Selma P. Wiertsema

Genetic polymorphisms in immunoresponse genes TNFA, IL6, IL10, and TLR4 are associated with recurrent acute otitis media.

OBJECTIVE. Cytokines and other inflammatory mediators are involved in the pathogenesis of otitis media. We hypothesized that polymorphisms in inflammatory response genes contribute to the increased susceptibility to acute otitis media in otitis-prone children. PATIENTS AND METHODS. DNA samples from 348 children with ≥2 acute otitis media episodes, who were participating in a randomized, controlled vaccination trial, and 463 healthy adult controls were included. Polymorphisms in TNFA, IL1B, IL4, IL6, IL10, IL8, NOS2A, C1INH, PARP, TLR2, and TLR4 were genotyped. Genotype distributions in children with recurrent acute otitis media were compared with those in controls. Within the patient group, the number of acute otitis media episodes before vaccination and the clinical and immunologic response to pneumococcal conjugate vaccinations were analyzed. RESULTS. The IL6-174 G/G genotype was overrepresented in children with acute otitis media when compared with controls. In the patient group, TNFA promoter genotypes −238 G/G and −376 G/G and the TLR4 299 A/A genotype were associated with an otitis-prone condition. Furthermore, lower specific anticapsular antibody production after complete vaccination was observed in patients with the TNFA-238 G/G genotype or TNFA-863 A allele carriage. Finally, the IL10-1082 A/A genotype contributed to protection from the recurrence of acute otitis media after pneumococcal vaccination. CONCLUSIONS. Variation in innate immunoresponse genes such as TNFA-863A, TNFA-376G, TNFA-238G, IL10-1082 A, and IL6-174G alleles in the promoter sequences may result in altered cytokine production that leads to altered inflammatory responses and, hence, contributes to an otitis-prone condition.

Molecular surveillance of true nontypeable Haemophilus influenzae: An evaluation of PCR screening assays

Background Unambiguous identification of nontypeable Haemophilus influenzae (NTHi) is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh); however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination. Methodology/Principal Findings Here we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22), Hh (n = 27) or equivocal (n = 11), were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3) and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction. Conclusions/Significance Our data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease.

Toll-like receptor 2 polymorphism is associated with preterm birth

Evidence is increasing for a role of polymorphisms in maternal or fetal innate immune response genes in preterm birth. Toll-like receptors (TLRs) are important receptors in the innate immunity. The genotype distribution of two TLR2 single nucleotide polymorphisms (SNPs) and one TLR4 SNP were determined among 524 neonates and associated with gestational age (GA). Genomic DNA was isolated from prospectively collected blood samples and polymorphisms in TLR2 (T-16934A, RS4696480 and Arg753Gln, RS5743708) and TLR4 (Thr399Ile, RS4986791) were determined using sequence specific primers by PCR. Allele frequencies of two TLR2 SNPs and one TLR4 SNP were analyzed according to prematurity. Analysis among 305 infants, after exclusion of infants born after multiple pregnancy or because of preeclampsia, revealed significantly shorter GAs for infants carrying two polymorphic TLR2 alleles (-16934TA/AA and 753ArgGln/GlnGln) compared with infants carrying one polymorphic and one wild-type allele or two wild-type alleles (median GA 30.6 wk versus 34.1–36.8 wk, respectively, p < 0.02). Carriage of two variant TLR2 alleles potentially leads to aberrant innate immune responses, which may have contributed to very preterm birth.

Predominance of nontypeable Haemophilus influenzae in children with otitis media following introduction of a 3 + 0 pneumococcal conjugate vaccine schedule

In Australia the 7-valent pneumococcal conjugate vaccine (PCV7) is administered at 2, 4 and 6 months of age, with no booster dose. Information on bacterial carriage and the aetiology of recurrent acute otitis media (rAOM) after introduction of PCV7 using the 3+0 schedule is required to evaluate the potential impact of second generation pneumococcal vaccines. We found that 2-4 years after introduction of PCV7 in the National Immunisation Program, nontypeable Haemophilus influenzae (NTHi) was the predominant pathogen isolated from the nasopharynx and middle ear of children with a history of rAOM. Compared with healthy controls (n=81), NTHi and Streptococcus pneumoniae carriage rates were significantly higher in children with a history of rAOM (n=186) (19% vs. 56% p<0.0001 and 26% vs. 41%, p=0.02, respectively). Carriage of PCV7 pneumococcal serotypes was rare, whereas PCV7-related and non-PCV7 serotypes were isolated of 38% of cases and 24% of controls. Serotype 19A was the most common serotype isolated from the nasopharynx and middle ear and accounted for 36% (14/39) of total pneumococcal isolates with reduced susceptibility to cotrimoxazole. Of the 119 children carrying NTHi, 17% of isolates were β-lactamase positive. The scarcity of PCV7 serotypes in children with and without a history of rAOM indicates that the 3+0 PCV7 schedule is preventing carriage and rAOM from PCV7 serotypes. Introduction of new vaccines in Australia with increased pneumococcal serotype and pathogen coverage, including 19A and NTHi, should decrease the circulation of antibiotic-resistant bacteria and reduce the burden of rAOM.

Multi-species bacterial biofilm and intracellular infection in otitis media

BackgroundBacteria which are metabolically active yet unable to be cultured and eradicated by antibiotic treatment are present in the middle ear effusion of children with chronic otitis media with effusion (COME) and recurrent acute otitis media (rAOM). These observations are suggestive of biofilm presence or intracellular sequestration of bacteria and may play a role in OM pathogenesis. The aim of this project is to provide evidence for the presence of otopathogenic bacteria intracellularly or within biofilm in the middle ear mucosa of children with COME or rAOM.MethodsMiddle ear mucosal biopsies from 20 children with COME or rAOM were examined for otopathogenic bacteria (either in biofilm or located intracellularly) using transmission electron microscopy (TEM) or species specific fluorescent in situ hybridisation (FISH) and confocal laser scanning microscopy (CLSM). One healthy control biopsy from a child undergoing cochlear implant surgery was also examined.ResultsNo bacteria were observed in the healthy control sample. In 2 of the 3 biopsies imaged using TEM, bacteria were observed in mucus containing vacuoles within epithelial cells. Bacterial species within these could not be identified and biofilm was not observed. Using FISH with CLSM, bacteria were seen in 15 of the 17 otitis media mucosal specimens. In this group, 11 (65%) of the 17 middle ear mucosal biopsies showed evidence of bacterial biofilm and 12 demonstrated intracellular bacteria. 52% of biopsies were positive for both biofilm and intracellular bacteria. At least one otopathogen was identified in 13 of the 15 samples where bacteria were present. No differences were observed between biopsies from children with COME and those with rAOM.ConclusionUsing FISH and CLSM, bacterial biofilm and intracellular infection with known otopathogens are demonstrated on/in the middle ear mucosa of children with COME and/or rAOM. While their role in disease pathogenesis remains to be determined, this previously undescribed infection pattern may help explain the ineffectiveness of current treatment strategies at preventing or resolving COME or rAOM.

Neutrophil Extracellular Traps and Bacterial Biofilms in Middle Ear Effusion of Children with Recurrent Acute Otitis Media – A Potential Treatment Target

Background Bacteria persist within biofilms on the middle ear mucosa of children with recurrent and chronic otitis media however the mechanisms by which these develop remain to be elucidated. Biopsies can be difficult to obtain from children and their small size limits analysis. Methods In this study we aimed to investigate biofilm presence in middle ear effusion (MEE) from children with recurrent acute otitis media (rAOM) and to determine if these may represent infectious reservoirs similarly to those on the mucosa. We examined this through culture, viability staining and fluorescent in situ hybridisation (FISH) to determine bacterial species present. Most MEEs had live bacteria present using viability staining (32/36) and all effusions had bacteria present using the universal FISH probe (26/26). Of these, 70% contained 2 or more otopathogenic species. Extensive DNA stranding was also present. This DNA was largely host derived, representing neutrophil extracellular traps (NETs) within which live bacteria in biofilm formations were present. When treated with the recombinant human deoxyribonuclease 1, Dornase alfa, these strands were observed to fragment. Conclusions Bacterial biofilms, composed of multiple live otopathogenic species can be demonstrated in the MEEs of children with rAOM and that these contain extensive DNA stranding from NETs. The NETs contribute to the viscosity of the effusion, potentially contributing to its failure to clear as well as biofilm development. Our data indicates that Dornase alfa can fragment these strands and may play a role in future chronic OM treatment.

Trivalent influenza vaccine and febrile adverse events in Australia, 2010: Clinical features and potential mechanisms

INTRODUCTION Increased numbers of children presenting with febrile adverse events following trivalent influenza vaccine (TIV) were noted in Australia in 2010. We describe the epidemiology and clinical features of the adverse events and explore the biological basis for the adverse events using an in vitro model. MATERIALS AND METHODS Children presenting to a tertiary paediatric hospital in 2010 with adverse events within 72 h of TIV were retrospectively reviewed. Demographics, clinical features, physiological variables and outcomes were examined. Plasma cytokine and chemokine levels were examined in a subgroup of children with vaccine-related febrile convulsions. Peripheral blood mononuclear cells of age-matched children were stimulated with different TIV preparations. Inflammatory cytokine and chemokine analysis was performed on cultured supernatants. RESULTS Vaccine-related febrile adverse events were identified in 190 children. Most occurred in healthy children (median age: 1.5 years) within 12 h of vaccination. Twenty-eight (14.7%) required hospital admission. High temperature ≥39.0 °C (101/190; 53%), vomiting (120/190; 63%) and convulsions (38/190; 20%) were common. All children presenting had received Fluvax(®) or Fluvax Junior(®). In the in vitro model, IFN-α, IL-1β, IL-6, IL-10, IP-10 and MIP-1α levels were significantly higher when measured at 6 and 24 h in cultures stimulated with Fluvax(®) compared with alternative 2010 TIV preparations. CONCLUSIONS Numerous febrile adverse events (including febrile seizures) were observed following Fluvax(®) or Fluvax Junior(®) in 2010. Clear differences in cytokine production were observed when peripheral blood mononuclear cells were stimulated with Fluvax(®) compared with alternate TIV preparations. Increased awareness of these potential adverse events is required to ensure earlier detection and prevention in the future.

Association of CD14 promoter polymorphism with otitis media and pneumococcal vaccine responses.

Innate immunity is of particular importance for protection against infection during early life, when adaptive immune responses are immature. CD14 plays key roles in innate immunity, including in defense against pathogens associated with otitis media, a major pediatric health care issue. The T allele of the CD14 C-159T polymorphism has been associated with increased serum CD14 levels. Our objective was to investigate the hypothesis that the CD14 C-159T allele is protective against recurrent acute otitis media in children. The association between the CD14 promoter genotype and the number of acute otitis media episodes was evaluated both retrospectively and prospectively in a cohort of 300 children. Serotype-specific immunoglobulin G (IgG) antibody responses after pneumococcal vaccinations were examined according to CD14 genotype to compare immune responsiveness across genotypes. An age-dependent association was found: compared with that for CC homozygotes aged between 12 to 24 months, TT homozygotes had fewer episodes of acute otitis media (79 versus 41%, respectively; P = 0.004); this relationship was absent in older children. Additionally, TT homozygotes showed higher serotype-specific anti-pneumococcal IgG antibody levels. Our data suggest that genetic variation in CD14, a molecule at the interface of innate and adaptive immune responses, plays a key role in the defense against middle ear disease in childhood and in pneumococcal vaccine responsiveness. These findings are likely to be important to these and other immune-mediated outcomes in early life.

Effect of Combined Pneumococcal Conjugate and Polysaccharide Vaccination on Recurrent Otitis Media With Effusion

BACKGROUND. Otitis media with effusion (OME) is very common during childhood. Because Streptococcus pneumoniae is one of the most common bacterial pathogens involved in OME, pneumococcal vaccines may have a role in the prevention of recurrent OME. OBJECTIVE. We sought to assess the effect of combined pneumococcal conjugate and polysaccharide vaccinations on the recurrence of OME. METHODS. A randomized, controlled trial was performed with 161 children, 2 to 8 years of age, with documented persistent bilateral OME. All subjects were treated with tympanostomy tubes (TTs). One half of the subjects were assigned randomly to additional vaccination with a 7-valent pneumococcal conjugate vaccine 3 to 4 weeks before and a 23-valent pneumococcal polysaccharide vaccine 3 months after tube insertion. Blood samples were drawn at the first vaccination, at the time of TT placement, and 1 and 3 months after the second vaccination. Levels of IgA and IgG serum antibody against the 7-valent pneumococcal conjugate vaccine serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F were measured with enzyme-linked immunosorbent assays. All children were monitored for recurrence of OME for 6 months after spontaneous extrusion of the TTs. RESULTS. The overall recurrence rate of bilateral OME was 50%. Pneumococcal vaccinations induced significant 4.6- to 24.4-fold increases in the geometric means of all conjugate vaccine serotype antibody titers but did not affect recurrence of OME. CONCLUSIONS. Combined pneumococcal conjugate and polysaccharide vaccination does not prevent recurrence of OME among children 2 to 8 years of age previously known to have persistent OME. Therefore, pneumococcal vaccines are not indicated for the treatment of children suffering from recurrent OME.

High detection rates of nucleic acids of a wide range of respiratory viruses in the nasopharynx and the middle ear of children with a history of recurrent acute otitis media

Both bacteria and viruses play a role in the development of acute otitis media, however, the importance of specific viruses is unclear. In this study molecular methods were used to determine the presence of nucleic acids of human rhinoviruses (HRV; types A, B, and C), respiratory syncytial viruses (RSV; types A and B), bocavirus (HBoV), adenovirus, enterovirus, coronaviruses (229E, HKU1, NL63, and OC43), influenza viruses (types A, B, and C), parainfluenza viruses (types 1, 2, 3, 4A, and 4B), human metapneumovirus, and polyomaviruses (KI and WU) in the nasopharynx of children between 6 and 36 months of age either with (n = 180) or without (n = 66) a history of recurrent acute otitis media and in 238 middle ear effusion samples collected from 143 children with recurrent acute otitis media. The co‐detection of these viruses with Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis was analyzed. HRV (58.3% vs. 42.4%), HBoV (52.2% vs. 19.7%), polyomaviruses (36.1% vs. 15.2%), parainfluenza viruses (29.4% vs. 9.1%), adenovirus (25.0% vs. 6.1%), and RSV (27.8% vs. 9.1%) were detected significantly more often in the nasopharynx of children with a history of recurrent acute otitis media compared to healthy children. HRV was predominant in the middle ear and detected in middle ear effusion of 46% of children. Since respiratory viruses were detected frequently in the nasopharynx of both children with and without a history of recurrent acute otitis media, the etiological role of specific viruses in recurrent acute otitis media remains uncertain, however, anti‐viral therapies may be beneficial in future treatment and prevention strategies for acute otitis media. J. Med. Virol. 83:2008–2017, 2011.