Acute purulent pericarditis in children caused by haemophilus influenzae

Acute purulent pericarditis caused by haemophilus influenzae is an unusual condition, especially in childhood. In most cases, respiratory symptoms are the presenting features, and children aged less than 4 years are most often affected. A high index of suspicion and aggresive microbiological and cardiological evaluation are often warranted to make an early diagnosis. We herein report two cases of pericarditis caused by H.influenzae in children aged less than two years. Pericardiocentesis was performed in each case. Early recognition, rapid diagnosis and aggressive medical and surgical therapy are paramount in the successful treatment of this condition (AU)

Acute purulent pericarditis in children caused by haemophilus influenzae

Acute purulent pericarditis caused by haemophilus influenzae is an unusual condition, especially in childhood. In most cases, respiratory symptoms are the presenting features, and children aged less than 4 years are most often affected. A high index of suspicion and aggresive micorbiological and cardiological evaluation are often warranted to make an early diagnosis. We herein reported two cases of pericarditis caused by H. influenzae in children aged less than two years. Pericardiocentesis was performed in each case. Early recognition, rapid diagnosis and aggressive medical and surgical therapy are paramount in the successful treatment of this condition.

Characterization of noncapsulate Haemophilus influenzae by whole-cell polypeptide profiles, restriction endonuclease analysis, and rRNA gene restriction patterns.

Thirty-four clinical isolates of noncapsulate Haemophilus influenzae representing isolates with either related or dissimilar patterns of whole-cell polypeptide profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were further characterized by restriction enzyme analysis (REA) and rRNA gene restriction patterns. Total cellular DNA was extracted by a rapid, microcentrifuge-scale method and digested with BamHI, which gave a pattern of about 18 discrete bands. This confirmed the five closely related groupings suggested by SDS-PAGE. Isolates dissimilar by SDS-PAGE were also distinguishable by REA. However, there was no correlation between the degrees of similarity estimated from whole-cell polypeptide profiles and those obtained from REA for the dissimilar isolates. Therefore, inferences of genetic relatedness made on the basis of these data should be interpreted with caution. rRNA gene restriction patterns also confirmed the groupings suggested by the other two techniques. We conclude that the three methods were highly discriminatory and that whole-cell polypeptide patterns or REA with BamHI would be appropriate techniques for epidemiological studies of noncapsulate H. influenzae.

Haemophilus influenzae polyarthritis in an adult: an analysis of serotype b strains.

A 61-year-old man who presented with clinical features suggestive of septic arthritis was found to have acute septic polyarthritis due to Haemophilus influenzae (type b). The clinical and laboratory diagnoses of the case are presented, and the isolates of H. influenzae are characterized. Four isolates recovered from different sites had identical minimal inhibitory drug concentrations, outer membrane protein patterns, and genomic DNA restriction digests. These observations indicate that the disseminated infection arose from a single source. The patient developed antibody to several outer membrane proteins, particularly the P6 protein.

Immunochemical characterization of variable epitopes of outer membrane protein P2 of nontypeable Haemophilus influenzae.

Monoclonal antibodies (MAbs) were elicited to the nontypeable Haemophilus influenzae variants d1 to d4, which differ in the outer membrane protein P2 to analyze the immunological properties of the variable parts of this protein. Five MAbs reacted in a whole-cell enzyme-linked immunosorbent assay (ELISA) only with the homologous strain and in some cases with its variants, but not with 69 unrelated nonencapsulated H. influenzae isolates; nine MAbs also reacted with some other H. influenzae isolates, and four MAbs showed broad cross-reactivity. All of the MAbs reacted with purified protein P2 in ELISAs and immunoblotting. The five MAbs which reacted with the homologous strain d3 and not with the variants d1, d2, and d4 promoted complement-dependent bactericidal activity against strain d3. These and four other MAbs reacted with the intact bacteria of strain d3 in immunogold electron microscopy, indicating that they were directed against surface-exposed epitopes of outer membrane protein P2. A mutant of strain d3 was isolated as a survivor from bacterial killing by complement and MAb 30DA5. This mutant had an altered P2 protein on sodium dodecyl sulfate-polyacrylamide gels and had lost its reactivity with all of the five H. influenzae d3-specific MAbs but not with the other MAbs. From these results, we conclude that the variable parts of outer membrane protein P2 of nonencapsulated H. influenzae from the sputum of patients with chronic obstructive pulmonary disease are immunogenic and mostly surface exposed. Only strain-specific MAbs promoted complement-dependent killing of the bacteria, which was abolished in a spontaneous mutant with an altered P2 protein.

Protein D, an immunoglobulin D-binding protein of Haemophilus influenzae: cloning, nucleotide sequence, and expression in Escherichia coli.

The gene for protein D, a membrane-associated protein with specific affinity for human immunoglobulin D, was cloned from a nontypeable strain of Haemophilus influenzae. The gene was expressed in Escherichia coli from an endogenous promoter, and the gene product has an apparent molecular weight equal to that of H. influenzae protein D (42,000). The complete nucleotide sequence of the gene for protein D was determined, and the deduced amino acid sequence of 364 residues includes a putative signal sequence of 18 amino acids containing a consensus sequence, Leu-Ala-Gly-Cys, for bacterial lipoproteins. The sequence of protein D shows no similarity to those of other immunoglobulin-binding proteins. Protein D is the first example of immunoglobulin receptors from gram-negative bacteria that has been cloned and sequenced.

Specific detection of Haemophilus influenzae type b lipooligosaccharide by immunoassay.

Three monoclonal antibody (MAb)-based immunoassays were developed for specific detection of Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS). (i) Hib LOS was captured onto microtiter plates by polyclonal Hib-directed antibodies and detected with MAbs to the oligosaccharide component of Hib LOS in an enzyme-linked immunosorbent assay, (ii) The high affinity of polymyxin B for lipid A was used to bind Hib LOS to microtiter wells, and the oligosaccharide-specific MAbs were used as the detection system in the polymyxin B-MAb assay. (iii) Hib LOS solubilized in detergent was captured by MAbs, and the immobilized LOS was detected with a chromogenic Limulus amebocyte lysate method in the immunolimulus assay. Endotoxin concentrations were measured in in vitro samples and cerebrospinal fluid samples from rabbits with experimental Hib meningitis. The results were compared with those obtained with the standard chromogenic Limulus amebocyte lysate assay. There were significant correlations between the results of all four assays. These new immunoassays provide methods for specific detection of Hib LOS in laboratory fluids and in research involving quantification of Hib endotoxin in experimental animal models.

Diversity of outer membrane protein profiles of nontypable Haemophilus influenzae from children from Papua New Guinea and the Philippines.

We determined capsular serotypes and, with use of SDS-PAGE, patterns of outer membrane proteins (OMP) of Haemophilus influenzae isolates from specimens of blood, lung, or CSF from children with acute respiratory tract infections or meningitis who were hospitalized in Papua New Guinea or the Philippines. Among 72 isolates from Papua New Guinea, 72% were type b, 14% were other encapsulated serotypes, and 14% were nontypable. The respective frequencies among 43 isolates from the Philippines were 56%, 7%, and 37%. The type b isolates could be subdivided into at least eight outer membrane subtypes, most of which have been described previously. In contrast, OMP profiles of the nontypable H. influenzae isolates from children in both countries were highly heterogeneous. These results differ markedly from those previously found for isolates from children with acute respiratory tract infections in Pakistan, for which encapsulated H. influenzae strains other than serotype b were not observed; 95% of type b isolates were of a single OMP subtype; and nontypable isolates showed evidence of clonal restriction. Candidate vaccines for use in developing countries as protection against disease caused by H. influenzae will need to include both capsular and noncapsular antigens and investigators must take into consideration regional differences among strains.

Structural models for the cell surface lipooligosaccharides of Neisseria gonorrhoeae and Haemophilus influenzae.

A structural model is proposed for the surface glycolipids, or lipooligosaccharides (LOS), of gram-negative pathogenic bacteria that colonize human mucosae, e.g. Neisseria gonorrhoeae and Haemophilus influenzae. The development of this model has involved analysis of a series of pyocin-resistant mutants with altered LOS and other recent immunochemical and structural data. A comprehensive approach to determining the necessary structural data has been constructed that utilizes liquid secondary ion mass spectrometry, tandem mass spectrometry, methylation analysis and nuclear magnetic resonance. To prepare purified oligosaccharides for these analyses, chromatographic and chemical techniques have been developed that include high-pH anion-exchange chromatography of underivatized oligosaccharides and reverse-phase chromatography after derivatization with hydrazino alkyl benzoates. The proposed LOS model has several unique features that distinguish it from models developed for the lipopolysaccharides of enteric bacteria. This information should lead to an understanding of the unique structure/function relationship of LOS and to the development of carbohydrate-based vaccines.

Protein D of Haemophilus influenzae. A novel bacterial surface protein with affinity for human IgD.

Protein D, a novel surface protein of the bacterial species Haemophilus influenzae with affinity for human IgD, was isolated after solubilization with sonication and Sarcosyl-extraction by a single SDS-PAGE step. From 1 ml of packed bacteria was prepared 0.25 mg of purified protein D. The apparent m.w. of protein D was estimated to 42,000 by SDS-PAGE and gel chromatography. Edman degradation cycles of protein D produced no amino acid phenylthiohydantoin derivatives and the amino-terminal end of the single protein D polypeptide chain is thus probably blocked. Protein D differs from all previously described outer membrane proteins (protein 1 to 6) of H. influenzae. Thus, protein D did not react with antibodies against protein 1 or protein 2 and the latter proteins did not bind IgD. Protein D was found to exhibit unique Ig-binding properties. Thus, in dot blots protein D bound four different human IgD myeloma proteins but not IgG, IgM, IgA, IgE, or some additional proteins. On the IgD molecule, constant parts of the H chains both in the Fab and Fc fragments appear responsible for the interaction with protein D. This novel Ig-binding reagent promises to be of theoretical and practical interest in immunologic and microbiologic research.

Biochemical characterization and worldwide distribution of serologically distinct lipopolysaccharides of Haemophilus influenzae type b.

The sugar composition and the electrophoretic mobility in SDS-polyacrylamide gel electrophoresis of the various lipopolysaccharides (LPS) from clinical isolates of Haemophilus influenzae type b (Hib) were determined to correlate epidemiologic data with compositional data. Rabbit sera specific in Ouchterlony immunodiffusion for 10 different LPS (LPS 1-10) reacted with 647 or 690 Hib strains isolated from patients with invasive disease in various continents. Serotype 1 was predominant and was found in 550 isolates (80%). None of the Hib isolates reacted with antisera specific for LPS of two nonencapsulated isolates (LPS 5 and 6). Sugar analysis by gas-liquid chromatography of trimethylsilylated methyl glycosides revealed that the LPS of the 10 serotypes contained glucose, galactose, L-glycero-D-mannoheptose, and glucosamine in various proportions. LPS 1, 2, 8, and 9 contained the highest amounts of glucose and galactose relative to L-glycero-D-mannoheptose, which is considered present in constant amounts in H. influenzae LPS. LPS 1, 2, and 9 were most frequently found in invasive disease isolates.

Cross-infection by non-encapsulated Haemophilus influenzae.

From October, 1988, to January, 1989, 18 patients admitted to an acute medical chest ward were infected with an ampicillin-resistant beta-lactamase-producing strain of Haemophilus influenzae. All 18 isolates were non-encapsulated strains of biotype III and showed identical cell envelope protein profiles, as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The organism was not isolated from repeated environmental samples but there was strong circumstantial evidence that a spirometer was a common iatrogenic source of the cross-infection.

[Haemophilus influenzae type B: subtyping of strains isolated from respiratory infections using the outer membrane protein profiles]. / Haemophilus influenzae tipo b: subtipificación de cepas aisladas de infecciones respiratorias mediante los perfiles de proteínas de la membrana externa.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) applied to outer membrane protein (OMP), extracted by a micromethod, was employed to subtype H. influenza b type I. A total of 37 H. influenzae b strains were isolated from children under 4 years of age, either with lower acute respiratory infection (LARI), or asymptomatic carriers matched according sex, socioeconomic level and seasonality. Twenty seven out of the 37 H. influenzae b strains belonged to biotype I. On the basis of OMP profiles, these 27 were classified into 8 subtypes (Figs. 2, 3, 4 and 5). The probability of two randomly chosen isolates having different OMP profiles was 0.733. The subtype termed "a" showed the greatest relative frequency and was detected both in invasive strains and in those isolated from throat samples of LARI cases and healthy children. The use of 14% SDS-PAGE allowed de detection either of a 51kD or a 49kD, as well as 25-40kD proteins, in a single run (Fig. 1). Most subtype profiles showed the 51 kD protein. Growth conditions and extraction of OMPs by our modified micromethod provide a single and inexpensive procedure within the means of the average clinical laboratory. Besides, this test is much less time-consuming than classical assays. Jointly, biotyping , serotyping and OMP profile determination, proved a useful epidemiological tool to survey H. influenzae b infection.

Haemocin, the bacteriocin produced by Haemophilus influenzae: species distribution and role in colonization.

Four hundred thirty-eight strains of Haemophilus influenzae were examined for production of and sensitivity to haemocin, a bacteriocin produced by some members of this species. Whereas 199 of 212 (94%) type b isolates produced haemocin, 131 of 134 (98%) nontypeable and 91 of 92 (99%) encapsulated non-type b isolates were sensitive to haemocin. Among strains previously genetically characterized by multilocus enzyme electrophoresis, haemocin production was detected in type b isolates belonging to 25 of 29 (86%) clonally distinct electrophoretic types. None of 60 clonally distinct nontypeable strains produced this substance, and all were sensitive to it in vitro. The genes encoding haemocin production were transformed independently of the genes necessary for capsule expression from a prototypic type b strain to a nontypeable strain. After intranasal inoculation of infant rats with an equal mixture of a non-haemocin-producing strain and its haemocin-producing transformant, organisms capable of haemocin production predominated in both nasopharyngeal and blood cultures. These data demonstrate that haemocin production is strongly associated with type b encapsulated members of this species and suggest a mechanism by which haemocin might play a role in host nasopharyngeal colonization by this pathogen.

Molecular size characterization of bacterial capsular polysaccharide vaccines by high performance liquid chromatography.

Bacterial capsular polysaccharides are major virulence factors and some are used as vaccinal antigens. Their molecular size is an important physicochemical criterion which correlates with immunogenicity. This article describes a new application of high performance liquid chromatography (HPLC), based on molecular sieving, for such an evaluation. This HPLC method is rapid, accurate, reproducible, requires only very low amounts of product and presents good correlation with conventional gel permeation chromatography.

The complete primary structure of pilin from Haemophilus influenzae type b strain Eagan.

Adherence of Haemophilus influenzae type b (Hib) to human oropharyngeal cells is mediated by pili which are proteinaceous filaments that extend outward from the bacterial cell surface. Pili from Hib strain Eagan were purified, and the primary structure of the major subunit, pilin, was determined. Sequencing of overlapping peptides showed the mature protein to be comprised of 196 amino acids and to have an Mr of 21,152. The amino terminal sequence was found to be homologous with the sequence previously reported for Hib strain M43 and also to have significant homology to pilins of other gram-negative pathogenic bacteria. Furthermore, Hib pilin had two cysteinyl residues in the amino terminal portion of the protein which were separated by 40 residues (positions 21 and 61); a motif found in other bacterial pilins. The data show that Hib pilin has structural features common to other bacterial pilins.

Relationship of high tissue concentrations of azithromycin to bactericidal activity and efficacy in vivo.

Measurement of killing kinetics of azithromycin against strains of Streptococcus pneumoniae and Klebsiella pneumoniae in vitro showed that it had a limited bactericidal activity (greater than 90% kill) for the first eight hours of incubation, but developed complete bactericidal activity (greater than 99.9% kill) by 24 h incubation. Since high and sustained tissue levels of azithromycin occur in animals and humans, it was proposed that it might produce a bactericidal effect in vivo. This was demonstrated in a lung infection model in mice, designed to mimic the in-vitro killing studies. A 25 mg/kg dose of azithromycin given 24 h before intranasal challenge reduced the recoverable Str. pneumoniae population by greater than 99.9%, in comparison with untreated controls. Erythromycin did not produce a bactericidal effect at 100 mg/kg, and roxithromycin only reduced the viable count by 96%, at a dose of 50 mg/kg. Against a K. pneumoniae lung infection, a 50 mg/kg dose of azithromycin reduced the bacterial count by 99%. The bactericidal effect was correlated with lung tissue concentrations of azithromycin. In a proliferating Escherichia coli paper disc infection model, extravascular fluid concentrations of azithromycin were correlated with a 99.9% reduction in bacterial count, while corresponding serum concentrations were always less than the MIC. Dosing with azithromycin eradicated Haemophilus influenzae from the bulla (middle ear) of gerbils, as was not the case with erythromycin and roxithromycin. This effect was correlated with the antibiotic concentration in bulla lavage.

Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

We utilized the recently described tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (TSDS-PAGE) system to study the lipooligosaccharides (LOS) and lipopolysaccharides (LPS) of gram negative bacteria. TSDS-PAGE resulted in a high degree of resolution of LOS and LPS in the 'mini-gel' format. TSDS-PAGE resulted in the LOS and LPS migrating as a function of their Mr during electrophoresis and allowed estimation of Mr from a protein standard. Several species of LOS were analyzed. The newly described procedure allowed a more rapid and accurate analysis of LOS and the core region of LPS.

Haemophilus influenzae tipo b: subtipificacíon de cepas aisladas de infecciones respiratorias mediante los perfiles de proteínas de la membrana externa / Haemophilus influenzae type B: subtyping of strains isolated from respiratory infections using the outer membrane protein profiles

Se utilizó la electroforesis en un gel desnaturalizante de poliacrilamida para separar las proteínas de la membrana externa (pme) obtenidas mediante un micrométodo de extracción. Ello sirvió para subclasificar a H. influenzae b biotipo I en distintos subtipos de acuerdo a los perfiles observados. Se analizaron 37 cepas de H. influenzae b aisladas de niños con infección respiratoria aguda inferior (IRAD) y 3 de las fauces de portadores de igual sexo, edad y nivel socioeconómico, menores de cuatro años y recuperadas con la misma estacionalidad. De ellas, 27 pertenecieron al biotipo I. De acuerdo al perfil de PME, los 27 H. influenzae b biotipo I se subdividieron en 8 subtipos. La probabilidad de que dos aislamientos tomados al azar presenten diferentes subtipos de acuerdo a los perfiles de PME fue de 0.733. El subtipo que presentó mayor frecuencia fue el denominado "a", observándose tanto en las cepas invasivas como en las aisladas de fauces de los casos con IRAI y de las fauces de los niños sanos. El empleo del gel desnaturalizante de poliacrilamida al 14% permitió evidenciar la presencia de la proteína P1 con su característica de presentar distintos pesos moleculares, así como las proteínas presentes en el rango de peso molecular 25-40 KD. La mayoría de los perfiles de PME presentaron la proteína P1 de mayor peso molecular. Las condiciones de crecimiento de las cepas y la adaptación del micrométodo de extracción de PME utilizados en este estudio son procedimientos simples y al alcance de todo laboratorio clínico. Además, requiere menor insumo de tiempo que los métodos clásicos. La combinación de biotipificación, serotipificación, y determinación del perfil de PME, mostró ser un arma epidemiológicamente útil para analizar la infección a H. influenzae b.

Haemophilus influenzae tipo b: subtipificacíon de cepas aisladas de infecciones respiratorias mediante los perfiles de proteínas de la membrana externa / Haemophilus influenzae type B: subtyping of strains isolated from respiratory infections using the outer membrane protein profiles

Se utilizó la electroforesis en un gel desnaturalizante de poliacrilamida para separar las proteínas de la membrana externa (pme) obtenidas mediante un micrométodo de extracción. Ello sirvió para subclasificar a H. influenzae b biotipo I en distintos subtipos de acuerdo a los perfiles observados. Se analizaron 37 cepas de H. influenzae b aisladas de niños con infección respiratoria aguda inferior (IRAD) y 3 de las fauces de portadores de igual sexo, edad y nivel socioeconómico, menores de cuatro años y recuperadas con la misma estacionalidad. De ellas, 27 pertenecieron al biotipo I. De acuerdo al perfil de PME, los 27 H. influenzae b biotipo I se subdividieron en 8 subtipos. La probabilidad de que dos aislamientos tomados al azar presenten diferentes subtipos de acuerdo a los perfiles de PME fue de 0.733. El subtipo que presentó mayor frecuencia fue el denominado "a", observándose tanto en las cepas invasivas como en las aisladas de fauces de los casos con IRAI y de las fauces de los niños sanos. El empleo del gel desnaturalizante de poliacrilamida al 14% permitió evidenciar la presencia de la proteína P1 con su característica de presentar distintos pesos moleculares, así como las proteínas presentes en el rango de peso molecular 25-40 KD. La mayoría de los perfiles de PME presentaron la proteína P1 de mayor peso molecular. Las condiciones de crecimiento de las cepas y la adaptación del micrométodo de extracción de PME utilizados en este estudio son procedimientos simples y al alcance de todo laboratorio clínico. Además, requiere menor insumo de tiempo que los métodos clásicos. La combinación de biotipificación, serotipificación, y determinación del perfil de PME, mostró ser un arma epidemiológicamente útil para analizar la infección a H. influenzae b. (AU)