Establishment and Characterization of an In Vitro Model for Cholesteatoma

Objectives

Experimental models are of importance to study the pathogenesis of middle ear cholesteatoma, however, they were not established until now. We aimed to develop in vitro model of middle ear cholesteatoma using primary keratinocytes and fibroblasts isolated from cholesteatoma tissue. HaCaT cell line was used as a "skin equivalent" and to compare the grade of homogeneity between cholesteatoma keratinocytes and HaCaT cells.

Methods

Primary keratinocytes were isolated from cholesteatoma tissue, co-cultured with preliminary prepared feeder layer from cholesteatoma fibroblasts and subsequently air-exposed. The protein profile of cholesteatoma keratinocytes and HaCaT cells was evaluated by means of immunoblot using monoclonal antibody against cytokeratin (CK) 13 and 16. Tissue localization of CK 13 and 16 was accomplished with immunohistochemistry.

Results

Different protein profile and stronger expression of CK 13 and 16 were demonstrated in cholesteatoma keratinocytes in comparison with HaCaT cells. Bigger stratification was observed in the 3D-in vitro systems when both cholesteatoma keratinocytes and HaCaT cells were respectively co-cultured with fibroblasts in comparison with the corresponding control groups without fibroblasts.

Conclusion

3D-model demonstrates the significance of intercellular interaction between components of cholesteatoma tissue.     

Genetic similarity between adenoid tissue and middle ear fluid isolates of Streptococcus pneumoniae,Haemophilus influenzae and Moraxella catarrhalis from Iranian children with otitis media with effusion

Background

Otitis media with effusion (OME) is a common disease among children, in the pathogenesis of which bacterial infections play a critical role. It was suggested that adenoid tissue could serve as a reservoir for bacterial infection, the eustachian tubes being the migration routes of bacteria into the middle ear cavity. The aim of this study was to investigate the genetic similarity between isolates of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis, obtained from adenoid tissue and middle ear fluid.

Methods

A total of 60 specimens of middle ear fluids (MEFs) and 45 specimens of adenoid tissue were obtained from 45 children with OME. All the samples were inoculated on culture media for bacterial isolation and identification. The genetic similarity between bacterial isolates was determined by pulsed-field gel electrophoresis (PFGE).

Results

The same bacterial species were simultaneously isolated from adenoid tissue and MEFs of 14 patients, among which, 6 pairs of M. catarrhalis, 5 pairs of S. pneumoniae and 3 pairs of H. influenzae were identified.

Conclusions

Based on the genetic similarities between isolate pairs, found by PFGE analysis, this study suggested that M. catarrhalis, S. pneumoniae and H. influenzae colonize the adenoid tissue, then migrate to the middle ear cavity and, hence, contribute to the total pathogenesis of OME.     

Frequency of Alloicoccus otitidis, Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae in children with otitis media with effusion (OME) in Iranian patients

Objective

To determine the presence of common bacterial agents of otitis media with effusion (OME), together with investigation these agent in the adenoid tissue and antimicrobial susceptibility pattern of isolated bacteria in Iranian children with OME.

Methods

Polymerase chain reaction (PCR) and bacterial culture methods were used for detection and isolation of Alloicoccus otitidis, Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae in 63 middle ear fluid samples and 48 adenoid tissues from 48 OME patients. Fifteen patients were bilaterally affected. Antimicrobial susceptibility of all bacterial isolates were determined by disk agar diffusion (DAD) method.

Results

Bacteria were isolated from 47% (n = 30) of the middle ear fluid samples and 79% (n = 38) of the adenoid tissue specimens in OME patients. A. otitidis was the most common bacterial isolated from the middle ear fluid 23.8% by culture and 36.5% by PCR method. S. pneumoniae was the most prevalent pathogen (35.5% and 31.2% by culture and PCR) in the adenoid tissues. In 10 patients the same organisms were isolated from the middle ear fluid and adenoid tissue. Antimicrobial susceptibility pattern showed taht most isolates of bacteria were sensitive to ampicillin, Amoxicillin/Clavulanate and fluoroquinolones.

Conclusion

The present study, being the first report on the isolation of A. otitidis by culture method in Iran and Asian countries, shows that A. otitidis is the most frequently isolated bacterium in Iranian children having otitis media with effusion. In this study A. otitidis, S. pneumoniae, H. influenzae and M. catarrhalis are the major bacterial pathogens in patients with OME and we found that ampicillin and Amoxicillin/Clavulanate have the excellent activity against bacterial agents in Iranian children with OME.     

Concurrent assay for four bacterial species including alloiococcus otitidis in middle ear, nasopharynx and tonsils of children with otitis media with effusion: a preliminary report

Objectives

To detect the prevalences of Alloiococcus otitidis, as well as Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis in children with chronic otitis media with effusion (OME) and to simultaneously investigate the colonization of these bacteria in the nasopharynx and palatine tonsils of these patients.

Methods

The study included 34 pediatric patients with OME, and 15 controls without OME. In the study group, A. otitidis, H. influenzae, S. pneumoniae, and M. catarrhalis were investigated in the samples obtained from middle ear effusions (MEE), nasopharyngeal swabs (NPS) and tonsillar swabs (TS), using multiplex polymerase chain reaction (PCR) and conventional culture methods. Only the samples obtained from NPS and TS were studied with the same techniques in the control group.

Results

A. otitidis was isolated only in MEE and only with multiplex PCR method. A. otitidis, S. pneumoniae, M. catarrhalis, H. influenzae were identified in 35%, 8.8%, 8.8%, and 2.9%, respectively, in 34 MEE. A. otitidis was not isolated in NPS or TS of the study and the control groups.

Conclusion

The prevalence of A.otitidis is high in children with OME and A.otitidis doesn''t colonize in the nasopharynx or tonsil.     

Early Sensorineural Hearing Loss in Ob/Ob Mouse, an Animal Model of Type 2 Diabetes

Objectives

There have been many studies on the relationship between diabetes mellitus and presbycusis. Microangiopathy and neuropathy that''s caused by chronic hyperglycemia may lead to damage to the inner ear. Several clinical studies on humans and animal studies have been performed to investigate the association between diabetes and hearing loss, however, this relationship is still a matter of debate. We investigated the association of diabetes and sensorineural hearing loss in an animal model of type-2 diabetes and obesity (the ob/ob mouse [OM]).

Methods

The auditory brainstem response (ABR) thresholds were obtained in the OM and the wild type mice (C57BL/6J mice) up to 25 weeks after birth. After the animals were sacrificed, their cochleae were retrieved and then subjected to histopathologic observations.

Results

The OM exhibited significantly elevated ABR thresholds at 21 weeks of age, yet the C57BL/6J mice exhibited no significant change until 25 weeks of age. On the histological findings, outer hair cell degeneration and loss of spiral ganglion cells were observed in the middle and basal turns of the OM. On the contrary, no degenerative change was observed until 25 weeks of age in the C57BL/6J mice.

Conclusion

This study suggests that chronic hyperglycemia and obesity may lead to early sensorineural hearing loss.     

Changing Patterns of Bacterial Strains in Adults and Children With Otitis Media in Korean Tertiary Care Centers

Objectives

Otitis media (OM) is an infectious disease that affects all age brackets. Aural discharge is a typical symptom, occurring in all subtypes of OM. We have compared the identity and antibiotic sensitivity of bacteria isolated from aural discharges of adults and children with various types of OM, including acute OM (AOM), OM with effusion (OME), chronic OM (COM), and cholesteatomatous OM (CSOM).

Methods

The study involved 2,833 patients who visited five tertiary hospitals between January 2001 and December 2010 and were diagnosed with AOM, OME, COM, or CSOM. The patients were divided into a pediatric group and an adult group, and the distribution of cultured bacteria and their antibiotic sensitivity were compared in the two groups.

Results

Bacterial detection rates were higher in adults than in children with OME and COM (P=0.000 each). The majority of the bacteria cultured from patients with AOM and OME bacteria were methicillin-susceptible Staphylococcus aureus (MSSA) and Streptococcus pneumoniae. Bacteria cultured from children were more susceptible to antibiotics (P=0.002) and had higher antibiotic sensitivity (P=0.001) than were bacteria cultured from adults. The majority of bacteria culture from patients with COM and CSOM were MSSA and pathogenic Pseudomonas aeruginosa. The frequency of methicillin-resistant Staphylococcus aureus was significantly higher in adults than in children, and more strains of bacteria isolated from adults were sensitive to the antibiotics septrin, vancomycin, and teicoplanin.

Conclusion

Bacteria cultured from children were more susceptible to antibiotics and had higher antibiotic sensitivity than did bacteria cultured from adults.     

The Influence of Incudostapedial Joint Separation on the Middle Ear Transfer Function

Objectives

One of the risks in middle ear surgery is high frequency hearing loss. It is believed that manipulations on the middle ear ossicles with the instruments may cause overstimulation of the inner ear and damage of the hear cells. Controversy arises whether temporary separation of the ossicles has any impact on middle ear transfer function and hearing threshold after surgery. The aim of the study is to evaluate the influence of incudostapedial joint (ISJ) separation on middle ear function in an experimental model.

Methods

With the use of single point laser Doppler vibrometer (LDV) stapes velocity in the intact chain and after ISJ separation was measured in 5 fresh human cadaver temporal bones.

Results

In all cases there was a decrease in stapes velocity after ISJ separation. Mead stapes velocity was reduced for 1 dB in 800 Hz to 9 dB in frequencies above 1,000 Hz. The decrease of velocity was greater in higher frequencies.

Conclusion

Separation of the ISJ does not reduce significantly the middle ear function.     

Clinical approaches for understanding the expression levels of pattern recognition receptors in otitis media with effusion

Objectives

Bacterial infections in the normally sterile environment of the middle ear cavity usually trigger host immune response, whereby the innate immune system plays a dominant role as the host''s first line of defense. We evaluated the expression levels of Toll-like receptors (TLRs) -2, -4, -5, -9, and nucleotide-binding oligomerization domain-containing proteins (NODs) -1 and -2, all of which are related to bacterial infection in pediatric patients with otitis media with effusion (OME).

Methods

The study sample consisted of 46 pediatric patients with OME, all of whom had ventilation tubes inserted. The expression levels of TLR-2, -4, -5, -9, NOD-1 and -2 mRNA in middle ear effusion were assessed by polymerase chain reaction (PCR). Difference of pattern recognition receptors (PRRs) expression level by presence of bacteria, ventilation tube insertion rate, and effusion fluid character was assessed.

Results

All effusion fluid samples collected from patients with OME showed expression of TLR-2, -4, -5, -9, NOD-1, and -2 mRNA by PCR. However, we found no differences among expression levels of PRRs in relation to characteristics of exudates, presence of bacteria, or frequencies of ventilation tube insertion (P>0.05).

Conclusion

Our findings suggest that exudates of OME patients show PRR expressions that are related to the innate immune response regardless of the characteristics of effusion fluid, presence of bacteria in exudates, or frequency of ventilation tube insertion.     

Lack of Chlamydophila pneumoniae and predominance of Alloiococcus otitidis in middle ear fluids of children with otitis media with effusion

Objective

To investigate the presence of Chlamydophila pneumoniae and other bacterial pathogens in middle ear effusion samples obtained from children with otitis media with effusion (OME).

Materials and methods

Twenty-eight children (mean age 7.03; standard deviation 2.18) with OME unresponsive to medical therapy were included in the study. All of the children underwent ventilation tube insertion under general anesthesia. Eighteen patients were bilaterally affected whereas 10 children had unilateral disease. The middle ear fluids (46 samples in total) were collected during ventilation tube insertion, and were evaluated subsequently for the presence of C. pneumoniae and other bacterial pathogens using polymerase chain reaction (PCR).

Results

Although all samples were negative for C. pneumoniae, bacterial DNA was detected in 21 of 46 samples. Overall 40% of the patients (4/10) with unilateral involvement, and 61% of the patients (11/18) with bilateral involvement were positive for bacterial DNA. In 6 patients with bilateral OME bilateral samples were positive, whereas 5 patients with bilateral OME showed only unilateral positivity. According to the results of DNA sequencing analysis, all of the positive samples harbored only one bacterial species. In 12 of 46 samples Alloiococcus otitidis DNA (26%), in 7 Haemophilus influenzae DNA (15%), in one Streptoccoccus pneumoniae DNA (2%) and in one Moraxella catarrhalis DNA (2%) were present.

Conclusions

Our findings support that C. pneumoniae does not seem to have a role in OME in children whereas A. otitidis was found to be more frequent than the other common pathogens. Further studies are required to elucidate the exact pathogenetic role of these microorganisms in OME.     

Mastoid obliteration with silicone blocks after canal wall down mastoidectomy

Objectives

To evaluate the usefulness of silicone blocks as graft material for mastoid cavity obliteration in the prevention of problematic mastoid cavities after canal wall down mastoidectomies.

Methods

Retrospective evaluation of 20 patients who underwent mastoid obliteration with silicone blocks between 2002 and 2009 at the Chonnam National University Hospital. The cases consisted of 17 patients with chronic otitis media with cholesteatoma and 3 patients with adhesive otitis media. The postoperative follow-up period was an average 49 months (range, 6 to 90 months). The surgical technique used at our institution composed four major steps: First, the canal wall down mastoidectomy was performed and the middle ear procedure was completed. The silicone blocks were used to fill up the mastoidectomized cavity. Then, a cortical bone pate was used to cover the surface of the silicone blocks. Finally, temporalis fascia and a split musculoperiosteal flap were used to surround the bone pate for reinforcement of the reconstructed canal wall. We examined postoperative success rate and hearing outcomes.

Results

In 19 cases (95%), the reconstructed canal wall maintained a cylindrical shape and the ear drum healed without perforation. In only 1 case (5%), the reconstructed canal wall was destroyed with ear drum perforation. The mean improvement in air-bone gap was about 12 dB (P<0.05), and the mean improvement in air-conduction was about 16 dB (P<0.05).

Conclusion

We suggest that silicone blocks could be valuable resources as graft materials for mastoid obliteration after canal wall down mastoidectomies.     

Expression of HSP70 and Its Relation with Other Cytokines in Human Middle Ear Effusion

Objectives

While other cytokines are known to be associated with otitis media with effusion (OME), the involvement of heat shock protein 70 (HSP70) in middle ear effusion (MEE) is unknown. This study was undertaken to investigate the possibility of there being a HSP70 expression in human MEE and to determine its potential role as a cytokine in OME.

Methods

The levels of HSP70, tumor necrosis factor-alpha and interleukin-1beta were measured by enzyme-linked immunosorbent assay in the effusion of different groups of OME patient following collection of the MEE using our new collection system. The clinical characteristics of the OME patients and the MEE status were analyzed.

Results

HSP70 was expressed in all the types of MEE. The mucous and seromucous effusions showed higher HSP70 levels than that of the serous effusion. The HSP70 level was correlated with the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the effusions. The positive correlations between HSP70, TNF-α and IL-1β were statistically significant (P<0.05).

Conclusion

The highly elevated level of HSP70 in the seromucous and mucous effusions implicates this protein in the chronicity of OME.     

The Value of Measuring Eustachian Tube Aeration on Temporal Bone CT in Patients with Chronic Otitis Media

Objectives

To evaluate that the cross-sectional area of the air space in the Eustachian tube (ET) on computed tomography (CT) images could be useful for predicting the postoperative aeration of the middle ear.

Methods

The patient group consisted of 80 patients (80 ears) with chronic otitis media and who underwent middle ear surgery from 2006-2007 and who were followed up for more than 1 yr. The control group consisted of 100 ears of 50 individuals with normal tympanic membranes and who underwent CT for other causes (such as tinnitus or hearing loss). The largest cross-sectional areas of the aerated ET were measured on the coronal images of the temporal bone CT by a single otologist using the computer-based "Region of Interest" picture archiving and communications system. The patient group was divided into two subgroups, 1) those with good postoperative aeration and 2) those with poor postoperative aeration. The largest cross-sectional areas of the aerated ETs were compared between the patients and the controls, and between the patients with good aeration and the patients with poor aeration.

Results

The mean cross-sectional areas significantly differed between the patient group and the control group, and between the good and poor aeration subgroups (P<0.05 each). The mean area of the poor aeration subgroup was smaller than that of the control group (P<0.05), but the mean area of the good aeration subgroup did not significantly differ from that of the controls.

Conclusion

The cross-sectional area of the aerated ET, as measured on the preoperative coronal images of temporal bone CT scans, may be useful for predicting the postoperative condition of the tympanic cavity.     

Sequence Variations and Haplotypes of the GJB2 Gene Revealed by Resequencing of 192 Chromosomes from the General Population in Korea

Objectives

Hearing impairment (HI) is the most common sensory deficit in human. The Gap Junction Protein, Beta-2 (GJB2) gene encodes the protein connexin 26, and this gene accounts for up to half of the cases of autosomal recessive nonsyndromic HI. This study was conducted to obtain a set of sequence variations (SVs) of the GJB2 gene among Koreans from the general population for making molecular genetic diagnoses and performing genetic counseling.

Methods

We resequenced the GJB2 gene in 192 chromosomes from 96 adult individuals of Korean descent and who were without a history of hearing difficulty. The data of the SVs was obtained and the haplotypes were reconstructed from the data.

Results

Five SVs were observed, including a novel one (c.558G>A; p.T186T), with the allele frequencies ranging from 0.5% (1/192) to 41% (79/192). The linkage disequilibrium study and haplotype construction showed that some of the SVs are in tight linkage, resulting in a limited number of haplotypes.

Conclusion

We observed SVs of the GJB2 gene with different allele frequencies, and a limited number of haplotypes were constructed. The data from this study can be used as reference data for GJB2-related hearing genetic studies, including studies on the founder effect and population genetics, and this data is particularly relevant to people of East Asian decent.     

Clinical Significance of β-Tricalcium Phosphate and Polyphosphate for Mastoid Cavity Obliteration during Middle Ear Surgery: Human and Animal Study

Objectives

Mastoid obliteration is used to obliterate the mastoid cavity following a mastoidectomy or to prevent the formation of a retraction pocket. This study evaluated the effectiveness of β-tricalcium phosphate and polyphosphate (β-TPP) for mastoid obliteration in middle ear surgeries in prospective human and animal studies.

Methods

Twenty patients with chronic otitis media underwent mastoid obliteration using β-TPP after a intact canal wall mastoidectomy or simple mastoidectomy. The clinical data were prospectively evaluated including: the diagnosis, temporal bone computed tomography (TBCT), otoscopic findings, pure tone audiogram, and complications. In the animal experiment, β-TPP was applied into the right bulla in five rats, and the opposite bulla was used as the control in the non-obliterated state. The skulls of five other rats were drilled out and the holes were obliterated with β-TPP. TBCT were obtained at 3, 6, and 9 months after the obliteration and histologic analysis was done at 3 and 9 months after surgery.

Results

In the human study, fourteen TBCTs were obtained at 12 months after the surgery. All demonstrated no bone resorption in the obliterated mastoids. Among the 15 cases displaying retracted tympanic membranes preoperatively, 11 showed no retraction, 2 showed retraction postoperatively, 1 was lost to follow-up and 1 was a case of postoperative wound infection. Among 20 cases, one case developed a postoperative infection that necessitated a second operation. Sixteen underwent ossiculoplasty; hearing improvements were obtained in 15 cases and 1 case showed decreased hearing. In the animal study, new bone formation without significant bone resorption in the radiologic and histologic findings were noted in both the skull and bulla groups.

Conclusion

Although β-TPP is a foreign material having the possibility of infection, mastoid obliteration with it can be a treatment option in middle ear surgeries to prevent retraction pockets or the recurrence of diseases.     

New morphometric findings in adhesive otitis media: Petroclival angle and eustachian tube-tympanic cavity ventilation angle

Objective

Observations during adhesive otitis media (OM) surgery led us to consider that morphometric variations might cause formation of the middle ear cavity with smaller bony boundaries during developmental process. Among the various factors mentioned in the literature, we thought that angle of the petrous bone relative to the midsagittal line and the eustachian tube-tympanic cavity ventilation angle might be factors in the course of adhesive otitis media.

Methods

Axial computerized tomography scans from 14 patients with adhesive OM and 19 cases (control group) with normal middle ear-tympanic membrane were retrospectively examined in terms of petroclival angle and eustachian tube-tympanic cavity ventilation angle. Measurements of these two angles were compared between the two groups. A correlation was also investigated between these two angles.

Results

The petroclival angle (54.5° vs 62.3°) and the eustachian tube-tympanic cavity ventilation angle (156° vs 162.6°) in the adhesive OM group were narrower than those of controls (p < 0.01). The correlation analysis showed that there was a strong association between two angles (r, 0.803; p < 0.01).

Conclusions

More medially positioning of the petrous bone and acutely angled-eustachian tube-tympanic cavity ventilation axis were observed in this study. It would be reasonable to conclude that these findings might take part in development of adhesive OM. We should also admit that these results do not decline the previous theories that account for development of the adhesive otitis media.     

Decreased Pattern-Recognition Receptor-Mediated Cytokine mRNA Expression in Obese Children With Otitis Media With Effusion

Objectives

To assess innate and humoral immune responses in middle ear effusion of obese pediatric patients with otitis media with effusion (OME).

Methods

We evaluated 219 children with OME, of whom 21 were obese and 198 were non-obese. We compared the expression in middle ear effusion of mRNAs encoding toll-like receptors (TLR) 2, 4, 5, and 9; nucleotide-binding oligomerization domains (NOD) 1 and 2; retinoic acid-inducible gene (RIG)-I; interleukins (IL)-6, -10, and -12; interferon (IFN)-γ; and tumor necrosis factor (TNF)-α mRNAs. We also compared the expression of immunoglobulins IgG, IgA, and IgM and the bacterial detection rate in the two groups.

Results

TLR2-mediated expression of IL-6 mRNA, TLR4-mediated expression of IL-6 and IL-10 mRNA, TLR5-mediated expression of IL-6, IL-10, and TNF-α mRNA, TLR9-mediated expression of IL-6 mRNA, and NOD2-mediated expression of IL-6, IL-12, and TNF-α mRNA were significantly lower in obese than in non-obese children (P<0.05). However, concentrations of IgG, IgA, and IgM in middle ear effusion were lower in obese than in non-obese children, but none of these differences was significant (P>0.05).

Conclusion

Mean body mass index was higher and pattern-recognition receptor-mediated cytokine mRNA expression was lower in obese than in non-obese children with OME.     

Facial Nerve Paralysis in Patients With Chronic Ear Infections: Surgical Outcomes and Radiologic Analysis

Objectives

The purpose of this study was to investigate the clinical features, radiologic findings, and treatment outcomes in patients of facial nerve paralysis with chronic ear infections. And we also aimed to evaluate for radiologic sensitivities on facial canal, labyrinth and cranial fossa dehiscences in middle ear cholesteatomas.

Methods

A total of 13 patients were enrolled in this study. Medical records were retrospectively reviewed for clinical features, radiologic findings, surgical findings, and recovery course. In addition, retrospective review of temporal bone computed tomography (CT) and operative records in 254 middle ear cholesteatoma patients were also performed.

Results

Of the 13 patients, eight had cholesteatomas in the middle ear, while two patients exhibited external auditory canal cholesteatomas. Chronic suppurative otitis media, petrous apex cholesteatoma and tuberculous otitis media were also observed in some patients. The prevalence of facial paralysis in middle ear cholesteatoma patients was 3.5%. The most common involved site of the facial nerve was the tympanic segment. Labyrinthine fistulas and destruction of cranial bases were more frequently observed in facial paralysis patients than nonfacial paralysis patients. The radiologic sensitivity for facial canal dehiscence was 91%. The surgical outcomes for facial paralysis were relatively satisfactory in all patients except in two patients who had petrous apex cholesteatoma and requiring conservative management.

Conclusion

Facial paralyses associated with chronic ear infections were observed in more advanced lesions and the surgical outcomes for facial paralysis were relatively satisfactory. Facial canal dehiscences can be anticipated preoperatively with high resolution CTs.     

Impact of 13-valent pneumococcal conjugate vaccine on otitis media bacteriology

Objectives

To determine the microbiology of otitis media (OM) since the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in February 2010.

Methods

Middle ear effusion from a pediatric Otolaryngology population undergoing pressure equalization tube (PET) placement was obtained and sent for aerobic culture and antibiotic susceptibility testing between August 2012 and April 2013. Vaccination records were obtained and statistical analysis was completed.

Results

During the 8-month period, 236 ears were evaluated, and of those 39 ears were found to have positive cultures. The single nonvaccine Streptococcus pneumoniae (serotype 16) isolate was obtained from a PCV7-only vaccinated patient and was penicillin susceptible. The three most common isolates were Staphylococcus coagulase negative (57%), Haemophilus influenzae (17%), and Moraxella catarrhalis (7%).

Conclusions

This study is the first to assess the bacteriology of OM in a pediatric population undergoing PET placement in the immediate post-PCV13 era. Our study is limited by sample size; however, the lack of S. pneumoniae cultures indicates that PCV13 has had a significant impact on pneumococcal infections during these initial years following licensure.     

Toll-like Receptors 2 and 4 and Their Mutations in Patients with Otitis Media and Middle Ear Effusion

Objectives

Toll-like receptors (TLRs) detect microbial infections and they can directly induce innate host defense responses. TLR 2 has been shown to be primarily involved in the recognition of peptidoglycans and lipoteichoic acid of gram positive bacteria. TLR 4 recognizes lipopolysaccharides and lipoteichoic acids from both gram-negative and gram-positive bacteria. Both mutations lead a reduced capacity to elicit inflammation and they increase the risk for gram-positive and negative infections. This study was performed to investigate the expressions of TLR 2 and 4 and their mutations in patients suffering with otitis media and middle ear effusion.

Methods

Middle ear fluid samples were collected from 40 otitis media effusion (OME) patients who had ventilating tubesinserted. Bacteria in the effusion fluid were detected by standard bacterial culture. The secreted IgG, IgA and IgM were measured by Enzyme-linked immunosorbent assay. TLR 2 and 4 were assessed by performing RT-PCR. The genomic DNA from each patient was isolated from the middle ear fluid samples that were collected from 60 OME patients, and the presence of mutations was determined by performing restriction digestion and DNA sequencing analysis.

Results

Among the 40 middle ear fluid samples, bacteria were detected in 13 middle ear fluid samples. The amounts of IgM, IgA, and IgG were 151.20±60.94 ng/mL, 21.59±7.96 ng/mL and 11.55±16.98 ng/mL, respectively. TLR 2 and 4 were expressed in the middle ear fluid and the expression of TLR 2 was higher than that of TLR 4. However, there was no correlation between the expressions of TLR 2 and 4, and the concentration of immunoglobulin or the presence of bacteria (P>0.05). There ware no mutations of TLR 2 (Arg753Gln, Arg677Trp) and TLR 4 (Asp299Gly, Thr399Ile).

Conclusion

TLR 2 and 4 were expressed in all the middle ear fluid samples of OME, but the mutations of TLR 2 and 4 were not detected. TLR 2 and 4 may play a vital role in the immunological responses of patients with OME.