perilabyrinthine air cells, and the petrous apex. The term otitis media is often used to describe any of a continuum of related diseases: Acute Otitis Media (AOM), Recurrent Acute Otitis Media (RAOM), Otitis Media with Effusion (OME), and Chronic Otitis Media with Effusion (COME). Acuteotitismedia(AOM)represents acute suppurative inflammation of the middle ear. It manifests with local symptoms of otalgia and otorrhea and can be associated with systemic symptoms including fever, irritability, anorexia, nausea, vomiting and even diarrhoea. In AOM without perforation, the tympanic membrane bulges into the external auditory canal and often appears opaque, with reduced mobility of the tympanic membrane upon pneumatic otoscopy. Recurrent AOM (rAOM) is defined as multiple discreet episodes of AOM, with greater than threeepisodes in 6months, or greater than 4 in 12 months (Kong and Coates, 2009). rAOM is further distinguished into twocategories: acute infection with total resolution of middle ear fluid between attacks and those with AOM with Underlying persistent otitis media with effusion (OME). Also, acute otitis media can result in a perforation of the tympanicmembrane in up to 30% of cases (Berger, 1989). AOMwith perforation describesAOM resulting in the rupture tympanic membrane, with subsequent discharge from the middle ear within 7 days of infection (Kong and Coates,2009). Otitismediawith effusion(OME), in contrast to AOM, is defined asfluid inthe middle ear withoutlocal or systemic illness. OME has been synonymously referred to as“glue ear” and “serous otitis media” in the past.. It is the most common cause of deafness (Kubba et al, 2000) and balance disorder in childhood (Casselbrant, 2008), and may result in developmental delaysand behavioural changes. 2.2 Epidemiologyi Otitis media is a global problem and is found to be slightly more common in males than in females. The specific number of cases per year is difficult to determine due to the lack of reporting and different incidences across many different geographical regions (Schilder et al, 2016) . The peak incidence of otitis media occurs between six and twelve months of life and declines after age five. Approximately 80% of all children will experience a case of otitis media during their lifetime, and between 80% and 90% of all children will experience otitis media with an effusion before school age. Otitis media is less common in adults than in children, though it is more common in specific sub-populations such as those with a childhood history of recurrent OM, cleft palate, immunodeficiency or immunocompromised status, and others (Usonis et al, 2016; Schilder et al, 2016). United States statistics OM, the most common specifically treated childhood disease, accounts for approximately 20 million annual physician visits. Various epidemiologic studies report the prevalence rate of AOM to be 17-20% within the first 2 years of life, and 90% of children have at least one documented MEE by age 2 years. OM is a recurrent disease. One third of children experience six or more episodes of AOM by age 7 years. International statistics Incidence and prevalence in other industrialized nations are similar to US rates. In less developed nations, OM is extremely common and remains a major contributor to childhood mortality resulting from late-presenting intracranial complications. International studies show increased prevalence of AOM and chronic OM (COM) among Micronesian and Australian aboriginal children. Age-related demographics Peak prevalence of OM in both sexes occurs in children aged 6-18 months. Some studies show bimodal prevalence peaks; a second, lower peak occurs at age 4-5 years and corresponds with school entry. Although OM can occur at any age, 80-90% of cases occur in children younger than 6 years. Children who are diagnosed with AOM during the first year of life are much more likely to develop recurrent OM and chronic OME than children in whom the first middle ear infection occurs after age 1 year. Sex-related demographics Several studies have now shown equal AOM prevalence in males and females; many previous studies had shown increased incidence in boys. Race-related demographics For some time, the prevalence of OM in the United States was reported to be higher in black and Hispanic children than in white children. However, a study that controlled for socioeconomic and other confounding factors showed equal incidence in blacks and whites. Hispanic children and Alaskan Inuit and other American Indian children have higher prevalence of AOM than white and black children in the United States. 2.3 Pathophysiology (from Acute Otitis bookshelf PDF) Otitis media begins as an inflammatory process following a viral upper respiratory tract infection involving the mucosa of the nose, nasopharynx, middle ear mucosa, and Eustachian tubes. Due to the constricted anatomical space of the middle ear, the edema caused by the inflammatory process obstructs the narrowest part of the Eustachian tube leading to a decrease in ventilation. This leads to a cascade of events resulting in an increase in negative pressure in the middle ear, increasing exudate from the inflamed mucosa, and build-up of mucosal secretions, which allows for the colonization of bacterial and viral organisms in the middle ear. The growth of these microbes in the middle ear then leads to suppuration and eventually frank purulence in the middle ear space. This is demonstrated clinically by a bulging or erythematous tympanic membrane and purulent middle ear fluid. This must be differentiated from chronic serous otitis media (CSOM), which presents with thick, amber-coloured fluid in the middle ear space and a retracted tympanic membrane on otoscopic examination. Both will yield decreased TM mobility on tympanometry or pneumatic otoscopy. Several risk factors can predispose children to develop acute otitis media. The most common risk factor is a preceding upper respiratory tract infection. Other risk factors include male gender, adenoid hypertrophy (obstructing), allergy, day-care attendance, environmental smoke exposure, pacifier use, immunodeficiency, gastroesophageal reflux, parental history of recurrent childhood OM, and other genetic predispositions. (Fireman et al, 1997; Kraemer et al 1983; Fireman et al, 1985) 2.4 ETIOLOGY ( Otitis media.... physiology PDF) A multitude of host, infectious, allergic, and environmental factors contribute to the development of OM. 2.4.1 Host factors 2.4.1.1 Immune system The immature immune systems of infants or the impaired immune systems of patients with congenital immune deficiencies, HIV infection, or diabetes may be involved in the development of OM. [2. (Appiah-Korang, et al, 2014) OM is an infectious disease that prospers in an environment of decreased immune defences. The interplay between pathogens and host immune defence plays a role in disease progression. Patel et al found higher interleukin (IL)-6 levels in patients with OM who also had influenza and adenoviral infections, whereas IL-1β levels were higher in patients who developed OM following URI. [3] In another study, Skovbjerg et al found that middle ear effusions with culturable pathogenic bacteria were associated with higher levels of IL-1β, IL-8, and IL-10 than sterile effusions. [4] 2.4.1.2 Familial (genetic) predisposition Although familial clustering of OM has been demonstrated in studies that examined genetic associations of OM, separating genetic factors from environmental influences has been difficult. No specific genes have been linked to OM susceptibility. As with most disease processes, effects of environmental exposures on genetic expression probably play an important role in OM pathogenesis. 2.4.1.3 Mucins The role of mucins in OME has been described. Mucins are responsible for gel-like properties of mucus secretions. The middle ear mucin gene expression is unique compared with the nasopharynx. Abnormalities of this gene expression, especially upregulation of MUC5B in the ear, may have a predominant role in OME. 2.4.1.4 Anatomic abnormality Children with anatomic abnormalities of the palate and associated musculature, especially the tensor veli palantini, exhibit marked ETD and have higher risk for OM. Specific anomalies that correlate with high prevalence of OM include cleft palate, Crouzon syndrome or Apert syndrome, Down syndrome, and Treacher Collins syndrome. 2.4.1.5 Physiologic dysfunction Abnormalities in the physiologic function of the ET mucosa, including ciliary dysfunction and edema, increase the risk of bacterial invasion of the middle ear and the resultant OME. Children with cochlear implants have a high incidence of OM, especially chronic OM and cholesteatoma formation. One study described a relation between laryngopharyngeal reflux and chronic OM (COM); the authors concluded that reflux workup should be performed as part of COM investigations and that if reflux is confirmed, reflux treatment should be initiated in addition to treatment of primary disease. [5] Other host factors include: Vitamin A deficiency is associated with pediatric upper respiratory infections and AOM. Obesity: Obesity has been linked to an increased incidence of OM, although the causal factor is unknown. Speculations include alteration of intrinsic cytokine profile, increased gastroesophageal reflux with alterations of the oral flora, and/or fat accumulation; all of these have been linked with an increased incidence of OM. Conversely, OM may increase the risk of obesity by altering the taste buds. [6] 2.4.2 Infectious factors 2.4.2.1 Bacterial pathogens The most common bacterial pathogen in AOM is Streptococcus pneumoniae, followed by nontypeable Haemophilus influenzae and Moraxella (Branhamella) catarrhalis. These three organisms are responsible for more than 95% of all AOM cases with a bacterial etiology. [7] In infants younger than 6 weeks, gram-negative bacilli (eg, Escherichia coli, Klebsiella species, and Pseudomonas aeruginosa) play a much larger role in AOM, causing 20% of cases. Streptococcus pneumoniae and Haemophilus influenzae are also the most common pathogens in this age group. Some studies also found Staphylococcus aureus as a pathogen in this age group, but subsequent studies suggested that the flora in these young infants may be that of usual AOM in children older than 6 weeks. Many experts had proposed that the MEE (Middle Ear Effusion) associated with OME was sterile because cultures of middle ear fluid obtained by tympanocentesis often did not grow bacteria. This view is changing as newer studies show 30-50% incidence of positive results in middle ear bacterial cultures in patients with chronic MEE. These cultures grow a wide range of aerobic and anaerobic bacteria, of which Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and group A streptococci are the most common. Moraxella catarrhalis–induced AOM differs from AOM caused by other bacterial pathogens in several ways. It is characterized by a higher proportion of mixed infections, younger age at the time of diagnosis, lower risk of spontaneous perforation of the tympanic membrane, and an absence of mastoiditis. [8] Further evidence for the presence of bacteria in the MEE of patients with OME was provided by studies using polymerase chain reaction (PCR) assay to detect bacterial DNA in MEE samples that were determined to be sterile with standard bacterial culture techniques. In one such study using PCR assay, 77.3% of the MEE samples had positive results for one or more common AOM pathogens (eg, Streptococcus pneumoniae, Hemophilia influenzae, Moraxella catarrhalis). In chronic suppurative OM, the most frequently isolated organisms include Pseudomonas aeruginosa, Staphylococcus aureus, Corynebacterium species, and Klebsiella pneumoniae. An unanswered question is whether these pathogens invade the middle ear from the nasopharynx via the ET (as do the bacteria responsible for AOM) or whether they enter through the perforated TM or a TT from the EAC. The role of Helicobacter pylori in children with OME has been increasingly recognized. [9] Evidence that this agent might be responsible for OME comes from its isolation from middle ear and tonsillar and adenoidal tissue in patients with OME. Alloiococcus otitidis is a species of gram-positive bacterium that has been discovered as a pathogen associated with OME. [10, 11] This organism is the most frequent bacterium in AOM, as well as in OME. It has also been detected in patients who had been treated with antibiotics, such as beta-lactams or erythromycin, suggesting that these agents may not be sufficiently effective to eliminate this organism. Further investigation is needed to reveal the clinical role of the organism in OM. 2.4.2.2 Viral pathogens Because acute viral URI is a prominent risk factor for AOM development, most investigators have suspected a role for respiratory viruses in AOM pathogenesis. Many studies have substantiated this suspicion by showing how certain respiratory viruses can cause inflammatory changes to the respiratory mucosa that lead to ETD, increased bacterial colonization and adherence, and, eventually, AOM. Studies have also shown that viruses can alter the host-immune response to AOM, thereby contributing to prolonged middle ear fluid production and development of chronic OME. The viruses most commonly associated with AOM are respiratory syncytial virus (RSV), influenza viruses, parainfluenza viruses, rhinovirus, and adenovirus. Human parechovirus 1 (HPeV1) infection is associated with OM and cough in pediatric patients. [12] OM developed in 50% of 3-month follow-up periods that yielded evidence of HPeV1 infection but in only 14% of the HPeV1-negative periods; in recurring OM, the middle ear fluid samples were positive for HPeV in 15% of episodes. 2.4.3 Allergy Factors The relation between allergies and OM remains unclear. In children younger than 4 years, the immune system is still developing, and allergies are unlikely to play a role in recurrent AOM in this age group. Although much evidence suggests that allergies contribute to the pathogenesis of OM in older children, extensive evidence refutes the role of allergies in the etiology of middle ear disease. The following is a brief list of evidence for and against the etiologic role of allergy in OM: Many patients with OM have concomitant allergic respiratory disease (eg, allergic rhinitis, asthma) Many patients with OM have positive results to skin testing or radioallergosorbent testing (RAST) Although mast cells are found in the middle ear mucosa, most studies fail to show significant levels of immunoglobulin E (IgE) or eosinophils in the MEE of patients with OM OM is most common in the winter and early spring, yet most major allergens (eg, tree and grass pollens) peak in the late spring and early fall Most patients with concomitant OM and allergy show no marked improvement in middle ear disease with aggressive allergy management, despite marked improvements to nasal and other allergy-related symptoms. 2.5 Diagnosis OME does not benefit from antibiotic treatment. Therefore, it is critical for clinicians to be able to distinguish normal middle ear status from OME or AOM. Doing so will avoid unnecessary use of antibiotics, which leads to increased adverse effects of medication and facilitates the development of antimicrobial resistance. 2.6 Examination Pneumatic otoscopy remains the standard examination technique for patients with suspected OM. In addition to a carefully documented examination of the external ear and tympanic membrane (TM), examining the entire head and neck region of patients with suspected OM is important. Every examination should include an evaluation and description of the following four TM characteristics: Color – A normal TM is a translucent pale gray; an opaque yellow or blue TM is consistent with middle-ear effusion (MEE) Position – In AOM, the TM is usually bulging; in OME, the TM is typically retracted or in the neutral position Mobility – Impaired mobility is the most consistent finding in patients with OME Perforation – Single perforations are most common Adjunctive screening techniques for OM include tympanometry, which measures changes in acoustic impedance of the TM/middle ear system with air pressure changes in the external auditory canal, and acoustic reflectometry, which measures reflected sound from the TM; the louder the reflected sound, the greater the likelihood of an MEE. 2.7 Prognosis US mortality is extremely low in this era of antimicrobial therapy (< 1 death per 100,000 cases). In developing nations with limited access to primary medical care and modern antibiotics, mortality figures are similar to those reported in the United States before antibiotic therapy. A study that examined the causes of death in Los Angeles County Hospital from-, years before the advent of sulfa, showed that 1 in 40 deaths was caused by intracranial complications of OM. Morbidity from this disease remains significant, despite frequent use of systemic antibiotics to treat the illness and its complications. Intratemporal and intracranial complications of OM are the two major types. Intratemporal complications include the following: Hearing loss (conductive and sensorineural) TM perforation (acute and chronic) Chronic suppurative OM (with or without cholesteatoma) Cholesteatoma Tympanosclerosis Mastoiditis Petrositis Labyrinthitis Facial paralysis Cholesterol granuloma Infectious eczematoid dermatitis Intracranial complications include the following [17] : Meningitis Subdural empyema Brain abscess Extradural abscess Lateral sinus thrombosis Otitic hydrocephalus The prognosis for almost all patients with OM is excellent [18] ; the exceptions are patients in whom OM involves intratemporal and intracranial complications (< 1%). Data on cognitive and educational outcomes of OM in the literature are limited. [19] The impact of OM on child development depends on numerous factors. OM in infants younger than 12 months predisposes to long-term speech and language problems. OM has also been reported to negatively affect pre-existing cognitive or language problems. Careful follow-up and early referral are key to management of the ailment 2.8 The Role ofBacteria inOtitisMedia (Chan Otitis) OM is a multi-factorial disease with many associated risk factors, both host factor and environmental. Despite this complexity, bacterialpathogens have been strongly associatedwith OM development. In the past, identification of bacteriawas limited due to the restraints present with traditional culture techniques, however, with advancements in culture-independent techniques such as polymerase chain reaction and16S rRNA pyrosequencing, bacteriaare almost universally found in aspirates of the middle ear. The following section discusses some of the recent advances in the Detection ofbacteria andsummarisesthe role of bacteriain OM, with a focuson OME. 2.8.1 Alloiococcus otitidis Alloiococcusotitidis is a strictly aerobic,gram-positive coccifirst documented by Faden andDryja in1989(Faden and Dryja, 1989). Itis weakly catalase positive and arranges in pairs, small clusters and tetrads. Alloiococcus otitidis grows slowly with smallalpha-haemolytic colonieswhen incubated on blood agar after 3-4 days of growth, which continue to remain small even after extended periods of growth. Due to the characteristic that Alloiococcus otitidis is salt resistant, isolation of clinical strains is achieved when incubated on Brain Heart Infusion (BHI) supplemented with 6.5%sodium chloride (Bosley, et Al, 1995).However, despite this,Polymerase Chain Reaction (PCR) has emerged as the goldstandard for identificationof Alloiococcusotitidis (Hendolin et Al, 1997; Hendolin et Al, 1999; Kalcioglu et al, 2002; Harimaya, et al, 2006; Harimaya et al, 2006) Whenusing PCR for identification, Alloiococcus otitidis is the most prevalent bacterial species found inpatients with non-purulentOME (19 to 64%) and has been identified as the single dominant bacterial species(Kaur et al, 2014). Moreover,it hasbeen proposed that the presence of Alloiococcus otitidis in the MEF correlate with a longer duration of OME (Leskinen et al, 2004) In contrast, in the setting of AOM, Alloiococcus otitidis is less frequently identified from MEF. Kaur et al.( Kaur et Al, 2014) found that in 97 children with AOM, no children had Alloiococcus otitidis within the middle ear effusions. However, Sillanpaa et al. (2016), reported an incidence of 6.7% in 79 children. Furthermore, Leskinen et al. (2004) reported the presence of Alloiococcus otitidis in up to 25%of children with AOM. However, spontaneous perforation occurs in 40% of AOM, which may have allowed Alloiococcus otitidis to translocate prior to sampling. This can be illustrated by the fact that Marsh et al.(2012) reported that 37% of Australian Aboriginal children with AOM with perforation were PCR-positive forAlloiococcus otitidis, suggesting the external ear canal bacteria hasthe opportunity to translocate into the middle ear in the presence of a tympanic membraneperforation. 2.8.2 Streptococcus pneumoniae Streptococcus pneumoniae is a Gram-positive, alpha haemolytic coccus that is often found to colonise the nasopharynx. It is a fastidiousbacterium, growing bestin 5 percentcarbon dioxide, andrequires a source ofcatalase (e.g., blood)to grow on agar plates. Virtually all strains of Streptococcus pneumoniae have a polysaccharide capsule, which is the basis for serotyping.There are at least 97 antigenicallydistinct serotypes of encapsulated Streptococcus pneumoniae (Geno et al, 2015). Of these serotypes, 20 account for 80% of invasive pneumococcal disease (Lynch et al,2009) Thepathogenesis of Streptococcus pneumoniae stems from its ability to colonise the nasopharynx. The pneumococcal capsule not only plays a protective role against opsonisation andphagocytosis, but also promotes colonization of the nasopharynx by reducing mucosal clearancefrom the nasopharynx (Nelson et al, 2007; Kadioglu et al, 2008; Magee and Yother, 2001) Once colonised within thenasopharynx, Streptococcus pneumoniae can migrate and cause a broad range of disease. The most common infections occur in the respiratory tract including: bronchitis, pneumonia and OM. Streptococcus pneumoniae can also invade and spread via the bloodstream, causing bacteraemia and meningitis (Shak et al, 2013). In AOM, Streptococcus pneumoniae has been identified in 30-40% of cases, with evidence that the same bacteria can be found to colonize the nasopharynx (Ngo et al, 2016; Tonnaer et al, 2005). 2.8.3 Haemophilusinfluenzae Haemophilusinfluenzae is a gram positive, oxidase positive, facultative anaerobic, and non-motile coccobacillus that is a commensal of the human pharynges. By 18 months of age, one-third of children have had Haemophilus influenzae nasopharyngeal colonizationwith both typeable and non-typeable Haemophilusinfluenzae (NTHi) (Rao et al, 1999; Aniansson et al, 1992) The name Haemophilus makesreference to theorganisms' absolute nutritional requirement for Haemin (factor X)and Nicotinamide Adenine Dinucleotide (factor V) for growth. Haemophilusinfluenzae may be involved in a variety of invasive infections, mostly within the respiratory tract and is commonly associated with pneumonia, bronchitis, epiglottitis, sinusitis andotitismedia. Outside of the respiratory tract, Haemophilus influenzae has been implicated in bacteraemia, cellulitis, septicarthritis andmeningitis. As discussed,Haemophilusinfluenzae alongwithStreptococcus pneumoniae is one of the traditional pathogensin OMand is particularlyassociated with AOM in older children and with recurrentdisease (Leibovitz et al, 2004). Historically, Haemophilusinfluenzae is second only to Streptococcus pneumoniae as the bacterial causeof AOM (23%vs 28% respectively). However compared to Streptococcus pneumoniae AOM, Haemophilusinfluenzae AOM is considered less severe (Rodriguez and Schwartz, 1999). In contrast,Haemophilusinfluenzae isthe most commonly cultured bacteria in cases ofOME (11.6%) (Ngo et al, 2016). 2.8.4 Moraxellacatarrhalis Moraxellacatarrhalis is a large Gram-negative coccobacillus that is exclusively present in humans.Moraxellacatarrhalis has beenreclassifiedin thelast40yearsandhaspreviously beencalledMima polymorha, Neisseria catarrhalis and Branhamella catarrhalis. Like Streptococcus pneumoniae and Haemophilus influenzae, Moraxellacatarrhalis had been considered a part of the normal flora of the respiratory tract, but is also associatedwithupperandlower respiratorytractinfections, particularly in exacerbations of chronic obstructivepulmonarydisease. Moraxellacatarrhalis is the thirdmost identified in AOM with an averagefrequency of detection of 7.0% and less frequentlyin rAOM (4.1%) (Ngo et al, 2016)Lesscommonly, Moraxella catarrhalis causes pneumonia, bacteraemia, periorbital cellulitis, neonatal meningitis, septic arthritis, and conjunctivitis. Like Haemophilus influenzae, Moraxellacatarrhalis otitis media infections appear to be milder than pneumococcal otitis media (Aniansson et al, 1992).In addition, comparedwith other bacterial pathogens,Moraxella catarrhalis otitis mediais characterizedby: a higherproportionof mixedbacterial infections, younger ageat diagnosis, a lower proportion ofspontaneous perforationof thetympanic membrane andnoassociatedmastoiditis (Broides et al, 2009) 2.8.5 Staphylococcusaureus Staphylococcus aureus , a Gram-positive coccus is a commensal bacteria of the upperrespiratory tract. It is found to colonize approximately one third of the population (Gorwitz et al, 2008; Van Belkum et al, 2009). Staphylococcus aureus, including methicillin resistant strains (MRSA),are not commonly associated with AOM (Hartnick et al, 2000), with only 1 casereported of MRSA OM in the literature for AOM (Heslop and Ovesen, 2006). In contrast, thereare reports that methicillin-susceptible Staphylococcus aureus (MSSA) AOM incidence ranges from 8– 21% (Heslop and Ovesen, 2006). Thereis evidence from observational studies that have shown that Staphylococcus aureus prevalence within the nasopharynx is negatively associated with the presence of Streptococcus pneumoniae (Van denBergh et al, 2012). Additionally, Staphylococcus aureus has been suggested to have an inverse relationship with Haemophilus influenzae (Reiss-Mandel and Regev-Yochay, 2016) . 2.8.6 Pseudomonasaeruginosa Pseudomonas aeruginosa is a Gram negative bacillus and is a facultative anaerobe. It is considered an opportunistic bacterium in patients who are immunocompromised, but is also associated with diseasefor immunocompetent patients. Pseudomonas aeruginosa is more commonlyassociatedwithcolonizing the external ear canal (Tano et al, 2008) and the nasopharynx. It has also been found in biofilm form on theadenoids,tonsils andfromthe nasal cavity (Liu et al, 2015; Swidsinski et al, 2007). Pseudomonas aeruginosa isnot commonly associated with AOM, but instead with CSOM andhas been identified as the bacterial cause in 27.9% of cases (Kim et al, 2015) Furthermore, Pseudomonas aeruginosa is the most common bacterial pathogen in otitisexterna (Roland and Stroman, 2002) in particular being isolated in 61% of cases of malignant otitis externa and also being the most common cause of petrositis (Stern et al, 2016). These characteristics suggest that Pseudomonas aeruginosa may primarily be a bacteria originating from the external auditory canal that can cause middle ear disease via a perforation in the tympanic membrane. CHAPTER THREE MATERIALS AND METHODOLOGY Note( Streak plate technique, gram staining, biochemical testing) Materials 3.0 CHAPTER THREE Materials and methods 3.1 Study site/area 3.2 Media preparation 3.3 Sample Collection 3.4 Biochemical identification of Bacterial pathogens 3.4 Antibiotics susceptibility testing 3.5 Molecular characterization of Bacterial pathogens