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Haemophilus influenzae is the most common cause of bacterial infection in the lungs of chronic obstructive pulmonary disease (COPD) patients and contributes to episodes of acute exacerbation which are associated with increased hospitalization and mortality. Due to the ability of H. influenzae to adhere to host epithelial cells, initial colonization of the lower airways can progress to a persistent infection and biofilm formation. This is characterized by changes in bacterial behaviour such as reduced cellular metabolism and the production of an obstructive extracellular matrix (ECM). Herein we discuss the multiple mechanisms by which H. influenzae contributes tothe pathogenesis of COPD. In particular, mechanisms that facilitate bacterial adherence to host airway epithelial cells, biofilm formation, and microbial persistence through immune system evasion and antibiotic tolerance will be discussed.

As a stepping stone toward evaluation in infants, the safety and immunogenicity of an investigational 12-valent pneumococcal non-typeable Haemophilus influenzae protein D-conjugate vaccine (12vPHiD...

Haemophilus are fastidious Gram negative bacilli, which require factor X (hemin), factor V (NAD), or both for their growth. Haemophilus influenzae is the type species, and is considered to be the most pathogenic. They are associated with many invasive infections including meningitis, epiglottitis, pneumonia, and otitis media. Serotype b is most commonly associated with infections. Haemophilus species isolated from patients in a tertiary care centre in South India were studied. Identification, serotyping and biotyping were done and antibiotic susceptibility test was performed. The incidence of H. influenzae infections in our study was 65.3 cases/100,000 persons. Serotype b was the most common (66.67%), followed by non typeable H.influenzae (NTHi) (25%). Most isolates from adults were type b, while all isolates from pediatric population were non typeable. The most common biotype was type II, followed by type I and type III. Three of 24 isolates were β lactamase producers (12.5%). One isolate was β lactamase negative Ampicillin resistant (BLNAR). Resistance to ampicillin was 16.67%. Resistance to cephalosporins and fluoroquinolones was low (4-10%). Co-trimoxazole resistance was found to be very high (75%). All isolates were susceptible to azithromycin, tetracycline, chloramphenicol and meropenem. No isolates of H.influenzae type b were obtained from the paediatric population which may be due to the introduction of Hib vaccine. The increase in resistance to commonly used antibiotics is worrisome, especially penicillins and co-trimoxazole. Use of co-trimoxazole in empirical therapy of upper and lower respiratory tract infections has a high chance of failure in the current scenario.

Objectives: The aims of this study were to analyse the serotypes of epidemic Haemophilus influenzae and changes in mechanisms of β-lactam resistance over the past decade. Results: Haemophilus influenzae isolates in Western Sichuan from 2013–2014 were non-typeable H. influenzae (NTHi). β-Lactam MICs for NTHi isolated during 2013–2014 were significantly higher than those from 2003–2004 (P < 0.05). Of 274 NTHi, 141 (51.5%) were β-lactamase-positive (TEM-1 type). There were 35 amino acid (AA) substitutions in ftsI among NTHi isolated from 2013–2014. However, NTHi isolates from 2003–2004 had only nine AA substitutions. Ordered multiple classification logistic regression analysis showed that different AA substitution patterns in ftsI had different effects on β-lactam MICs. The main factors affecting the ampicillin MIC were the mutations R517H (OR = 6.999), L389F (OR = 7.128), N526K (OR = 4.660) and D350N (OR = 0.450). The main factor influencing the amoxicillin/clavulanic acid MIC was an N526K mutation (OR = 9.349). The main factors affecting the cefuroxime MIC were the mutations S357N (OR = 37.453) and N526K (OR = 14.816). Compared with 2003–2004, gBLNAR and gBLPAR isolated from 2013–2014 increased significantly from 13.0% (7/54) and 9.3% (5/54) to 38.2% (84/220) and 45.5% (100/220), respectively (P < 0.001). In the ‘others’ group of ftsI gene mutations, 13 NTHi had the same ftsI gene mutation pattern and 24 AA substitutions. Conclusion: These results confirm that β-lactam-resistant NTHi isolates increased rapidly. AA substitutions in ftsI were more complex and diversified in 2013–2014.

Australian Aboriginal populations have unacceptably high rates of bronchiectasis. This disease burden is associated with high rates of detection of pathogenic bacteria, particularly non-typeable Haemophilus influenzae (NTHi). While there is evidence to suggest that exposure to inorganic particulate matter (PM) is associated with worse respiratory infections, no studies have considered the direct effect of this PM on bacterial growth. Nine clinical isolates of pathogenic NTHi were used for this study. Isolates were exposed to two common iron oxides, haematite (Fe2O3) or magnetite (Fe3O4), or quartz (SiO2), as the main constituents of environmental inorganic PM. NTHi isolates were exposed to PM with varying levels of heme to identify whether the response to PM was altered by iron availability. The maximal rate of growth and maximum supported growth were assessed. We observed that inorganic PM was able to modify the maximal growth of selected NTHi isolates. Magnetite and quartz were able to increase maximal growth, while haematite could both increase and suppress the maximal growth. However, these effects varied depending on iron availability and on the bacterial isolate. Our data suggest that inorganic PM may directly alter the growth of pathogenic NTHi. This observation may partly explain the link between exposure to high levels of crustal PM and chronic bacterial infection in Australian Aboriginals.

In the post-Haemophilus influenzae serotype b (Hib) vaccine era, invasive H. influenzae serotype a (Hia) disease emerged in Canadian First Nation, Inuit, and Alaskan Indigenous populations. Previous studies by our group found a high incidence of invasive Hia disease in northwestern Ontario. We retrospectively reviewed 24 cases (4 pediatric and 20 adult) of invasive H. influenzae disease hospitalized at the northwestern Ontario regional hospital between August 2011 and June 2018. The objectives were to further document the changing epidemiology of invasive H. influenzae disease in the region and to discuss potential control measures. Twenty-two H. influenzae isolates were serotyped and characterized using molecular-biological methods. Of the serotyped cases, there were 2 Hib, 9 Hia, and 11 non-typeable (NTHi). All Hia isolates belonged to the most common sequence types (ST) found in Canada (ST-23 and ST-929); 8 out of 9 were pan susceptible to antibiotics. One (11%) of 9 Hia and 5 (45%) of 11 NTHi cases were fatal. Our data on the consistent presence of serious invasive H. influenzae disease, with 41% prevalence of Hia (9 out of 22 serotyped isolates) and 50% prevalence of NTHi strains (11 out of 22), emphasize the importance of continued surveillance of H. influenzae in the post-Hib vaccine era and are critical information to inform potential vaccine development.

The implementation of pneumococcal conjugate vaccines (PCVs) has led to a decline in vaccine-type disease. However, there is evidence that the epidemiology of non-typeable Haemophilus influenzae (NTHi) carriage and disease can be altered as a consequence of PCV introduction. We explored the epidemiological shifts in NTHi carriage using whole genome sequencing over a 5-year period that included PCV13 replacement of PCV7 in the UK's National Immunization Programme in 2010. Between 2008/09 and 2012/13 (October to March), nasopharyngeal swabs were taken from children5 years of age. Significantly increased carriage post-PCV13 was observed and lineage-specific associations with Streptococcus pneumoniae were seen before but not after PCV13 introduction. NTHi were characterized into 11 discrete, temporally stable lineages, congruent with current knowledge regarding the clonality of NTHi. The increased carriage could not be linked to the expansion of a particular clone and different co-carriage dynamics were seen before PCV13 implementation when NTHi co-carried with vaccine serotype pneumococci. In summary, PCV13 introduction has been shown to have an indirect effect on NTHi epidemiology and there exists both negative and positive, distinct associations between pneumococci and NTHi. This should be considered when evaluating the impacts of pneumococcal vaccine design and policy.

Background: In chronic obstructive pulmonary disease (COPD), exacerbations cause acute inflammatory flare-ups and increase the risk for hospitalization and mortality. Exacerbations are common in all disease stages and are often caused by bacterial infections e.g., non-typeable Heamophilus influenzae (NTHi). Accumulating evidence also associates vitamin D deficiency with the severity of COPD and exacerbation frequency. However, it is still unclear whether vitamin D deficiency when combined with cigarette smoking would worsen and prolong exacerbations caused by repeated infections with the same bacterial strain. Methods: vitamin D sufficient (VDS) and deficient (VDD) mice were exposed to nose-only cigarette smoke (CS) for 14 weeks and oropharyngeally instilled with NTHi at week 6, 10 and 14. Three days after the last instillation, mice were assessed for lung function, remodeling, inflammation and immunity. The impact of VDD and CS on inflammatory cells and immunoglobulin (Ig) production was also assessed in non-infected animals while serum Ig production against NTHi and dsDNA was measured in COPD patients before and 1 year after supplementation with Vitamin D3. Results: VDD enhanced NTHi eradication, independently of CS and complete eradiation was reflected by decreased anti-NTHi Ig’s within the lung. In addition, VDD led to an increase in total lung capacity (TLC), lung compliance (Cchord), MMP12/TIMP1 ratio with a rise in serum Ig titers and anti-dsDNA Ig’s. Interestingly, in non-infected animals, VDD exacerbated the CS-induced anti-NTHi Ig’s, anti-dsDNA Ig’s and inflammatory cells within the lung. In COPD patients, serum Ig production was not affected by vitamin D status but anti-NTHi IgG increased after vitamin D3 supplementation in Vitamin D insufficient patients before treatment. Conclusion: During repeated infections, VDD facilitated NTHi eradication and resolution of local lung inflammation through production of anti-NTHi Ig, independently of CS but it also produced autoantibodies. In COPD patients, vitamin D supplementation might be protective against NTHi infections in vitamin D insufficient patient. Future research is needed to decipher the protective or self-destructive effects of VDD on adaptive immunity.Trail registration: ClinicalTrails, NCT00666367. Registered 23 April 2008, https://www.clinicaltrials.gov/ct2/show/study/NCT00666367

Dipeptidyl peptidase 4 (DPP4) expression is increased in the lungs of chronic obstructive pulmonary disease (COPD). DPP4 is known to be associated with inflammation in various organs, including LPS-induced acute lung inflammation. Since non-typeable Haemophilus influenzae (NTHi) causes acute exacerbations in COPD patients, we examined the contribution of DPP4 in NTHi-induced lung inflammation in COPD. Pulmonary macrophages isolated from COPD patients showed higher expression of DPP4 than the macrophages isolated from normal subjects. In response to NTHi infection, COPD, but not normal macrophages show a further increase in the expression of DPP4. COPD macrophages also showed higher expression of IL-1β, and CCL3 responses to NTHi than normal, and treatment with DPP4 inhibitor, diprotin A attenuated this response. To examine the contribution of DPP4 in NTHi-induced lung inflammation, COPD mice were infected with NTHi, treated with diprotin A or PBS intraperitoneally, and examined for DPP4 expression, lung inflammation, and cytokine expression. Mice with COPD phenotype showed increased expression of DPP4, which increased further following NTHi infection. DPP4 expression was primarily observed in the infiltrated inflammatory cells. NTHi-infected COPD mice also showed sustained neutrophilic lung inflammation and expression of CCL3, and this was inhibited by DPP4 inhibitor. These observations indicate that enhanced expression of DPP4 in pulmonary macrophages may contribute to sustained lung inflammation in COPD following NTHi infection. Therefore, inhibition of DPP4 may reduce the severity of NTHi-induced lung inflammation in COPD.

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide. The major bacterial cause of COPD exacerbations is non-typeable Haemophilus influenzae (NTHi). This susceptibility to infection involves a defective production of interleukin (IL)-22 which plays an important role in mucosal defense. Prophylactic administration of flagellin, a Toll-like receptor 5 (TLR5) agonist, protects healthy mice against respiratory pathogenic bacteria. We hypothesized that TLR5-mediated stimulation of lung immunity might prevent COPD exacerbations due to NTHi. Mice were chronically exposed to cigarette smoke and then infected with NTHi. According our preventive or therapeutic protocol, flagellin was administered intraperitoneally. Cigarette smoke-exposed mice treated with flagellin showed a lower bacterial load in the airways, the lungs and the blood. This protection was associated with an early neutrophilia, a lower production of pro-inflammatory cytokines and an increased IL-22 production. Flagellin treatment decreased the recruitment of inflammatory cells and the lung damages related to exacerbation. Protective effect of flagellin against NTHi was altered by treatment with anti-IL-22 blocking antibodies in cigarette smoke-exposed mice and in Il22−/− mice. Flagellin treatment also amplified the production of the β-defensin2 anti-bacterial peptides. This study shows that stimulation of innate immunity by a TLR5 ligand is a potent antibacterial treatment in cigarette smoke exposed mice, suggesting innovative therapeutic strategies against acute exacerbation in COPD.

Objectives Typing of Chlamydia trachomatis (CT) is traditionally performed by characterising the ompA gene, resulting in more than a dozen different genovars, A to L. Type L is associated with Lymphogranuloma venereum (LGV) and commonly screened for using PCR, targeting the chromosomal pmpH gene. We aimed to develop and validate a new CT/LGV plasmid-based typing assay targeting the pgp3 gene, to increase sensitivity and thus reduce the number of non-typeable results. Methods The new pgp3 PCR assay using LNA probes to detect point mutations was analytically and prospectively validated in a routine diagnostic laboratory setting. For the analytical tests, quantified nucleotide constructs (gBlocks) were used to perform limit of detection analyses. Quality control panel samples from 2018 and 2019 for CT were also tested. For the clinical study patient samples which were collected in two months in 2018 were tested simultaneously using the pmpH PCR and the pgp3 PCR. Results Analytically, the assay proved to be 100% specific relative to the previously used LGV typing assay targeting the single copy pmpH gene but it was much more sensitive to detect non-LGV CT. In the quality control panel 2 nonLGV samples and 7 LGV samples were solely positive with the pgp3 PCR and not with the pmpH PCR. None of the samples from analytical specificity panels were positive, indicating 100% specificity. In a prospective panel of 152 clinical samples, 142 (93%) were successfully typed with the pgp3 PCR compared to 78% with the pmpH PCR. The pgp3 PCR was fully concordant with the pmpH PCR to identify all LGV subtypes and detected an increased number of clinical samples of non-LGV subtype. Conclusion We developed and validated a sensitive and specific plasmid-based typing assay to discriminate LGV from non-LGV CT subtypes. This is useful in a clinical setting to quickly determine the optimal treatment for Chlamydia trachomatis infections.

In 2010, Korea introduced 10-valent pneumococcal conjugate vaccine for children aged 6 weeks to 5 years against invasive disease caused by Streptococcus pneumoniae serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 23F and cross-reactive 19A. The aim of this 6-year real-world study of 646 healthy Korean children from 16 centers vaccinated in routine practice is to monitor vaccine safety, as per Ministry of Food and Drug Safety regulations. Around 50% had a past or existing medical condition, 19.3% an existing condition and 7.6% received concomitant medication). Total of 489 recorded adverse events (AEs) were reported in 274 infants; 86% were mild and the rest moderate, only three were reported as serious. Most AEs (97.8%) were not related to vaccination; one case of injection-site swelling and of fever was related, two cases of fever were probably related, five cases of fever and one case each of diarrhea and coughing were possibly related. None of the serious AEs were related to vaccination. Of 11 adverse drug reactions (ADRs) in 10 subjects, none were serious. Overall, 263 subjects (40.7%) received medication (mainly antibiotics or antipyretics) for the treatment of an AE, of which 6 subjects were treated for an ADR. There was no difference in the incidence of AEs according to age, sex or concomitant vaccination. Subjects with an existing medical condition had significantly more AEs than those without any conditions (p = 0.03), but no differences regarding ADRs. Four-dose vaccination with PHiD-CV appears to have a clinically-acceptable safety profile for Korean children. ClinicalTrials.gov identifier: NCT01248988

Non-typeable Haemophilus influenzae (NTHi) has attracted more interest in recent years due to an increased prevalence of infections caused by the pathogen. This upsurge is at least partly ascribed to the introduction of the pneumococcal conjugated vaccines that has resulted in an aetiological shift in NTHi's favor with respect to upper respiratory tract infections. Moreover, an increased antimicrobial resistance has been associated with the pathogen, a fact that further strengthens the case for novel vaccine development.A background to NTHi-mediated diseases and pathogenesis is outlined. The literature in the field of NTHi vaccine antigens and clinical trials is reviewed with focus on data added to scientific databases in the last two years. Various vaccine development strategies are conceptually discussed.Several promising vaccine antigens have been defined in recent years. A multicomponent protein-based vaccine, potentially boosted with extracellular vesicles, would constitute a suitable path going forward. Of note, however, a clinical trial investigating the efficacy of a combined NTHi/Moraxella catarrhalis vaccine to prevent infections in chronic obstructive pulmonary disease (COPD) patients has been initiated. But, as this clinical trial has not yet concluded, and its results are thus unknown, investigations of NTHi pathogenesis must determinedly continue.

We previously reported 10-valent pneumococcal non-typeable Haemophilus influenzae (NTHi) protein D conjugate vaccine (PHiD-CV) efficacy in a double-blind randomized trial (ClinicalTrials.gov: NCT00466947) against various diseases, including acute otitis media (AOM). Here, we provide further analyses. In the Panamanian subset, 7,359 children were randomized (1:1) to receive PHiD-CV or control vaccine at age 2/4/6 and 15–18 months. Of these, 2,000 had nasopharyngeal swabs collected. AOM cases were captured when parents sought medical attention for children with AOM symptoms; surveillance was enhanced approximately 2 y into the study through regular telephone calls or home visits by study personnel, who advised parents to visit the clinic if their child had AOM symptoms. Mean follow-up was 31.4 months. Clinical AOM (C-AOM) cases were assessed by physicians and confirmed by otorhinolaryngologists. Middle ear fluid samples, taken from children with C-AOM after specific informed consent, and nasopharyngeal samples were cultured for pathogen identification. For 7,359 children, 2,574 suspected AOM cases were assessed by a primary healthcare physician; 649 cases were C-AOM cases as per protocol definition. From the 503 MEF samples collected, 158 resulted in a positive culture. In the intent-to-treat cohort (7,214 children), PHiD-CV showed VE against first C-AOM (24.0% [95% CI: 8.7, 36.7]) and bacterial (B-AOM) episodes (48.0% [20.3, 66.1]) in children

Loss of airway microbial diversity is associated with non-typeable Haemophilus influenzae (NTHi) infection and increased risk of exacerbation in chronic obstructive pulmonary disease (COPD). We assessed the safety and immunogenicity of an investigational vaccine containing NTHi antigens, recombinant protein D (PD) and combined protein E and Pilin A (PE-PilA), and AS01 adjuvant in adults with moderate/-severe COPD and prior exacerbations. In this phase 2, observer-blind, controlled trial (NCT02075541), 145 COPD patients aged 40-80 years randomly (1:1) received two doses of NTHi vaccine or placebo 60 days apart, on top of standard care. Reactogenicity in the 7-day post-vaccination period was higher following NTHi vaccine than placebo. Most solicited adverse events (AEs) were mild/moderate. At least one unsolicited AE was reported during the 30-day post-vaccination period by 54.8% of NTHi vaccine and 51.4% of placebo recipients. One serious AE (placebo group) was assessed by the investigator as vaccine-related. Anti-PD, anti-PE and anti-PiIA geometric mean antibody concentrations increased up to 30 days after each NTHi vaccine dose, waned thereafter, but remained higher than baseline (non-overlapping confidence intervals) up to 13 months post-dose 2. The frequency of specific CD4(+) T cells increased following two doses of NTHi vaccine and remained higher than baseline. Exploratory analysis showed a statistically non-significant lower yearly rate of moderate/severe exacerbations in the NTHi vaccine group than following placebo (1.49 versus 1.73) in the one-year period post-dose 2, with estimated vaccine efficacy of 13.3% (95% confidence interval -24.2 to 39.5; p = 0.44). The NTHi vaccine had an acceptable safety and reactogenicity profile and good immunogenicity in adults with COPD.

The efficacy of chocolate agar, versus bacitracin, vancomycin, clindamycin, chocolate agar (BVCCA) for the isolation of non-typeable Haemophilus influenzae (NTHi) from nasopharyngeal swabs was determined. BVCCA cultured NTHi from 97.3% of NTHi-positive swabs, compared to 87.1% for chocolate agar. To maximise culture sensitivity, the use of both media is recommended.

Haemophilus influenzae typically causes illness and infection in the paediatric population. We report a case of a 53-year-old man who developed invasive non-typeable H. influenzae infection associated with purpura fulminans and multiorgan failure. On review of the literature, this is the first reported case of non-typeable H. influenzae causing purpura fulminans. The patient was treated with intravenous ceftriaxone 2 g/day and was eventually discharged from the hospital almost 2 months after admission. We discuss the role that infection/sepsis plays in disturbances to the coagulation cascade leading to purpura fulminans and the virulence factors that make non-typeable H. influenzae unique. Finally, we review other cases of H. influenzae associated with purpura fulminans and discuss the similarities with our case.

Late Breaking Abstract - Safety and immunogenicity of non-typeable H. influenzae (NTHi) adjuvanted vaccine in older adults with chronic obstructive pulmonary disease (COPD)

The implementation of pneumococcal conjugate vaccines (PCVs) has led to a decline in vaccine-type disease. However, there is evidence that the epidemiology of non-typeable Haemophilus influenzae (NTHi) carriage and disease can be altered as a consequence of PCV introduction. We explored the epidemiological shifts in NTHi carriage using whole genome sequencing over a 5-year period that included PCV13 replacement of PCV7 in the UK’s National Immunization Programme in 2010. Between 2008/09 and 2012/13 (October to March), nasopharyngeal swabs were taken from children

Our analysis has considered the most up-to-date clinical evidence, placing emphasis on data from randomized double-blind controlled trials, rather than having relied on theoretical assumptions. In addition, we have consulted an external group of experts to validate each parameter that was used as base-case input into the model. We hope that the evidence provided here, in addition to what is reflected in our original paper, testifies that we have considered the strengths and weaknesses of all available data to construct a sensible and conservative base-case analysis as well as have meticulously addressed any areas of uncertainty using appropriate sensitivity and scenario analyses.

Early airway colonization and infection with Haemophilus influenzae in children with cystic fibrosis (CF) is common. Although the pathogenicity of non-typeable H. influenzae (NTHi) in patients with CF is controversial, this organism can cause both upper and lower respiratory tract infections. Extra-pulmonary disease, however, is rare. Purulent pericarditis is a suppurative complication of bacterial infection of the pericardial space that can arise as a result of direct extension from an adjacent infection. We describe a case of purulent pericarditis due to NTHi in a young child with CF that developed as a complication of inadequately treated bronchopneumonia.

[This corrects the article DOI: 10.1371/journal.pone.0167250.].

High molecular weight (Hmw) proteins 1 and 2, type IV pilin protein (PilA), outer-membrane protein P5 (OmpP5), Haemophilus protein D (Hpd) and Haemophilus adhesive protein (Hap) are surface proteins involved in the adherence of non-typeable Haemophilus influenzae. One hundred clinical isolates were evaluated for the presence of the genes encoding these proteins by PCR and for their adherence capacity (AC) to Detroit 562 nasopharyngeal cells (D562). The majority of isolates were from blood (77/100); other sites were also represented. Confluent D562 monolayers (1.2×105 cells per well) were inoculated with standardized minimal infective doses (m.o.i.) of 102, 103 or 104 c.f.u. per well. The AC was categorized as low (hmw1A and/or hmw2A were detected in 69/100 (69 %) of isolates. The presence of hmw1A and/or hmw2A was associated with increased adherence to D562 cells (P≤0.001). Dot immunoblots were performed to detect protein expression using mAbs 3D6, AD6 and 10C5. Among the high-adherence isolates (n = 69), 72 % reacted with 3D6 and 21 % with 10C5. Our data indicate that the absence of Hmw1 and/or Hmw2 was associated with decreased adherence to D562 cells.

We report the novel branching pattern in lipopolysaccharide (LPS) expressed by non-typeable Haemophilus influenzae (NTHi) strain 1232. The strain expressed the β-D-Glcp-(1→4)-[α-D-Galp-(1→4)-β-D-Galp- (1→7)]-D-α-D-Hepp-(1→6)-β-D-Glcp chain linked to the proximal heptose (HepI) of the conserved triheptosyl inner-core moiety of NTHi LPS: L-α-D-HepIIIp-(1→2)-[PEtn→6]-L-α-D-HepIIp-(1→3)- L-α-DHepIp-( 1→5)-[PPEtn→4]-α-Kdop-(2→6)-lipid A. The structure has been elucidated using NMR spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis coupled to electrospray ionization tandem mass spectrometry (CE-ESI-MSn) on O-deacylated LPS and core oligosaccharide (OS) materials, as well as HPLC-ESI-MSn on permethylated, dephosphorylated OS. It was also found that a tetrasaccharide unit bearing sialic acid [α-Neu5Ac-(2→3)-β-D-Galp-(1→4)- β-D-GlcNAcp-(1→3)-β-D-Galp-(1→] could substitute O-4 of the β-D-Glcp linked to HepI. In addition, the distal heptose (HepIII) was substituted by PCho→6-β-D-Galp-(1→ at the O-2 position. © 2013 Published by Elsevier Ltd.

Non-typeable Haemophilus influenzae (NTHi)-associated ear and respiratory diseases (including pneumonia) represent a major health burden in many parts of the world. NTHi strains retrieved from the upper airways commonly reflect those found in the lower airways. Despite growing genomic and genotyping data on NTHi, there remains a limited understanding of global and regional NTHi population structures. The aim of this study was to determine whether nasopharyngeal carriage in four Australian paediatric groups at varying risk of NTHi colonisation was dominated by the same NTHi genotypes. Genotyping data generated by PCR-ribotyping were evaluated for 3070 NTHi isolates colonising the nasopharynges of Aboriginal and non-Aboriginal children enrolled in four longitudinal studies in three separate urban and remote regions of Australia. Several NTHi PCR-ribotypes dominated in nasopharyngeal carriage, irrespective of study setting. Principal coordinates analysis confirmed a cluster of common PCR-ribotypes among all cohorts. In conclusion, we identified dominant PCR-ribotypes common to geographically disparate Australian paediatric populations. Future genomic analyses will shed further light on the precise factors underlying the dominance of certain NTHi strains in nasopharyngeal carriage.

Nontypeable Haemophilus influenzae (NTHi) is increasingly recognized as emerging pathogen. The routine immunization of infants with conjugated vaccines against H. influenzae type b (Hib) has greatly reduced the incidence of invasive Hib disease; however a marked change in the predominant invasive serotype from Hib to NTHi has occurred. Localized infections where the role of H. influenzae is important, such as otitis media in children and acute exacerbations in chronic obstructive pulmonary disease (COPD) in adults, are almost exclusively associated with NTHi isolates. The implementation of pneumococcal conjugate vaccines has resulted in changes in frequency of nasopharynx colonizing pathogens with an increase of NTHi, although this data is yet under debate. An effective vaccine against NTHi is not currently available. The major challenge in developing a successful vaccine is the intrinsic heterogeneity of NTHi. H. influenzae protein D is used as carrier protein in the licensed 10-valent pneumococcal conjugate vaccine (Synflorix, GlaxoSmithKline), but no robust evidences for protective efficacy against NTHi otitis have been until now obtained. Several other vaccine candidates are under investigations and we hope that significant advancements in vaccine development will be achieved in the next future. Genome-based vaccine strategy might provide an additional useful tool for discovering further vaccine antigens.

In India, pneumococcal diseases are major causes of child mortality, and effective vaccines against Streptococcus pneumoniae are needed. This single-blind, randomized study assessed the immunogenicity, reactogenicity, and safety of the 10-valent pneumococcal non-typeable Hemophilus influenzae (NTHi) protein D conjugate vaccine (PHiD-CV) co-administered with DTPw-HBV/Hib in Indian infants as 3-dose primary vaccination course. A total of 360 infants were randomized (2:1) to receive either PHiD-CV co-administered with DTPw-HBV/Hib (PHiD-CV group) or a Hib vaccine co-administered with DTPw-HBV (control group) at 6, 10, and 14 weeks of age. For each vaccine pneumococcal serotype, the percentage of infants in the PHiD-CV group with antibody concentrations ≥ 0.2 µg/mL one month after the third vaccine dose was at least 98.3%, except for serotypes 6B (77.7%) and 23F (89.5%), and opsonophagocytic activity titers ≥ 8 were measured in at least 95.7% of infants, except for serotypes 1 (90.5%) and 6B (84.5%). In addition, all the infants in the PHiD-CV group were seroprotected against diphtheria, tetanus, Hib, and hepatitis B or seropositive for antibodies against pertussis and NTHi protein D (except one infant). Incidences of solicited local and general symptoms were comparable between groups, except for fever (axillary temperature ≥ 37.5°C), which seemed to occur more frequently in the PHiD-CV group. In conclusion, PHiD-CV was shown to be immunogenic and well-tolerated when co-administered with DTPw-HBV/Hib in Indian infants.

The epidemiology of non-typeable Haemophilus influenzae (NTHi) remains poorly understood. We therefore sought to determine the genetic relationship of 25 NTHi isolated from various states in Malaysia using multilocus sequence typing (MLST). The majority of isolates were obtained from sputum. There were 24 novel sequence types (STs). Eight isolates were single-locus variants, the remainder being singletons. Clustering was not based on clinical site of isolation or geographical origin. Despite the limited number of isolates examined in this study, we demonstrate that NTHi isolates in Malaysia are diverse and warrant further investigation.

ResumenObjetivoAnálisis de coste-efectividad de la vacunación antineumocócica pediátrica en España.MétodosSe utilizó un modelo determinístico poblacional en forma de árbol de decisión con un horizonte temporal de 1 año para estimar el impacto de la vacunación con Synflorix® o Prevenar13® sobre la población menor de 2 años en España, bajo la perspectiva del Sistema Nacional de Salud. Los datos epidemiológicos se obtuvieron del Conjunto Mínimo Básico de Datos al alta hospitalaria y de la literatura. Los datos sobre costes se obtuvieron de bases de datos nacionales de costes sanitarios. Los principales resultados en salud medidos fueron los casos evitados de enfermedad neumocócica invasora, otitis media aguda (OMA), miringotomías y hospitalizaciones por neumonía. Todos los costes se expresaron en euros de 2010. La efectividad se midió en años de vida ajustados por calidad (AVAC).ResultadosEl potencial demostrado por Synflorix® para prevenir episodios de OMA causados por Haemophilus influenzae no tipificable podría traducirse en la prevención adicional de 41.513 episodios de OMA, 36.324 prescripciones de antibióticos y 382 miringotomías, y supondrían la ganancia de 143 AVAC frente a Prevenar13®. El coste total de la vacunación con Synflorix® resultaría unos 22 millones de euros menor.ConclusionesLa vacunación antineumocócica de la población menor de 2 años en España con Synflorix®, en comparación con la vacunación con Prevenar13®, podría resultar en un mayor número de AVAC, así como en una reducción sustancial del coste total, resultando una estrategia dominante en términos de coste-efectividad.AbstractObjectiveTo perform a cost-effectiveness analysis of pediatric pneumococcal vaccination in Spain.MethodsA deterministic population-based model in the form of a decision-tree with a 1-year time horizon was used to estimate the impact of vaccination with Synflorix® or Prevenar13® in children aged less than 2 years in Spain from the perspective of the National Health System. Epidemiological data were obtained from the hospital discharge minimum data set (MDS) and the literature. Data on costs were obtained from national health costs databases. The main outcomes measured were the number of cases avoided of invasive pneumococcal disease, acute otitis media (AOM) and myringotomies, and hospital admissions for pneumonia. All costs were expressed in 2010 euros. Effectiveness was measured as the number of quality-adjusted life years (QALYs) gained.ResultsThe efficacy of Synflorix® in preventing episodes of AOM caused by non-typeable Haemophilus influenzae could lead to additional prevention of 41,513 episodes of AOM, 36,324 antibiotic prescriptions and 382 myringotomy procedures and 143 QALYs gained compared with Prevenar13®. The total vaccination cost with Synflorix® would result in savings of 22 million euros.ConclusionsPneumococcal vaccination with Synflorix® in comparison with Prevenar13® in children aged less than 2 years old in Spain could achieve a higher number of QALYs and a substantial cost offset. Vaccination with Synflorix® would be a dominant strategy in terms of cost-effectiveness.

Identifcation and prioritisation of candidate antigens on which novel vaccines can be based, or the effcacy of existing vaccines improved, are critically dependent on characterising the strategies utilised by microbial pathogens to evade host defences, and, in particular, the key virulence factors involved. In this review, we have focused on the immune evasion strategies utilised by two important bacterial respiratory pathogens, viz Streptococcus pneumoniae and non-typeable Haemophilus infuenzae, with particular emphasis on key virulence factors and their potential to serve as candidate immunogens.

Vaccines to prevent acute otitis media (AOM) caused by non-typeable Haemophilus influenzae (NTHi) are under development. Because NTHi is highly variable and colonization rates are high, special vaccine characteristics and trial designs might be needed. We examined in mathematical models the equilibrium NTHi-caused AOM rate given hypothetical vaccines that generated immunity identical to corresponding maximal naturally acquired immunity. Vaccines were examined with single effects and combinations of immunity affecting (1) AOM rates given colonization (pathogenicity), (2) susceptibility to colonization, and (3) contagiousness given colonization. Percent reductions in AOM across all preschool children were (1) 34%, (2) 31%, (3) 9%, (1 and 2) 57%, (2 and 3) 50%, and (1, 2, and 3) 75%. Effects on children in daycare vs. not in daycare were (1) 18 vs. 48%, (2) -1 vs. 57%, (3) 13 vs. 5%, (1 and 2) 30 vs. 79%, (2 and 3) 33 vs. 60%, and (1, 2, and 3) 64 vs. 85%. Pure pathogenicity effects (1 alone) will need to be supplemented by transmission effects. The effects of susceptibility (2 alone) are diminished or negative because children protected against colonization have lower levels of immunity to (1) and (3) than unvaccinated children. For trials to predict population effects, both colonization and AOM outcomes must be studied and all three effects must be evaluated. This need arises because, unlike H. influenzae type B, high NTHi exposure diminishes cumulative vaccine effects and high colonization rates generate rapid accumulation of natural immunity that alters the indirect effects of vaccine immunity on transmission differently by age and daycare status.

Because all important assumptions used in the model are simultaneously biased toward PCV10, the model results are erroneous and misleading. Routine infant pneumococcal vaccination in Japan would undoubtedly bring substantial reductions in morbidity and mortality. However, given the current epidemiologic landscape in Japan and the current evidence, the clinical and economic gains from the use of PCV13 would, undoubtedly, far exceed those potentially observed from the use of PCV10. We urge those who conduct, critique, and consider cost-effectiveness studies to evaluate the strength of the evidence of clinical claims for the products and the influence these assumptions have on the overall findings. In addition, when performing economic predictive modeling, it is critical to provide a balanced perspective by weighing the strengths and weaknesses of all available data to construct the base case analysis.

Nontypeable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus are closely related bacteria that reside in the upper respiratory tract. NTHi is associated with respiratory tract infections that frequently result in antibiotic prescription whilst H. haemolyticus is rarely associated with disease. NTHi and H. haemolyticus can be indistinguishable by traditional culture methods and molecular differentiation has proven difficult. This current review chronologically summarizes the molecular approaches that have been recently developed for differentiation of NTHi from H. haemolyticus, highlighting the advantages and disadvantages of each target and/or technique. We also provide suggestions for the development of new tools that would be suitable for clinical and research laboratories.