Panel 1: Epidemiology and Diagnosis.

Objective To create a literature review between 2011 and June 1, 2015, on advances in otitis media (OM) epidemiology and diagnosis (including relevant audiology studies). Data Sources Electronic search engines (PubMed, EMBASE, and Cochrane Library) with a predefined search strategy. Review Methods Articles with appropriate epidemiologic methodology for OM, including acute mastoiditis and eustachian tube dysfunction. Items included OM worldwide and in high-risk populations, OM-related hearing loss, news in OM diagnostics, prenatal risk factors and comorbidities, postnatal risk factors, genetics, microbiological epidemiology, guidelines, and quality of life. Conclusions Diagnostic evidence and genetic studies are increasing; guidelines are introduced worldwide; and there is evidence of benefit of pneumococcal conjugate vaccines. New risk factors and comordities are identified in the study period, and quality of life is affected in children and their families. Implications for Practice Chronic suppurative OM occurs worldwide and contributes to lifelong hearing loss. Uniform definitions are still lacking and should be provided. An association between HIV and chronic suppurative OM has been found. Tympanometry is recommended for diagnosis, with or without pneumatic otoscopy. Video otoscopy, algorithms, and validated questionnaires may assist clinicians. Childhood obesity is associated with OM. Heritability accounts for 20% to 50% of OM diagnoses. OM-prone children seem to produce weaker immunologic responses to pneumococcal conjugate vaccines. Clinicians tend to individualize treatment without adhering to guidelines.

Emerging Streptococcus pneumoniae Strains Colonizing the Nasopharynx in Children After 13-valent Pneumococcal Conjugate Vaccination in Comparison to the 7-valent Era, 2006-2015.

BACKGROUND: After introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) in the United States in 2000, emergence of replacement serotypes occurred, leading to the introduction of a 13-valent pneumococcal conjugate vaccine (PCV13) in 2010 that contained all PCV7 serotypes plus 6 additional serotypes (1, 3, 5, 6A, 7F and 19A). Here, we describe a 9-year prospective, longitudinal study characterizing Streptococcus pneumoniae strains colonizing the nasopharynx (NP) of young children based on serotype, sequence type (ST) and antibiotic susceptibility during the PCV7 and PCV13 eras. METHODS: NP samples were obtained for pneumococcal identification from prospectively followed children at 6, 9, 12, 15, 18, 24 and 30 months of age. A total of 1072 visits during the PCV7 era (June 2006 to September 2010) and 2044 visits during the PCV13 era (October 2010 to September 2015) were included from 665 children. Serotyping and multilocus sequence typing types of Streptococcus pneumoniae isolates were evaluated along with their antibiotic resistance pattern. RESULTS: A total of 1045 Streptococcus pneumoniae were isolated; 350 during the PCV7 era and 685 during the PCV13 era. The most common serotypes identified during the PCV7 era were 19A and 23B compared with 35B, 23B and 21 in PCV13 era. Serotypes 15A/B/C emerged in equal proportion during the PCV13 era. Serotypes 16 and 20 were only observed in the PCV13 era. NP carriage of 19A persisted 5 years after PCV13 introduction (5% of all isolates). Multilocus ST 199 remained a dominant ST during both the PCV7 and PCV13 eras, and ST558 and ST62 emerged after PCV13. Antibiotic resistance to penicillin, ceftriaxone, cefotaxime, erythromycin, tetracycline and trimethoprim/sulfamethoxazole significantly decreased from the PCV7 to the PCV13 era. CONCLUSIONS: Serotypes 35B, 23B, 21 and 15A/B/C rapidly emerged as NP colonizers in the early PCV13 era. Genetically divergent strains with ST558 and ST62 emerged. Resistance to common antibiotics declined after the introduction of PCV13.

Functional Immune Cell Differences Associated With Low Vaccine Responses in Infants.

BACKGROUND: We sought to understand why some children respond poorly to vaccinations in the first year of life. METHODS: A total of 499 children (6-36 months old) provided serum and peripheral blood mononuclear cell samples after their primary and booster vaccination. Vaccine antigen-specific antibody levels were analyzed with enzyme-linked immunosorbent assay, and frequency of memory B cells, functional T-cell responses, and antigen-presenting cell responses were assessed in peripheral blood mononuclear cell samples with flow cytometric analysis. RESULTS: Eleven percent of children were low vaccine responders, defined a priori as those with subprotective immunoglobulin G antibody levels to ≥66% of vaccines tested. Low vaccine responders generated fewer memory B cells, had reduced activation by CD4(+) and CD8(+) T cells on polyclonal stimulation, and displayed lower major histocompatibility complex II expression by antigen-presenting cells. CONCLUSIONS: We conclude that subprotective vaccine responses in infants are associated with a distinct immunologic profile.

Otitis-prone Children Have Immunologic Deficiencies in Naturally Acquired Nasopharyngeal Mucosal Antibody Response after Streptococcus pneumoniae Colonization.

OBJECTIVES: Acute otitis media (AOM) is the most common pediatric bacterial infection, and stringently defined otitis-prone (sOP) children have immunologic deficiencies. We recently found that nasopharyngeal (NP) colonization by Streptococcus pneumoniae (Spn) elicits a NP mucosal antibody response to vaccine candidate pneumococcal proteins that correlate with protection from AOM in non-sOP (NOP) children. Here, we sought to determine if sOP children experience significantly higher colonization rates with Spn than NOP children, develop lower naturally acquired NP mucosal antibody responses to those same pneumococcal proteins after colonization by Spn, and suffer greater frequency of AOM as a consequence. METHODS: NP samples were collected from 130 NOP and 45 sOP children during 270 healthy visits and 201 AOM visits between 6 and 24 months of age. Spn were identified by standard culture. NP mucosal IgG and IgA levels to vaccine candidate proteins pneumococcal histidine triad protein D, pneumococcal choline binding protein A (PcpA) and pneumolysin D1 were measured by quantitative enzyme-linked immunosorbent assay. RESULTS: sOP children had significantly higher colonization frequency by Spn (P < 0.0001) and significantly lower IgG and IgA levels to all 3 vaccine candidate proteins studied compared with NOP children (all P values <0.05) except IgG to Ply D1 (P = 0.31). Spn colonization in NOP children led to 2-fold to 5-fold increase in mucosal IgG and IgA levels to all 3 proteins (all P values <0.01), whereas Spn colonization in sOP children generally failed to elicit antibody responses (all P values >0.05). PcpA was unique in inducing significant increases in mucosal IgA (P = 0.02). When high mucosal IgG levels to all 3 proteins and IgA to PcpA were measured, they correlated with reduced AOM in sOP children. CONCLUSION: sOP children experience significantly higher colonization rates with Spn, develop lower naturally acquired NP mucosal antibody responses to pneumococcal vaccine candidate proteins pneumococcal histidine triad protein D, PcpA and pneumolysin D1 after colonization by Spn, and suffer greater frequency of AOM if they do not generate high mucosal antibody to the studied proteins.

Familial and microbiological contribution to the otitis-prone condition.

OBJECTIVE: Our group has an ongoing clinical research project investigating the immunology of the otitis-prone (OP) phenotype. In light of evidence that this condition arises from underlying immunological defects, we examined our sample population of stringently defined OP (sOP) children suffering 3 episodes of acute otitis media within 6 months or 4 within a year for a familial association with the sOP phenotype. METHODS: We analyzed the frequency of sOP within and between families and the nasopharyngeal (NP) otopathogen colonization patterns within and between families. RESULTS: The presence of sOP siblings significantly predicted that additional children in the same family would likewise become sOP, with an odds ratio of 3.7 (95% CI 0.77-15.2, 95% lower bound 0.95). We further present evidence for an environmental contribution to this effect by means of prolonged exposure to otopathogens within family units. CONCLUSION: sOP children have a significant familial association. The tendency of siblings to share similar patterns of microbial NP colonization contributes to this association. Further research is necessary to determine whether and to what extent genetics are involved.

Serum antibody response to Moraxella catarrhalis proteins OMP CD, OppA, Msp22, Hag, and PilA2 after nasopharyngeal colonization and acute otitis media in children.

BACKGROUND: There is no licensed vaccine for Moraxella catarrhalis (Mcat), which is a prominent bacterium causing acute otitis media (AOM) in children and lower respiratory tract infections in adults. Nasopharyngeal (NP) colonization caused by respiratory bacteria results in natural immunization of the host. To identify Mcat antigens as vaccine candidates, we evaluated the development of naturally induced antibodies to 5 Mcat surface proteins in children 6-30 months of age during Mcat NP colonization and AOM. METHODS: Human serum IgG against the recombinant Mcat proteins, outer membrane protein (OMP) CD, oligopeptide permease (Opp)A, hemagglutinin (Hag), Moraxella surface protein (Msp)22, and PilA clade 2 (PilA2) was quantitated by using an ELISA assay. RESULTS: There were 223 Mcat NP colonization episodes documented in 111 (60%) of 184 children in the study. Thirty five Mcat AOM episodes occurred in 30 (16%) of 184 children. All 5 Mcat candidate vaccine antigens evaluated stimulated a significant rise in serum IgG levles over time from 6 to 36 months of age (P<0.001), with a rank order as follows: Msp22=OppA>OMP CD=Hag=PilA2. Children with no detectable Mcat NP colonization showed a higher serum IgG level against OppA, Hag, and Msp22 compared to those with Mcat NP colonization (P<0.05). Individual data showed that some children responded to AOM with an antibody increase to one or more of the studied Mcat proteins but some children failed to respond. CONCLUSIONS: Serum antibody to Mcat candidate vaccine proteins OMP CD, OppA, Msp22, Hag, and PilA2 increased with age in naturally immunized children age 6-30 months following Mcat NP colonization and AOM. High antibody levels against OppA, Msp22, and Hag correlated with reduced carriage. The results support further investigation of these vaccine candidates in protecting against Mcat colonization and infection.

Effect of pH and oxygen on biofilm formation in acute otitis media associated NTHi clinical isolates.

OBJECTIVES/HYPOTHESIS: Biofilms occur in animal models of acute otitis media (AOM) and in children with recurrent AOM (rAOM) and chronic otitis media with effusion (OME). We therefore studied the ability of nontypeable Haemophilus influenzae (NTHi) strains from children to form biofilms in vitro under conditions we presumed occurred in the middle ear during AOM, rAOM, and OME. STUDY DESIGN: Evaluate NTHi isolates for biofilm formation across a pH range under aerobic, microaerophilic, and anaerobic conditions. METHODS: Using a crystal violet biofilm assay we studied 12 NTHi pediatric clinical isolates to investigate biofilm formation over a pH range of 4.5 to 10 under aerobic, microaerophilic, and anaerobic conditions. RESULTS: Our findings included: 1) not all clinical NTHi strains form biofilms (75% did); 2) the pH of middle ear fluid collected from AOM (n = 170; age range, 4-36 months), rAOM (n = 54; age range, 7-36 months), and OME (n = 30; age range, 9-60 months) subjects tested immediately after withdrawal was similar (mean = 8.0;range 7.0-9.0); 3) biofilms formed optimally at pH 8.0, a finding that is consistent with previous studies by other investigators; 4) biofilms did not form under aerobic conditions as likely occurs in AOM, whereas under microaerophilic and anaerobic conditions biofilm formation was observed as likely occurs during rAOM and OME. CONCLUSIONS: We concluded that biofilm formation by NTHi does not occur in all strains, occurs best where the pH = 8.0 and in anaerobic conditions as likely occurs in children during rAOM and OME. However, biofilm formation is limited or absent under aerobic conditions as likely occurs during AOM. LEVEL OF EVIDENCE: NA.

Correlation of nasopharyngeal cultures prior to and at onset of acute otitis media with middle ear fluid cultures.

BACKGROUND: We sought to determine if nasopharyngeal (NP) cultures taken at times of healthy visits or at onset of acute otitis media (AOM) could predict the otopathogen mix and antibiotic-susceptibility of middle ear isolates as determined by middle ear fluid (MEF) cultures obtained by tympanocentesis. METHODS: During a 7-year-prospective study of 619 children from Jun 2006-Aug 2013, NP cultures were obtained from 6-30 month olds at healthy visits and NP and MEF (by tympanocentesis) at onset of AOM episodes. RESULTS: 2601 NP and 530 MEF samples were collected. During healthy visits, S. pneumoniae (Spn) was isolated from 656 (31.7%) NP cultures compared to 253 (12.2%) for Nontypeable Haemophilus influenzae (NTHi) and 723 (34.9%) for Moraxella catarrhalis (Mcat). At onset of AOM 256 (48.3%) of 530 NP samples were culture positive for Spn, 223 (42%) for NTHi and 251 (47.4%) for Mcat, alone or in combinations. At 530 AOM visits, Spn was isolated from 152 (28.7%) of MEF compared to 196 (37.0%) for NTHi and 104 (19.6%) for Mcat. NP cultures collected at onset of AOM but not when children were healthy had predictive value for epidemiologic antibiotic susceptibility pattern assessments. CONCLUSIONS: NP cultures at onset of AOM more closely correlate with otopathogen mix than NP cultures at healthy visits using MEF culture as the gold standard, but the correlation was too low to allow NP cultures to be recommended as a substitute for MEF culture. For epidemiology purposes, antibiotic susceptibility of MEF isolates can be predicted by NP culture results when samples are collected at onset of AOM.

Payment analysis of two diagnosis and management approaches of acute otitis media.

We determined the cost of care for 2 diagnosis and management approaches for acute otitis media (AOM) among children 6 to 30 months old. A case-control design was used. Cases included 208 children diagnosed with AOM based on a bulging tympanic membrane (TM) and treated with amoxicillin/clavulanate. Controls (5:1 ratio) included 1020 children with AOM diagnosed not requiring bulging of the TM and treated with amoxicillin. Fewer cases (49%) than controls (69%) were diagnosed with AOM (P < .001), fewer were diagnosed with recurrent AOM or AOM treatment failure (0.34 vs 1.6/child; P < .0001), and fewer had insertion of tympanostomy tubes (6.3% vs 14.8%) due to recurrent AOM (P < .0001). The combined direct payments and indirect costs for management of AOM were $539/case versus $1,023/control. Using Rochester NY payments generalized to the US birth cohort, this case diagnosis and treatment strategy could save $1.008 billion per year.

Nonprotective responses to pediatric vaccines occur in children who are otitis prone.

OBJECTIVE: We recently found that children who experience recurrent otitis media despite individualized care (stringently-defined otitis prone [sOP]) do not develop an antibody response to several vaccine candidate protein antigens expressed by Streptococcus pneumonia (Spn) and Haemophilus influenzae. Here we sought to determine if these same children also failed to develop antibody to routine pediatric vaccinations. STUDY DESIGN: One hundred forty sera collected from children age 6-24 months were analyzed. sOP (n=34) and age-matched non-sOP (n=34) children were assessed for IgG concentrations to diphtheria toxoid, tetanus toxoid, pertussis toxoid, filamentous hemagglutinin, pertactin (DTaP), polio, hepatitis B, H. influenzae type b capsule polyribosyl-ribitol-phosphate (PRP) and Spn capsular polysaccharide conjugate vaccine. RESULTS: IgG protective titers to diphtheria toxoid (P=0.006), tetanus toxoid (P<0.0001), pertussis toxoid (P<0.0001), filamentous hemagglutinin (P=0.001), pertactin (P=0.005), hepatitis B (P<0.0001), polio 3 (P=0.03) and Spn 23F (P=0.01) but not polio 1,2, PRP or Spn 6B, and 14 were decreased in sOP versus non-sOP children using generalized estimating equations. A high percentage of sOP children had nonprotective antibody values that persisted until 24 months of age despite routine boosters. CONCLUSION: sOP children may fail to achieve protective antibody concentrations after several routine vaccinations.

Acute otitis media otopathogens during 2008 to 2010 in Rochester, New York.

BACKGROUND: The otopathogen distribution colonizing the nasopharynx (NP) and causing acute otitis media (AOM) is in flux following the introduction of pneumococcal conjugate vaccine 7 (PCV7) and will continue to change. METHODS: Two hundred seventy-seven children were followed prospectively; tympanocentesis was performed during AOM and 208 NP samples were collected to compare with middle ear fluid (MEF) isolates. Eight hundred sixty-three NP samples were collected at 7 healthy visits between 6 and 30 months of age. All children received PCV7 until April 2010 when it was substituted by PCV13. Multilocus sequence typing was used to speciate Streptococcus pneumoniae. RESULTS: The distribution of otopathogens in the MEF during the study time frame was stable. PCV7 serotypes of pneumococci were virtually absent. The frequency of isolation of S. pneumoniae was 26-36% compared with 28-34% for nontypeable Haemophilus influenzae. Moraxella catarrhalis isolation was less common, 7-18%. The proportion of S. pneumoniae that were penicillin nonsusceptible was stable during the 3 years, 40-52%. All M. catarrhalis and 34% of nontypeable H. influenzae were ß-lactamase producing. NP isolates of otopathogens at onset of AOM included the isolate from the MEF and was dissimilar from the distribution at times of health. Sequence types 320 and 199 of S. pneumoniae expressing serotypes 19A and 15 most often caused AOM. CONCLUSIONS: The otopathogen distribution, antibiotic susceptibility and the diversity of strains within the S. pneumoniae species during 2008 through late 2010 were stable. NP isolation of otopathogens at onset of AOM better reflected, albeit incompletely, likely MEF isolates compared with NP isolates at times of health.

Relationship with original pathogen in recurrence of acute otitis media after completion of amoxicillin/clavulanate: bacterial relapse or new pathogen.

OBJECTIVE: We sought to determine whether recurrent acute otitis media (rAOM) occurring within 30 days of amoxicillin/clavulanate treatment was caused by bacterial relapse or new pathogens. METHODS: Pneumococcal conjugate vaccinated children, age 6-36 months, enrolled in a prospective, longitudinal study experiencing rAOM<1 month after completing amoxicillin/clavulanate therapy were studied. AOM episodes occurred between June 2006 and November 2012. Multilocus sequence typing was used to genotype isolates. RESULTS: Sixty-six children were in the study cohort; 63 otopathogens were recovered from middle ear fluid after tympanocentesis. Nontypeable Haemophilus influenzae (NTHi) accounted for 47% of initial AOMs versus 15% by Streptococcus pneumoniae (Spn), P<0.0001. NTHi accounted for 42% of rAOM versus 24% by Spn (P value=0.04). NTHi was the main otopathogen that caused true bacteriologic relapses (77%). ß-lactamase-producing NTHi and penicillin nonsusceptible Spn were not more common in rAOM than initial AOM infections. Among 21 paired (initial and rAOM events) NTHi isolates genotyped, 13 (61.9%) were the same organism; 1 of 9 (11.1%) of paired Spn isolates was the same (P value=0.017). rAOM occurring within a week of stopping amoxicillin/clavulanate was a different pathogen in 21% of cases, 8-14 days later in 33%, 15-21 days in 41% and 22-30 days in 57% (P=0.04). CONCLUSIONS: In amoxicillin/clavulanate-treated children, NTHi was the main otopathogen that caused true bacteriologic relapses. New pathogens causing rAOM versus persistence of the initial pathogen significantly increased week to week. Neither relapses nor new infections were caused more frequently by ß-lactamase producing NTHi or penicillin nonsusceptible Spn.

Reducing the frequency of acute otitis media by individualized care.

OBJECTIVE: We sought to determine if use of more stringent diagnostic criteria for acute otitis media (AOM) than currently advocated by the American Academy of Pediatrics, tympanocentesis and pathogen-specific antibiotic treatment (individualized care) would result in reducing the incidence of recurrent AOM and consequent tympanostomy tube surgery. METHODS: A 5-year longitudinal, prospective study in Rochester, NY, was conducted from July 2006 to July 2011 involving 254 individualized care children. When this individualized care group developed symptoms of AOM, strict diagnostic criteria were applied and a tympanocentesis was performed. Pathogen resistance to empiric high-dose amoxicillin/clavulanate (80 mg/kg of amoxicillin component) caused a change in antibiotic to an optimized choice. Legacy controls (n = 208) were diagnosed with the same diagnostic criteria by the same physicians as the individualized care group and received the same empiric amoxicillin/clavulanate (80 mg/kg of amoxicillin component) but no tympanocentesis or change in antibiotic. Community control children (n = 1020) were diagnosed according to current American Academy of Pediatrics guidelines and treated with high-dose amoxicillin (80 mg/kg) without tympanocentesis as guideline recommended. RESULTS: 5.9% of children of the individualized care group compared with 14.4% of Legacy controls and 27.3% of community controls became otitis prone, defined as 3 episodes of AOM within a 6-month time span or 4 AOM episodes within a 12-month time span (P < 0.0001). 2.4% of the individualized care group compared with 6.3% of Legacy controls, and 14.8% of community controls received tympanostomy tubes (P < 0.0001). CONCLUSIONS: Individualized care of AOM significantly reduces the frequency of AOM and tympanostomy tube surgery. Use of strict diagnostic criteria for AOM and empiric antibiotic treatment using evidence-based knowledge of circulating otopathogens and their antimicrobial susceptibility profile also produces improved outcomes.

Comparison of amoxicillin/clavulanic acid high dose with cefdinir in the treatment of acute otitis media.

BACKGROUND: 10 days of amoxicillin/clavulanic acid high dose and 5 days of cefdinir have been the preferred first- or second-line antibiotics for treatment of children with acute otitis media (AOM) since 2004, as recommended by the American Academy of Pediatrics in the USA, but no head-to-head comparison study has been done. OBJECTIVE: The purpose of the study was to compare the clinical efficacy of amoxicillin/clavulanic acid high-dose therapy for 10 days with cefdinir therapy for 5 days for AOM at recommended doses. METHODS: This was an investigator-blind trial in young children 6-24 months old with no history of recurrent AOM who were randomly assigned to amoxicillin/clavulanic acid (80 mg/kg/day amoxicillin) or cefdinir (14 mg/kg/day), both in two divided doses. The diagnosis of AOM was based on specific clinical criteria by validated otoscopists at two AOM research centres. The outcome measure for clinical cure was resolution of all symptoms and signs of AOM except for persistence of middle-ear effusion at test-of-cure (TOC) 11-14 days after initiation of antibiotic treatment. Clinical failure was defined as persistence of symptoms and signs of AOM and the need for additional antibiotic therapy. Subjects lost to follow up or who had not taken at least 80% of the prescribed medication were classified as having an indeterminate response. Compliance was monitored using Medical Electronic Monitoring System (MEMS) caps and antibiotic bottle volume measurement at the TOC visit. A logistic regression model was used to estimate the association of age with cure rate. Full interactions in terms of age with treatment were included to estimate any age gradient differential. RESULTS: A total of 330 children (average age 13.1 months) with AOM were studied. At TOC, 256 children had clinical cure, 69 had clinical failure, and 5 were lost to follow-up. High-dose amoxicillin/clavulanic acid-treated children had a better cure rate (86.5%) than cefdinir-treated patients (71.0%; p = 0.001). Cefdinir was correlated with less frequent cure outcomes as children increased in age between 6 and 24 months. The odds ratios for clinical cure per increasing month of age estimated from a logistic regression model for amoxicillin/clavulanic acid high dose and cefdinir treatment groups was 0.992 (95% CI 0.932, 1.056), p > 0.05 and 0.932 (95% CI 0.881, 0.986), p = 0.01. The differences in the odds ratios are significant at p < 0.002, indicating a stable clinical cure rate across the ages of children studied for amoxicillin/clavulanic acid and decreasing clinical cure rates as children increased in age for cefdinir. CONCLUSION: In children with bona fide AOM for whom clinical outcomes are assessed by validated otoscopists, 10 days of high-dose amoxicillin/clavulanic acid is significantly more effective than 5 days of cefdinir as therapy for AOM. Because of the identified age effect (correlated to child weight), higher doses of cefdinir may have led to a different conclusion; 10 days of cefdinir may also have led to a different conclusion.

Nasopharyngeal bacterial interactions in children.

Antimicrobial treatments and vaccines can alter bacterial interactions in the nasopharynx, thereby altering disease processes. To better understand these interactions, we examined colonization rates of 3 respiratory bacterial pathogens among 320 children when healthy and at onset of acute otitis media (AOM). Bacterial interactions were analyzed with a repeated measures logistic regression model. Among healthy children, Streptococcus pneumoniae and Moraxella catarrhalis were synergistically (positively) associated. Colonization with S. pneumoniae when healthy, but not at onset of AOM, was competitively (negatively) associated with Staphylococcus aureus. Among children with AOM, competitive associations were found between Haemophilus influenzae and S. pneumoniae and between H. influenzae and M. catarrhalis; rates of colonization with H. influenzae were higher. Bacterial interactions result in differing pathogen prevalence during periods of health and at onset of AOM. H. influenzae might become a more common cause of AOM among children who receive pneumococcal conjugate vaccine.

Antibody in middle ear fluid of children originates predominantly from sera and nasopharyngeal secretions.

The human middle ear is devoid of any immunocompetent cells in normal mucosa. We sought to determine the source of antibody present in the middle ear of children. Total IgG, IgA, and secretory IgA antibodies were determined by enzyme-linked immunosorbent assay from the nasopharyngeal, middle ear, and serum samples of children with acute otitis media. The two-dimensional gel electrophoresis pattern of the entire array of IgA antibodies in the nasal wash (NW) and middle ear fluid (MEF) was compared from the MEF and NW samples using isoelectric focusing and Western blotting. The total IgG and IgA antibodies in the MEF and NW samples of 137 children were compared. The ratio of IgG to IgA in the MEF was significantly different (P < 0.008) compared to NW because IgA levels were higher and IgG levels lower in NW. The IgG/IgA ratio of MEF resembled serum consistent with transudation to the MEF. Small amounts of secretory IgA were detected in MEF but the electrophoresis patterns of the entire array of IgA antibodies in the MEF and NW were virtually identical in each child evaluated; thus, IgA in MEF derived predominantly from serum and the nasopharynx by reflux via the Eustachian tube. The IgG/IgA antibody levels in the MEF and the same composition of IgA antibody in the MEF and NW identifies the predominant source of antibody in the MEF as a transudate of serum combined with nasal secretions refluxed from the nasopharynx in children.

Antibody response to Streptococcus pneumoniae proteins PhtD, LytB, PcpA, PhtE and Ply after nasopharyngeal colonization and acute otitis media in children.

We prospectively compared serum antibody levels of 5 Streptococcus pneumoniae (Spn) proteins: PcpA PhtD, PhtE Ply and LytB associated with nasopharyngeal (NP) colonization and acute otitis media (AOM) infection in a cohort of 6-30 mo old children. Antigen-specific antibody titers were determined by ELISA. A total of 731 visits among 168 children were studied. There were 301 Spn NP colonization episodes documented in 109 (65%) children and 42 Spn AOM episodes in 34 (20%) children. IgG antibody titers to the 5 proteins were significantly different among children over time (p < 0.001), with a rank order as follows: PcpA > PhtE = PhtD > Ply > LytB Characterization of IgG and IgM acute and convalescent serum antibody levels of Spn AOM infection showed the kinetics of the response differed among children, with the same rank order of antibody levels over time. Individual data showed that some children responded to AOM with an antibody increase to one or more of these Spn proteins but some children failed to respond. We conclude that antibody levels to Spn proteins PcpA PhtD, PhtE, Ply and LytB, all rise over time in children age 6 to 30 mo following natural exposure to Spn after NP colonization and AOM; however, there were significant differences in quantity of antibody elicited among these potential vaccine antigens.

Reduced serum IgG responses to pneumococcal antigens in otitis-prone children may be due to poor memory B-cell generation.

A low level of serum antibody to antigens expressed by Streptococcus pneumoniae has been proposed to explain the susceptibility of children to recurrent episodes of acute otitis media (hereafter, "otitis-prone children"). By use of enzyme-linked immunospot assays, the percentages of memory B cells to pneumococcal protein antigens PhtD, LytB, PcpA, PhtE, and Ply were compared between otitis-prone and non-otitis-prone children at the time of acute otitis media or nasopharyngeal colonization with S. pneumoniae. We found significantly lower percentages of memory B cells to 3 pneumococcal protein antigens (PhtD, PhtE, and Ply) and reduced antigen-specific immunoglobulin G concentrations in otitis-prone children, compared with non-otitis-prone children.

When co-colonizing the nasopharynx haemophilus influenzae predominates over Streptococcus pneumoniae except serotype 19A strains to cause acute otitis media.

Of 368 acute otitis media (AOM) cases among 7-valent pneumococcal conjugate-vaccinated children, 43.5% were colonized by multiple otopathogens in the nasopharynx but only 7.1% experienced polymicrobial AOM. When co-colonization occurred, Haemophilus influenzae predominated over all Streptococcus pneumoniae strains except 19A strains to cause AOM. Haemophilus influenzae and Streptococcus pneumoniae both predominated over Moraxella catarrhalis to cause AOM.

Phylogenetic relatedness and diversity of non-typable Haemophilus influenzae in the nasopharynx and middle ear fluid of children with acute otitis media.

The phylogenetic relationships of non-typable Haemophilus influenzae (NTHi) strains prospectively isolated from healthy children and children with acute otitis media (AOM) were analysed using multilocus sequence typing (MLST). A total of 165 NTHi isolates were collected over a 3.5 year time frame during 2006 through 2009. The strains were tested for ß-lactamase production; 28.5% were positive. Seventy different NTHi sequence types (STs) were identified of which 29 (41.4%) were novel. NTHi strains did not show any phylogenetic grouping or clustering among asymptomatic colonizing strains or strains that caused AOM, or based on ß-lactamase enzyme production. Evaluation of triplets and other siblings over time demonstrated relatively frequent genetic exchanges in NTHi isolates in vivo in a short time frame and subsequent transfer among children in a family. Comparison of the MLST STs isolated at different time points showed that in ~85% of the nasopharynx (NP) colonizations, NTHi strains cleared from the host within 3 months, that sequential colonization in the same child involved different strains in all cases except one, and that NP and middle ear isolates were identical STs in 84% of cases. In this first study of its type to our knowledge, we could not identify predominant MLST types among strains colonizing the NP versus those causing AOM or expressing a ß-lactamase enzyme conferring penicillin resistance in children.