Endotoxin and tumor necrosis factor-alpha in middle ear effusions in relation to upper airway infection

OBJECTIVES/HYPOTHESIS: This study was performed to elucidate the role of endotoxin and tumor necrosis factor-alpha (TNF-alpha) in the middle ear effusions (MEEs) of children with otitis media with effusion (OME) in relation to the chronicity of the disease and the presence of upper respiratory tract infection (URTI). STUDY DESIGN: In a retrospective study 140 MEEs were collected from 101 children between 2 and 12 years of age, and evaluated for the cytokine TNF-alpha and the lipopolysaccharide endotoxin. The amounts were quantified and correlated with the type of MEE, OME duration, and the presence of URTI. METHODS: Endotoxin levels were measured using a limulus amebocyte lysate assay and TNF-alpha levels were measured with an enzyme-linked immunosorbent assay (ELISA). Means of the different variables were compared using the one-way ANOVA least significance difference test with P<.05. RESULTS: In MEEs classified as mucopurulent (22.8%) both endotoxin and TNF-alpha levels (11.9+/-3 ng/mg total protein and 61.1+/-21 pg/mg total protein, respectively) were significantly higher compared with serous- (23.6%) or mucoid- (53.6%) type effusions. Fifty-five percent of the children who were classified as having chronic OME also had significantly higher amounts of endotoxin and TNF-alpha. The majority of the children (61%) had no URTI, although children with URTI (36%) did also have significantly higher levels of endotoxin and TNF-alpha in their middle ears. CONCLUSIONS: These results indicate that there is a strong correlation between the endotoxin and the TNF-alpha concentration in the middle ear and the type of MEE, the presence of URTI, and the chronicity of the disease.     

Middle ear problems in children hospitalised because of lower respiratory tract infections: A comparison between two cohorts in Burundi and Italy

Objectives

To compare the prevalence of acute otitis media (AOM) and otitis media with effusion (OME) in children hospitalized for acute lower respiratory infections (LRTIs) in Burundi and Italy.

Methods

The study, which was conducted from 1 February to 30 April 2011 at the hospital of Kiremba (Burundi, Africa) and at Paediatric Clinic 1 of the University of Milan (Italy), enrolled patients aged <5 years who were hospitalised because of LRTIs. Upon admission, the children underwent an otological examination (pneumatic otoscopy and tympanometry), and middle ear diseases were compared between the two groups.

Results

A total of 108 children enrolled in Burundi (44 males; median age 17 months) were compared to 108 patients enrolled in Italy (53 males; median age 19 months). About one-third of the children in Burundi (33, 30.6%) had normal middle ears. AOM was never diagnosed, whereas OME was detected in 74 children (68.5%: bilateral in 51, 68.9%, and unilateral in 23, 31.1%). The prevalence of OME decreased with increasing age: it was 86.5% in children aged <12 months, 73.7% in those aged 12–24 months, and 43.8% in those aged >24 months (p < 0.001). There was no difference in the proportion of children with OME in Burundi (68.5%) and Italy (63.9%; p = 0.47). OME was significantly more frequent in the children with pneumonia admitted in Burundi than in the children with pneumonia admitted in Italy (p = 0.004).

Conclusions

In children hospitalized for lower respiratory tract infection, OME is almost as frequently seen in developing countries like Burundi, Africa, as in developed countries like Italy. Follow-up monitoring of these children might be required to assess if OME is just a transient phenomenon.     

Prostaglandin content in human middle ear effusions

Summary The concentrations of prostaglandins (PGs) were measured by a radioimmunoassay in human middle ear effusions (MEEs) obtained from patients with otitis media with effusions. Each sample of MEE was divided into two groups: serous effusions and mucoid ones. The main PG in both the serous and mucoid effusions was TXB₂ (375 and 857pg/ml, respectively), followed by PGE₂ (173 and 459pg/ml, respectively). Smaller quantities of PGD₂ (35 and 64 pg/ml, respectively), PGF_2a (139 and 183 pg/ml, respectively), and 6keto PGF_1a (129 and 201 pg/ml, respectively) were also found in the MEEs. The amounts of each PG in the mucoid effusions were 2 or 3 times higher than those found in the serous effusions, while protein concentrations and the profile of these PGs in the MEEs were almost the same in the mucoid and serous groups. These results suggest that PGs may play an important role as a mediator of the inflammatory responses in the pathogenesis of MEEs.     

Proinflammatory and immunoregulatory cytokines in the middle ear effusions

OBJECTIVE: The recognition of inflammatory mediators in middle ear effusions and their correlation with clinical parameters may allow better understanding of many complex events leading to development of permanent sequelae of otitis media and hopefully help to develop future interventions. The aim of the study was to evaluate the presence and level of proinflammatory interleukin (IL) 1beta, IL-6 and immunoregulatory IL-10 in the middle ear effusions, their mutual correlation and relationship with age, duration of the illness, number of episodes of acute otitis media, and presence of retraction pockets. METHODS: The study included 25 children (41 ears), who had been scheduled for myringotomy with the insertion of tympanostomy tubes due to otitis media with effusion. The interview (duration of the illness, number of episodes of acute otitis media), clinical and audiological examination was conducted according to the developed examination check list. Middle ear effusions (MEE) were collected aseptically, the samples centrifuged and supernatant frozen at a temperature of -80 degrees C. The IL-1beta, IL-6 and IL-10 were assayed using enzyme-linked immunosorbent assay (ELISA) kits incorporating monoclonal antibodies and the ETI system reader. The nonparametric Mann-Whitney U test was used for statistical analysis and Pearson's linear correlation coefficient was calculated. RESULTS: IL-1beta was detected in 80% of examined effusions, IL-6 in 78%, and IL-10 in 88%. There was a strong statistical correlation between IL-1beta and IL-6 concentrations (r=0.627, P=0.000), as well as IL-6 and IL-10 (r=0.66, P=0.000) No statistically significant correlation was found between levels of cytokines and clinical parameters. CONCLUSIONS: Proinflammatory and immunoregulatory cytokines participate in middle ear inflammatory response. The lack of direct correlation between IL-1beta and IL-10 may be responsible for chronic character of the disease. As no correlation between the levels of cytokines and clinical parameters was found it seems that the time of duration of inflammation rather than its intensity measured by levels of cytokines is the main factor in development of middle ear mucosa pathology.     

The effect of middle ear effusions from children on in vitro ciliary activity

Summary In an attempt to clarify the effect of middle ear effusions (MEEs) on ciliary activity, the ciliary beat frequency (CBF) of nasal epithelium from normal subjects was determined following incubation in Hanks' solution containing effusions from pediatric otitis media with effusion. Both serous and mucoid types of effusions increased CBF 15 min after incubation. The mucoid type of effusion was then found to decrease CBF 6 h after incubation. Since the pH and osmotic pressure of the solution containing the effusions were within the physiological ranges for normal ciliary beating, our findings show that MEEs can influence CBF in vitro. Offprint requests to: Y. Sakakura     

Factors associated with positive bacteriological cultures of chronic middle ear effusions

Pathogenic bacteria have been isolated from middle ear effusions in a number of studies. Our aim was to identify factors which predispose to patients having positive cultures. Over a 1-year period, prospective data were collected on patients admitted for myringotomy. Middle ear effusions were collected at the time of surgery using specially designed traps, and underwent microscopy and culture. Data on local weather parameters were obtained from the Meteorological Office in Edinburgh, UK and compared with the patterns of positive cultures. A higher proportion of cultures were positive in October, November and December and in February and May than in the other months of the year. We were not able to relate these variations to any of the meteorological data, with the possible exception of relative humidity, or to any other variable.     

The natural course of otitis media with effusion in preschool children

Summary In order to gain an insight into the natural course of otitis media with effusion (OME), a prospective study was carried out on 1328 children out of a cohort of 1439 preschool children. These children were seen in follow-up for their OME every 3 months, at which times tympanometry was performed. The results show a fairly constant rate of improvement of about 50% every 3 months. The cumulative rate of recurrence of OME was about 50% in the study period. Two-thirds of all OME with flat tympanograms had a duration of less than 3 months. Some factors could be identified as having an effect on the natural history of OME. The season and patients' age and sex were found to be associated with the duration and the recovery rate. Risk factors for recurrences were the same as those already reported in the literature.     

中耳共振频率对鼓室积液的诊断价值

目的:探讨226 Hz静态鼓室图与中耳共振频率诊断鼓室积液的价值,观察两者结合可否提高诊断效能.方法:前瞻性研究对比观察分泌性中耳炎组和对照组,以耳镜检查、纯音测听及GSI-33型中耳分析仪测试结果为标准,将对照组共振频率90%的可信区间(5%～95%)设为正常范围,对分泌性中耳炎组的共振频率进行正常或不正常归类,并对其鼓室图形为B、C型或异型者进行鼓膜穿刺抽液,以抽出液体者为阳性.结果:中耳共振频率在900～1100 Hz之间为正常,分泌性中耳炎组的共振频率明显低于对照组(P＜0.01).B型曲线的抽液阳性率为83%,明显高于C型和异型曲线抽液阳性率(P＜0.05).共振频率正常者,其抽液阳性率为0.≤500 Hz者的鼓室积液阳性率明显高于＞500 Hz者(P＜0.01).结论:通过测试中耳共振频率对判断鼓室是否积液,尤其是对鼓室图形为C型或异型者有重要临床意义.     

The accuracy of otomicroscopy for the diagnosis of paediatric middle ear effusions

Objective

To determine the diagnostic accuracy of otomicroscopy performed by otolaryngology specialists for the diagnosis of paediatric middle ear effusions.

Methods

Prospective study of consecutive children receiving tympanostomy tubes between December 2004 and February 2005 within the Department of Otolaryngology—Head & Neck Surgery, Manukau Surgery Centre, Middlemore Hospital, New Zealand. Eighty-six children were included in the study. Intraoperative otomicroscopic examination of the ears was performed under general anaesthesia by two otolaryngology specialists. The presence or absence of middle ear effusion was predicted prior to myringotomy. Intraoperative myringotomy findings were used as the diagnostic reference standard.

Results

The prevalence of middle ear effusions was 52.6% (71/135 ears). Otomicroscopy had a sensitivity 94.4% (95% CI: 85.5-98.2), and specificity 93.8% (95% CI: 84.0-98.0). The positive predictive and negative predictive values of otomicroscopy for the identification of middle ear effusions were 94.4% (95% CI: 85.5-98.2), and 93.8% (95% CI: 84.0-98.0) respectively. The overall accuracy of otomicroscopy was 94.1%. There was an excellent level of agreement between otomicroscopy and myringotomy findings (kappa = 0.88, 95% CI: 0.80-0.96).

Conclusions

Otomicroscopy performed by the specialist otolaryngologist is an accurate tool for the diagnosis of middle ear effusions. Otomicroscopy performed on the anaesthetised child achieves greater diagnostic accuracy than tympanometry and pneumatic otoscopy.     

Determination of lipid A in human middle ear effusions with the competitive ELISA technique

Summary Lipid A is a covalently bound component of gram-negative bacterial lipopolysaccharides and is the biologically active component in endotoxin. In order to detect and quantitate lipid A in middle ear effusions (MEE) from patients with otitis media with effusion (OME), antisera against lipid A were produced in mice by repeated immunization with conjugates using lipid A from Salmonella minnesota Re595 and bovine serum albumin. As a result, a competitive enzyme-linked immunosorbent assay (ELISA) was developed that was specific for lipid A. From the ELISA results, 10 out of 15 samples from the serous type MEE (mean, 13.57±2.17 g/ml) and 5 out of 6 from the mucoid type MEE (17.73±4.34 g/ml) were positive. These findings indicate that lipid A may contribute to the pathogenesis of both types of human OME.     

Vascular permeability of the middle ear mucosa in otitis media with effusion

Summary Vascular permeability (VP) of the middle ear mucosa (MEM) in chronic otitis media with effusion (OME) was estimated in both pediatric and adult patients by calculating the middle ear effusion (MEE) to serum concentration ratios of albumin and of four protease inhibitors: 1-antitrypsin (1-AT), 1-antichymotrypsin (1-X), inter--trypsin inhibitor (I--I) and 2-macroglobulin (2-M). The levels of albumin and 1-AT in MEE were higher while those of I--I and 2-M were lower than their serum levels in both adult serous and pediatric mucoid groups. There was a negative correlation between molecular weight and the mean value of the ratio (MEE/serum) of the four inhibitors in both serous (r=–0.989) and mucoid (r=–0.924) groups. Vascular permeability of the MEM seems to be variable in both serous and mucoid groups during middle ear inflammation. Selective leakage of proteins by molecular weight appears to occur in MEM. Our findings further indicate that a high level of the high-molecular-weight inhibitor 2-M in MEE may be a significant index reflecting the remarkably enhanced VP of the MEM.     

中耳积液中纤维蛋白原含量与分泌性中耳炎疗效的关系

目的探讨中耳积液中纤维蛋白原含量与分泌性中耳炎病情迁延的关系及巴曲酶治疗分泌性中耳炎的可能机制。方法用凝固法对156例分泌性中耳炎患者治疗过程中的中耳积液中纤维蛋白原含量进行动态检测。鼓室抽液2次（间隔1周），积液复发者随机分成巴曲酶组及地塞米松组，穿刺后分别用0．5ml巴曲酶（2BU／ml）或0．5ml地塞米松（2mg／ml）行鼓室内注射治疗，观察治疗效果。结果积液复发组第1、2次复发中耳积液中纤维蛋白原含量均明显高于痊愈组，且第2次复发者的中耳积液纤维蛋白原含量更高（P〈0．01）。巴曲酶组治疗有效率为91．6％，较地塞米松组（有效率为62．5％）差异有统计学意义（P〈0．001）。治疗后6个月巴曲酶组平均气导听阈值（0．5、1．0、2．0kHz）变化与地塞米松组比较，治疗1周后2组中耳积液中纤维蛋白原含量差异，均有统计学意义（P〈0．01）。结论纤维蛋白原可能在分泌性中耳炎发生发展中起重要作用；鼓室内注射巴曲酶比注射地塞米松治疗分泌性中耳炎，疗效差异有统计学意义，其机制可能是通过降解纤维蛋白原以解除其对咽鼓管表面活性物质的抑制作用或（和）阻止其转变成不溶性纤维蛋白聚合体。     

Direct measurement of middle ear pressure through the eustachian tube

Summary We report a method for measuring middle ear pressure through the eustachian tube. We used a 1-mm-diameter micro-tip catheter pressure transducer (Mikro-tip) and inserted this into the tympanic cavity through the eustachian tube. In preliminary studies, we measured four normal ears, two ears with tubal dysfunction, one ear with a dry perforation and 13 ears with otitis media with effusion (OME). Among those ears with OME, three showed negative middle ear pressure, three slight positive pressure and one normal pressure. These findings suggest that our transtubal method is reliable and useful for measuring middle ear pressure.     

Glucocorticoids reduce nitric oxide concentration in middle ear effusion from lipopolysaccharide induced otitis media

Objective

Otitis media with effusion (OME) is a common childhood disease that is characterized by an accumulation of fluid in the middle ear. Chronic OME can also lead to sensorineural hearing loss (SNHL). Nitric oxide (NO), an inflammatory mediator (IM) of OME, is a free radical known to regulate cell proliferation, cell death, and angiogenesis. Previous studies have shown that nitric oxide may cause SNHL through outer hair cell (OHC) cytotoxicity. This experiment was designed to determine whether glucocorticoids, dexamethasone, fluticasone propionate, or rimexolone, can reduce the concentration of NO in middle ear effusion (MEE).

Methods

Fifty-three chinchillas were divided into 7 groups, vehicle vs. each glucocorticoid at 0.1% and 1.0% concentrations. Due to anesthesia complications, N ranged from 6 to 9 per group. Two hundred microlitres of each test article was injected into the bullae of each animal. Two hours later, lipopolysaccharide (LPS) (0.3 mg in solution) was added. Test articles were re-administered at 24 and 48 h post-LPS induction. After 96 h, animals were euthanized and the MEE was collected.

Results

All three glucocorticoids numerically reduced NO concentration in the middle ear when administered at 0.1%, but only FP showed a significant reduction. At 1.0% concentrations, all 3 steroids significantly reduced NO concentration.

Conclusion

This study suggests that glucocorticoid treatment reduces NO concentration in the MEE and may protect the ear from the SNHL caused by NO.     

Comparison of study designs for acute otitis media trials

BACKGROUND: A framework for evaluating the efficacy of antibiotics in development as well as those currently approved for acute otitis media (AOM) is needed. OBJECTIVE: Review strengths and limitations of various antibiotic trial designs and their outcome measures. METHODS: A review of 157 published trials involving 36,710 subjects for the treatment of AOM. RESULTS: AOM trials have three designs: (1) clinical, clinical diagnosis and assessment of outcomes; (2) single tympanocentesis, microbiologic diagnosis (by middle ear fluid culture) and clinical assessment of outcomes; and (3) double tympanocentesis, microbiologic diagnosis and microbiologic outcome assessment. Identifiable strengths and limitations of each design are reviewed. Case definitions for entry of children in trials of AOM vary widely. The lack of stringent diagnostic criteria in a clinical design allows for inclusion of a significant proportion of children with a non-bacterial etiology (i.e., viral AOM or otitis media with effusion). Tympanocentesis increases diagnostic accuracy at study entry; however, the procedure is confounding because of its potentially therapeutic benefit and the procedure is not performed in a uniform manner. A second tympanocentesis allows a high sensitivity to detect microbiologic eradication, but it does not correlate with clinical outcomes in half of the cases. The timing of outcome assessment also varies widely among trials. CONCLUSIONS: Improved clinical diagnosis criteria for AOM are needed to enhance specificity; emphasis on a bulging tympanic membrane has the best evidence base. Tympanocentesis within study designs has merits. At study entry it assures diagnostic accuracy but may alter outcomes and it is useful to document microbiologic outcomes but lacks specificity for clinical outcomes. For all designs, test of cure assessment 2-7 days after completion of therapy seems most appropriate.     

Ciliary beat frequency of nasal and middle ear mucosa in children with otitis media with effusion

Impaired mucociliary function of respiratory tract mucosa is associated with secretory otitis media in some well recognized syndromes. Ciliary activity per se may now be assessed directly by determination of ciliary beat frequency by a photoelectric technique.^1,2 49 children with otitis media with effusion undergoing surgical treatment were studied. Middle ear mucosa and nasal epithelial cells were obtained by biopsy and cytological brushings respectively at the time of surgery (myringotomy ± grommet insertion under general anaesthesia). From these samples mean nasal ciliary beat frequency was 11.0 Hz and mean middle ear ciliary beat frequency was 11.2 Hz. A positive correlation exists between mean ciliary beat frequency of nasal and middle ear samples from individual patients. A comparison of mean ciliary beat frequency between children who were effusion positive and effusion negative at the time of surgery revealed no statistically significant difference. In addition, no difference existed between those children with recurrent otitis media with effusion and newly presenting cases. No prima facie evidence exists of impaired ciliary function in this population of children with otitis media with effusion.     

Effect of bacterial endotoxin and middle ear effusion on ciliary activity: implications for otitis media

OBJECTIVES/HYPOTHESIS: Otitis media with effusion (OME) is the most common cause of childhood deafness. The pathogenesis is not fully understood, especially the reasons for failure of mucociliary clearance of the middle ear. It is not clear whether the cilia function normally in the middle ear and eustachian tube in the chronic phase of otitis media with effusion. However, impaired ciliary function in primary ciliary dyskinesia is known to be frequently associated with the development of otitis media with effusion. We hypothesized that endotoxin or the bacterial products in middle ear fluid in otitis media with effusion would adversely affect ciliary activity, thereby contributing to the pathogenesis of the disease. STUDY DESIGN: Laboratory-based study of human ciliary activity with reference to otitis media with effusion. METHODS: We have studied the activity of human adenoidal cilia under various conditions. Ciliary activity in the presence of Haemophilus influenzae endotoxin additions (at varying concentrations) to cultured adenoidal explants has been measured. In addition, ciliary activity of these explants was also observed after addition of middle ear effusion aspirated from patients. RESULTS: We have shown that endotoxin in concentrations far in excess of those found in the middle ear with chronic otitis media with effusion had no effect on ciliary activity. Furthermore, ciliary activity was completely unaffected by the presence of middle ear effusion. CONCLUSION: There is no evidence that ciliary activity is reduced by the constituents of middle ear fluid in chronic otitis media with effusion.     

Results of middle ear ventilation with ‘Mangat’ T-tubes

Tympanostomy tube placement has been shown to be an effective treatment for recurrent acute otitis media and chronic otitis media with effusion. The Senior author, (K.S. Mangat), considered stiffness and the longer inner limbs of the Goode (Xomed) or Treace (Treace Medical) T-tubes as important factors in the high incidence of complications, and used smaller soft silicone, Mangat-tube (Xomed) with shorter inner limbs. A prospective study was undertaken over a five year period (July 1987–July 1992) which was a continuation of a previous retrospective study of Goode and Treace T-tubes (Mangat, K.S, Morrison, G.A.J., and Ganiwalla, T.M. (1993) Int. J. Pediatr. Otorhinolaryngol. 25, 119–125). 322 Mangat tubes (M-tubes) were inserted in 191 patients with persistent otitis media with effusion. The peak ages for insertion were between 4 and 6 years. Spontaneous extrusion occurred in 240 ears (66.5%; 154 patients) at a mean time of 29.3 months. Of these, there were 60 perforations at three months follow-up (18.6%) which fell to 31 perforations after six months (9.6%). Surgical extraction of the M-tube was necessary in 82 ears (22.7%; 50 patients) following persistent otorrhoea or resolution of the condition. Otorrhoea, requiring treatment, was noted in 36 ears (11%). No association was found between the occurrence of infection and the incidence of perforation persisting after a year. There was a higher incidence of persistent perforation in those requiring surgical extraction. The overall persistent perforation rate of only 9.6% would appear to be less than that experienced with Goode or Treace T-tubes.     

Magnesium levels in middle ear fluids and sera in otitis media with effusion

Summary Middle ear fluids (MEE) and matched sera (S) were obtained from 50 patients with serous otitis media and magnesium levels were measured to determine if magnesium concentration was distinctly varied in otitis media with effusion (OME). The MEE/S ratio was considerably raised along with transient sensory hearing loss in chronic OME when compared with acute OME. The higher magnesium level found in the MEE implies that it is probably produced locally by the middle ear mucosa and may contribute to the hearing loss found. We also regard the MEE/S ratio as a prognostic factor in OME. Correspondence to: W. L. Yue     

Characteristic differences in the lipid composition of middle ear effusions in adult and pediatric patients: phosphatidylethanolamine and phosphatidylserine levels

Summary Middle ear effusions (MEEs) from adult patients with otitis media with effusion are usually serous in nature, but those from pediatric patients younger than 8 years old are frequently mucous in consistency. MEEs contain substances secreted by the epithelial cells of the middle ear and eustachian tube to regulate surface tensions as well as those produced as the result of inflammation. Since the biochemical bases of serous and mucous MEEs have not been clearly established, we analyzed all lipid components of MEEs from seven children and seven adults by thin-layer and gas-liquid chromatography, and also compared them with those from sera. Although no significant difference in the lipid composition was observed between adult and pediatric sera, the relative concentration of phosphatidylethanolamine (PE) in the pediatric MEEs was 26.5%, while that in the adult MEEs was 9.2% and was significantly different. A similar high concentration of phosphatidylserine (PS) was also observed in the pediatric MEE. Since phospholipids are major components of surfactants secreted from the epithelial cells of the middle ear, significantly high concentrations of both PE and PS as charged phospholipids may be responsible in part for the mucoid characteristics seen in pediatric MEEs.This work was supported in part by a Grant-in-Aid for Acute Profound Deafness from the Ministry of Health and Welfare, Japan Offprint requests to: R. Yabe