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.

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.

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.

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.

Reduced memory CD4+ T-cell generation in the circulation of young children may contribute to the otitis-prone condition.

BACKGROUND: An explanation for the immunologic dysfunction that causes children to be prone to repeated episodes of acute otitis media (AOM) has long been sought. Poor antibody response has been associated with the otitis-prone condition; however, there is no precise mechanistic explanation for this condition. METHODS: Non-otitis-prone and otitis-prone children with AOM or nasopharyngeal (NP) colonization caused by either Streptococcus pneumoniae or Haemophilus influenzae were compared for pathogen-specific CD4(+) T-helper memory responses by stimulating peripheral blood mononuclear cells using 6 vaccine candidate S. pneumoniae and 3 H. influenzae protein antigens. Samples were analyzed by multi-parameter flow cytometry. RESULTS: Significantly reduced percentages of functional CD45RA(Low) memory CD4(+) T cells producing specific cytokines (interferon γ, interleukin [IL]-2, IL-4 and IL-17a) were observed in otitis-prone children following AOM and NP colonization with either S. pneumoniae or H. influenzae. Immunoglobulin (Ig) G responses to the studied protein antigens were reduced, which suggests that antigen-specific B-cell function may be compromised as a result of poor T-cell help. Staphylococcal enterotoxin B stimulated similar cytokine patterns in memory CD4(+)T cells in both groups of children. CONCLUSIONS: Otitis-prone children have suboptimal circulating functional T-helper memory and reduced IgG responses to S. pneumoniae or H. influenzae after colonization and after AOM; this immune dysfunction causes susceptibility to recurrent AOM infections.

Haemophilus influenzae vaccine candidate outer membrane protein P6 is not conserved in all strains.

An outer membrane protein of nontypeable Haemophilus influenzae (NTHi), P6, is a vaccine candidate because it has been characterized as conserved among all H. influenzae strains. Among 151 isolates from children, age 6 to 30 months, evaluating NTHi nasopharyngeal (NP) and oropharyngeal (OP) colonization and tympanocentesis confirmed acute otitis media we identified 14 strains (9.3%) that had variant protein sequences of P6. One atypical omp P6 isolate had sequence mutations in the binding site of a proposed major antigenic epitope of omp P6 identified by monoclonal antibody 7F3. Eight strains (5.3%) had non-homologous variations in amino acids that could result in significant changes to the protein structure of P6, and 5 other strains had amino acid substitutions at four previously described key residue sites. These results show that NTHi omp P6 is not invariant in its structure among respiratory isolates from children.

Novel type of Streptococcus pneumoniae causing multidrug-resistant acute otitis media in children.

After our recent discovery of a Streptococcus pneumoniae 19A "superbug" (Legacy strain) that is resistant to all Food and Drug Administration-approved antimicrobial drugs for treatment of acute otitis media (AOM) in children, other S. pneumoniae isolates from children with AOM were characterized by multilocus sequence typing (MLST). Among 40 isolates studied, 16 (40%) were serotype 19A, and 9 (23%) were resistant to multiple antimicrobial drugs. Two others had unreported sequence types (STs) that expressed the 19A capsule, and 8 (88%) of the 9 multidrug-resistant strains were serotype 19A, including the Legacy strain with the new ST-2722. In genetic relatedness, ST-2722 belonged to a cluster of reported strains of S. pneumoniae in which all strains had 6 of the same alleles as ST-156. The multidrug-resistant strains related to ST-156 expressed different capsular serotypes: 9V, 14, 11A, 15C, and 19F.

Breast-feeding is associated with a reduced frequency of acute otitis media and high serum antibody levels against NTHi and outer membrane protein vaccine antigen candidate P6.

Nontypeable Haemophilus influenzae (NTHi) causes acute otitis media (AOM) in infants. Breast-feeding protects against AOM and/or nasopharyngeal (NP) colonization; however, the mechanism of protection is incompletely understood. Children with AOM and healthy children were studied according to feeding status: breastfed,breast/formula fed, or formula fed. Cumulative episodes of AOM, ELISA titers of serum IgG antibodies to whole-cell NTHi and vaccine candidate outer membrane protein P6, bactericidal titers of serum and NP colonization by NTHi were assessed. A lower incidence of AOM was found in breast- versus formula-fed children. Levels of specific serum IgG antibody to NTHi and P6 were highest in breast-fed, intermediate in breast/formula fed, and lowest in formula-fed infants. Serum IgG antibody to P6 correlated with bactericidal activity against NTHi. Among children with AOM, the prevalence of NTHi in the NP was lower in breast- versus nonbreast-fed infants. We conclude that breast-feeding shows an association with higher levels of antibodies to NTHi and P6, suggesting that breast-feeding modulates the serum immune response to NTHi and P6. Higher serum IgG might facilitate protection against AOM and NP colonization in breast-fed children.

A comparison of 2 white blood cell count devices to aid judicious antibiotic prescribing.

A low or normal white blood cell (WBC) count is usually associated with viral illnesses. This study evaluated the reliability of a new point-of-care, inexpensive, WBC count device which requires only 10 microL (1 drop) of whole blood from a finger stick to an automated Cell-Dyn counter in a busy office practice setting and assessed its reliability to assist in avoiding antibiotic prescribing. A total of 120 acutely ill children and potential antibiotic recipients were studied from October 2007 to March 2008. The mean WBC count was 7.4x10(9)/L and 8.1x10( 9)/L for the new WBC device and the automated Cell-Dyn counter, respectively. The correlation between the 2 devices was high (r=.988, P=.005). A total of 88 children (73%) did not receive antibiotics and mean WBC was 7.2x10(9)/L. In all, 32 children (27%) received an antibiotic and 1 (3%) returned for a follow-up office visit for the same or a related illness. Of the 88 children with a low blood count who did not receive an antibiotic, 3 (3%) had return visit within 30 days and received an antibiotic. A simple and quick point-of-care WBC count device produces similar results as achievable with a Cell-Dyn counter for total WBCs and may assist in judicious antibiotic prescribing.

Diagnostic inaccuracy and subject exclusions render placebo and observational studies of acute otitis media inconclusive.

BACKGROUND: Diagnostic accuracy and appropriate inclusion/exclusion criteria representative of children at greatest risk is of paramount importance in trials to evaluate placebo or observation as an option for acute otitis media (AOM) management. METHODS: Twelve observational studies spanning the time frame 1958-2005 and 13 natural history studies spanning the time frame 1968-2006 were evaluated for the diagnostic criteria, inclusion criteria, and exclusion criteria applied within the study design. RESULTS: Although a bulging or full tympanic membrane (TM) with effusion is the best indication of a diagnosis of bacterial AOM based on tympanocentesis findings, few observational and natural history studies required a bulging TM. Examination of subject inclusion criteria showed that many subjects did not have AOM but rather had no middle ear disease at all or they had otitis media with effusion. Exclusion criteria of subjects were also remarkable. Frequently children <2 years old were excluded; mean age among the studies reflected an older age group, unlike the true epidemiology of AOM. Otitis prone children, those with severe disease, with a bulging TM, with fever, with a definite need for antibiotics, with recent antibiotic treatment, with recent AOM, or with perforation of the TM were often excluded. CONCLUSIONS: Guidelines and some authorities have overlooked or discounted the importance of the issues of inaccurate diagnosis on study entry, broad inclusion criteria, and the creation of bias in exclusion criteria among placebo/natural history trials in AOM. The current data favoring observation of children with AOM should be reconsidered until better studies are conducted.

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.

Pathogens causing recurrent and difficult-to-treat acute otitis media, 2003-2006.

This study sought to determine the microbiology of recurrent acute otitis media (AOM) and AOM treatment failure (AOMTF) in the context of widespread use of heptavalent pneumococcal conjugate vaccine (PCV7). In this retrospective cohort study, 244 AOM isolates obtained by tympanocentesis during 3 respiratory seasons--2003-2004 (n = 126), 2004-2005 (n = 52), 2005-2006 (n = 66)--from three geographically diverse pediatric populations were compared. Most isolates were from children less than 2 years old, who had received PCV7. For the 3 seasons the proportion of Streptococcus pneumoniae isolates was 35%, 35%, and 46% and for Haemophilus influenzae was 55%, 58%, and 39%, respectively (change in trend, P = .09). A total of 37%, 39%, and 50% of S. pneumoniae were penicillin nonsusceptible (PNSP) and 48%, 67%, and 50% of H. influenzae produced beta-lactamase, respectively. Although H. influenzae remains the most frequently isolated pathogen in children with AOMTF or recurrent AOM, S. pneumoniae that are PNSP are reemerging as important organisms.

Frequency of symptomatic relapses of group A beta-hemolytic streptococcal tonsillopharyngitis in children from 4 pediatric practices following penicillin, amoxicillin, and cephalosporin antibiotic treatment.

The objective was to determine the frequency of early symptomatic relapses following antibiotic treatment for group A beta-hemolytic streptococcal (GABHS) tonsillopharyngitis in children from Rochester, New York; Houston, Texas; Spokane, Washington; and Los Angeles, California (2004--2006). The study included 4278 patients. The proportion with a bacteriologic relapse of GABHS tonsillopharyngitis within 1 to 5 days of completing a 10-day treatment course was 8% (penicillin and bicillin), 6% (amoxicillin), 2% (first-generation cephalosporin), and 1% (second-generation and third-generation cephalosporin; P = .0001); symptomatic relapses occurred within 6 to 20 days after completion of therapy in 16%, 14%, 9%, and 7% of cases (P = .0001). Cases from New York and Washington had higher penicillin or amoxicillin failure rates than cases from Texas and California. The frequency of symptomatic relapses of GABHS tonsillopharyngitis, therefore, differs according to the antibiotic treatment selected; the trend for such relapses being penicillin or amoxicillin > cephalosporins although geographic differences may occur.

Evolving microbiology and molecular epidemiology of acute otitis media in the pneumococcal conjugate vaccine era.

The addition of the 7-valent pneumococcal conjugate vaccine (PCV7) to the routine immunization schedule in the United States for infants has produced a much more favorable impact on the incidence of acute otitis media (AOM) than anticipated. Because the serotypes included in PCV7 were those most frequently expressing antibiotic resistance in 2001, predictions were made that up to 98% of pneumococcal AOM episodes would be caused by penicillin susceptible strains. However, recent studies have shown that the benefits of PCV7 are becoming eroded. Replacement serotypes of pneumococci have emerged, expressing polysaccharide capsules different from those included in PCV7, with increasing frequency. These replacement strains are coming to dominate in the nasopharynx and in AOM isolates (and in invasive disease). Expansion in the isolation of serotypes 3, 7F, 15B/C/F, 19A, 22F, 33F, and 38 has been described in various surveillance systems. Pneumococcal strains expressing non-PCV7 capsular serotypes also appear to be rapidly acquiring resistance to penicillin and other antibiotics. Emergence of strains of pneumococci expressing non-PCV7 capsular serotypes is occurring by multiple mechanisms including capsular switching as suggested by molecular epidemiology studies. Expansion of the number of serotypes included in pneumococcal conjugate vaccines is needed to sustain a long-term benefit from immunization against these bacteria.

The evidence base for cephalosporin superiority over penicillin in streptococcal pharyngitis.

Current treatment guidelines from the Infectious Diseases Society of America, the American Heart Association, and the American Academy of Pediatrics recommend only oral penicillin V or intramuscular benzathine penicillin G as the drugs of choice for treatment of group A beta-hemolytic streptococcal (GABHS) pharyngitis. Ten-day treatment courses with 1st-generation oral cephalosporins or erythromycin are recommended as suitable alternatives in patients who are allergic to penicillin. Despite these recommendations, oral cephalosporins are used as drugs of choice for many patients with GABHS pharyngitis. Simpler and/or short-course regimens of cephalosporins that have been approved by the Food and Drug Administration offer alternatives with the potential for unchanged patient compliance. Increasing cephalosporin use in patients with GABHS pharyngitis has followed from numerous reports and metaanalyses of cephalosporin superiority over penicillin for bacteriologic eradication and clinical response. This review examines the evidence supporting the use of cephalosporins as a first choice of treatment for many patients with GABHS pharyngitis.

Bacterial eradication rates with shortened courses of 2nd- and 3rd-generation cephalosporins versus 10 days of penicillin for treatment of group A streptococcal tonsillopharyngitis in adults.

In a meta-analysis of 5 randomized controlled trials involving 1030 adults, the likelihood of bacteriologic eradication in the treatment of group A beta-hemolytic streptococcal (GAS) tonsillopharyngitis with 5 days of select cephalosporins (cefpodoxime, cefuroxime, cefotiam, and cefdinir) was noninferior to 10 days of penicillin (odds ratio, 1.46; 95% confidence interval, 0.96-2.22, P = 0.08).

Safe use of selected cephalosporins in penicillin-allergic patients: a meta-analysis.

BACKGROUND: Recent analysis of clinical data and a clearer understanding of the role of chemical structure in the development of cross-reactivity indicate that the increased risk of an allergic reaction to a cephalosporin in penicillin-allergic patients is smaller than previously postulated. METHOD: Medline and EMBASE databases were searched with the keywords: cephalosporin, penicillin, allergy, and cross-sensitivity for the years 1960 through 2005. Among 219 articles retrieved, 9 served as source material for this evidence-based meta-analysis. RESULTS: A significant increase in allergic reactions to cephalothin (odds ratio [OR] = 2.5; 95% confidence interval [CI] = 1.1 to 5.5), cephaloridine (OR = 8.7; CI = 5.9 to 12.8), and cephalexin (OR = 5.8; CI = 3.6 to 9.2), and all first generation cephalosporins plus cefamandole (OR = 4.8; CI = 3.7 to 6.2) were observed in penicillin allergic patients; no increase was observed with second generation cephalosporins (OR = 1.1; CI, 0.6 to 2.1) or third generation cephalosporins (OR = 0.5; CI = 0.2 to 1.1). Clinical challenges, skin testing, and monoclonal antibody studies point to the paramount importance of similarities in side chain structure to predict cross-allergy between cephalosporins and penicillins. CONCLUSION: First-generation cephalosporins have cross-allergy with penicillins, but cross-allergy is negligible with second- and third-generation cephalosporins. Particular emphasis should be placed on the role of chemical structure in determining the risk of cross-reactivity between specific agents.

Systematic review of factors contributing to penicillin treatment failure in Streptococcus pyogenes pharyngitis.

OBJECTIVE: Review the evidence for various explanations for microbiologic treatment failure following use of penicillin in group A streptococcal (GAS) tonsillopharyngitis. DATA SOURCE: Systematic review of the literature based on Medline and EMBASE searches, and review of reference lists of included studies. RESULTS: The explanations for penicillin treatment failure in GAS tonsillopharyngitis include 1) carrier state, 2) lack of compliance, 3) recurrent exposure, 4) in vivo copathogenicity of beta-lactamase-producing normal pharyngeal flora, 5) in vivo bacterial coaggregation, 6) poor antibiotic penetration to tonsillopharyngeal tissue, 7) in vivo eradication of normal protective flora, 8) early initiation of antibiotic therapy resulting in suppression of an adequate host immune response, 9) intracellular localization of GAS, 10) GAS tolerance to penicillin, 11) contaminated toothbrushes or orthodontic appliances, and 12) transmission from the family pet. There is very little type I or II evidence to support any of the above-cited explanations for treatment failure in GAS tonsillopharyngitis; available studies are mostly observational (in patients) or laboratory-based without clinical confirmation. CONCLUSION: Multiple explanations have been offered by investigators to explain penicillin treatment failures in GAS tonsillopharyngitis, but the evidence base to support the proposed explanations is generally weak by current standards. Further research is needed to better understand the mechanism(s) of penicillin treatment failure in GAS tonsillopharyngitis.

Symptomatic relapse of group A beta-hemolytic streptococcal tonsillopharyngitis in children.

The frequency of symptomatic relapses following various antibiotic treatments for group A beta-hemolytic streptococcal tonsillopharyngitis was evaluated in 1080 pediatric patients. Within 5 days of completing therapy, the rank-order frequency of treatment failures was (1) penicillin, (2) amoxicillin, (3) first-generation cephalosporins, (4) beta-lactamase stable cephalosporins and amoxicillin-clavulanate ( P = .005). Retreatment of symptomatic failures resulted in another symptomatic relapse more often with penicillin than with cephalosporins (P = .02). Clinicians should be aware that the rate of symptomatic failures after antibiotic therapy for group A beta-hemolytic streptococcal tonsillopharyngitis differs by drug and is not an uncommon event.

Emergence of a multiresistant serotype 19A pneumococcal strain not included in the 7-valent conjugate vaccine as an otopathogen in children.

CONTEXT: Concern has been raised about the possible emergence of a bacterial strain that is untreatable by US Food and Drug Administration (FDA)-approved antibiotics and that causes acute otitis media (AOM) in children. OBJECTIVE: To monitor continuing shifts in the strains of Streptococcus pneumoniae that cause AOM, with particular attention to capsular serotypes and antibiotic susceptibility, following the introduction of a pneumococcal 7-valent conjugate vaccine (PCV7). DESIGN, SETTING, AND PATIENTS: Prospective cohort study using tympanocentesis to identify S pneumoniae strains that caused AOM in children receiving PCV7 between September 2003 and June 2006. All children were from a Rochester, New York, pediatric practice. MAIN OUTCOME MEASURE: Determination of serotypes and antibiotic susceptibility of S pneumoniae causing AOM. RESULTS: Among 1816 children in whom AOM was diagnosed, tympanocentesis was performed in 212, yielding 59 cases of S pneumoniae infection. One strain of S pneumoniae belonging to serotype 19A was a new genotype and was resistant to all antibiotics approved by the FDA for use in children with AOM. This strain was identified in 9 cases (2 in 2003-2004, 2 in 2004-2005, and 5 in 2005-2006). Four children infected with this strain had been unsuccessfully treated with 2 or more antibiotics, including high-dose amoxicillin or amoxicillin-clavulanate and 3 injections of ceftriaxone; 3 had recurrent AOM; and for 2 others, the infection was their first in life. The first 4 cases required tympanostomy tube insertion after additional unsuccessful antibiotic therapies. Levofloxacin was used in the subsequent 5 cases, with resolution of infection without surgery. CONCLUSION: In the years following introduction of PCV7, a strain of S pneumoniae has emerged in the United States as an otopathogen that is resistant to all FDA-approved antibiotics for treatment of AOM in children.