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Article Reference Outer membrane protein D15 is conserved among Haemophilus influenzae species and may represent a universal protective antigen against invasive disease.
We have cloned and sequenced the d15 gene from two strains of Haemophilus influenzae type b (Hib) and two strains of nontypeable H. influenzae (NTHI). The nucleotide and deduced protein sequences of d15 are highly conserved, with only a small variable region identified near the carboxyl terminus of the protein. Analysis of upstream sequences revealed that the H. influenzae d15 gene may be part of a large potential operon of closely spaced open reading frames, including one with significant homology to the Escherichia coli cds gene encoding CDP-diglyceride synthetase. Southern blot analysis demonstrated that the d15 gene is also present in H. influenzae types a, c, d, e, and f and in Haemophilus parainfluenzae. A recombinant D15 (rD15) protein was expressed in good quantity in E. coli from the inducible T7 promoter, and monospecific anti-rD15 antibodies were raised. Immunoblot analysis of H. influenzae serotypes a, b, c, d, e, and f, NTHI, and H. parainfluenzae lysates revealed that they all expressed a cross-reactive D15-like protein. Purified rD15 was found to be highly immunogenic in mice, guinea pigs, and rabbits, and passive transfer of anti-rD15 antibodies protected infant rats from challenge with H. influenzae type b or type a in infant rat models of bacteremia. Thus, D15 is a highly conserved antigen that is protective in animal models and it may be a useful component of a universal subunit vaccine against Haemophilus infection and disease.
Article Reference Invasive non-typeable Haemophilus influenzae diseases in children.
The current approach to patients with invasive non-typeable H. influenza disease is based upon past experience with the type b strains. In areas where clinicians cannot obtain typing information in a timely manner, issues concerning treatment and prophylaxis should be approached as if the patients were infected with a type b strain. This approach will not change until further information becomes available on invasive non-typeable H. influenzae infections in children.
Article Reference A virulent nonencapsulated Haemophilus influenzae.
Nontypeable Haemophilus influenzae strain INT1 was isolated from the blood of a young child with clinical signs of meningitis following acute otitis media. No immunologic or anatomic predisposition of this child for invasive bacterial infection with an unusual organism was documented. Sensitive ELISA proved the absence of intra- or extracellular capsular polysaccharide production by INT1 and Southern blot analysis confirmed the lack of an intact capsulation (cap) gene locus within the chromosome. Nevertheless, INT1 established bacteremia and meningitis in infant and weanling rat models of invasive H. influenzae infection. High-molecular-weight DNA isolated from INT1 was shown to confer an invasive phenotype on transformation of a nonencapsulated, avirulent laboratory strain of H. influenzae. Together these findings imply the presence of one or more as-yet-undiscovered, noncapsular virulence factors of H. influenzae that are capable of mediating invasive disease and resistance to immunologic clearance.
Article Reference Prospective epidemiological study of invasive Haemophilus influenzae disease in adults.
The incidence and characteristics of invasive Haemophilus influenzae disease were studied in 43 adult patients admitted to the acute care hospitals in El Vall├ęs County (Barcelona, Spain) between January 1987 and June 1992. The annual incidence of Haemophilus influenzae disease was 1.2 per 100,000 inhabitants. Pneumonia occurred in 24 patients, meningitis in five, intraabdominal infections in three, obstetric infections in two, epiglottitis in two and cellulitis in one. In six patients the source of infection was unknown. Ten (23%) of the infections were hospital acquired. Underlying conditions were diagnosed in 30 (70%) patients. Nontypeable Haemophilus influenzae strains predominated in all adult age groups. Sixty-one percent of type b and 34% of nontypeable strains were ampicillin resistant (p = 0.08). Multiple antibiotic resistance was also high among type b (53%) and nontypeable (18%) strains. The mortality rate was significantly higher in patients with pneumonia, bacteremia from an unidentified focus or shock at presentation.
Article Reference Neonatal, urogenital isolates of biotype 4 nontypeable Haemophilus influenzae express a variant P6 outer membrane protein molecule.
The P6 outer membrane protein is a highly conserved molecule which is present on the surface of all strains of Haemophilus influenzae. Sixty strains of nontypeable H. influenzae which caused invasive disease or colonized the female urogenital tract were studied with monoclonal antibodies 7F3 and 4G4, which recognize different surface-exposed epitopes on the P6 molecule. All 60 strains expressed the epitope recognized by 4G4, whereas 47 of 60 strains expressed the epitope recognized by antibody 7F3. The 7F3-nonreactive strains were all biotype 4 and were recovered from the blood of neonates or postpartum women or from the female urogenital tract. The P6 genes from two 7F3-nonreactive strains were cloned, and the nucleotide sequences were determined. Analysis of amino acid sequences, immunoassays with synthetic peptides, and site-directed mutation of the P6 gene indicate that the epitope recognized by antibody 7F3 is conformational and that the sequence Asp-Ile-Thr is critical in maintaining the conformation of the epitope. We conclude that the unusually virulent clone family of biotype 4 strains of nontypeable H. influenzae express a variant P6 molecule which has an alteration in a highly conserved surface-exposed epitope.
Article Reference Immunoprophylaxis of otitis media.
Prospects for an effective otitis media vaccine are bolstered by a number of encouraging observations. Results of pneumococcal polysaccharide vaccine trials beginning in 1975, the enormously enhanced immunogenicity of protein-Hib polysaccharide coupled vaccines in infants, and the apparent effectiveness of a protein-PCP coupled vaccine in experimental otitis media suggest that a pneumococcal vaccine targeted to prevent invasive and middle ear infections is not too distant. The identification of several conserved surface antigens on NTHi and demonstration of otitis media protection elicited by these antigens in an animal model give promise for the development of H. influenzae vaccines for otitis media. Evidence that attenuated influenza A virus vaccination may also be an effective strategy for otitis media prevention, at least in an animal model, suggests that priority should be given to testing the efficacy of influenza, parainfluenza and respiratory syncytial virus vaccines with respect to otitis media prevention. It seems quite likely that not one but several immunoprophylaxis approaches will be necessary to reduce the overall incidence of otitis media given the multifactorial nature of the disease. Increasing parent and physician concern with the high incidence of otitis media and its morbidity suggests high participation rates in vaccine trials and high utilization of vaccines shown to be protective. Even if a vaccine could reduce the incidence of otitis media by 30%, an annual health care savings of $300-750 million would be achieved.
Article Reference Bacteremia due to nontypable Haemophilus influenzae–three cases in a community hospital.
Nontypable Haemophilus influenzae is a normal inhabitant of the upper respiratory tract and a common pathogen in diseases limited to mucosal surfaces. Nontypable H. influenzae has only occasionally been reported to cause invasive disease locally or systemically. In a period of two years, three patients of 17 with positive blood cultures for H. influenzae were found to have nontypable strains, two of which were resistant to ampicillin. The presumed sites of entry were an oral mucosal lesion, sinus mucosa, and female genital tract. All three patients responded rapidly to antibiotics to which their isolates were susceptible.
Article Reference Haemophilus influenzae serotype b and a capsule-deficient type mutant (b-)invasive disease on a partially vaccinated child in Brazil.
We report a rare case of infection by two different forms of Haemophilus influenzae strains on a child who received only one dose of the of Hib conjugate vaccine (DTwP+Hib). The strains were recovered from blood and CSF and were phenotypically identified as H. influenzae serotype b (Hib) and non-typeable H. influenzae (NTHi) respectively after serological tests. The two strains were characterized by PCR capsular typing, Multilocus Sequence Typing (MLST) and Pulsed-Field Gel Electrophoresis (PFGE). Our results suggest that the infection was caused by the invasion of the bloodstream by a single Hib strain following the entry of the Blood-Brain Barrier and the diffusion of the bacteria into the CSF. The strain recovered from CSF however, was identified as a capsule-deficient type mutant (b-) strain. Despite the high efficacy of Hib conjugate vaccine, the arise of strains able to escape from the immune system attack of vaccinated population, advocates for continued surveillance.
Article Reference Pediatric invasive Haemophilus influenzae infections in Israel in the era of Haemophilus influenzae type b vaccine: a nationwide prospective study.
The conjugated Haemophilus influenzae (Hi) type b vaccine caused a marked decrease in invasive Hi disease rates. Nonencapsulated Hi infection now constitutes most invasive Hi morbidity and mortality. This study examines invasive Hi infection incidence in Israel in the postvaccine era years, 2003-2012, and characterizes the epidemiology, clinical diagnosis and case fatality rates of invasive Hi disease in children <15 years of age.
Article Reference Comparative genomic analysis reveals distinct genotypic features of the emerging pathogen Haemophilus influenzae type f.
The incidence of invasive disease caused by encapsulated Haemophilus influenzae type f (Hif) has increased in the post-H. influenzae type b (Hib) vaccine era. We previously annotated the first complete Hif genome from a clinical isolate (KR494) that caused septic shock and necrotizing myositis. Here, the full genome of Hif KR494 was compared to sequenced reference strains Hib 10810, capsule type d (Hid) Rd Kw20, and finally nontypeable H. influenzae 3655. The goal was to identify possible genomic characteristics that may shed light upon the pathogenesis of Hif.
Invasive Disease
Article Reference Clinical and molecular epidemiology of haemophilus influenzae causing invasive disease in adult patients.
The epidemiology of invasive Haemophilus influenzae (Hi) has changed since the introduction of the Hi type b (Hib) vaccine. The aim of this study was to analyze the clinical and molecular epidemiology of Hi invasive disease in adults.
Article Reference Case of invasive nontypable Haemophilus influenzae respiratory tract infection with a large quantity of neutrophil extracellular traps in sputum.
Haemophilus influenzae type b was once the most common cause of invasive H. influenzae infection, but the incidence of this disease has decreased markedly with introduction of conjugate vaccines to prevent the disease. In contrast, the incidence of invasive infection caused by nontypable H. influenzae has increased in the US and in European countries. Neutrophil extracellular traps (NETs) are fibrous structures released extracellularly from activated neutrophils during inflammation, including in pneumonia, and rapidly trap and kill pathogens as a first line of immunological defense. However, their function and pathological role have not been fully investigated. Here, we report a case of fatal nontypable H. influenzae infection with severe pneumonia and bacteremia in an adult found to have a vast amount of NETs in his sputum. The patient had a two-day history of common cold-like symptoms and was taken to the emergency room as a cardiopulmonary arrest. He recovered temporarily, but died soon afterwards, although appropriate antibiotic therapy and general management had been instituted. Massive lobular pneumonia and sepsis due to nontypable H. influenzae was found, in spite of H. influenzae type b vaccine being available. His sputum showed numerous bacteria phagocytosed by neutrophils, and immunohistological staining indicated a number of NETs containing DNA, histone H3, and neutrophil elastase. This case highlights an association between formation of NETs and severe respiratory and septic infection. An increase in severe nontypable H. influenzae disease can be expected as a result of ``pathogen shift'' due to increased use of the H. influenzae type b vaccine in Japan.
Article Reference Increased biofilm formation by nontypeable Haemophilus influenzae isolates from patients with invasive disease or otitis media versus strains recovered from cases of respiratory infections.
Biofilm formation by nontypeable (NT) Haemophilus influenzae remains a controversial topic. Nevertheless, biofilm-like structures have been observed in the middle-ear mucosa of experimental chinchilla models of otitis media (OM). To date, there have been no studies of biofilm formation in large collections of clinical isolates. This study aimed to investigate the initial adhesion to a solid surface and biofilm formation by NT H. influenzae by comparing isolates from healthy carriers, those with noninvasive respiratory disease, and those with invasive respiratory disease. We used 352 isolates from patients with nonbacteremic community-acquired pneumonia (NB-CAP), chronic obstructive pulmonary disease (COPD), OM, and invasive disease and a group of healthy colonized children. We then determined the speed of initial adhesion to a solid surface by the BioFilm ring test and quantified biofilm formation by crystal violet staining. Isolates from different clinical sources displayed high levels of biofilm formation on a static solid support after growth for 24 h. We observed clear differences in initial attachment and biofilm formation depending on the pathology associated with NT H. influenzae isolation, with significantly increased biofilm formation for NT H. influenzae isolates collected from patients with invasive disease and OM compared with NT H. influenzae isolates from patients with NB-CAP or COPD and healthy colonized subjects. In all cases, biofilm structures were detached by proteinase K treatment, suggesting an important role for proteins in the initial adhesion and static biofilm formation measured by crystal violet staining.