Middle Ear Effusion Samples Research Articles

Bacterial quantification—a necessary complement for the comprehension of middle ear inflammations

Quantification of bacteria in various types of middle ear effusion (MEE) obtained during current acute otitis media (AOM), otitis media with effusion (OME) and chronic suppurative otitis media (COM) was performed. The bacteria were stained with acridine orange and their number per ml effusion evaluated under the fluorescence microscope according to a method described in detail elsewhere. During AOM, 53% of the MEE samples were culture-positive and contained 10 6–10 8 bacteria per ml (median value 10 7 per ml). During OME, serous effusion and 78% of the mucoid effusions contained no bacteria whatsoever, whereas the remaining mucoid effusions contained 10 4–5 × 10 5 bacteria per ml (median value 10 4 per ml). Mucopurulent effusions contained 6 × 10 5–10 8 bacteria per ml (median value 5 × 10 6 per ml). During COM, purulent MEE had 6 × 10 6–10 9 bacteria per ml (median value 10 8 per ml). Quantification of bacteria involved in middle ear diseases provides further information about the etiopathogenesis and appropriate management of various pathological conditions of the middle ear.

Implication of Surfactant Apoprotein in Otitis Media with Effusion

A two-site simultaneous immunoassay using monoclonal antibodies against human surfactant apoprotein (SAP) was used to measure SAP in middle ear effusions (MEEs). In 130 MEE samples from children with otitis media with effusion, SAP was detected in 54 samples (SAP-positive cases, 41.5%). In the remainder, the SAP concentration was below the sensitivity of the immunoassay (SAP-negative cases, 58.5%). A significant difference in periods of observation was found between the SAP-positive cases (17.3 +/- 16.8 months) and the SAP-negative cases (26.2 +/- 22.5 months) (p less than .01). The percentage of positive cases was highest in the serous MEE group (81.2%) and decreased in the purulent MEE group (57%), the mucoid MEE group (30%), and the hyperviscous MEE group (13.6%), in that order. In the purulent MEE group and the mucoid MEE group, the period of observation was significantly shorter in the SAP-positive cases (18.3 +/- 20.4 months and 20.2 +/- 19.4 months) than in the SAP-negative cases (35.9 +/- 24.5 months and 25.4 +/- 18.7 months) (p less than .05). These results suggest that SAP is present in the middle ear cleft and may be a good prognostic predictor of otitis media with effusion in children.

Immunoglobulin-coated bacteria in effusions from secretory and chronic suppurative otitis media

Samples of middle ear effusions from 10 children with secretory otitis media and from 10 children with chronic suppurative otitis media were subjected to qualitative and quantitative bacteriologic analysis. In addition, secretory immunoglobulin A (SlgA)- and IgG-coated bacteria were evaluated using the immunofluorescence technique. Secretory otitis media effusions harbored few, if any, immunoglobulin-coated bacteria, whereas chronic otitis media effusions as a rule had heavily IgG- and SlgA-coated bacteria. However, those chronic otitis media effusions that were culture positive for Pseudomonas aeruginosa had no immunoglobulin-coated bacteria. The effusions of very young children were completely devoid of SlgA-coated bacteria. This study demonstrates that, based on the immunoglobulin coating of bacteria obtained from the middle ear cleft, one can evaluate immunologic response during otitis media.

Occurrences of Antibodies against Streptococcus Pneumoniae, Haemophilus Influenzae and Branhamella Catarrhalis in Middle Ear Effusion and Serum during the Course of Acute Otitis Media

The occurrence of IgG, IgM and IgA class antibodies against a type-specific capsular polysaccharide of Streptococcus pneumoniae (Pn) and against a whole cell antigen of Haemophilus influenzae (Hi) and Branhamella catarrhalis (Br) was studied using the ELISA method on middle ear effusion (MEE) samples of 85 patients and paired serum samples of 40 patients during the course of acute otitis media (AOM). Although specific antibodies to all of these three bacteria appeared in MEE during the course of an AOM episode, antibodies against the infecting bacteria of that particular AOM episode were more often prominent. The antibodies were also detectable in the MEE without simultaneous presence in the serum. The middle ear infection was prolonged more often if specific antibodies to the infecting bacterium could not be detected in the MEE obtained at the beginning of the AOM attack. The present study indicates that AOM caused by Pn, Hi or Br may induce both a systemic and a local production of specific antibodies against the causative organisms during the course of otitis media. The occurrence of such antibodies in MEE seems to play a major role in the resolution of AOM.

Cellular Events in Relation to Bacteria-specific Antibodies in Middle Ear Effusion during Acute Otitis Media

The proportions of neutrophils and lymphocytes, the total number of viable lymphocytes and their spontaneous proliferating activity as well as the proportion of B-cells were determined in 238 middle ear effusion (MEE) samples from 124 ears and in 40 serum samples of 85 children in relation to the presence of bacteria-specific antibodies in MEE and the clinical outcome of acute otitis media (AOM) during the course of AOM. The percentage of lymphocytes was higher, and that of the neutrophils lower, in the ears with bacteria-specific antibodies than in the ears with no antibodies. The higher proportion of MEE lymphocytes and the presence of antibodies correlated to the faster resolution of AOM. Moreover, the total number of viable lymphocytes and the proliferating activity of these cells were related to the presence of specific antibodies in MEE. The findings of this study underline the importance of local mucosal immunity taking place in the middle ear in connection with bacteria-specific antibodies in resting AOM.

Secretory Antibodies Specific to Streptococcus pneumoniae, Haemophilus influenzae and Branhamella catarrhalis in Middle Ear Effusion during Acute Otitis Media

The occurrence of specific secretory antibodies against the type-specific capsular polysaccharide of Streptococcus pneumoniae (Pn) and against the whole cell antigen of Haemophilus influenzae (Hi) and Branhamella catarrhalis (Br) were measured by the ELISA method in 211 middle ear effusion (MEE) samples of 85 children with acute otitis media (AOM) during the course of the disease. Antibodies against at least one of those bacteria were detected at the initial visit in 33.6% of the ears and later in 20%. All in all, such antibodies could be found in 50% of the ears during the follow-up. Pneumococcal secretory antibodies were found in 5 out of 116 ears only, anti-Hi antibodies in 52 and anti-Br antibodies in 42 ears. The specific secretory antibodies were detected against all these bacteria regardless of the bacterial etiology of the AOM attack in question. The AOM attack was prolonged more often if such antibodies were not found in the MEE sample taken at the initial visit. The appearance of such antibodies during the disease seemed to imply termination of the AOM episode in question. The conclusions of this study are that during an AOM attack a local production of antibodies in middle ear against the three most common bacteria. Pn, Hi and/or Br, causing AOM may be induced. The appearance of such antibodies in MEE seems to be beneficial for the resolution of AOM.

Sequential analysis of serum proteins in middle ear effusions

Two otomicroscopically well-defined types of otitis media (OM)--one culture-negative serous (SOM) and the other purulent (POM)--were induced in rats by mechanically obstructing the eustachian tube or by splitting the soft palate. Samples of middle ear effusions (MEE) produced and sera were characterized electrophoretically and immunochemically at 1, 2, 3, 4 and 6 weeks after inducing middle ear fluid. The levels of albumin, transferrin, IgG, IgG1, IgG2, IgM, IgA, and secretory IgA (S-IgA) were determined. All proteins increased 3- to 10-fold in the MEE of the SOM group, whereas the concentrations in the POM group were low. Concentrations of some of the MEE proteins were seen to increase during the course of SOM, while concentrations in the POM group appeared to diminish. The protein content of the MEE in SOM and POM changed dynamically during the observation period. These alterations differed between the two types of OM, reflecting the differing mechanisms that produce the MEE content. These findings indicate that sequential characterization of effusion material in animal OM models may elicit information which might allow staging of this complex disease clinically.

Quantitative analysis of the bacterial findings in otitis media

Qualitative and quantitative bacterial analysis of 200 samples of middle ear effusions collected from patients with current otitis media was performed. When middle ear pathogens (S. pneumoniae, H. influenzae and B. catarrhalis) were found during current acute otitis media or otitis media with effusion infection, the quantity of these bacteria was of the magnitude 10(6)-10(8)/ml and 0-5 x 10(5)/ml effusion material, respectively. Mucopurulent effusion material contained 6 x 10(5)-10(8) bacteria per ml whereas effusion from chronically discharging ears exceeded 10(9) bacteria per ml. Serous effusions did not harbour middle ear pathogens. The appearance of the effusion material was dependent on the number of bacteria involved. Quantification of bacteria in various middle ear effusions offers opportunities to make the diagnosis of various otitis media infections more accurate and readily comparable.

High Performance Liquid Chromatographic Analysis of Amoxicillin in Microliter Volumes of Chinchilla Middle Ear Effusion and Plasma

Abstract A reversed-phase high-performance liquid chromatographic procedure was developed to analyze 75 μ/m1 volumes of chinchilla middle ear effusion and plasma for amoxicillin. The small sample volumes were dictated by the chinchilla model we use to study otitis media and our need to collect multiple samples over an 8-h dosing interval. Amoxicillin was separated on an octylsilane column using methanol-10 mM sodium dihydrogen phosphate-acetonitrile, (88:10:2, v/v), pH 3. Amoxicillin and the internal standard were detected at 230 nm. Middle ear effusion and plasma samples were precipitated with perchloric acid and neutralized prior to injecting 6 μ onto the column. The limit of quantitation in plasma and middle ear effusion was 0.5 μ/m1 (coefficient of variation 14.8% and 18.2%, respectively), and 99% of amoxicillin was recovered.

Effect of Prostaglandin, Leukotriene, and Arachidonic Acid on Experimental Otitis Media with Effusion in Chinchillas

Although previous studies have shown that prostaglandins (PGs), leukotrienes (LTs), and other arachidonic acid (AA) metabolites play an important role in the pathogenesis of otitis media with effusion (OME), the exact role of each AA metabolite in OME is still unknown. The purpose of this study was to examine the effect of several individual AA metabolites alone or in combination and AA itself on experimental otitis media in chinchillas. Normal chinchillas were inoculated daily with normal saline, PGE2, LTC4, LTC4 + PGE2, and AA through the superior bullae over 7 days. Animals were followed by otoscopy and tympanometry, samples of middle ear effusion were collected for biochemical assay, and temporal bones were processed for histopathology. The highest number of ears that developed OME was in the group inoculated with PGE2 + LTC4. The degree of inflammatory change was more pronounced in groups injected with LTC4 than any other group. The findings of this study suggest that eicosanoids PGE2, LTC4, and AA alone or in combination inoculated into the middle ear can induce OME.

Endotoxin and Lysosomal Protease Activity in Acute and Chronic Otitis Media with Effusion

Endotoxin levels and lysosomal protease (collagenase, cathepsin B, and lysozyme) activity were measured in 104 middle ear effusions (MEEs) from patients with otitis media with effusion (OME). The MEE samples were classified into four groups: pediatric serous, mucoid, and acute, and adult serous. Endotoxin levels and lysosomal protease activity in MEEs were significantly different in the following order: adult less than serous less than mucoid less than acute groups, indicating that both endotoxin and lysosomal proteases are more closely related to the pathogenesis of pediatric chronic OME than to adult OME. In pediatric serous and mucoid effusions, endotoxin level had a significant correlation with activity of the lysosomal proteases. In conclusion, endotoxin enhances leukocyte infiltration into the middle ear, and lysosomal proteases released from leukocytes damage the middle ear mucosa and thereby prolong mucosal inflammation, which may be responsible for delayed recovery from acute OME.

Penetration of Enoxacin into Middle Ear Effusion

An oral dose of 400 mg enoxacin was given to each of 19 patients with long-standing secretory otitis media (SOM). A total of 27 samples of middle ear effusion (MEE) and blood were collected at different intervals during a 6-h period after administration of the drug. The concentration of enoxacin in MEE and serum was determined by a microbiological agar-well diffusion method. Enoxacin was found to penetrate rapidly into MEE but was eliminated slowly. The concentration in MEE was about the same at one hour as at 6 h (median value 0.6 mg/l) with a slight peak at 4 h (median value 0.9 mg/l). The peak in blood occurred after one hour (median value 1.8 mg/l), followed by a slow decrease in the concentration during the 6 h studied. The concentration of enoxacin in MEE exceeded the MICs of some (but not all) pathogens usually responsible for infections in chronic otitis media.

Antibodies againststreptococcus pneumoniae, haemophilus influenzaeandbranhamella catarrhalisin middle ear effusion during early phase of acute otitis media

Serum type (IgG, IgM and IgA-class) and secretory type antibodies specific to Streptococcus pneumoniae (Pn), Haemophilus influenzae (Hi) and Branhamella catarrhalis (Br) were measured by enzyme-linked immunosorbent assay (ELISA) in 46 serum and 114 middle ear effusion (MEE) samples from 85 children with acute otitis media (AOM). The samples were obtained within 12 h from the onset of the ear symptoms. Serum (but not secretory) type antibodies to the infecting Pn serotype were found in 24% of the MEE samples of the patients with Pn AOM and, correspondingly, serum and/or secretory type antibodies to Hi and Br were seen in 54% and 63% of the MEE samples of the patients with Hi or Br AOM, respectively. Moreover, antibodies against bacteria other than the causative one could also be found in the MEE. The occurrence of the serum type antibodies against these bacteria in the MEE was closely correlated with their serum levels. The findings of this study indicate that during the very early phase of AOM, the MEE contains both serum type antibodies originating from the serum, and secretory antibodies of middle ear origin. Among them there are antibodies specific to the three most common bacteria causing AOM (Pn, Hi, and Br) regardless of the bacterial etiology of the AOM attack in question.

Beta 2-microglobulin in otitis media with effusion.

beta 2-Microglobulin (beta 2M) is a low-molecular-weight protein present in serum and other fluids during various autoimmune or chronic inflammatory diseases. beta 2-Microglobulin was measured in middle ear effusion (MEE) and serum samples obtained from 36 patients with chronic otitis media with effusion. Using a quantitative competitive enzyme immunoassay, we were able to demonstrate beta 2M in 98% of the MEE samples. The mean concentration of beta 2M was higher in the MEE samples than in the serum samples. There was considerable variability between ears in those patients with bilateral MEE. There was no correlation between beta 2M concentration and the patients' age, sex, MEE type, and culture results, or cytologic profiles of the MEEs. This increased beta 2M may reflect earlier lymphocyte activity during the inflammatory process.

浸出性中耳炎とプロスタグランジン 特に炎症の観点から

The biochemical characteristics of middle ear effusion reflect the inflammatory changes in the middle ear cavity. In this study, prostaglandins (PGE2, TXB2, 6-keto-PGF1α) in middle ear effusions of patients with secretory otitis media were measured by radioimmunoassay (RIA).Fifty samples of middle ear effusion were obtained from 45 patients aged 3 to 76 years who were divided into three groups; 1) children (23 samples; mucoid 19, serous 4), 2) adults (18 samples; mucoid 2, serous 16), and 3) patients with cholesterol granuloma (9 samples).Middle ear effusions in groups 1 and 3 contained higher concentrations of pros taglandins (PGE2, TXB2) than these in group 2. Even in group 2, however, the concentration of PGE2 was higher in the middle ear effusions than in the serum.These results suggest that prostaglandins play an active role in maintaining the inflammatory response in the middle ear, especially in children and in patients with cholesterol granuloma

High-performance liquid chromatographic analysis of trimethoprim and sulfamethoxazole in microliter volumes of chinchilla middle ear effusion and serum

A reversed-phase high-performance liquid chromatographic procedure was developed to analyze 25-μl volumes of chinchilla middle ear effusion and 50-μl volumes of serum for trimethoprim and sulfamethoxazole. The small sample volumes were dictated by the chinchilla model of otitis media and our need to collect multiple samples over a 12-h drug dosing interval. The drugs were separated on a cyanopropylsilane column using acetonitrile-40 mM sodium phosphate, (16:84, v/v), pH 4.8. Trimethoprim and the internal standard were detected at 230 nm while sulfamethoxazole was detected at 250 nm. Middle ear effusion and serum samples were extracted with ethyl acetate-dichloromethane (25:75, v/v). The limit of quantitation was 0.5 μg/ml for sulfamethoxazole and 0.1 μg/ml for trimethoprim (coefficient of variation <20%), the limit of detection 0.25 and 0.05 μg/ml, respectively. Middle ear and serum samples of a chinchilla with experimentally induced otitis media receiving 10 mg/kg trimethoprim and 50 mg/kg sulfamethoxazole intramuscularly were collected over a 12-h period and analyzed. All statistics that validate the analytic method are reported.

Antibody response in experimental Haemophilus influenzae otitis media.

Because Haemophilus influenzae is one of the most common pathogens in otitis media with effusion, we have investigated the antibody response in the serum and middle ear effusion (MEE) in nontypable H influenzae-induced experimental acute otitis media in the chinchilla, using an enzyme-linked immunosorbent assay. During acute otitis media, a reasonable antibody titer was observed: local antibody titers in the MEEs were equal to or greater than those of the serum samples for four of five MEE samples obtained at one week after inoculation. By two weeks, titers in both serum and MEE samples were increased significantly. However, the bacteria persisted in the middle ear cavity, even in the presence of increasing antibody titers, for up to five weeks after infection. The reason for this persistence of the bacteria in the middle ear is unknown.

A cytological study of effusions in children with otitis media with effusion

A cytological analysis in 330 samples of middle ear effusions (MEEs) from the children with otitis media with effusion (OME) was performed with the use of cytocentrifuge method. MayGiemsa stained smears were classified into five types on the bases of the inflammatory cellular patterns, i. e., neutrophil dominant type (N), mononuculear cell dominant type (M), mixed type (Mix), mucous and cellular remnant type (M. CR), and too few cells type (FC). And the cellular types such classified were compared with their clinical data in order to obtain the definitive information about the inflammatory state and process in the middle ear cavity (MIC). The results were as follows:1) A large descrepancy was observed between the global appearance and cytological findings of the MEEs.2) In acute group OME, almost sixty per cent of the MEEs showed (N) type smears. In chronic group, the percentage of (N) type smears were decreased to 23.5% and (M) type, (Mix) type, (M. CR) type, and (FC) type smears were increased to 18.5%, 14.5%, 31.2% and 12.4%, respectively.3) On the other hand, in chronic group MEEs of cases who had a recent episode of acute exaservation showed (N) type smears in 55.6%.4) Long term examination of smears in each case revealed the presence of continuous changes in the cytological patterns.From the results described above, the cytological analysis of MEEs may be one of the useful breakthrough in order to clarify the mechanism of chronicity of OME.

Lysosomal Thiol Proteases (Cathepsin B-like Proteases) in Serous Middle Ear Effusions from Adult Patients

Hydrolytic activity of cathepsins B, H and trypsin-like proteases was measured in 38 serous middle ear effusion (MEE) samples. The concentrations of (alpha 1-AT) and alpha 2-macroglobulin (alpha 2-M) were also quantitated. The mean value of cathepsin B activity was 25.0 +/- 20.7 RFU and that of cathepsin H was 14.3 +/- 3.0--both significantly higher than those in plasma (1.8 +/- 0.4 RFU, 1.2 +/- 0.3 RFU, p less than 0.005). Very low trypsin-like protease activity could be observed. The mean concentrations of alpha 1-AT and alpha 2-M were 368 +/- 94.8 mg/dl and 57.5 +/- 57.3 mg/dl. The bulk of alpha 1-AT in MEEs was occupied by free alpha 1-AT, which can saturate exogenous trypsin. Due to the very low molar concentration of alpha 2-M in MEEs, thiol proteases (mainly cathepsin B) could be a possible major factor inflicting proteolytic injury on the middle ear mucosa and reflecting the severity of the inflammatory process.

Bacterial and Polymorphonuclear Leukocyte Contribution to Middle Ear Inflammation in Chronic Otitis Media with Effusion

Bacteria can be cultured from approximately one third of chronic middle ear effusions, yet the contribution of these bacteria to the pathogenesis of chronic otitis media with effusion (OME) is not clear due to the absence of signs and symptoms of acute infection in most children with this disease. To explore the role of bacteria in chronic OME, lysozyme, lactoferrin, serum complement factors C3 and C5a, and polymorphonuclear leukocyte (PMNL) chemotaxin content was measured in 21 chronic middle ear effusion samples. Concentrations of lysozyme, lactoferrin, and chemotaxin were significantly higher in culture-positive than in sterile effusions. Lysozyme appeared to be contributed by both PMNL and non-PMNL sources in the middle ear space. These non-PMNL sources, presumably middle ear epithelial cells, accounted for 50% to 80% of the lysozyme variation in middle ear effusion. Although C3 and C5a were present in effusion, chemotaxin content correlated poorly with the C3 and C5a content, suggesting that chemotaxins were derived from bacterial peptides rather than from complement activation products. These results suggest that bacteria contribute to chronic middle ear inflammation with effusion. The eradication of bacteria from chronic middle ear effusion might disrupt the host responses which maintain chronic OME.