Low genetic diversity of Haemophilus influenzae type b compared to nonencapsulated H. influenzae in a population in which H. influenzae is highly endemic

Immunization with Haemophilus influenzae type b (Hib) conjugate polysaccharide vaccines has dramatically reduced Hib disease worldwide. As in other populations, nasopharyngeal carriage of Hib declined markedly in Aboriginal infants following vaccination, although carriage has not been entirely eliminated. In this study, we describe the genetic characteristics and the carriage dynamics of longitudinal isolates of Hib, characterized by using several typing methods. In addition, carriage rates of nonencapsulated H. influenzae (NCHi) are high, and concurrent colonization with Hib and NCHi is common; we also observed NCHi isolates which were genetically similar to Hib. There is a continuing need to promote Hib immunization and monitor H. influenzae carriage in populations in which the organism is highly endemic, not least because of the possibility of genetic exchange between Hib and NCHi strains in such populations.     

Systemic Haemophilus influenzae infection in childhood

Forty-nine children who had systemic Haemophilus infection and were treated at the Westmead Centre, Sydney, over a two-year period are described. The majority (29 of 49 children) were aged two years or less. Epiglottis and meningitis accounted for 77% of these infections. All H. influenzae isolates associated with clinical disease were of the capsular type b. Eight per cent (four of 50) of H. influenzae infections were caused by beta-lactamase producing strains. There was no geographic clustering or seasonal variation. There was no mortality. Major morbidity included two patients who had epiglottis and required tracheostomy, and two patients who had meningitis developed bilateral profound sensorineural deafness. No secondary cases were detected in household contacts of 21 patients with H. influenzae meningitis during the study period. Epiglottis frequently occurs in very young children. The rapid response to antibiotic treatment suggests that early cases of epiglottis may be undiagnosed, but treated with antibiotic agents in the community.     

Nontypeable Haemophilus influenzae: pathogenesis and prevention

In this paper, we describe the ability of nontypeable Haemophilus influenzae (NTHi) to coexist with the human host and the devastating results associated with disruption of the delicate state of balanced pathogenesis, resulting in both acute and chronic respiratory tract infections. It has been seen that the strains of NTHi causing disease show a marked genetic and phenotypic diversity but that changes in the lipooligosaccharide (LOS) and protein size and antigenicity in chronically infected individuals indicate that individual strains of NTHi can remain and adapt themselves to avoid expulsion from their infective niche. The lack of reliance of NTHi on a single mechanism of attachment and its ability to interact with the host with rapid responses to its environment confirmed the success of this organism as both a colonizer and a pathogen. In vitro experiments on cell and organ cultures, combined with otitis media and pulmonary models in chinchillas, rats, and mice, have allowed investigations into individual interactions between NTHi and the mammalian host. The host-organism interaction appears to be a two-way process, with NTHi using cell surface structures to directly interact with the mammalian host and using secreted proteins and LOS to change the mammalian host in order to pave the way for colonization and invasion. Many experiments have also noted that immune system evasion through antigenic variation, secretion of enzymes and epithelial cell invasion allowed NTHi to survive for longer periods despite a specific immune response being mounted to infection. Several outer membrane proteins and LOS derivatives are discussed in relation to their efficacy in preventing pulmonary infections and otitis media in animals. General host responses with respect to age, genetic makeup, and vaccine delivery routes are considered, and a mucosal vaccine strategy is suggested.     

Mathematical models of Haemophilus influenzae type b

Electron microscopic anterograde autoradiography has been used to analyze the morphology and postsynaptic relationships of area 17 cortical terminals in the lateral division of the lateral posterior nucleus (LPl) of the cat and medial division of the inferior pulvinar nucleus (IPm) of the owl monkey. Such terminals are thought to arise exclusively from layer 5 in the cat and primate (Lund et al. [1975] J. Comp. Neurol. 164:287-304; Abramson and Chalupa [1985] Neuroscience 15:81-95). All labeled terminals in both nuclei exhibited the morphology of ascending "lemniscal" afferents. That is, they contained round vesicles, were large, made asymmetrical synaptic and filamentous nonsynaptic contacts, and were classified as RLs. These cortical RLs also exhibited the postsynaptic relationships of lemniscal afferents. Thus, they were presynaptic to large dendrites within glial encapsulated glomeruli, where a majority was involved in complex synaptic arrangements called triads. They also were found adjacent to terminal profiles with pleomorphic vesicles but never adjacent to small terminals containing round vesicles. Our results suggest that the layer 5 projection from area 17 provides a functional "drive" for some LPl and IPm neurons. Information carried over this "re-entrant" pathway (Guillery [1995] J. Anat. 187:583-592) could be modified within the LPl and IPm by both cortical and subcortical pathways and subsequently conveyed to higher visual cortical areas, where it could be integrated with messages carried through the well-documented corticocortical pathways (Casagrande and Kaas [1994] Cerebral cortex New York: Plenum Press).     

A region of Haemophilus influenzae Rd hybridizes with repetitive human DNA

A 17.7 kbp EcoR1 fragment of Haemophilus influenzae Rd (wild-type strain) DNA containing repeats has been cloned together with the DNA repair gene uvr3. The region containing repetitive DNA was subcloned on a 13 kbp EcoR1 fragment (pHi13). The insert hybridizes with restriction enzyme digests of H. influenzae Rd and H. parainfluenzae and exhibits a repetitive pattern. Discrete bands superimposed upon a polydisperse background were observed when pHi13 hybridizes with restriction digests of human DNA. The hybridization patterns indicate that H. parainfluenzae and human DNA contain cross-hybridizing sequences. The repeats of pHi13 are present on a 2.6 kbp PvuII fragment.     

Clonality of multidrug-resistant nontypeable strains of Haemophilus influenzae

The genetic structure of a population of multidrug-resistant nontypeable (unencapsulated) Haemophilus influenzae strains isolated at a hospital in Barcelona, Spain, was investigated by using multilocus enzyme electrophoresis to determine the allelic variation in 15 structural loci. In our study we have also included some antimicrobial agent-susceptible strains isolated at the same hospital. All enzymes were polymorphic for two to eight electromorphs, and the analysis revealed 43 distinct electrophoretic types among the 44 isolates. The mean genetic diversity of the entire population was 0.55. Multilocus linkage disequilibrium analysis of the isolates revealed a strong association between alleles, suggesting little possibility of recombination. Furthermore, the dendrogram and the allele mismatch distribution are typical of a population with no extensive genetic mixing.     

Nasopharyngeal colonization with nontypeable Haemophilus influenzae in chinchillas

Colonization of the nasopharynx by a middle ear pathogen is the first step in the development of otitis media in humans. The establishment of an animal model of nasopharyngeal colonization would therefore be of great utility in assessing the potential protective ability of candidate vaccine antigens (especially adhesins) against otitis media. A chinchilla nasopharyngeal colonization model for nontypeable Haemophilus influenzae (NTHI) was developed with antibiotic-resistant strains. This model does not require coinfection with a virus. There was no significant difference in the efficiency of NTHI colonization between adult (1- to 2-year-old) and young (2- to 3-month-old) animals. However, the incidence of middle ear infection following nasopharyngeal colonization was significantly higher in young animals (83 to 89%) than in adult chinchillas (10 to 30%). Chinchillas that had recovered either from a previous middle ear infection caused by NTHI or from an infection by intranasal inoculation with NTHI were completely protected against nasopharyngeal colonization with a homologous strain and were found to be the best positive controls in protection studies. Systemic immunization of chinchillas with inactivated whole-cell preparations significantly protected animals not only against homologous NTHI colonization but also partially against heterologous NTHI infection. In all protected animals, significant serum anti-P6 and anti-HMW antibody responses were observed. The outer membrane P6 and high-molecular-weight (HMW) proteins appear to be promising candidate vaccine antigens to prevent nasopharyngeal colonization and middle ear infection caused by NTHI.     

Human lactoferrin receptor activity in non-encapsulated Haemophilus influenzae

We recently reported that attenuation of vasoactive agent-induced calcium signal and cell contraction of mesangial cell by insulin-like growth factor 1 (IGF-1), observed in normal mesangial cells, is totally abolished in spontaneously hypertensive rat (SHR) mesangial cells. This phenomenon might be related to the well-known aberrant regulation of SHR glomerular hemodynamics. Since it has been reported that in vivo IGF-1 infusion increases renal plasma flow (RPF) and glomerular filtration rate (GFR), we examined whether the modulation of renal function by IGF-1 is altered in SHR. We performed in vivo renal clearance studies using eight-week-old SHR and control Wistar Kyoto rats (WKY) before and after IGF-1 (5 micrograms/kg) infusion into the left renal artery for 20 minutes. Mean arterial pressure was not affected by IGF-1 in both WKY and SHR. In WKY, IGF-1 increased GFR and RPF, and decreased renal vascular resistance (RVR). However, GFR, RPF, and RVR were not altered by IGF-1 in SHR, while systemic infusion of angiotensin II antagonist, CV-11974, increased GFR and RPF. The present data show that the modulation of renal hemodynamics by IGF-1 is absent in SHR. This might be related the pathophysiology of the development of hypertension.     

A new transformation-deficient mutant of Haemophilus influenzae Rd with normal DNA uptake

Haemophilus influenzae Rd is a gram-negative natural transformer. A mutant strain, RJ248, that has normal DNA uptake and translocation but whose transformation frequency is 300 times lower than that of wild-type H. influenzae and whose phage recombination is 8 times lower was isolated. The affected gene, comM, is induced during competence development in wild-type H. influenzae but not in RJ248.     

Polymerase chain reaction-based strain characterization of noncapsulate Haemophilus influenzae

A polymerase chain reaction-based typing method for noncapsulate Haemophilus influenzae was developed. Randomly amplified polymorphic DNA fingerprints were generated from boiled supernatants prepared directly from bacterial colonies without the need for DNA extraction. The technique was applied to isolates obtained during putative outbreaks of chest infection and validated by comparison with sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis of outer membrane protein-enriched preparations and rRNA gene restriction analysis. There was complete concordance between the three techniques. The results show that randomly amplified polymorphic DNA analysis provides a highly discriminatory method of characterizing strains of noncapsulate H. influenzae which is eminently suitable as an epidemiological tool for the rapid investigation of outbreaks of infection.     

Early onset Haemophilus influenzae sepsis in the newborn infant

Neonatal sepsis caused by Haemophilus influenzae is characterized by an early onset syndrome associated with pneumonia, shock and neutropenia. Over a 30-month period 13 infants referred to this hospital had early onset H. influenzae sepsis. Obstetric complications included preterm labor (92%), prolonged rupture of membranes > 12 hours (63%), maternal fever (64%), chorioamnionitis (43%), vaginal discharge (44%) and premature rupture of membranes (15%). All 13 infants were symptomatic at delivery and 7 required immediate intubation. Pneumonia and respiratory distress were the prominent clinical findings. H. influenzae was isolated from infant blood, maternal blood, placenta and genital tract. Isolates were predominantly non-type b, beta-lactamase-negative. A study to determine the prevalence of H. influenzae colonization of the genital tract among women attending clinic at the hospital with the most cases showed a rate of 0.3%. Perinatal risk factors and clinical findings in the infants are similar to disease caused by other organisms associated with early onset sepsis.     

Functional characterization of the Haemophilus influenzae 4.5S RNA

General registration of congenital malformations was established in Denmark in the early 1960s as a consequence of the thalidomide tragedy. The aims of the present paper are to describe the registration of congenital malformations in Denmark since the thalidomide tragedy, to discuss the limitations of these data and to point towards possibilities for improving the registration of congenital malformations in the future. Important components in a surveillance system are compulsory detailed reporting of malformations shortly after the diagnoses have been made and continuous expert evaluation of all reports. However, since 1995 the registration of congenital malformations in Denmark is based mainly on routine discharge diagnoses from hospitals. A high quality registration of congenital malformations in Denmark would be particularly valuable, because such a register together with existing health related registers and an ongoing large scale cohort study of pregnant women would provide a unique resource for etiological research in congenital malformations. Considering the consequences of congenital malformations for the affected children, their families and society, the establishment of a high quality registration of congenital malformations seems justifiable--and the Swedish experience shows that it is feasible.     

Subdural effusion in type B Haemophilus influenzae meningitis. A study of 38 cases

OBJECTIVE: Studying clinical, laboratory and radiologic findings, as well as outcome, observed in patients with meningitis caused by Hib, and its relationship with subdural effusion. MATERIAL AND METHODS: Retrospective study of 38 meningitis caused by Hib. Patients were aged between 3 months and 5 years. Imaging was performed in 26 cases (68%): CT in 21 children (55%) and cranial sonography in 11 cases (29%). EEG was made in 29 patients (76%) and auditory-evoked potentials in 13 (34%). The mean follow-up period after discharge was 24 months. RESULTS: Sixty-six per cent were male and 34% female. Eight cases had subdural effusion. These patients showed higher white cell counts in blood and CSF, higher levels of proteins in CSF, and lower levels of glucose in the same medium. They also had seizures before or during hospitalization, with higher frequency than those without subdural effusion (50% vs 26%) as well as more prolonged fever (127 vs 73 hours). No specific treatment was required in any case. CONCLUSIONS: Subdural effusion is one of the most frequent complications observed in meningitis. Patients frequently present more important clinical and laboratory alterations. This finding is not related with neurologic sequelae and they resolve spontaneously with time.     

Evolution of the major pilus gene cluster of Haemophilus influenzae

Haemophilus influenzae is a ubiquitous colonizer of the human respiratory tract and causes diseases ranging from otitis media to meningitis. Many H. influenzae isolates express pili (fimbriae), which mediate adherence to epithelial cells and facilitate colonization. The pilus gene (hif) cluster of H. influenzae type b maps between purE and pepN and resembles a pathogenicity island: it is present in invasive strains, absent from the nonpathogenic Rd strain, and flanked by direct repeats of sequence at the insertion site. To investigate the evolution and role in pathogenesis of the hif cluster, we compared the purE-pepN regions of various H. influenzae laboratory strains and clinical isolates. Unlike Rd, most strains had an insert at this site, which usually was the only chromosomal locus of hif DNA. The inserts are diverse in length and organization: among 20 strains, nine different arrangements were found. Several nontypeable isolates lack hif genes but have two conserved open reading frames (hicA and hicB) upstream of purE; their inferred products are small proteins with no data bank homologs. Other isolates have hif genes but lack hic DNA or have combinations of hif and hic genes. By comparing these arrangements, we have reconstructed a hypothetical ancestral genotype, the extended hif cluster. The hif region of INT1, an invasive nontypeable isolate, resembles the hypothetical ancestor. We propose that a progenitor strain acquired the extended cluster by horizontal transfer and that other variants arose as deletions. The structure of the hif cluster may correlate with colonization site or pathogenicity.     

The decline of Haemophilus influenzae type b disease in Australia

Between July 1993 and June 1996, there were 412 cases of invasive Haemophilus influenzae type b (Hib) disease reported to the Hib Case Surveillance Scheme, 71% in children under the age of five years. Meningitis was the most frequent illness reported, followed by epiglottitis, septicaemia and pneumonia. There were 18 deaths. Thirty-four cases were classified as vaccine failures. The number of vaccine failures increased over time and the total number of cases of Hib disease fell, consistent with an increase in Hib vaccine coverage. Based on an estimated vaccine coverage of 50% in April 1995, the vaccine efficacy for all vaccines in the period was estimated to be 89%. Invasive Hib is a serious illness of childhood which is being significantly reduced by the use of Hib vaccines, and has the potential to be eliminated from this country. Vaccination providers should aim to immunise all children against Hib disease on time and according to the National Health and Medical Research Council Standard Vaccination Schedule.     

Haemophilus influenzae type b-specific antibody in infants after maternal immunization

OBJECTIVE: To study the kinetics of Haemophilus influenzae type b (Hib)-specific antibody in infants born to mothers immunized with an Hib polysaccharide or one of two Hib conjugate vaccines. STUDY DESIGN: Serum antibody to the polyribosylribitol (PRP) moiety of Hib was measured by radioimmunoassay and enzyme-linked immunosorbent assay at birth and at 2 and 6 months of age in infants born to women immunized with Hib polysaccharide or conjugate vaccine (PRP-D and HbOC). A subset of infants > or = 6 months of age was immunized with Hib conjugate vaccine after licensure of this vaccine for infants. A comparison group of 18 infants born to unimmunized women received the same Hib conjugate vaccine on a similar schedule. RESULTS: Total PRP antibody concentrations were 1.50, 14.4 and 20.4 microg/ml in 2-month-old infants born to mothers immunized with polysaccharide, PRP-D and HbOC vaccines, respectively, and 2.54, 1.35 and 2.46 microg/ml in 6-month-old infants. Infants born to mothers immunized with polysaccharide vaccine had significantly less PRP antibody at 2 months of age but similar antibody concentrations at 6 months of age. Persistence or increases in total PRP antibody during 6 months were noted in 21 of 47 (44.6%) study infants. A subset of study and comparison infants was immunized with a mean of 2.6 doses of Hib vaccines between 6 months and 2 years of age, and all infants had total PRP antibody concentrations > or = 0.15 microg/ml. CONCLUSION: Conjugate Hib vaccines administered during the last trimester of pregnancy resulted in significantly higher PRP antibody titers in infants at birth and 2 months of age than did polysaccharide vaccine. A subset of infants born to immunized mothers was subsequently immunized with Hib conjugate vaccine and had antibody concentrations similar to those in infants born to nonimmunized women.