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Although vaccination has reduced the incidence of Haemophilus influenzae type b, nontypeable H. influenzae and other encapsulated types remain a health threat. Little is known regarding the contemporary molecular epidemiology of these organisms. We conducted multilocus sequence typing on invasive H. influenzae during a period of increasing incidence., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Development of vaccines to prevent disease and death from Streptococcus pneumoniae , and nontypeable Haemophilus influenzae (NTHi), the main pathogens that cause otitis media, pneumonia, meningitis and sepsis, are a global priority. Children living in low and lower-middle income settings are at the highest risk of contracting and dying from these diseases. Improved vaccines with broader coverage are required. Data on the natural development of antibodies to putative vaccine antigens, especially in high-risk settings, can inform the rational selection of the best antigens for vaccine development., Methods: Serum IgG titres to four pneumococcal proteins (PspA1, PspA2, CbpA, and Ply) and five NTHi antigens (P4, P6, OMP26, rsPilA and ChimV4) were measured in sera collected from 101 Papua New Guinean children at 1, 4, 9, 10, 23 and 24 months of age using multiplexed bead-based immunoassays. Carriage density of S. pneumoniae and H. influenzae were assessed by quantitative PCR on genomic DNA extracted from nasopharyngeal swabs using species-specific primers and probes. All data were log-transformed for analysis using Student's unpaired t-tests with geometric mean titre (GMT) or density (GMD) calculated with 95% confidence intervals (CI)., Results: Serum -pneumococcal protein-specific IgG titres followed a "U" shaped pattern, with a decrease in presumably maternally-derived IgG titres between 1 and 4 months of age and returning to similar levels as those measured at 1 month of age by 24 months of age. In contrast, NTHi protein-specific IgG titres steadily increased with age. There was no correlation between antibody titres and carriage density for either pathogen., Conclusion: This longitudinal study indicates that the waning of maternally- derived antibodies that is usually observed in infants, after infants does not occur for NTHi antigens in Papua New Guinean infants. Whether NTHi antigen IgG can be transferred maternally remains to be determined. Vaccines that are designed to specifically increase the presence of protective NTHi antibodies in the first few months of life may be most effective in reducing NTHi disease., Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT01619462., Competing Interests: WP has received funding from Pfizer Australia to attend a conference. AB conducts part-time consultancy work for vaccine companies on projects not related to this study. L-AK has received investigator-initiated research grants, educational grants and travel support from Pfizer, GlaxoSmithKline and Merck, Sharp & Dohme, and is an inventor on patents for a pneumococcal protein vaccine antigen. DL is an investigator on an investigator-initiated research grant that was funded by Pfizer Australia. PR has received nonfinancial support from Pfizer, grants from GlaxoSmithKline and Pfizer, and nonfinancial support from GlaxoSmithKline for work outside the submitted work. The Papua New Guinea Institute of Medical Research received sponsorship from Pfizer Australia to host a national Medical Symposium in 2014. AC is a member of the Seqirus Australia Pneumococcal Advisory Board and the Merck & Co Global Pneumococcal Advisory Board. LB has received grants from GlaxoSmithKline to develop NTHi type IV pilus-derived vaccine antigens. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Martinovich, Rahman, de Gier, Seppanen, Orami, Granland, Francis, Yoannes, Corscadden, Ford, Jacoby, van den Biggelaar, Bakaletz, Cripps, Lehmann, Richmond, Pomat, Kirkham and Thornton.)

Cigarette smoke is a potent proinflammatory trigger contributing to acute lung injury and the development of chronic lung diseases via mechanisms that include the impairment of inflammation resolution. We have previously demonstrated that secondhand smoke (SHS) exposure exacerbates bacterial infection-induced pulmonary inflammation and suppresses immune responses. It is now recognized that resolution of inflammation is a bioactive process mediated by lipid-derived specialized proresolving mediators that counterregulate proinflammatory signaling and promote resolution pathways. We therefore hypothesized that proresolving mediators could reduce the burden of inflammation due to chronic lung infection following SHS exposure and restore normal immune responses to respiratory pathogens. To address this question, we exposed mice to SHS followed by chronic infection with nontypeable Haemophilus influenzae (NTHI). Some groups of mice were treated with aspirin-triggered resolvin D1 (AT-RvD1) during the latter half of the smoke exposure period or during a period of smoking cessation and before infection. Treatment with AT-RvD1 markedly reduced the recruitment of neutrophils, macrophages, and T cells in lung tissue and bronchoalveolar lavage and levels of proinflammatory cytokines in the bronchoalveolar lavage. Additionally, treatment with AT-RvD1 improved Ab titers against the NTHI outer membrane lipoprotein Ag P6 following infection. Furthermore, treatment with AT-RvD1 prior to classically adjuvanted immunization with P6 increased Ag-specific Ab titers, resulting in rapid clearance of NTHI from the lungs after acute challenge. Collectively, we have demonstrated that AT-RvD1 potently reverses the detrimental effects of SHS on pulmonary inflammation and immunity and thus could be beneficial in reducing lung injury associated with smoke exposure and infection., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Background: The widespread administration of the Haemophilus influenzae type b vaccine has led to the predominance of non-typable H. influenzae (NTHi). However, the occurrence of invasive NTHi infection based on gynecologic diseases is still rare., Case Presentation: A 51-year-old Japanese woman with a history of adenomyoma presented with fever. Blood cultures and a vaginal discharge culture were positive with NTHi. With the high uptake in the uterus with 67 Ga scintigraphy, she was diagnosed with invasive NTHi infection. In addition to antibiotic administrations, a total hysterectomy was performed. The pathological analysis found microabscess formations in adenomyosis., Conclusions: Although NTHi bacteremia consequent to a microabscess in adenomyosis is rare, this case emphasizes the need to consider the uterus as a potential source of infection in patients with underlying gynecological diseases, including an invasive NTHi infection with no known primary focus.

Background: The epidemiologic characteristics of invasive Haemophilus influenzae type b disease (HIBD) have markedly changed since the introduction of the Haemophilus influenzae type b (Hib) conjugate vaccine worldwide. The immunization schedule against Haemophilus influenzae type b differs in Europe., Methods: This is a retrospective observational study which evaluates all the data included in the molecular surveillance register for invasive infectious diseases at the Laboratory of Molecular Diagnosis at Meyer Children's University Hospital from December 2008 to December 2018 with a diagnosis of invasive HIBD in children <5 years of age., Results: We identified 4 cases of HIBD: all the cases presented signs or symptoms of invasive infection and the H. influenzae type b was identified in cerebrospinal fluid, or blood or bronchoalveolar lavage by molecular test. The crude incidence for Hib invasive disease in Tuscany is 0.26/100,000 p-y in children younger than 5 years, significantly different from the incidence rate before the introduction of the Hib vaccination. Vaccination effectiveness can be estimated at 97.9% and the impact of hexavalent (2p+1) vaccine at 99.6%., Conclusions: This work confirms the high impact of the hexavalent vaccine 2p+1 schedule for HIBD in children <5 years, emphasizing the role of molecular test for HIBD diagnosis and surveillance.

Haemophilus parasuis is the etiological agent of Glässer's disease which is characterized by fibrinous polyserositis, arthritis and meningitis. The pathogenesis of this bacterium remains largely unknown. Genes expressed in vivo may play an important role in the pathogenicity of H. parasuis. The development of in vivo-induced antigen technology (IVIAT) has provided a valuable tool for the identification of in vivo-induced genes during bacterial infection. In this study, IVIAT was applied to identify in vivo-induced antigens of H. parasuis. Pooled swine H. parasuis-positive sera, adsorbed against in vitro-grown cultures of H. parasuis SH0165 and Escherichia coli BL21 (DE3), were used to screen the inducible expression library of genomic proteins from whole genome sequenced H. parsuis SH0165. Finally, 24 unique genes expressed in vivo were successfully identified after secondary and tertiary screening with IVIAT. These genes were implicated in cell surface proteins, metabolism, stress response, regulation, transportation and other processes. Quantitative real-time PCR showed that the mRNA levels of 24 genes were all upregulated in vivo relative to in vitro, with 13 genes were detected significantly upregulated in H. parasuis infected pigs. Several potential virulence-associated genes were found to be uniquely expressed in vivo, including espP, lnt, hutZ, mreC, vtaA, pilB, tex, sunT and aidA. The results indicated that the proteins identified using IVIAT may play important roles in the pathogenesis of H. parasuis infection in vivo., Competing Interests: Declaration of Competing Interest The authors declare that there are no financial or other relationships that might lead to a conflict of interest. All authors have seen and approved the manuscript., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Moraxella catarrhalis (Mcat) is a frequent pathogen of acute otitis media (AOM) in young children. Here we prospectively assessed naturally-induced serum antibodies to four Mcat vaccine candidate proteins in stringently defined otitis prone (sOP) and non-otitis prone (NOP) children age 6-36months old following nasopharyngeal (NP) colonization, at onset of AOM and convalescence from AOM., Methods: Serum IgG and IgM antibody against recombinant Mcat proteins, oligopeptide permease A (OppA), outer membrane protein (OMP) CD, hemagglutinin (Hag), and PilA clade 2 (PilA2), were quantitated by ELISA., Results: During NP colonization by Mcat all four antigens were immunogenic in both sOP and NOP children. However, sOP children had lower antibody responses than NOP children across age 6-36months, similar to our findings for protein vaccine candidates of Streptococcus pneumoniae (Spn) and Nontypeable Haemophilus influenzae (NTHi). sOP children displayed a later and lower peak of antibody rise than NOP children for all four antigens during NP colonization of Mcat. The age-dependent increase of antibody ranked as OppA>Hag5-9>OMP CD>PilA2 in both sOP and NOP children. Lower serum antibody levels to the Mcat antigens were measured in sOP compared to NOP children at the onset of AOM. We did not find a consistent significant increase of antibody at the convalescence phase after an AOM event., Conclusions: sOP children is a highly vulnerable population that mount lower serum antibody responses to Mcat candidate vaccine proteins compared to NOP children during asymptomatic NP carriage and at onset of AOM., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The clinical features of Guillain-Barré syndrome (GBS) are highly variable, according to the type of antecedent infection. Although a major GBS phenotype, Fisher syndrome (FS), has been shown to be preceded by infections similar to those preceding GBS, whether or not the clinical features in FS also vary according to antecedent infection remains unclarified. Frequent antecedent infections among this study of 70 FS patients included Haemophilus influenzae [n = 15 (21%)], Campylobacter jejuni [n = 10 (14%)], and cytomegalovirus (CMV) [n = 6 (8.6%)]. Compared with other FS patients, H. influenzae-seropositive FS patients more frequently had a history of prior upper respiratory tract infection; double vision as the initial symptom; and, except for oculomotor disturbance, more rarely showed cranial nerve involvement. C. jejuni-related FS occurred predominantly in younger male patients and characteristically presented with blurred vision. According to GBS disability scale, CMV-related FS tended to be more severe, although every patient received immunotherapy. Serum anti-GQ1b IgG antibodies were detected in most cases, regardless of antecedent infection type. At the nadir of illness, the most frequent diagnosis in H. influenzae-related cases was "pure FS" without limb weakness or central nervous system involvement (71%), in C. jejuni-related cases "incomplete FS" such as acute ophthalmoparesis with or without ataxia (60%), and in CMV-related cases (50%) advanced conditions such as GBS overlap and Bickerstaff brainstem encephalitis. These findings indicate that the type of preceding infection determined the neurological features of FS. CMV-related FS appeared to be similar to H. influenzae- and C. jejuni-related FS regarding anti-GQ1b antibody-mediated pathogenesis, as opposed to CMV-related GBS.

Haemophilus influenzae type b (Hib) are one of most dangerous microbes that occupies the paediatric nasopharyngeal as a commensal opportunistic bacterium, which may lead to meningitis in uncontrolled infection. Colonisation of pharyngeal tissues is the starting point for most H. influenzae infections, which may develop into invasive diseases, such meningitis. The vaccination against Hib in specific, as well as against most of vaccines preventable diseases; in general, play a major role in reducing children (< 5 years old) Hib meningitis from 57/100,000 to the lowest known Hib meningitis incidents in the history. First invented Hib vaccine was licensed in 1985 and contained Hib capsular polysaccharide (CPS); afterward, conjugate vaccines have been innovated and licensed on the road to improve Hib vaccine efficacy. Polyribosylribitol phosphate (PRP) is the main vaccine unite structure. Since anti-CPS antibodies in the serum reflect the extent of the acquired immunity against Hib infections, the concentration of ⩾ 0.15 g/ml of anti-CPS is believed to be an indicator for short-term protection from invasive Hib diseases, whereas one-month post-completion of primary Hib immunization concentration of ⩾ 1.0 g/ml is trusted to be immunological protective. As considered that serum anti-CPS antibodies are effectively linked to protection, the evaluation of antibodies concentration and reconsideration of published worldwide populations antibodies concentration are consider vital strides on the way to accurate valuation of Hib immunity that induced by vaccination; either direct or herd. As documented, some populations; worldwide, still susceptible to invasive Hib infections. Several populations worldwide remain vulnerable to Hib-related infections. We believe that up-to-date review article regarding circulated Hib immunology, represented in anti-Hib antibodies and worldwide Hib incidences will provide a precious information for microbiologists, public health officials, epidemiologists, immunologists, and strategic preventive healthcare executives.

Pathogens such as non-typeable Haemophilus influenzae (NTHi) evade the immune system by presenting host-derived sialic acids. NTHi cannot synthesize sialic acids and therefore needs to utilize sialic acids originating from host tissue. Here we report sialic acid-based probes to visualize and inhibit the transfer of host sialic acids to NTHi. Inhibition of sialic acid utilization by NTHi enhanced serum-mediated killing. Furthermore, in an in vitro model of the human respiratory tract, we demonstrate efficient inhibition of sialic acid transfer from primary human bronchial epithelial cells to NTHi using bioorthogonal chemistry., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

H. haemolyticus is often misidentified as NTHi due to their close phylogenetic relationship. Differentiating between the two is important for correct identification and appropriate treatment of infective organism and to ensure any role of H. haemolyticus in disease is not being overlooked. Speciation however is not completely reliable by culture and PCR methods due to the loss of haemolysis by H. haemolyticus and the heterogeneity of NTHi. Haemophilus isolates from COPD as part of the AERIS study (ClinicalTrials - NCT01360398) were speciated by analysing sequence data for the presence of molecular markers. Further investigation into the genomic relationship was carried out using average nucleotide identity and phylogeny of allelic and genome alignments. Only 6.3% were identified as H. haemolyticus. Multiple in silico methods were able to distinguish H. haemolyticus from NTHi. However, no single gene target was found to be 100% accurate. A group of omp2 negative NTHi were observed to be phylogenetically divergent from H. haemolyticus and remaining NTHi. The presence of an atypical group from a geographically and disease limited set of isolates supports the theory that the heterogeneity of NTHi may provide a genetic continuum between NTHi and H. haemolyticus.

During the last two decades, Haemophilus influenzae serotype a (Hia) emerged as an important cause of invasive disease in Canadian First Nations and Inuit, and Alaskan Native populations, with the highest rates reported in young children. Immunocompetent adults, in contrast to children, do not typically develop invasive Hia disease. To clarify factors responsible for an increased burden of invasive Hia disease in certain population groups we studied serum bactericidal activity (SBA) against Hia and quantified IgG and IgM specific to Hia capsular polysaccharide in healthy adult members of two First Nations communities: 1) with reported cases of invasive Hia disease (Northern Ontario, NO), and 2) without reported cases (Southern Ontario, SO), in comparison to non-First Nations living in proximity to the NO First Nations community, and non-First Nations elderly non-frail Canadians from across the country (total of 110 First Nations and 76 non-First Nations). To elucidate the specificity of bactericidal antibodies, sera were absorbed with various Hia antigens. Naturally acquired SBA against Hia was detected at higher rates in First Nations (NO, 80%; SO, 96%) than non-First Nations elderly Canadians (64%); the SBA titres in First Nations were higher than in non-First Nations elderly Canadians (P<0.001) and NO non-First Nations adults (P>0.05). Among First Nations, SBA was mediated predominantly by IgM, and by both antibodies specific to Hia capsular polysaccharide and lipooligosaccharide., Conclusions: The SBA against Hia is frequently present in sera of First Nations adults regardless of the burden of Hia disease observed in their community; it may represent part of the natural antibody repertoire, which is potentially formed in this population under the influence of certain epigenetic factors. Although the nature of these antibodies deserves further studies to understand their origin, the data suggest that they may represent important protective mechanism against invasive Hia disease., Competing Interests: The authors have declared that no competing interests exist.

We studied the epidemiology of Haemophilus influenzae type b infections among children with cancer admitted to Canadian pediatric hospitals. From 1991 to 2014, 13 cases among children with cancer were identified through active surveillance. Average age was 6.7 years. Six of 7 cases eligible for infant immunization were age-appropriately immunized (vaccine failures). Children with cancer may benefit from booster Hib immunization.

Haemophilus influenzae type b (Hib) conjugate vaccines have drastically reduced disease incidence worldwide. Protection against Hib infection has relied on the serum bactericidal activity (SBA) of antibodies to the Hib capsular polysaccharide (polyribosylribitol phosphate). However, licensure usually relies on measuring induction of antibodies to PRP as a surrogate for SBA. In a phase III clinical trial we compared a PRP-conjugate vaccine using the nontoxic diphtheria toxin mutant, CRM 197 , as carrier protein with the licensed tetanus toxoid conjugate when administered subcutaneously as a three dose primary series in Japanese infants. As an addition to the phase III study, we have now evaluated SBA and show PRP-CRM 197 induces higher levels of SBA than PRP-T four weeks after the primary series, with a statistically significant correlation with anti-PRP titers. This data confirms the superior immunogenicity of PRP-CRM 197 compared with PRP-T assessed as SBA following a three-dose primary series by subcutaneous administration. Clinical trial registry: Registered on ClinicalTrials.gov (NCT01379846)., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Haemophilus influenzae remains a common cause of illness in children throughout the world. Before the introduction of vaccination, H influenzae type b (Hib) disease was the leading cause of bacterial meningitis in young children and a frequent cause of pneumonia, epiglottitis, and septic arthritis. Clinicians should remain diligent in counseling parents on the dangers of Hib and provide vaccination starting at 2 months of age. The epidemiology of invasive H influenzae disease is shifting. It is imperative that clinicians recognize the changing epidemiology and antibiotic resistance patterns for H influenzae to optimize care in hospital and ambulatory settings., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objectives: Haemophilus influenzae is a human-specific Gram-negative coccobacillus responsible for a significant number of respiratory tract infections and severe invasive infections such as meningitis and sepsis. The purpose of this study was to characterise the mechanisms of β-lactam resistance among Polish H. influenzae isolates and to evaluate the resistance detection methods applied., Methods: This study was conducted on 117 Polish H. influenzae isolates collected in 2012. Minimum inhibitory concentrations were assessed by broth microdilution. All strains were evaluated using the disk diffusion method and the algorithm proposed by the Nordic Committee on Antimicrobial Susceptibility Testing (NordicAST). To detect changes in penicillin-binding protein 3 (PBP3), PCR screening was performed, followed by ftsI gene sequencing., Results: Neither β-lactamase production nor PBP3 alterations were demonstrated in 76 isolates (65.0%). Susceptibility to ampicillin, amoxicillin, amoxicillin/clavulanic acid, cefuroxime (intravenous) and ceftriaxone was observed in 70.9%, 78.6%, 98.3%, 82.9% and 100% of the isolates, respectively. β-Lactamase production characterised 21 isolates (17.9%). Screening PCR identified 20 isolates (17.1%) with PBP3 alterations, and according to subsequent ftsI sequencing all these strains were finally recognised as gBLNAR (genetically β-lactamase-negative, ampicillin-resistant), among which 65.0% were ampicillin-resistant. According to molecular classification of PBP3 alterations, 95.0% of gBLNAR belonged to group II, representing four subgroups IIa-IId., Conclusions: Haemophilus influenzae resistance to antibiotics requires continuous attention, effective detection methods and a rational policy of antibiotic usage. The algorithm proposed by NordicAST can be applied in routine laboratory work, whereas sequencing of the ftsI gene may be useful in molecular epidemiology studies., (Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Background: C-reactive protein (CRP) measurement has proven valuable for detecting exacerbations, but its usefulness in predicting etiology remains controversial. Likewise, its potential value as a marker of severity, which is well established in patients with pneumonia, remains unproven in chronic obstructive pulmonary disease (COPD) exacerbations., Methods: A cohort study of 118 patients with severe COPD and acute infectious exacerbations were included and followed up over 1 year. Episodes of exacerbations meeting Anthonisen's criteria type I-II were evaluated, analyzing the etiology and inflammatory response as measured by CRP in blood., Results: A total of 380 episodes were recorded. Full microbiological analysis was available in 265 samples. Haemophilus influenzae was the most commonly isolated bacteria and rhinovirus the most common virus. Median CRP levels from the 265 episodes were higher in the cases with positive cultures for bacteria (58.30 mg/L, interquartile range [IQR] 21.0-28.2) than in episodes only positive for viruses (37.3 mg/L, IQR 18.6-79.1) and cases negative for any microorganism (36.4 mg/L, IQR 10.8-93.7) ( P <0.014). H. influenzae and Streptococcus pneumoniae reached the highest CRP levels of 74.5 mg/L (IQR 23.9-167.9) and 74.1 mg/L (IQR 42.0-220.7), respectively. In the 380 exacerbations studied, 227 (~60%) were community-managed, while 153 (~40%) required hospital admission. In the multivariate analysis to assess the influence of inflammatory response on exacerbation severity, baseline hypercapnia (odds ratio [OR]: 2.70, 95% confidence interval [CI]: 1.46-4.9) and CRP levels >100 mg/L (OR: 4.23, 95% CI: 2.12-8.44) were independent predictors after adjustment for baseline characteristics., Conclusion: CRP level was higher in bacterial infections, especially when H. influenzae and S. pneumoniae were isolated. CRP values >100 mg/L were associated with a fourfold increased risk of hospital admission. Therefore, CRP blood levels may be a useful biomarker in the management of exacerbations appearing in patients with severe disease., Competing Interests: The authors report no conflicts of interest in this work.

Haemophilus parasuis is a Gram-negative bacterium that colonizes the upper respiratory tract of swine and is capable of causing a systemic infection, resulting in high morbidity and mortality. H. parasuis isolates display a wide range of virulence and virulence factors are largely unknown. Commercial bacterins are often used to vaccinate swine against H. parasuis, though strain variability and lack of cross-reactivity can make this an ineffective means of protection. Outer membrane vesicles (OMV) are spherical structures naturally released from the membrane of bacteria and OMV are often enriched in toxins, signaling molecules and other bacterial components. Examination of OMV structures has led to identification of virulence factors in a number of bacteria and they have been successfully used as subunit vaccines. We have isolated OMV from both virulent and avirulent strains of H. parasuis, have examined their protein content and assessed their ability to induce an immune response in the host. Vaccination with purified OMV derived from the virulent H. parasuis Nagasaki strain provided protection against challenge with a lethal dose of the bacteria.

GSK1322322 is a novel inhibitor of peptide deformylase (PDF) with good in vitro activity against bacteria associated with community-acquired pneumonia and skin infections. We have characterized the in vivo pharmacodynamics (PD) of GSK1322322 in immunocompetent animal models of infection with Streptococcus pneumoniae and Haemophilus influenzae (mouse lung model) and with Staphylococcus aureus (rat abscess model) and determined the pharmacokinetic (PK)/PD index that best correlates with efficacy and its magnitude. Oral PK studies with both models showed slightly higher-than-dose-proportional exposure, with 3-fold increases in area under the concentration-time curve (AUC) with doubling doses. GSK1322322 exhibited dose-dependent in vivo efficacy against multiple isolates of S. pneumoniae, H. influenzae, and S. aureus. Dose fractionation studies with two S. pneumoniae and S. aureus isolates showed that therapeutic outcome correlated best with the free AUC/MIC (fAUC/MIC) index in S. pneumoniae (R(2), 0.83), whereas fAUC/MIC and free maximum drug concentration (fCmax)/MIC were the best efficacy predictors for S. aureus (R(2), 0.9 and 0.91, respectively). Median daily fAUC/MIC values required for stasis and for a 1-log10 reduction in bacterial burden were 8.1 and 14.4 for 11 S. pneumoniae isolates (R(2), 0.62) and 7.2 and 13.0 for five H. influenzae isolates (R(2), 0.93). The data showed that for eight S. aureus isolates, fAUC correlated better with efficacy than fAUC/MIC (R(2), 0.91 and 0.76, respectively), as efficacious AUCs were similar for all isolates, independent of their GSK1322322 MIC (range, 0.5 to 4 μg/ml). Median fAUCs of 2.1 and 6.3 μg · h/ml were associated with stasis and 1-log10 reductions, respectively, for S. aureus., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Memory B cells are long-lived and could contribute to persistence of humoral immunity by maintaining the plasma-cell pool or making recall responses upon re-exposure to an antigen. We determined the ability of a pneumococcal conjugate vaccine to induce anti-pneumococcal memory B cells. Frequencies of memory B cells against pneumococcal capsular polysaccharides from serotypes 1, 6B, 14, 19F and 23F were determined by cultured B cell enzyme-linked immunospot (ELISPOT) in 35 children aged 12-23 months who received pneumococcal non-typeable Haemophilus influenzae protein-D conjugate vaccine (PHiD-CV). The relationships between plasma antibodies and memory B cell frequencies were also assessed. After two doses of PHiD-CV, the proportion of subjects with detectable memory B cells against pneumococcal capsular polysaccharides increased significantly for serotypes 1 (3-45%; P < 0·01), 19F (21-66%; P < 0·01) and 23F (13-36%; P = 0·02), but not serotypes 6B (24-42%; P = 0·24) and 14 (21-40%; P = 0·06). Correlations between antibodies and memory B cells were weak. Carriage of serotype 19F at enrolment was associated with poor memory B cell responses against this serotype at subsequent time-points (day 30: non-carriers, 82% versus carriers, 0%, P < 0·01; day 210: non-carriers, 72% versus carriers, 33%, P = 0·07). PHiD-CV is capable of inducing memory B cells against some of the component pneumococcal capsular polysaccharides., (© 2015 British Society for Immunology.)

Non-typeable Haemophilus influenzae (NTHi) is commonly associated with chronic suppurative lung disease in children. We have previously shown that children with chronic suppurative lung disease have a reduced capacity to produce IFN-γ in response to NTHi compared with healthy control children. The aim of this study was to determine if deficient NTHi-specific IFN-γ production is associated with heightened systemic or airway inflammation. We measured a panel of cytokines (IFN-γ, IL-1β, IL-6, IL-8, IL-12 p70), antimicrobial proteins (LL-37, IP-10) as well as cellular and clinical factors associated with airway and systemic inflammation in 70 children with chronic suppurative lung disease. IFN-γ was measured in peripheral blood mononuclear cells challenged in vitro with live NTHi. Regression analysis was used to assess the association between the systemic and airway inflammation and the capacity to produce IFN-γ. On multivariate regression, NTHi-specific IFN-γ production was significantly negatively associated with the BAL concentrations of the inflammatory cytokines IL-6 (β=-0.316; 95%CI -0.49, -0.14; p=0.001) and IL-1β (β=-0.023; 95%CI -0.04, -0.01; p=0.001). This association was independent of bacterial or viral infection, BAL cellularity and the severity of bronchiectasis (using modified Bhalla score on chest CT scans). We found limited evidence of systemic inflammation in children with chronic suppurative lung disease. In summary, increased local airway inflammation is associated with a poorer systemic cell-mediated immune response to NTHi in children with chronic suppurative lung disease. These data support the emerging body of evidence that impaired cell-mediated immune responses and dysregulated airway inflammation may be linked and contribute to the pathobiology of chronic suppurative lung disease.

Nontypeable Haemophilus influenzae (NTHi), a typical mucosal pathogen largely responsible for respiratory infections and pediatric otitis media, has been increasingly recognized as a significant cause of invasive disease, especially in immunocompromised individuals. Lipooligosaccharide (LOS) is a conserved molecule with an important role in H. influenzae virulence and immune evasion, and it may be considered as a vaccine candidate. However, abilities of H. influenzae LOS to induce protective immune response are poorly understood. The goal of this study was to determine whether antibodies against LOS isolated from H. influenzae strains Eagan, Rd and NTHi 375 are present in the sera of normal individuals. Antigen specific IgG and IgM were studied in sera of 71 and 30 healthy adults, respectively. IgG specific for LOS of all three strains was ubiquitously present in our sample population while IgM specific for Eagan, Rd and NTHi 375 LOS compounds was detected in 37%, 63%, and 40% of samples, respectively. All tested serum samples exhibited bactericidal activity against all three H. influenzae strains; the removal of anti-LOS antibodies from the sera resulted in significant increases in bacterial survival of the corresponding strain. NTHi 375 exhibited the highest serum resistance, whereas the Rd strain was the least resistant. Serum bactericidal activity of anti-LOS antibody was mediated via the classical complement pathway. These findings suggest that in healthy adults, naturally acquired complement-activating anti-LOS antibodies significantly contribute to the overall serum bactericidal activity against both encapsulated and non-encapsulated strains of H. influenzae., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Patients with beta-thalassaemia major and asplenia have an increased risk of encapsulated bacterial infections. The aim of this study was to determine the Haemophilus influenza type b (Hib) antibody concentrations in beta-thalassaemia patients with or without spleens. The Hib antibody concentrations were investigated in 850 patients with thalassaemia major, of whom 437 had undergone splenectomy. Hib antibody levels equal or greater than 1.0 μg mL(-1) were classified as long-term protection, those between 0.15 and less than 1.0 μg mL(-1) as short-term protection and those less than 0.15 μg mL(-1) as no protection. The mean Hib antibody level was lower in asplenic subjects than in non splenectomised subjects (0.39 ± 0.5 vs. 1.08 ± 0.55 μg mL(-1), p < 0.001). The protective antibody level prevalence in asplenic patients was significantly lower than that in patients with spleens (32.3% vs. 85.7%, p < 0.001). Protection against Hib decreased as the time interval after splenectomy increased from 57.2% at a less than 60 months interval to 10.8% at a greater than 120 months interval (p = 0.001). Nearly 30% of the 437 splenectomised subjects had long-term protection against Hib, whereas 64.4% of the 413 non splenectomised subjects had long-term protection (p < 0.001). Asplenic subjects had lower Hib antibody levels than non splenectomised subjects. Additionally, the antibody levels decreased as the time interval increased after splenectomy. A Hib vaccine recommendation for splenectomised thalassaemia major seems essential.

Background: The impact on carriage and optimal schedule for primary vaccination of older children with 10-valent pneumococcal non-typeable Haemophilus influenzae protein-D conjugate vaccine (PHiD-CV) are unknown., Methods: 600 Kenyan children aged 12-59 months were vaccinated at days 0, 60 and 180 in a double-blind randomized controlled trial according to the following vaccine sequence: Group A: PHiD-CV, PHiD-CV, diphtheria/tetanus/acellular pertussis vaccine (DTaP); Group B: PHiD-CV, DTaP, PHiD-CV; Group C: hepatitis A vaccine (HAV), DTaP, HAV. Nasopharyngeal carriage of Streptococcus pneumoniae was measured at five timepoints. In 375 subjects, serotype-specific responses were measured by 22F-inhibition ELISA and opsonophagocytic killing assays (OPA) one month after vaccination., Results: Following one dose of PHiD-CV, >90% of recipients developed IgG≥0.35 µg/mL to serotypes 1, 4, 5, 7F, 9V and 18C and OPA≥8 to serotypes 4, 7F, 9V, 18C, 23F. After a second dose >90% of recipients had IgG≥0.35 µg/mL to all vaccine serotypes and OPA≥8 to all vaccine serotypes except 1 and 6B. At day 180, carriage of vaccine-type pneumococci was 21% in recipients of two doses of PHiD-CV (Group A) compared to 31% in controls (p = 0.04). Fever after dose 1 was reported by 41% of PHiD-CV recipients compared to 26% of HAV recipients (p<0.001). Other local and systemic adverse experiences were similar between groups., Conclusions: Vaccination of children aged 12-59 months with two doses of PHiD-CV two to six months apart was immunogenic, reduced vaccine-type pneumococcal carriage and was well-tolerated. Administration of PHiD-CV would be expected to provide effective protection against vaccine-type disease., Trial Registration: ClinicalTrials.gov NCT01028326.

Haemophilus parasuis is an economically important swine pathogen with 15 recognized serovars. An enzyme-linked immunosorbent assay (ELISA) was developed that detects serum antibodies to the oligopeptide permease A (OppA) polypeptide membrane protein present in the reference strains for 13 of the H. parasuis serovars. Using the OppA-ELISA, H. parasuis serologic profiles were assessed on 2 swine farms, with seroconversion defined as an OppA-ELISA sample-to-positive (S/P) ratio ≥0.5. Ten gilts from each farm were vaccinated for H. parasuis using either a live avirulent culture vaccine (farm 1) or an inactivated autogenous vaccine (farm 2). Seroconversion occurred in 100% of farm 1 gilts and 90% of farm 2 gilts, with a mean S/P ratio (MSPR) of 3.36 and 1.43, respectively. The OppA-ELISA MSPRs were determined for 2 piglets, 1 male and 1 female, randomly selected from 10 first-parity (P1), 10 second-parity (P2), and 10 third-parity (P3) litters farrowed by respective vaccinated gilts on each farm. On both farms, postfarrowing MSPRs and rate of seropositivity were highest in P1 versus P2 and P3 dams. Parity 1 piglets had higher MSPRs and rates of seropositivity versus later parities, with the difference being significant (P < 0.05) on farm 2. Polymerase chain reaction analysis of nasal swabs indicated that 100% of farm 1 piglets and 47-84%, depending on parity, of farm 2 piglets were H. parasuis-colonized at weaning. The results indicated that H. parasuis vaccination of gilts will not maintain serologic responses in the OppA-ELISA over their reproductive lifetimes, and that maternally derived antibodies do not prevent H. parasuis colonization of piglets.

Background: Dried blood spot (DBS) is a reliable blood collection method for storing samples at room temperature and easily transporting them. We have previously validated a Real-Time PCR for detection of Streptococcus pneumoniae in DBS. The objective of this study was to apply this methodology for the diagnosis of S. pneumoniae and Haemophilus influenzae b (Hib) in DBS samples of children with pneumonia admitted to two hospitals in Mozambique and Morocco., Methods: Ply and wzg genes of S. pneumoniae and bexA gene of Hib, were used as targets of Real-Time PCR. 329 DBS samples of children hospitalized with clinical diagnosis of pneumonia were tested., Results: Real-Time PCR in DBS allowed for a significant increase in microbiological diagnosis of S. pneumoniae and Hib. When performing blood bacterial culture, only ten isolates of S. pneumoniae and none of Hib were detected (3·0% positivity rate, IC95% 1·4-5·5%). Real-Time PCR from DBS samples increased the detection yield by 4x fold, as 30 S. pneumoniae and 11 Hib cases were detected (12·4% positivity rate, IC95% 9·0-16·5%; P<0·001)., Conclusion: Real-Time PCR applied in DBS may be a valuable tool for improving diagnosis and surveillance of pneumonia caused by S. pneumoniae or Hib in developing countries.

Objectives/hypothesis: Acute otitis media (AOM) involves an inflammatory response to microbes in the middle ear that facilitates clearance of otopathogens. Clinically, Streptococcus pneumoniae (Spn) infections of the respiratory tract are characterized by greater inflammatory responses than nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat). Interleukin 10 (IL-10) plays an important role in down-regulating the inflammatory response. We compared serum IL-10 levels in children before onset, at onset, and after recovery from AOM caused by Spn, NTHi, and Mcat. We sought to determine if IL-10 could serve as a biomarker to distinguish AOM caused by Spn versus NTHi and Mcat., Study Design: Prospective, longitudinal study in a primary care pediatric practice in Rochester, NY., Methods: Participants were 54 children, 6 to 30 months of age. Outcomes measured were serum IL-10 levels when healthy, at onset of AOM, and after recovery from AOM., Results: Serum IL-10 was elevated when children developed AOM (P=0.013) due to infections caused by Spn (P=0.011) but not AOM caused by NTHi or Mcat. Middle ear fluid levels of IL-10 mirrored those seen in serum but were 10-fold higher (P=0.02). Other effector cytokines in serum: IL-4, IFN-γ, and TNF-alpha, did not show the same increases as IL-10 at onset of AOM., Conclusion: Our study indicates that AOM caused by Spn elicits a significantly higher IL-10 response compared to NTHi and Mcat and may prove to be a biomarker of AOM infections by Spn., (Copyright © 2013 The American Laryngological, Rhinological and Otological Society, Inc.)

Tebipenem pivoxil, an oral carbapenem antibiotic for pediatric use, exhibits excellent clinical effects on acute otitis media (AOM). The present study was conducted to assess the pharmacokinetic profile of tebipenem in middle ear effusion and to examine the clinical efficacy of tebipenem pivoxil by calculating the values of the pharmacokinetic-pharmacodynamic parameters (AUC/MIC, C max/MIC, and T > MIC) of tebipenem at the site of action. Twenty-three pediatric outpatients diagnosed with AOM were enrolled. Ear discharge or nasopharyngeal swabs collected before the onset of oral administration were used to conduct bacteriological examinations, and subjects were then treated by twice-a-day oral administration of tebipenem pivoxil 6 mg/kg. The clinical isolates of Streptococcus pneumoniae and Haemophilus influenzae were obtained from 10 and 19 pediatric patients (8 overlapped), respectively. On day 2 of administration, blood and middle ear effusion were collected from 20 pediatric patients to measure plasma and middle ear concentrations of tebipenem. Consequently, the C max and the AUC0-∞ in plasma were 5.3 ± 1.6 μg/ml (mean ± SD) and 7.9 ± 0.2 μg h/ml, respectively. The C max in middle ear effusion of tebipenem was 1.2 ± 0.1 μg/ml, exceeding its MIC for these pathogens. The ratio of AUC0-∞ in middle ear effusion to AUC0-∞ in plasma was 0.36, showing the good transfer of tebipenem into the effusion; this result corroborated the known high rate of clinical efficacy of tebipenem pivoxil for patients with AOM and the low incidence of recurrence in them as manifested by the healing rate of 94.1 % (16/17).

Introduction: Infection markers are appreciated supplements in the clinical diagnosis of ear, nose and throat (ENT) infections. We aimed to examine the differential diagnostic usefulness of C-reactive protein (CRP), white blood cell count (WBC) and absolute neutrophil count (ANC) according to severity of middle ear and tonsillar infections., Material and Methods: This was a retrospective study including all patients admitted to the ENT Department, Aarhus University Hospital, from January 2001 to December 2008 and diagnosed with acute otitis media, mastoidismus, acute mastoiditis, acute tonsillitis, peritonsillar abscess, parapharyngeal abscess and necrotizing fasciitis., Results: A total of 1,773 patients were included. Between the tonsil subgroups, significant differences were found in CRP (p < 0.001), WBC (p < 0.001) and ANC (p < 0.001) levels. However, sensitivities and specificities related to differential diagnostics were low. In the middle ear group, no differences in CRP (p = 0.84), WBC (p = 0.46), and ANC (p = 0.72) levels were found. The number of CRP levels above the reference value was significantly higher than the corresponding number of WBC and ANC levels. A trend (non-significant) was found towards lower parameter levels in acute tonsillitis and peritonsillar abscess patients who grew Staphylococcus aureus compared with patients infected with other bacteria., Conclusion: CRP and ANC levels were related to severity of tonsillar-derived infections, but no such relation was found in infections with middle ear origin. None of the infection markers studied were useful for differential diagnostics., Funding: not relevant., Trial Registration: not relevant.

This study was aimed at determining the effect of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) on Haemophilus parasuis (HPS) in co-infection. A quantitative real-time PCR targeting infB gene, which is conserved among different HPS serotypes, was developed to improve the accuracy and speed of the detection of HPS. A total of 32 four-week-old conventional pigs were distributed randomly into four groups: pigs in group I were intranasally infected with HP-PRRSV first, and were then intraperitoneally inoculated with HPS on 5 days after HP-PRRSV infection; pigs in group II were intranasally inoculated with HP-PRRSV alone; pigs in group III were intraperitoneally inoculated with HPS alone; pigs in group IV were intraperitoneally inoculated with physiological saline. The amount of HPS in serum on 0, 3, 6, 9 and 12 days post-inoculation (dpi) with HPS were detected using the established quantitative real-time PCR. Clinical signs, pathological changes and histopathological lesions were observed. The amount of HPS in serum reached 10(6)copies/μl at 3 dpi with HPS in pigs of group I, while it arrived 10(5.7)copies/μl at 9 dpi with HPS in pigs of group III. The HPS loads in hearts and lungs were much higher than in other tissues. The study showed that HP-PRRSV was able to accelerate HPS infection and loads., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The bactericidal antibody response to three nontypeable Haemophilus influenzae (NTHi) outer membrane proteins (D, P6, and OMP26) was studied in 24 otitis-prone children (aged 7-28 months) after an acute otitis media (AOM) caused by NTHi. The study was carried out to understand the contribution of antigen-specific bactericidal antibody responses in the class of children who are most vulnerable to recurrent otitis media infections. Levels of protein D (P = 0.005) and P6 (P = 0.026) but not OMP26 antibodies were higher in bactericidal sera compared with nonbactericidal sera. For five (24%) and 16 (76%) of 21 bactericidal sera tested, removal of anti-protein D and P6 antibody, respectively, resulted in a two- to fourfold drop in bactericidal antibody. Antibodies to OMP26 did not make any contribution to the overall bactericidal activity in any serum samples. Eleven of 21 sera (52%) had bactericidal activity against a heterologous NTHi (86-028 NP) strain but the titers were significantly lower (P < 0.05) as compared to the homologous strains. Future studies of protein D, P6, OMP26, and other potential NTHi vaccine antigens should include studies of bactericidal antibody in children who are otitis prone as a possible correlate of protection., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

We herein present a case of Good syndrome complicated by diffuse pulmonary lesions similar to diffuse panbronchiolitis (DPB). A 45-year-old Japanese man was referred to our department due to recurrent lower respiratory tract infections that had started and ameliorated nine months after thymectomy for pure red cell aplasia and myasthenia gravis. Diffuse centrilobular opacities on chest computed tomography and positivity for HLA-B54 were consistent with DPB. Additionally, hypogammaglobulinemia and a marked decrease of B-lymphocytes were observed, and therefore Good syndrome was considered. Combination therapy with azithromycin and clarithromycin alleviated the patient's respiratory symptoms and reduced the exacerbation of chronic bronchitis.

Haemophilus parasuis is the etiological agent of Glässer's disease, which is characterized by fibrinous polyserositis, polyarthritis and meningitis in pigs. This study was focused on the characterization of the acute-phase response after immunization and infection of colostrum-deprived pigs with H. parasuis serovar 5, by measuring serum concentrations of three positive acute-phase proteins (APPs) (pig major acute-phase protein pig, MAP; haptoglobin, HPG; C-reactive protein, CRP) and one negative APP (apolipoprotein A-I, ApoA-I). Six experimental groups were established: a non-immunized but infected control group (CTL); two groups immunized with either a recombinant transferrin-binding protein (Tbp) A or TbpB fragment from H. parasuis Nagasaki strain (rTbpA and rTbpB, respectively); two groups immunized with native outer membrane proteins with affinity to porcine transferrin (NPAPT), one of them inoculated intramuscularly (NPAPTim) and the other intratracheally (NPAPTit), and the last group receiving a commercially available bacterin (PG). The greatest concentrations of the three positive APPs and the lowest concentration of the negative APP were detected in CTL group, as well as in those animals belonging to rTbpA or rTbpB groups that died in response to challenge. Significant differences (P<0.005) were found in these groups when comparing challenge with the following days after it. However, no significant differences were seen for the remaining vaccinated groups (NPAPTim, NPAPTit and PG), which were effectively protected against Glässer's disease. Therefore, APPs could be used as useful biomarkers for both evaluating disease progression and determining vaccination effectiveness., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Preterm infants are at greater risk of morbidity from vaccine-preventable diseases. Therefore, their responses to vaccination are of particular interest., Methods: In this open, controlled, Spanish multicenter study, we assessed immunogenicity and safety following primary vaccination of 163 preterm infants (n = 56, <31 weeks' gestation; n = 107, 31-36 weeks' gestation) and 150 full-term infants (>36 weeks' gestation), with Haemophilus Influenzae type B (Hib)-MenC-TT, DTaP(diphtheria-tetanus-acellular pertussis vaccine)-HepB-IPV, and PCV7 at 2 to 4-6 months of age followed by booster vaccination at 16 to 18 months of age. Serum bactericidal activity (rabbit complement) against MenC, and antibodies to Hib and hepatitis b (anti-HBs) were determined. Local/general symptoms were assessed after each vaccination via diary cards. Serious adverse events were recorded throughout the study., Results: There were no statistically significant differences between preterm and full-term infants in either Hib or MenC seroprotection rates or geometric mean concentrations at 1 month postdose 3, before or 1 month postbooster. Postdose 3, >99% of participants had seroprotective anti-HBs antibody concentrations. Anti-HBs geometric mean concentrations was significantly lower in the <31-week group compared with other groups and this difference persisted until 16 to 18 months of age. Hib-MenC-TT vaccine was well tolerated at all ages. There was one death caused by meningococcal serogroup-B sepsis (full term). No serious adverse events were assessed by the investigator as being vaccine related., Conclusions: Hib-MenC-TT vaccine had a similar immunogenicity and safety profile in preterm and full-term infants. These results demonstrate that preterm infants can be safely vaccinated with Hib-MenC-TT at the recommended chronologic age without impacting the responses to the Hib and MenC antigens.

The influence of age and maternal antibodies on the development and duration of postvaccinal antibody response against Glässer's disease were investigated. Pigs born to immune (MDA-positive) and non-immune (MDA-negative) sows were vaccinated with inactivated vaccine. Vaccination was done according to three different protocols: at 1 and 4, at 2 and 5 or at 4 and 7 weeks of age. There were also two control groups for MDA-negative and MDA-positive pigs. The level of Haemophilus parasuis (Hps) specific antibodies were determined using commercial ELISA test. No serological responses were seen in any of the groups after the first vaccination. Maternally derived antibodies (MDA) against Hps were above the positive level until approximately 3 weeks of life in MDA-positive pigs. In those pigs the strongest postvaccinal humoral response was observed in piglets vaccinated at 4 and 7 weeks of age. In the remaining MDA-positive piglets only slight seroconversion was noted but levels of antibodies never exceeded values considered as positive. All MDA-negative pigs produced Hps-specific antibodies after the second vaccination. The results of the present study indicated that MDA may alter the development and duration of active postvaccinal antibody response. Age of pigs at the moment of vaccination was not associated with the significant differences in the magnitude of antibody response, however influenced the kinetics of decline of Hps-specific antibodies.

Aim: Comparative assessment of immunobiological characteristics of 3 antigenic preparations containing capsular polysaccharide of Haemophilus influenzae type b (CPS Hib)., Materials and Methods: The following preparations were assessed: CPS Hib obtained by using cetavlon; hydroxylamine preparation of Hib (HAP Hib); mixture of CPS Hib and lipooligosaccharide of non-typeable H. influenzae (LOS NTHi) detoxified by hydroxylamine hydrochloride. Effects of these preparations on immunophenotype of mononuclear leukocytes of mice spleen as well as on spectrum and level of cytokines in serum were studied., Results: It was shown that mixture of CPS Hib and detoxified LOS NTHi has low toxicity and most protective activity during Hib challenge leading to activation of innate immunity effectors and initiation of adaptive immune response., Conclusion: Obtained data provide perspective for development of preparation able to protect from infections caused by both capsular and acapsular strains of H. influenzae.

In Bamako, Mali, where surveillance revealed a high incidence of Haemophilus influenzae type b (Hib) invasive disease, Hib conjugate vaccine was introduced into the Expanded Program on Immunization and the impact assessed. Annual confirmed Hib hospitalizations for infants 0-11 months of age fell from 175/10(5) to 44/10(5) (P < 0.001); among infants 6-7 months of age Hib hospitalizations fell from 377/10(5) to 69/10(5), (82% decrease, P < 0.001). Invasive Streptococcus pneumoniae hospitalizations remained unchanged. In a baseline serosurvey, only 3/200 infants 6-7 months of age (1.5%) had protective anti-polyribosylribitol phosphate (PRP) titers > or = 0.15 microg/mL and 1(0.5%) had >or = 1.0 microg/mL. In serosurveys 18 and 30 months after vaccine introduction, 168/201 (84%) and 184/200 (92%) infants, respectively, had titers > or = 0.15 microg/mL and 141/201 (70%) and 163/200 (82%) had titers > or = 1.0 microg/mL. Introduction of Hib vaccine led to rises in anti-PRP seroprevalence, significant reductions in Hib disease, and all-cause hospitalizations, whereas S. pneumoniae disease remained unchanged.

The serum antibody response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, was characterized by ELISA measuring IgM and IgGt levels against whole-cells and outer-membrane-proteins (OMPs) as antigens. Five groups of pigs were studied, four of those were previously immunized with different formulations, and the fifth was maintained as non-immunized control. All groups were challenged with 5x10(9) CFU of H. parasuis. The non-commercial bacterin induced a full protection against disease, the OMP-vaccine and the exposure to a sublethal dose of 10(5) CFU protected only partially, and the recombinant TbpB-vaccine conferred no protection. The humoral response in the pigs that died after infection (all controls, all those vaccinated with the recombinant TbpB, and two of both those inoculated with OMPs and those exposed to the sublethal dose) could be only measured before it, but it was irrelevant in all cases. However, a specific IgM and IgGt production was observed before challenge in all the surviving pigs, irrespective of the type of immunization received. This antibody response was even greater after H. parasuis infection, especially in those survivors receiving the sublethal dose. These results suggest a role of the antibodies developed after the different immunization protocols in preventing infection and death; therefore, the humoral immunity is protective against experimental Glässer's disease.

In 176 patients with Guillain-Barré syndrome the subclass and cross-reactivity of serum IgG antibodies to motor gangliosides was related to preceding infections and clinical phenotypes. Two subgroups of patients were identified. Presence of only IgG1 antibodies was related to diarrhea, positive Campylobacter serology, cross-reactive antibodies to C. jejuni lipo-oligosaccharides and poor outcome. In contrast, having both IgG1 and IgG3 antibodies was related to upper respiratory tract infections, cross-reactive antibodies to Haemophilus influenzae lipo-oligosaccharides and better outcome. These findings support a model in which C. jejuni and H. influenzae infections induce two distinct patterns of cross-reactive antibodies with different clinical outcome.

Background: Despite the excellent immunogenicity of Haemophilus influenzae type b (Hib) conjugate vaccines, breakthrough cases of Hib disease still affect a small proportion of vaccinated children in the United Kingdom. We performed a retrospective study to compare the avidity of antibody directed against the Hib polysaccharide capsule (PRP) in children who experienced Hib vaccine failure in the United Kingdom among 3 historical cohorts and with age-matched healthy control subjects., Methods: Serum samples from vaccinated children with invasive Hib disease were collected beginning in 1992 as part of enhanced surveillance for Hib disease following vaccine introduction. A total of 251 children who experienced Hib vaccine failure were identified from 3 historical cohorts (1992-1995, 1996-1999, and 2000-2003). The anti-PRP antibody concentration and avidity from healthy age-matched control subjects was obtained for the 3 contemporary time points (1995, 1999, and 2002). Serum anti-PRP antibody concentration was measured in each of the samples using a standard Hib ELISA, and antibody avidity was determined using thiocyanate elution., Results: Within the first 60 days after disease onset, there was no change in the anti-PRP antibody avidity, and there was no statistically significant difference in the geometric mean Hib antibody avidity over the 3 study periods. However, the children who experienced Hib vaccine failure had significantly lower Hib antibody avidity than did healthy control subjects, despite a marked antibody response following infection., Conclusions: Children who experience Hib disease despite vaccination appear to have a defect in immunological priming, leading to a qualitative difference in Hib-specific memory B cells. Low anti-PRP antibody avidity decreases the functional activity of anti-PRP antibody in the sera of these children experiencing vaccine failure, leading to disease susceptibility.

The bacterial thyA gene encodes the enzyme thymidylate synthase, which is essential for dTMP synthesis and, consequently, for DNA replication. In this work, a Haemophilus parasuis thyA mutant was constructed in order to analyze its colonization characteristics and its capacity to generate serum bactericidal activity in infected guinea pigs. The data showed that colonization by the H. parasuis thyA mutant was much less than that of the wild-type strain. Nevertheless, the mutant generated a strong immunogenic response in the host, as detected by measuring serum bactericidal activity.

We are investigating a nontypeable Haemophilus influenzae (NTHI) strain, R2866, isolated from a child with meningitis. R2866 is unusually resistant to killing by normal human serum. The serum 50% inhibitory concentration (IC50) for this strain is 18%, approaching that of encapsulated H. influenzae. R3392 is a derivative of R2866 that was found to have increased sensitivity to human serum (IC50, 1.5%). Analysis of tetrameric repeat regions within lipooligosaccharide (LOS) biosynthetic genes in both strains indicated that the glycosyltransferase gene lgtC was out of frame ("off") in most colonies of R3392 but in frame with its start codon ("on") in most colonies of the parent. We sought antigenic and biochemical evidence for modification of the LOS structure. In a whole-cell enzyme-linked immunosorbent assay, strain R3392 displayed reduced binding of the Galalpha1,4Gal-specific monoclonal antibody 4C4. Mass spectrometry analysis of LOS from strain R2866 indicated that the primary oligosaccharide glycoform contained four heptose and four hexose residues, while that of R3392 contained four heptose and three hexose residues. We conclude that the R2866 lgtC gene encodes a galactosyltransferase involved in synthesis of the 4C4 epitope, as in other strains, and that expression of lgtC is associated with the high-level serum resistance that has been observed for this strain. This is the first description of the genetic basis of high-level serum resistance in NTHI, as well as the first description of LOS composition in an NTHI strain for which the complete genome sequence has been determined.

A total of 16 isolates of Streptococcus pneumoniae and 18 isolates of Haemophilus influenzae were obtained from the blood of children admitted to the pediatric wards of hospitals in Hokkaido Kamikawa subprefecture between January 2003 and December 2005. The ages of the patients with S. pneumoniae or H. influenzae infection ranged from 2 months to 9 years and from 1 month to 4 years, respectively. The diagnoses of S. pneumoniae infection were as follows: pneumonia in 8 patients, occult bacteremia in 5 patients, and meningitis in 3 patients. The diagnoses of H. influenzae were: meningitis in 6 patients, pneumonia in 4 patients, occult bacteremia in 4 patients, epiglotitis in 2 patients, and facial cellulitis in 2 patients. Out of 16 S. pneumoniae isolates, penicillin-resistant strains with a mutation of 3 genes were observed in 7 children, and penicillin intermediate-resistant strains with a mutation of 1 or 2 genes were observed in 8 children. Out of 18 H. influenzae isolates, the beta-lactamase-negative ampicillin-resistant strain with a substitution of 2 points in the ftsI gene was revealed in 2 children, the beta-lactamase-negative ampicillin-resistant strain with a substitution of 1 point in the ftsI gene was observed in 4 children, the beta-lactamase-positive amoxicillin/clavulanic acid-resistant strain with blaTEM-1 and ftsI with 2 substitutions in the ftsI gene was observed in 3 children, and the beta-lactamase-positive ampicillin-resistant strain with blaTEM-1was not observed. The MBC90s of ampicillin, ceftriaxone, cefotaxime, meropenem, panipenem, and vancomycin against S. pneumoniae were 8 microg/ml, 1 microg/ml, 1 microg/ml 1 microg/ml, 0.25 microg/ml, and 0.5 microg/ml, respectively. Those of ampicillin, piperacillin, ceftriaxone, cefotaxime, meropenem, and panipenem against H. influenzae were >128 microg/ml, >128 microg/ml, 0.25 microg/mL, 1 microg/ml, 0.12 microg/ml, and 0.5 g/ml, respectively. It is suggest that the minimum bactricidal concentration (MBC) was dissociated from the minimum inhibitory concentration (MIC) in S. pneumoniae and H. influenzae with abnormal pbp genes.

From September 2002 to December 2004, a total of 281 strains of Haemophilus parasuis were isolated from 17 provinces of China. All these isolates were serotyped by both the gel diffusion (GD) and the indirect haemagglutination (IHA) tests. By combining the GD and IHA results, serovar 4 (24.2%) and serovar 5 (19.2%) were the most prevalent serovars, followed by serovars 13 (12.5%), 14 (7.1%) and 12 (6.8%), while 12.1% of the isolates could not be assigned to a serovar (nontypable). A comparison of the number of isolates obtained from the respiratory tract of swine without polyserositis with those obtained from swine with polyserositis revealed an increased frequency of serovar 4 and a significantly decreased frequency of serovar 13 among isolates from the respiratory tract of swine without polyserositis, whereas the frequency of isolation of serovars 5, 12, 14 and nontypable from swine with or without polyserositis were similar. Co-infection of H. parasuis and other bacterial agents was studied in 183 cases examined from June 2003 to December 2004. Streptococcus suis (30.6%; 56), Escherichia coli (21.9%; 40), Bordetella bronchiseptica (21.3%; 39) and Pasteurella multocida (14.2%; 26) were the bacterial agents frequently co-isolated with H. parasuis in China.

The microbiological and virological examination of 87 acute bronchitis patients (36 patients with the prolonged course and 31 patients with the relapsing course of the disease) was carried out. All forms of bronchitis were characterized by a high degree of infection with respiratory viruses and pneumococci. Haemophilus influenzae (type b) infection was registered rather rarely and only in combination with pneumococcal one. The highest characteristics of viro-bacterial associations were found in patients with acute bronchitis and with prolonged form of acute bronchitis, viral associations--with the prolonged and relapsing forms of the course of acute bronchitis in the presence of the bronchoobstructive syndrome.

Objectives: To analyze the peripheral leukocyte counts of children with acute otitis media (AOM) in relation to etiology, age, clinical symptoms and signs, prior antibiotic treatment, previous AOM history and pathogen eradication during antibiotic therapy., Patients and Methods: Leukocyte counts were determined at diagnosis and on days 4 to 6 of therapy in patients age 3 to 36 months with AOM enrolled in double tympanocentesis studies. Clinical status was determined by a clinical score evaluating severity of fever, irritability and tympanic membrane redness and bulging., Results: Of 771 enrolled patients, culture-positive middle ear fluid was reported in 590 (77%): 294 (50%) Haemophilus influenzae; 150 (25%) Streptococcus pneumoniae; 127 (21%) H. influenzae and S. pneumoniae together; 9 (2%) Moraxella catarrhalis; and 10 (2%) others. Mean leukocyte count +/- sd in patients with AOM caused by S. pneumoniae (15.7 +/- 6.7 cells x 10/mm) was significantly higher than those of patients with AOM caused by H. influenzae (13.7 +/- 5.8 cells x 10/mm) and patients with culture-negative AOM (13.8 +/- 5.6 cells x 10/mm), P < 0.01 for each comparison. Mean absolute neutrophil count (ANC) +/- sd was higher in patients with AOM caused by S. pneumoniae (8.6 +/- 5.1 cells x 10/mm) than in patients with AOM caused by H. influenzae (6.5 +/- 4.0 cells x 10/mm) or culture-negative patients (6.5 +/- 4.4 cells x 10/mm), P < 0.05 for each comparison. When multivariant regression analysis was used, S. pneumoniae etiology and fever (body temperature > or = 38 degrees Celsius) were independent factors, each significantly associated with leukocyte and ANCs. A significant correlation was found between leukocyte and ANCs and higher clinical scores in patients with pneumococcal AOM (P = 0.01, r = 0.21 and P < 0.01, r = 0.27, respectively). The mean leukocyte count on Days 4 to 6 was lower than on Day 1 (11.5 +/- 4.1 compared with 14.2 +/- 6.0 cells x 10/mm, P < 0.01). When paired (Day 1 and Days 4 to 6) examinations were analyzed, the leukocyte counts were lower on Days 4 to 6 irrespective of bacterial eradication or persistence., Conclusions: Significantly higher leukocyte counts and ANCs were found in pneumococcal AOM than in AOM caused by H. influenzae or in culture-negative AOM. A significant decrease in leukocyte counts was found during antibiotic therapy for AOM, regardless of etiology and bacteriologic outcome. Isolation of S. pneumoniae and fever were each significantly associated with increased peripheral leukocyte and ANCs.