Monitoring of Haemophilus influenzae isolated from carriage, lower respiratory tract infections and blood over a six-month period in Belgium.

INTRODUCTION: H. influenzae carriage may evolve into respiratory or systemic infections. However, no surveillancesystem is in place in Belgium to monitor carriage strains. MATERIAL AND METHODS: This study provides a detailed description of H. influenzae strains isolated from both carriage and lower respiratory infections, collected during a six-month national surveillance. Subsequently, a comparison is conducted with invasive isolates collected during the same period at the National Reference Centre (NRC). RESULTS AND DISCUSSION: From November 2021 to April 2022, 39 clinical laboratories collected 142 and 210 strains of H. influenzae from carriage and infection, respectively, and 56 strains of blood were submitted to the NRC. In each group, the biotype II comprised more than 40%, followed by biotypes III and I. The majority of strains were non-typeable H. influenzae, with a notable increase in the number of encapsulated strains in the invasive group (14.3% vs. 1-2%). A beta-lactamase was identified in 18.5% and 12.5% of surveillance and invasive strains, respectively. Resistance to the amoxicillin-clavulanic acid combination accounted for 7% in the surveillance strains and 10.7% in invasive strains. The overall resistance to third-generation cephalosporins at 1.2% is consistent with rates observed in other European countries. Of particular significance is the identification of mutations in the ftsI gene in both carriage and infected strains, which are associated with high-level beta-lactam resistance. CONCLUSION: NRC must engage in regular and systematic monitoring of beta-lactam susceptibility of H. influenzae to guarantee safe empiric therapy in severe cases and identify potential transitions from low-level to high-level resistance in the future.

Characterization of bacterial and viral pathogens in the respiratory tract of children with HIV-associated chronic lung disease: a case-control study.

INTRODUCTION: Chronic lung disease is a major cause of morbidity in African children with HIV infection; however, the microbial determinants of HIV-associated chronic lung disease (HCLD) remain poorly understood. We conducted a case-control study to investigate the prevalence and densities of respiratory microbes among pneumococcal conjugate vaccine (PCV)-naive children with (HCLD +) and without HCLD (HCLD-) established on antiretroviral treatment (ART). METHODS: Nasopharyngeal swabs collected from HCLD + (defined as forced-expiratory-volume/second < -1.0 without reversibility postbronchodilation) and age-, site-, and duration-of-ART-matched HCLD- participants aged between 6-19 years enrolled in Zimbabwe and Malawi (BREATHE trial-NCT02426112) were tested for 94 pneumococcal serotypes together with twelve bacteria, including Streptococcus pneumoniae (SP), Staphylococcus aureus (SA), Haemophilus influenzae (HI), Moraxella catarrhalis (MC), and eight viruses, including human rhinovirus (HRV), respiratory syncytial virus A or B, and human metapneumovirus, using nanofluidic qPCR (Standard BioTools formerly known as Fluidigm). Fisher's exact test and logistic regression analysis were used for between-group comparisons and risk factors associated with common respiratory microbes, respectively. RESULTS: A total of 345 participants (287 HCLD + , 58 HCLD-; median age, 15.5 years [IQR = 12.8-18], females, 52%) were included in the final analysis. The prevalence of SP (40%[116/287] vs. 21%[12/58], p = 0.005) and HRV (7%[21/287] vs. 0%[0/58], p = 0.032) were higher in HCLD + participants compared to HCLD- participants. Of the participants positive for SP (116 HCLD + & 12 HCLD-), 66% [85/128] had non-PCV-13 serotypes detected. Overall, PCV-13 serotypes (4, 19A, 19F: 16% [7/43] each) and NVT 13 and 21 (9% [8/85] each) predominated. The densities of HI (2 × 104 genomic equivalents [GE/ml] vs. 3 × 102 GE/ml, p = 0.006) and MC (1 × 104 GE/ml vs. 1 × 103 GE/ml, p = 0.031) were higher in HCLD + compared to HCLD-. Bacterial codetection (≥ any 2 bacteria) was higher in the HCLD + group (36% [114/287] vs. (19% [11/58]), (p = 0.014), with SP and HI codetection (HCLD + : 30% [86/287] vs. HCLD-: 12% [7/58], p = 0.005) predominating. Viruses (predominantly HRV) were detected only in HCLD + participants. Lastly, participants with a history of previous tuberculosis treatment were more likely to carry SP (adjusted odds ratio (aOR): 1.9 [1.1 -3.2], p = 0.021) or HI (aOR: 2.0 [1.2 - 3.3], p = 0.011), while those who used ART for ≥ 2 years were less likely to carry HI (aOR: 0.3 [0.1 - 0.8], p = 0.005) and MC (aOR: 0.4 [0.1 - 0.9], p = 0.039). CONCLUSION: Children with HCLD + were more likely to be colonized by SP and HRV and had higher HI and MC bacterial loads in their nasopharynx. The role of SP, HI, and HRV in the pathogenesis of CLD, including how they influence the risk of acute exacerbations, should be studied further. TRIAL REGISTRATION: The BREATHE trial (ClinicalTrials.gov Identifier: NCT02426112 , registered date: 24 April 2015).

Comparison of bacterial culture with BioFire® FilmArray® multiplex PCR screening of archived cerebrospinal fluid specimens from children with suspected bacterial meningitis in Nigeria.

BACKGROUND: Diagnosis of bacterial meningitis remains a challenge in most developing countries due to low yield from bacterial culture, widespread use of non-prescription antibiotics, and weak microbiology laboratories. The objective of this study was to compare the yield from standard bacterial culture with the multiplex nested PCR platform, the BioFire® FilmArray® Meningitis/Encephalitis Panel (BioFire ME Panel), for cases with suspected acute bacterial meningitis. METHODS: Following Gram stain and bacterial culture on cerebrospinal fluid (CSF) collected from children aged less than 5 years with a clinical suspicion of acute bacterial meningitis (ABM) as defined by the WHO guidelines, residual CSF specimens were frozen and later tested by BioFire ME Panel. RESULTS: A total of 400 samples were analyzed. Thirty-two [32/400 (8%)] of the specimens were culture positive, consisting of; three Salmonella spp. (2 Typhi and 1 non-typhi), three alpha hemolytic Streptococcus, one Staphylococcus aureus, six Neisseria meningitidis, seven Hemophilus influenzae, 11 Streptococcus pneumoniae and 368 were culture negative. Of the 368 culture-negative specimens, the BioFire ME Panel detected at least one bacterial pathogen in 90 (24.5%) samples, consisting of S. pneumoniae, N. meningitidis and H. influenzae, predominantly. All culture positive specimens for H. influenzae, N. meningitidis and S. pneumoniae also tested positive with the BioFire ME Panel. In addition, 12 specimens had mixed bacterial pathogens identified. For the first time in this setting, we have data on the viral agents associated with meningitis. Single viral agents were detected in 11 (2.8%) samples while co-detections with bacterial agents or other viruses occurred in 23 (5.8%) of the samples. CONCLUSIONS: The BioFire® ME Panel was more sensitive and rapid than culture for detecting bacterial pathogens in CSF. The BioFire® ME Panel also provided for the first time, the diagnosis of viral etiologic agents that are associated with meningoencephalitis in this setting. Institution of PCR diagnostics is recommended as a routine test for suspected cases of ABM to enhance early diagnosis and optimal treatment.

Invasive Haemophilus influenzae Infections after 3 Decades of Hib Protein Conjugate Vaccine Use.

Haemophilus influenzae serotype b (Hib) was previously the most common cause of bacterial meningitis and an important etiologic agent of pneumonia in children aged <5 years. Its major virulence factor is the polyribosyl ribitol phosphate (PRP) polysaccharide capsule. In the 1980s, PRP-protein conjugate Hib vaccines were developed and are now included in almost all national immunization programs, achieving a sustained decline in invasive Hib infections. However, invasive Hib disease has not yet been eliminated in countries with low vaccine coverage, and sporadic outbreaks of Hib infection still occur occasionally in countries with high vaccine coverage. Over the past 2 decades, other capsulated serotypes have been recognized increasingly as causing invasive infections. H. influenzae serotype a (Hia) is now a major cause of invasive infection in Indigenous communities of North America, prompting a possible requirement for an Hia conjugate vaccine. H. influenzae serotypes e and f are now more common than serotype b in Europe. Significant year-to-year increases in nontypeable H. influenzae invasive infections have occurred in many regions of the world. Invasive H. influenzae infections are now seen predominantly in patients at the extremes of life and those with underlying comorbidities. This review provides a comprehensive and critical overview of the current global epidemiology of invasive H. influenzae infections in different geographic regions of the world. It discusses those now at risk of invasive Hib disease, describes the emergence of other severe invasive H. influenzae infections, and emphasizes the importance of long-term, comprehensive, clinical and microbiologic surveillance to monitor a vaccine's impact.

Antimicrobial activity of ceftobiprole and comparator agents when tested against gram-positive and -negative organisms collected across China (2016-2018).

BACKGROUND: Ceftobiprole is a fifth-generation cephalosporin which has been reported to have broad antibacterial spectrum when tested against bacteria collected from other countries except China. This study evaluated the in vitro activity of ceftobiprole in comparison with other comparators against clinically significant isolates collected across from China. RESULTS: Susceptibility testing of ceftobiprole and comparators against 1163 clinically isolated Gram-positive and Gram-negative bacteria was performed with broth micro dilution method following the CLSI guidelines. All 110 S. aureus were susceptible to ceftobiprole with MIC50/90 of 1/2 mg/L for MRSA and 0.5/1 mg/L for MSSA. For Coagulase-negative staphylococci (CNS), MIC50/90 of ceftobiprole for MRCNS and MSCNS was 1/2 mg/L and 0.25/0.5 mg/L. Ceftobiprole demonstrated good potency against E. faecalis (MIC50/90 of 0.5/1 mg/L) but limited activity against E. faecium (MIC50/90 of > 32/ > 32 mg/L). Ceftobiprole demonstrated potent activity against all 39 ß-hemolytic Streptococcus spp. with MIC50/90 ≤ 0.015/ ≤ 0.015-2 mg/L and 110 of PSSP with 98.2% susceptibility. Ceftobiprole inhibited all isolates of H. influenzae and M. catarrhalis at ≤ 1 mg/L. 91.8% and 98.2% of the ESBL-negative E. coli and K. pneumoniae were susceptible to ceftobiprole, but most of the ESBL-positive or carbapenem-resistant strains were also resistant to ceftobiprole. Ceftobiprole inhibited 84.2% of carbapenem-susceptible P. aeruginosa and 94.1% of carbapenem-susceptible A. baumannii at ≤ 8 mg/L, but only 52.6% of carbapenem-resistant P. aeruginosa and 5.3% of carbapenem-resistant A. baumannii. CONCLUSION: Ceftobiprole demonstrated good in vitro activity against a broad range of clinically relevant contemporary Gram-positive and Gram-negative bacterial isolates.

Use of trans-complementation method to determine the effects of various ftsI mutations on ß-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae strains.

Haemophilus influenzae is a causative agent of serious infections, especially among children. ß-lactam antibiotics are commonly used for the treatment of these infections. Among H. influenzae isolates, ß-lactam resistance is due to the presence of ß-lactamase, or to mutations in the ftsI gene that generate altered PBP3 (penicillin-binding protein 3) with reduced affinity for ß-lactams (BLNAR-ß-lactamase-negative, ampicillin-resistant). Wild-type ftsI gene encoding for PBP3 was amplified in whole from ß-lactam susceptible H. influenzae Rd and cloned in pLS88 plasmid to obtain pADUTAS17, which was then used to transform known BLNAR strains, susceptible strains, and a strain (CF55) with wild-type ftsI but unexplained reduced ß-lactam susceptibility. Ampicillin and cefotaxime MICs (minimum inhibitory concentration) were determined after transformation with pLS88 and pADUTAS17 plasmids. The results showed that antibiotic susceptibilities were not affected by trans-complementation for isolates carrying wild-type ftsI gene. However, trans-complementation for all BLNAR strains showed decreases between - 0.957 and 0.5-fold for ampicillin and cefotaxime, confirming the role of the PBP3 substitutions in the BLNAR phenotype of these isolates. The first article showed that trans-complementation might be a useful tool in the investigation of decreased ß-lactam susceptibility in H. influenzae.

Respiratory quinolones can eradicate amoxicillin-induced mature biofilms and nontypeable Haemophilus influenzae in biofilms.

OBJECTIVES: Biofilm is thought to be involved in the persistent bacterial infections caused by nontypeable Haemophilus influenzae (NTHi). This study aims to evaluate the efficacy of antibiotics against NTHi biofilms. METHODS: A 96-wells pin replicator assay was applied for evaluation of antimicrobial efficacies against NTHi biofilms. The NTHi IH-202 strain for the standard and 10 clinical strains were evaluated, as well as the viability of NTHi in biofilms after antimicrobial exposures. RESULTS: Biofilms formed by IH-202 strain accumulated during incubation. AMPC if not high concentrations, neither reduce or inhibit biofilm formation, nor eradicate matured NTHi biofilms. The NTHi in matured biofilm were alive after exposure to amoxicillin (AMPC). Even high concentration of AMPC produced live NTHi after suspension of exposure, while tosufloxacin and garenoxacin inhibited biofilm formation of NTHi and eradicated matured biofilms. The respiratory quinolones, but not AMPC, killed NTHi in biofilms even at sub-MIC. CONCLUSIONS: NTHi persists in biofilms, even after exposure to AMPC. These findings may eventually lead to a better understanding of effective use of antibiotics to eradicate NTHi growing as biofilms, or even to the development of novel therapeutic agents for treating patients with mucosal NTHi biofilm infections. Meanwhile, respiratory quinolones are attractive agents in reducing NTHi biofilm formation and destroying established biofilm.

A Prospective, Population-based Study to Determine the Incidence and Bacteriology of Bacterial Conjunctivitis in Children <2 Years of Age Following 7-Valent and 13-Valent Pneumococcal Conjugate Vaccine Sequential Implementation.

BACKGROUND: Bacterial conjunctivitis is most commonly caused by nontypeable Haemophilus influenzae (NTHi), followed by Streptococcus pneumoniae. No population-based data on the impact of pneumococcal conjugate vaccines (PCVs) on the incidence of bacterial conjunctivitis have been published. We assessed rate dynamics of overall, pneumococcal, and NTHi conjunctivitis in children aged 2-23 months in southern Israel before and after PCV implementation. METHODS: This is a 12-year prospective, population-based surveillance, from July 2004 through June 2017. Our medical center serves a captive population of approximately 30 000 children < 2 years of age, and its clinical microbiology laboratory processes > 80% of all community-derived cultures, enabling incidence calculation. The 7-valent and 13-valent PCVs (PCV7 and PCV13, respectively) were implemented in the national immunization program in July 2009 and November 2010, respectively. Pneumococci, NTHi, Moraxella catarrhalis, and Streptococcus pyogenes were considered pathogens. Continuous annual incidences and incidence rate ratios comparing the PCV13 period (2015-2017) to the pre-PCV period (2004-2008) were calculated. RESULTS: Disease caused by PCV13 serotypes declined by 93%, without significant replacement with non-PCV13 serotypes. Rates of pneumococcal, NTHi, and overall culture-positive episodes declined by 59%, 41%, and 42%, respectively, while rates of culture-negative and other pathogens episodes did not change significantly. An overall reduction in all submitted culture rates of 35% was observed. This pattern was seen across all ages, including infants aged 2-5 months. CONCLUSIONS: PCV7/PCV13 implementation resulted in a marked and significant decline in pneumococcal, NTHi, and overall conjunctivitis rates in children < 2 years of age. The impact on NTHi episodes alludes to the role of pneumococcus-NTHi interaction in conjunctivitis. The impact in infants aged < 6 months suggests herd protection.

Epidemiology of Haemophilus influenzae in the Republic of Ireland, 2010-2018.

The purpose of this study was to investigate H. influenzae epidemiology in the Republic of Ireland. We performed serotyping, multi-locus sequence typing (MLST) and susceptibility testing on H. influenzae isolates received by the Irish Meningitis and Sepsis Reference Laboratory from 2010 to 2018. Three hundred sixty-seven invasive and 41 non-invasive infection (NII) isolates were received. Invasive isolates were mostly recovered from paediatric (21%) and elderly (42%) populations. Invasive disease was more prevalent in females of childbearing age (72%) compared with males the same age (28%). Non-typeable H. influenzae (NTHi) predominated among invasive (83%) and NII (95%). Invasive Hib disease isolates were infrequent (4%, n = 15). Among invasive disease, Hif was the commonest encapsulated serotype (10%, n = 37), and the only encapsulated serotype detected in NII (5%, 2/41). The first PCR-confirmed serotypes d and a in Ireland were characterised among invasive disease in 2017 and 2018, respectively. MLST revealed a diverse NTHi population, while encapsulated serotypes were clonal. Sequence type (ST) 103 (n = 14) occurred exclusively in invasive NTHi disease. Ampicillin resistance (AmpR) was 18% among invasive isolates and 22% in NII. ß-Lactamase production was the main source of ampicillin resistance in invasive and NII isolates. We detected ß-lactamase negative ampicillin resistance (BLNAR) among invasive isolates. We report an NTHi fluoroquinolone-resistant clone: ST1524 among invasive (n = 2) and NII isolates (n = 2). The Hib vaccine has positively impacted on Hib disease in Ireland, given the low frequency of Hib. The dominance of NTHi, emergence of serotypes a and d and BLNAR suggest a changing H. influenzae epidemiology in Ireland.

Diagnosis of Haemophilus influenzae Pneumonia by Nanopore 16S Amplicon Sequencing of Sputum.

We used deep sequencing of the 16S rRNA gene from sputum to identify Haemophilus influenza in a patient with community-acquired pneumonia. This method may be more effective than conventional diagnostic tests in pneumonia patients because of its speed and sensitivity.

Azithromycin Pharmacodynamics against Persistent Haemophilus influenzae in Chronic Obstructive Pulmonary Disease.

The pharmacodynamic profile of azithromycin against persistent strains of nontypeable Haemophilus influenzae (NTHi) from chronic obstructive pulmonary disease (COPD) patients was characterized. Azithromycin displayed differential concentration-dependent activities (R2 ≥ 0.988); the pharmacodynamic response was attenuated when we compared the "first" and "last" strains of NTHi that persisted in the airways of the same patient for 819 days (the 50% effective concentration [EC50] increased more than 50 times [0.0821 mg/liter versus 4.23 mg/liter]). In the hollow-fiber infection model, NTHi viability was maintained throughout simulated azithromycin (Zithromax) Z-Pak regimens over 10 days.

Molecular characterization of fluoroquinolones, macrolides, and imipenem resistance in Haemophilus influenzae: analysis of the mutations in QRDRs and assessment of the extent of the AcrAB-TolC-mediated resistance.

The aims of the present study were to characterize the mechanisms of resistance to fluoroquinolones, macrolides, and imipenem in Haemophilus influenzae, to assess the extent of the AcrAB-TolC-mediated resistance, and to define a core genome multilocus sequence typing (cgMLST) scheme for H. influenzae by using whole-genome sequencing. Four amino acid substitutions in GyrA (at Ser84 and Asp88), ParC (at Ser84), and ParE (at Asp420) were found to be closely associated to the MICs. We did not find any amino acid substitution surrounding the three highly conserved amino acid motifs in PBP3 related to imipenem resistance. All the isolates possessed the ermB gene. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased the MIC of imipenem by twofold for FQR-6 and fourfold for GE47 and GE88 strains. For erythromycin, the MICs were decreased by twofold. We found that the six FQR isolates were clustered in two groups. The number of different loci within FQR-1_FQR-3_FQR-5 cluster was 6, while FQR-2 and FQR-4 differed for 21 loci. FQR-1_FQR-3_FQR-5 and FQR-2_FQR-4 clusters were distant among each other and compared to 19 genomes downloaded from NCBI, to 8 strains heteroresistant to imipenem, and to 4 strains monoresistant to ciprofloxacin isolated in Denmark. We confirmed that specific amino acid substitutions in GyrA, ParC, and ParE are implicated in quinolone resistance. Additionally, the degree of resistance is related to the number of these amino acid substitutions. We provide robust evidence that drug efflux is one of the substantial mechanisms of imipenem and erythromycin resistance in H. influenzae.

Epidemiological analysis and rapid detection by one-step multiplex PCR assay of Haemophilus influenzae in children with respiratory tract infections in Zhejiang Province, China.

BACKGROUND: Haemophilus influenzae (H. influenzae) is one of the most important pathogenic bacteria causing respiratory tract infection diseases in children. There are two main types of H. influenzae, encapsulated H. influenzae and nontypeable H. influenzae (NTHi). Serotype b of H. influenzae (Hib) used to be the main epidemic type of H. influenzae, causing the invasive infection. However, the epidemiology of invasive H. influenzae disease has changed substantially, and most invasive diseases are now caused by NTHi and other serotypes of H. influenzae. The aim of this study was to determine the main epidemic strains of H. influenzae in Zhejiang Province in China, and establish a one-step multiplex PCR assay to distinguish H. influenzae from other bacteria associated with respiratory tract infections, and distinguish encapsulated H. influenzae from NTHi. METHOD: In this study, bacterial culture and serum agglutination testing were used to determine the most prevalent serotype of H. influenzae, and the results have served as a gold standard for clinical diagnosis. We also designed a one-step multiplex PCR assay using several kinds of standard strains of respiratory tract infection bacteria, to examine the stability, specificity, and detection limit of the PCR assays. We then used 1514 nasopharyngeal secretion (NPS) samples collected from children with respiratory tract infections to verify the specificity and sensitivity of the PCR assay. RESULTS: The bacterial culture and serum agglutination test results showed that the positive rates of H. influenzae and encapsulated H. influenzae were 18.49 and 1.18%, respectively. The PCR results showed that the detection limit of the multiplex PCR assay was 1.89 × 103 copies /µL, the ompP6 positive rate was 19.35%, and the bexA positive rate was 1.32%. The sensitivity and specificity of the multiplex PCR were 100 and 99.86%, respectively. CONCLUSIONS: According to our study, the most prevalent H. influenzae subtype in Zhejiang Province was NTHi, account for 93.57%; the one-step multiplex PCR assay we established can be used as the differential detection of clinical H. influenzae strains, replacing routine bacterial culture and serum agglutination testing.

Nonantibiotic macrolides restore airway macrophage phagocytic function with potential anti-inflammatory effects in chronic lung diseases.

We reported defective efferocytosis associated with cigarette smoking and/or airway inflammation in chronic lung diseases, including chronic obstructive pulmonary disease, severe asthma, and childhood bronchiectasis. We also showed defects in phagocytosis of nontypeable Haemophilus influenzae (NTHi), a common colonizer of the lower airway in these diseases. These defects could be substantially overcome with low-dose azithromycin; however, chronic use may induce bacterial resistance. The aim of the present study was therefore to investigate two novel macrolides-2'-desoxy-9-(S)-erythromycylamine (GS-459755) and azithromycin-based 2'-desoxy molecule (GS-560660)-with significantly diminished antibiotic activity against Staphylococcus aureus, Streptococcus pneumonia, Moraxella catarrhalis, and H. influenzae We tested their effects on efferocytosis, phagocytosis of NTHi, cell viability, receptors involved in recognition of apoptotic cells and/or NTHi (flow cytometry), secreted and cleaved intracellular IL-1ß (cytometric bead array, immunofluorescence/confocal microscopy), and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) using primary alveolar macrophages and THP-1 macrophages ± 10% cigarette smoke extract. Dose-response experiments showed optimal prophagocytic effects of GS-459755 and GS-560660 at concentrations of 0.5-1 µg/ml compared with our findings with azithromycin. Both macrolides significantly improved phagocytosis of apoptotic cells and NTHi (e.g., increases in efferocytosis and phagocytosis of NTHi: GS-459755, 23 and 22.5%, P = 0.043; GS-560660, 23.5 and 22%, P = 0.043, respectively). Macrophage viability remained >85% following 24 h exposure to either macrolide at concentrations up to 20 µg/ml. Secreted and intracellular-cleaved IL-1ß was decreased with both macrolides with no significant changes in recognition molecules c-mer proto-oncogene tyrosine kinase; scavenger receptor class A, member 1; Toll-like receptor 2/4; or CD36. Particulate cytoplasmic immunofluorescence of NLRP3 inflammasome was also reduced significantly. We conclude that GS-459755 and GS-560660 may be useful for reducing airway inflammation in chronic lung diseases without inducing bacterial resistance.

Multi-site placebo-controlled randomised clinical trial to assess protection following oral immunisation with inactivated non-typeable Haemophilus influenzae in chronic obstructive pulmonary disease.

BACKGROUND: Previous studies identified factors that modify response to an oral non-typeable Haemophilus influenzae (NTHi) vaccine in chronic obstructive pulmonary disease (COPD): severe COPD, moderate-severe exacerbations as end-point and a threshold prevalence of NTHi in the study population. More data are needed to confirm parameters that influence clinical outcomes. AIMS: The primary aim was to determine the efficacy of an oral NTHi vaccine (HI-164OV) in reducing the rate of exacerbations requiring systemic corticosteroids or hospitalisation in COPD. Secondary aims included effect on the proportion of patients experiencing such exacerbations, severity of infections and quality of life (St George Respiratory Questionnaire for COPD patients (SGRQ-C)). METHODS: This multi-centre, double-blind, placebo-controlled study was conducted at 21 Australian sites for 9 months in 2011. RESULTS: Three-hundred and twenty subjects with COPD, FEV1 <60% predicted and ≥1 moderate-severe exacerbations in the previous 12 months were recruited. The primary and secondary end-points for the intention-to-treat population aged 40-88 years were not achieved, and only 5% of subjects had an H. influenzae-positive sputum sample. Subsequent exploratory analysis of patients <65 years (91 subjects) indicated protection with respect to the primary and most of the secondary end-points, with SGRQ-C symptom scores lower at 3 and 6 months. CONCLUSION: Patients aged 40-88 years with moderate to severe COPD and low rates of H. influenzae-positive sputum were not protected against exacerbations by HI-1640V. Further studies are needed to confirm protection in subjects aged <65 years. Older age and low colonisation rates appear to affect adversely response to this vaccine.

The outcome of H. influenzae and S. pneumoniae inter-species interactions depends on pH, nutrient availability and growth phase.

Haemophilus influenzae and Streptococcus pneumoniae exist together as common commensals of the healthy human nasopharynx, but both are important aetiological agents of different diseases, including the paediatric disease otitis media. It was recently shown that the formation of a multispecies biofilm of H. influenzae and S. pneumoniae is the cause of chronic forms of otitis media. However, the interactions between the two species are not clearly defined. Using a defined and kinetic analysis, our study has shown that while co-existence of the two species occurs, S. pneumoniae is also able to convert H. influenzae to a non-culturable state. We determined that this process was dependent on growth phase and pH. To analyse the H. influenzae/S. pneumoniae interactions in more depth, we investigated the growth and transcriptional profile in a pH-defined batch culture model, as well as in a growth phase independent flow cell system. Transcriptomics has shown that there are changes in gene expression in each of the species when grown in co-culture, intriguingly inducing the S. pneumoniae bacteriocin transport genes, and phage-associated genes in both species. Importantly, we have shown vast changes in gene expression in a group of S. pneumoniae metabolic genes, including those encoding lactose utilisation, glycerol utilisation and sugar transport proteins; we have shown that the expression of these genes depends not only on the presence of H. influenzae, but also on the growth system utilised.

Trends in the epidemiology of invasive Haemophilus influenzae disease in Queensland, Australia from 2000 to 2013: what is the impact of an increase in invasive non-typable H. influenzae (NTHi)?

Following the introduction of vaccination against Haemophilus influenzae type b (Hib), cases of invasive encapsulated Hib disease have decreased markedly. This study aimed to examine subsequent epidemiological trends in invasive H. influenzae disease in Queensland, Australia and in particular, assess the clinical impact and public health implications of invasive non-typable H. influenzae (NTHi) strains. A multicentre retrospective study was conducted from July 2000 to June 2013. Databases of major laboratories in Queensland including Queensland Forensic and Scientific Services (jurisdictional referral laboratory for isolate typing) were examined to identify cases. Demographic, infection site, Indigenous status, serotype, and mortality data were collected. In total, 737 invasive isolates were identified, of which 586 (79·5%) were serotyped. Hib, NTHi and encapsulated non-b strains, respectively, constituted 12·1%, 69·1% and 18·8% of isolates. The predominant encapsulated non-b strains were f (45·5%) and a (27·3%) serotypes. Of isolates causing meningitis, 48·9% were NTHi, 14·9% Hib, 14·9% Hie, 10·6% Hif, 6·4% Hia and 4·3% were untyped. During the study period, there was an increase in the incidence of invasive NTHi disease (P = 0·007) with seasonal peaks in winter and spring (P 0·001) and Hib (P = 0·039) than non-Indigenous patients. In Queensland, invasive H. influenzae disease is now predominantly encountered in adults and most commonly caused by NTHi strains with demonstrated pathogenicity extending to otherwise young or immunocompetent individuals. Routine public health notification of these strains is recommended and recent available immunization options should be considered.

Detection of Bacterial Biofilms in Chronic Pharyngitis Resistant to Medical Treatment.

OBJECTIVE: To evaluate the role played by adenoids as a reservoir for infection in children assigned for adenoidectomy. METHODOLOGY: The study included 35 children with adenoid hypertrophy. All patients underwent clinical examination and adenoidectomy, adenotonsillectomy, or myringotomy with insertion of aeration tube according to indications. Surgical specimens were processed for conventional bacterial culture examination and to assay for biofilm formation. The obtained adherence values using spectrophotometer at 595 nm (OD595) was used to classify isolates according to its biofilm forming capacity. RESULTS: We did adenotonsillectomy and myringotomy with insertion of aeration tube in 5 patients having adenotonsillitis with otitis media with effusion. We did adenotonsillectomy in 12 patients having adenotonsillitis and adenoidectomy in 18 patients having adenoid hypertrophy. Thirty-one surgical specimens showed bacterial growth on conventional media, while 4 specimens failed to give growth. The predominant organism was H influenzae then Staph aureus and Strept pneumoniae. Thirty-two specimens showed biofilm forming capacity (BFC) of variable extent, while others showed no BFC. CONCLUSION: Adenoids act as a bacterial reservoir secondary to bacterial biofilm formation so could induce chronicity and initiate development of complications. Determination of BFC using the proposed protocol is feasible, inexpensive, and available and spares the need for sophisticated instruments or approaches.