J Pediatr Orthop, 1999 Nov-Dec, 19(6), 705 - 9 Reduction in osteomyelitis and septic arthritis related to Haemophilus influenzae type B vaccination; Howard AW et al.; This retrospective cohort study compares organisms responsible for septic arthritis and osteomyelitis before and after Haemophilus influenzae type b vaccination . Before vaccination, Haemophilus influenzae type b was responsible for 5% of culture positive osteomyelitis and 41% of culture positive septic arthritis . Since the administration of the conjugated vaccine PRP-T began in 1992, no case of osteomyelitis or septic arthritis has been caused by Haemophilus influenzae type b (p < 0.005, t test) . Vaccination has succeeded in eliminating Haemophilus influenzae type b as an infective agent in hematogenous septic arthritis and osteomyelitis . Current empirical antibiotic therapy for hematogenous septic arthritis and osteomyelitis need only cover gram-positive agents in vaccinated infants and children of all age groups. Rev Panam Salud Publica, 1999 Oct, 6(4), 234 - 41 {Monitoring antibiotic resistance in Argentina . The WHONET program, 1995-1996}; Rossi A et al.; The World Health Organization has implemented a surveillance program for antimicrobial resistance that is known as WHONET . In Argentina the program was developed through a network of 23 public and private hospitals that participate in national and international quality-control programs . Between January 1995 and December 1996, the antimicrobial susceptibility of 16,073 consecutive clinical isolates was determined, using the recommended standards of the National Committee for Clinical Laboratory Standards of the United States of America . More than half of the Escherichia coli urinary isolates were resistant to ampicillin and more than 30% to trimethoprim/sulfamethoxazole (SXT) . When the percentage of resistant isolates from outpatients (OPs) was compared to that observed in hospitalized patients (HPs), a marked difference in antimicrobial activity was noted in the case of gentamicin (2% from OPs resistant vs . 8% from HPs resistant), norfloxacin (2% vs . 6%), and third-generation cephalosporins (7% vs . 15%) . Of the Klebsiella pneumoniae isolates recovered from blood cultures, 71% and 60% showed resistance to third-generation cephalosporins and to gentamicin, respectively . The overall rate of oxacillin resistance in Staphylococcus aureus was 39% . Around half of the Enterococcus spp . isolates showed high resistance to aminoglycosides, but resistance to glycopeptides was not found . In Argentina, ampicillin and SXT were not suitable for treating diarrhea . Shigella flexneri had a higher number of isolates resistant to both of those drugs (87% and 74%, respectively) than Sh . sonnei did (47% and 71%, respectively) . About 40% of the Salmonella spp . isolated in pediatric hospitals were resistant to third-generation cephalosporins . When microorganisms causing bacterial meningitis were examined, Streptococcus pneumoniae showed a resistance rate of 18% to penicillin and Haemophilus influenzae a resistance rate of 19% to ampicillin . These rates are within the intermediate range reported for other countries of the Americas and for Europe. Nucleic Acids Res, 1999 Dec 15, 27(24), 4743 - 50 Conservation of a tRNA core for aminoacylation; Hou YM et al.; The core region of Escherichia coli tRNA(Cys)is important for aminoacylation of the tRNA . This core contains an unusual G15:G48 base pair, and three adenosine nucleotides A13, A22 and A46 that are likely to form a 46:{13:22} adenosine base triple . We recently observed that the 15:48 base pair and the proposed 46:{13:22} triple are structurally and functionally coupled to contribute to aminoacylation . Inspection of a database of tRNA sequences shows that these elements are only found in one other tRNA, the Haemophilus influenzae tRNA(Cys) . Because of the complexity of the core, conservation of sequence does not mean conservation of function . We here tested whether the conserved elements in H . influenzae tRNA(Cys)were also important for aminoacylation of H . influenzae tRNA(Cys) . We cloned and purified a recombinant H . influenzae cysteine-tRNA synthe-tase and showed that it depends on 15:48 and 13, 22 and 46 in a relationship analogous to that of E . coli cysteine-tRNA synthetase . The functional conservation of the tRNA core is correlated with sequence conservation between E.coli and H.influenzae cysteine-tRNA synthetases . As the genome of H . influenzae is one of the smallest and may approximate a small autonomous entity in the development of life, the dependence of this genome on G15:G48 and its coupling with the proposed A46:{A13:A22} triple for aminoacylation with cysteine suggests an early role of these motifs in the evolution of decoding genetic information. J Bacteriol, 1999 Dec, 181(23), 7298 - 307 Direct selection of IS903 transposon insertions by use of a broad-host-range vector: isolation of catalase-deficient mutants of Actinobacillus actinomycetemcomitans; Thomson VJ et al.; Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events . We have developed an IS903-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency . The transposon, IS903phikan, carries a cryptic kan gene, which can be expressed only after successful transposition . This allows the stable introduction of the transposon delivery vector into the host . Generation of insertion mutants is then limited only by the frequency of transposition . IS903phikan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters . After transposase induction, IS903phikan insertion mutants were readily selected in Escherichia coli by their resistance to kanamycin . We used IS903phikan to isolate three catalase-deficient mutants of the periodontal pathogen Actinobacillus actinomycetemcomitans from a library of random insertions . The mutants display increased sensitivity to hydrogen peroxide, and all have IS903phikan insertions within an open reading frame whose predicted product is closely related to other bacterial catalases . Nucleotide sequence analysis of the catalase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of Haemophilus influenzae . Our results demonstrate the utility of the IS903phikan mutagenesis system for the study of A . actinomycetemcomitans . Because IS903phikan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species. Biochim Biophys Acta, 1999 Oct 8, 1455(2-3), 241 - 53 Terminal glycosylation in cystic fibrosis; Scanlin TF et al.; Cystic fibrosis (CF) is a common genetic disease for which the gene was identified within the last decade . Pulmonary disease predominates in this ultimately fatal disease and current therapy only slows the progression . CF transmembrane regulator (CFTR), the gene product, is an integral membrane glycoprotein that normally functions as a chloride channel in epithelial cells . The most common mutation, deltaF508, results in mislocalization and altered glycosylation of CFTR . Altered fucosylation and sialylation are hallmarks of both membrane and secreted glycoproteins in CF and the focus here is on these investigations . Oligosaccharides from CF membrane glycoproteins have the Lewis x, selectin ligand in terminal positions . In addition, two major bacterial pathogens in CF, Pseudomonas aeruginosa and Haemophilus influenzae, have binding proteins, which recognize fucose in alpha1,3 linkage and asialoglycoconjugates . We speculate that the altered terminal glycosylation of airway epithelial glycoproteins in CF contributes to the chronic infection and robust inflammatory response in the CF lung . Understanding the effects of mutant CFTR on glycosylation may provide further insight into the regulation of glycoconjugate processing as well as therapy for CF. Gene, 1999 Sep 30, 238(1), 143 - 55 Studies of codon usage and tRNA genes of 18 unicellular organisms and quantification of Bacillus subtilis tRNAs: gene expression level and species-specific diversity of codon usage based on multivariate analysis; Kanaya S et al.; We examined codon usage in Bacillus subtilis genes by multivariate analysis, quantified its cellular levels of individual tRNAs, and found a clear constraint of tRNA contents on synonymous codon choice . Individual tRNA levels were proportional to the copy number of the respective tRNA genes . This indicates that the tRNA gene copy number is an important factor to determine in cellular tRNA levels, which is common with Escherichia coli and yeast Saccharomyces cerevisiae . Codon usage in 18 unicellular organisms whose genomes have been sequenced completely was analyzed and compared with the composition of tRNA genes . The 18 organisms are as follows: yeast S . cerevisiae, Aquifex aeolicus, Archaeoglobus fulgidus, B . subtilis, Borrelia burgdorferi, Chlamydia trachomatis, E . coli, Haemophilus influenzae, Helicobacterpylori, Methanococcusjannaschii, Methanobacterium thermoautotrophicum, Mycobacterium tuberculosis, Mycoplasma genitalium, Mycoplasma pneumoniae, Pyrococcus horikoshii, Rickettsia prowazekii, Synechocystis sp., and Treponema pallidum . Codons preferred in highly expressed genes were related to the codons optimal for the translation process, which were predicted by the composition of isoaccepting tRNA genes . Genes with specific codon usage are discussed in connection with their evolutionary origins and functions . The origin and terminus of replication could be predicted on the basis of codon usage when the usage was analyzed relative to the transcription direction of individual genes. Infect Immun, 1999 Dec, 67(12), 6335 - 40 Expression of sialylated or paragloboside-like lipooligosaccharides are not required for pustule formation by Haemophilus ducreyi in human volunteers; Young RS et al.; The lipooligosaccharide (LOS) of Haemophilus ducreyi, the etiologic agent of chancroid, chemically and immunologically resembles human glycosphingolipid antigens . To test whether LOS that contains paragloboside-like structures was required for pustule formation, an isogenic mutant (35000HP-RSM2) was constructed in losB, which encodes D-glycero-D-manno-heptosyltransferase . 35000HP-RSM2 produces a truncated LOS whose major glycoform terminates in a single glucose attached to a heptose trisaccharide core and 2-keto-3-deoxyoctulosonic acid . Five human subjects were inoculated with 35000HP and 35000HP-RSM2 in a dose-response trial . For estimated delivered doses (EDDs) of >/=25 CFU, the pustule formation rates were 80% for 35000HP and 58% for 35000HP-RSM2 . Preliminary data indicated that a previously described Tn916 losB mutant made a minor glycoform that does not require DD-heptose to form the terminal N-acetyllactosamine . If 35000HP-RSM2 made this glycoform, then 35000HP-RSM2 could theoretically make a sialylated glycoform . To test whether sialylated LOS was required for pustule formation, a second trial comparing an isogenic sialyltransferase mutant (35000HP-RSM203) to 35000HP was performed in five additional subjects . For EDDs of >/=25 CFU, the pustule formation rates were 30% for both 35000HP and 35000HP-RSM203 . The histopathology and recovery rates of H . ducreyi from surface cultures and biopsies obtained from mutant and parent sites in both trials were similar . These results indicate that neither the expression of a major glycoform resembling paragloboside nor sialylated LOS is required for pustule formation by H . ducreyi in humans. Infect Immun, 1999 Dec, 67(12), 6518 - 25 Microtubules are associated with intracellular movement and spread of the periodontopathogen Actinobacillus actinomycetemcomitans; Meyer DH et al.; Actinobacillus actinomycetemcomitans SUNY 465, the invasion prototype strain, enters epithelial cells by an actin-dependent mechanism, escapes from the host cell vacuole, and spreads intracellularly and to adjacent epithelial cells via intercellular protrusions . Internalized organisms also egress from host cells into the assay medium via protrusions that are associated with just a single epithelial cell . Here we demonstrate that agents which inhibit microtubule polymerization (e.g., colchicine) and those which stabilize polymerized microtubules (e.g., taxol) both increase markedly the number of intracellular A . actinomycetemcomitans organisms . Furthermore, both colchicine and taxol prevented the egression of A . actinomycetemcomitans from host cells into the assay medium . Immunofluorescence microscopy revealed that protrusions that mediate the bacterial spread contain microtubules . A . actinomycetemcomitans SUNY 465 and 652, strains that are both invasive and egressive, interacted specifically with the plus ends (growing ends) of the filaments of microtubule asters in a KB cell extract . By contrast, neither A . actinomycetemcomitans 523, a strain that is invasive but not egressive, nor Haemophilus aphrophilus, a noninvasive oral bacterium with characteristics similar to those of A . actinomycetemcomitans, bound to microtubules . Together these data suggest that microtubules function in the spread and movement of A . actinomycetemcomitans and provide the first evidence that host cell dispersion of an invasive bacterium may involve the usurption of host cell microtubules. Infect Immun, 1999 Dec, 67(12), 6394 - 402 Cytolethal distending toxin of Haemophilus ducreyi induces apoptotic death of Jurkat T cells; Gelfanova V et al.; The immune response to Haemophilus ducreyi is mediated in part by T cells infiltrating the site of infection . In this study, we show that H . ducreyi antigen preparations inhibited the proliferation of peripheral blood mononuclear cells and primary human T-cell lines . H . ducreyi also inhibited Jurkat T-cell proliferation and induced apoptosis of Jurkat T cells, confirmed through the detection of DNA degradation and membrane unpacking . The cytotoxic product(s) was present in cell-free culture supernatant and whole-cell preparations of H . ducreyi and was heat labile . H . ducreyi produces two known heat-labile toxins, a hemolysin and a cytolethal distending toxin (CDT) . Whole cells and supernatants prepared from a hemolysin-deficient mutant had the same inhibitory and apoptotic effects on Jurkat T cells as did its isogenic parent . Preparations made from an H . ducreyi cdtC mutant were less toxic and induced less apoptosis than the parent . The toxic activity of the cdtC mutant was restored by complementation in trans . CdtC-neutralizing antibodies also inhibited H . ducreyi-induced toxicity and apoptosis . The data suggest that CDT may interfere with T-cell responses to H . ducreyi by induction of apoptosis. Acta Paediatr Suppl, 1999 Aug, 88(430), 83 - 8 Dietary nucleotides: effects on the immune and gastrointestinal systems; Carver JD; Nucleotides (NT) and their related metabolic products play key roles in many biological processes . NT can be synthesized endogenously and thus are not considered essential nutrients . Studies have demonstrated, however, that dietary NT can have beneficial effects; the term "conditionally essential" has been used to describe their role in human nutrition . These nutrients may become essential when the endogenous supply is insufficient for normal function, even though their absence from the diet does not lead to a classic clinical deficiency syndrome . Most dietary NT are rapidly metabolized and excreted . However, some are incorporated into tissues, particularly at younger ages and with fasting . Under conditions of limited NT intake, rapid growth or certain disease states, dietary NT may spare the cost of de novo NT synthesis and optimize the function of rapidly dividing tissues such as those of the gastrointestinal and immune systems . Animals fed NT-supplemented versus non-NT supplemented diets have enhanced gastrointestinal growth and maturation, and improved recovery following small and large bowel injury . Indices of humoral and cellular immunity are enhanced, and survival rates are higher following infection with pathogens . Infants receive NT in human milk, where they are present as nucleic acids, nucleosides, nucleotides and related metabolic products . The NT content of human milk is significantly higher than most cow's milk-based infant formulae . Dietary NT are reported to enhance the gastrointestinal and immune systems of formula-fed infants . Infants fed NT-supplemented versus non-supplemented formula have a lower incidence of diarrhea, higher antibody titers following Haemophilus influenzae type b vaccination and higher natural killer cell activity . These data suggest that human milk NT may contribute to the superior clinical performance of the breastfed infant. Rev Clin Esp, 1999 Sep, 199(9), 576 - 82 {A prospective study of meningitis diagnosed in a 3rd-level hospital during a 1-year period}; Elvira J et al.; OBJECTIVE: One-year prospective observational study of meningitis diagnosed at a third level hospital . PATIENTS AND METHODS: All patients with a cerebrospinal fluid (CSF) specimen with cyto-biochemical characteristics and clinical picture consistent with meningitis were included in the study . They were followed from admission to hospital up to discharge or exitus . The epidemiologic characteristics of patients, etiology, related risk factors and predisposing situations, CSF characteristics, clinical manifestations, clinical course, and antibiotic susceptibility of the causative agents were analyzed . RESULTS: Ninety-five cases were included . Seventy-six (69.4%) were community acquired and 29 (30.5%) nosocomially acquired meningitis . Among community acquired meningitis, 31 (46.9%) were of bacterial origin (8 N . meningitidis, 3 H . influenzae, 2 S . pneumoniae, 1 Streptococcus group B, 1 Listeria monocytogenes, 1 Staphylococcus aureus, and 1 Brucella spp.); CSF culture was negative in 14 cases (41.2%) . In most cases neither risk factor nor predisposing situations were detected . Patients with purulent meningitis and negative CSF culture had a significantly lower number of complications than patients with positive CSF culture . Among patients previously treated with beta-lactam antibiotics (8 cases) the probability of a negative CSF culture was greater than among not treated patients (OR 16.00, 95% CI 1.45-764.68; p = 0.011) . The remaining cyto-biochemical characteristics were similar in both groups . Thirty-five cases (53.03%) of community acquisition were lymphocytic meningitis (31 viral, 3 tuberculous, and 1 luetic meningitis) . Among nosocomial cases (29 cases, 30.5%), most were caused by gram-negative bacilli and microorganisms of the Staphylococcus genus . Fourteen cases (48.2%) were related to some type of neurosurgical procedure . Overall, only two exitus cases were recorded . CONCLUSIONS: The etiologic agents of community acquired meningitis are mainly N . meningitidis, S . pneumoniae and Haemophilus influenzae . The previous antibiotic therapy did not influence thy cyto-biochemical characteristics of CSF but it did influence the yielding of culture . Meningitis with negative CSF culture has a significantly lower number of complications . The availability of a Neurosurgery Department at a hospital confers a change in the epidemiologic spectrum of diagnosed meningitis, with a higher incidence of nosocomial meningitis . In our environment, a substantial proportion of cases due to Staphylococcus microorganisms was observed. Neurochirurgie, 1999 Sep, 45(3), 219 - 24 {Neurosurgical complications of purulent meningitis in the tropical zone}; Eholie SP et al.; Our retrospective study concerned 35 cases of surgical complications related to bacterial meningitis in 16 adults and 19 children . The mean age was 28 years for adults (15-56 years), and 6 months for children (1-12 months) . Portal of entry for meningitis was found in 12 cases (35%): 8 sinusitis and 4 otitis . Delay to appearance of complications was 4.5 days, and to diagnosis confirmation 9 days with CT scan (17 cases), and transfontanellar ultrasonography (19 cases) . The complications were: hydrocephalus, 19 cases (54%), brain empyemas, 7 cases (20%), abscesses, 10 cases (28.5%), ventriculitis, 2 cases (6%) . Twenty-two bacteria were isolated from the CSF: Streptococcus pneumoniae (15 cases), Haemophilus influenzae (5 cases), Neisseria meningitidis (1 case), and Escherichia coli (1 case) . Fourteen patients underwent neurosurgical treatment based on aspiration in case of suppuration and external drainage in case of hydrocephalus . The associated medical treatment was antibiotics combining third-generation cephalosporins, fluoroquinolone, and metronidazol, with a mean duration of 12 days . Recovery rate was 89%, letality 11%, and after effect rate were 33% . Our results confirm the low frequency of neurosurgical complications related to bacterial meningitis, but it emphasizes the role of an early CT-scan for diagnosis and prognosis. Dev Biol Stand, 1999, 101, 177 - 83 New methods for the characterisation of biopharmaceuticals: conjugate vaccines against Haemophilus influenzae type b; Lemercinier X et al.; Modern physicochemical methods allow biological pharmaceuticals, particularly those arising from recombinant DNA technology, to be characterised with a degree of precision not previously possible . These techniques, which tell us what a material is (rather than what it does) provide an approach complementary to traditional bioassays for the control of biological pharmaceuticals . As we come to understand the mechanisms by which structural variation modulates the various biological activities of a product, structure-based assays will be able to replace biological identity and potency assays, although replacement of safety tests to find trace impurities (such as endotoxin) may be more difficult. Dev Biol Stand, 1999, 101, 49 - 56 Use of animal testing for evaluating glycoconjugate vaccine immunogenicity; Madore DV et al.; Most animal species respond with high antibody levels to polysaccharide antigens after they have been covalently linked to a protein carrier, converting a T-cell independent to a T-cell dependent antigen . This chemical modification has enabled the development of glycoconjugate vaccines for Haemophilus influenzae type b, Neisseria meningitidis, and multivalent Streptococcus pneumoniae . This new generation of vaccines can be well characterized physically and chemically to ensure consistent vaccine manufacture . Such analytical tests provide an alternative to animal models for Quality Control Laboratories; biological models can be difficult and costly to develop and use on a routine basis . If animal tests are used, they need to be refined, defined, and validated for their intended purpose. J Clin Pathol, 1999 Jun, 52(6), 424 - 9 Northern region asplenia register--analysis of first two years; Spickett GP et al.; OBJECTIVES: To assess the feasibility of setting up a register of patients with asplenia within a defined geographical area; to ensure that guidelines on best practice were implemented; to obtain information on antibody levels to pneumococcal capsular polysaccharides and Haemophilus influenzae type b capsular polysaccharide, before and after immunisation and annually thereafter; to raise awareness of risks among clinicians and to offer advice on management . DESIGN: Prospective recruitment using multiple sources of recruitment . Annual follow up reminders sent from Registration Centre . SUBJECTS: Population of (old, pre-1995) Northern Health Region: approximately 3.1 million . MAIN OUTCOME MEASURES: Data were obtained on reasons for asplenia, duration of asplenia, use of prophylactic antibiotics, Medic-Alert bracelets, immunisations, antibody levels, death . RESULTS: The register was initiated at the beginning of April 1995 and ran to the end of March 1997 . After two years of operation, 1111 cases had been registered but the response from some health districts was poor . Major primary causes of asplenia were trauma (264), other surgical (198), lymphoproliferative disease (154), and idiopathic thrombocytopenic purpura (147) . There were 664 patients on prophylactic antibiotics, of whom 498 were on continuous antibiotics . Only 18 had any type of warning bracelet . Antibody measurements were carried out at least once on 75% of patients; 306 patients had satisfactory antibody levels on first blood sample in year 1, rising to 405 in year 2; 43 patients failed to make any antibody response to Pneumovax despite multiple immunisations, and three patients failed to respond to Hib vaccine . Sixteen patients with satisfactory antibody levels in year 1 had low levels in year 2 requiring vaccine boosters . Sixteen deaths were reported, two of which were directly attributable to overwhelming sepsis . CONCLUSIONS: Registration has been successful and has raised awareness of the management of asplenia . Compliance with antibiotic prophylaxis and immunisation was initially poor . A potential high risk group of vaccine non-responders has been identified and poor persistence of pneumococcal antibodies has been identified which is likely to alter approaches to immunisation in asplenic patients. Glycobiology, 1999 Dec, 9(12), 1371 - 80 The structural heterogeneity of the lipooligosaccharide (LOS) expressed by pathogenic non-typeable Haemophilus influenzae strain NTHi 9274; Rahman MM et al.; Nontypeable Haemophilus influenzae (NTHi) is an important pathogen responsible for otitis media in children and of pneumonitis in adults with depressed resistance . NTHi is acapsular and, therefore, capsular polysaccharide-based vaccines are ineffective for preventing infections by this pathogen . Recently it was found that a detoxified lipooligo-saccharide (LOS) conjugate from NTHi 9274 induced bactericidal antibodies effective against a large number of NTHi isolates, and conferred protection against NTHi otitis media in chinchillas (X.-X.Gu et al., 1996, Infect . Immun.,64, 4047-4053; X . -X.Gu et al., 1997., Infect . Immun.,65, 4488-4493) . In this paper we report the chemical character-ization of the LOS from NTHi 9274 LOS . NTHi is capable of expressing a heterogenous population of LOS exhibited by multiple oligosaccharide (OS) epitopes . OSs released from the LOS of NTHi 9274 by mild acid hydrolysis were purified using Bio-Gel P4 gel permeation chromatography . The OSs were characterized by glycosyl composition analysis, glycosyl linkage analysis, nuclear magnetic resonance spectroscopy (NMR), fast atom bombardment mass spectro-metry (FAB-MS), matrix-assisted laser desorption time of flight mass spectro-metry (MALDITOF-MS), and tandem MS/MS . At least 17 different OS molecules were observed . These contained variable glycosyl residues, phosphate (P), and phospho-ethanolamine (PEA) substituents . These molecules contained either three, four, or five hexoses, and all contained four heptosyl residues . The four heptosyl residues consisted of one D,D-Hep and three L,D-Hep . Dephosphorylation of the OSs with aqueous 48% hydrofluoric acid (HF) reduced the number of molecules to about to seven; Hex(1)-(7)Hep(4)Kdo(1) . Of these seven, Hex(2)Hep(4)Kdo(1), Hex(3)Hep(4)Kdo(1), and Hex(4)Hep(4)Kdo(1)were the major constituents . Thus, this NTHi LOS preparation is very heterogeneous, and contains structures different from those previously published for Haemophilus influenzae . The tandem MS/MS analysis and glycosyl linkage data suggest that the LOS oligosaccharides have the following structures where Hex is either a Glc or Gal residue. Onderstepoort J Vet Res, 1999 Mar, 66(1), 55 - 7 Confirmation that PCR can be used to identify NAD-dependent and NAD-independent Haemophilus paragallinarum isolates; Miflin JK et al.; Seventy five bacteria tentatively identified as Haemophilus paragallinarum (the causative agent of infectious coryza), eight identified as Ornithobacterium rhinotracheale and 13 identified as NAD-independent Pasteurella species were isolated from chickens with respiratory infection in various provinces in South Africa . The isolates were characterized by conventional biochemical and serological methods . A polymerase chain reaction (PCR) assay specific for H . paragallinarum was used to identify the cultures directly from colonies . The PCR assay gave positive results for all isolates that were identified by conventional methods as H . paragallinarum, irrespective of whether they were nicotinamide adenine dinucleotide (NAD)-dependent (43 isolates) or NAD-independent (32 isolates) . The eight isolates that were identified by conventional methods as O . rhinotracheale and the 13 isolates identified as various Pasteurella species gave negative results in the PCR assay . This study has demonstrated that colony PCR is a rapid method for uniquely identifying both NAD-dependent and NAD-independent strains of H . paragallinarum and distinguishing them from other bacteria, such as O . rhinotracheale and Pasteurella species. Am J Manag Care, 1999 Aug, 5(11 Suppl), S651 - 61 Emergence of antibiotic resistance in upper and lower respiratory tract infections; Jacobs MR; The increase in antibiotic resistance is of great concern to the medical community . The treatment of respiratory tract infections are significantly impacted by resistance, as 67% of antibiotic use in adults and 87% in children is for the treatment of such infections . The most common pathogens implicated in these infections are Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, and isolates of all 3 have developed resistance to some of the antibiotics currently on the market . In 1997, one third of S . pneumoniae strains were classified as penicillin resistant, up to 50% of H . influenzae strains produced beta-lactamase, and all M . catarrhalis strains produced beta-lactamase . As resistance can vary with geographic region and specific populations, one way to determine the-most effective antibiotic for an infection is to ascertain the resistance pattern of these pathogens from local laboratories or national surveillance studies . Breakpoints using pharmacodynamic data based on drug concentration present for at least 40% of the dosing interval, or area under the serum concentration curve:minimum inhibitory concentration ratios have been valuable for comparing the activities of oral agents . Of the currently available beta-lactams and macrolides, only amoxicillin/clavulanate and daily intramuscular ceftriaxone are active against more than 90% of all 3 respiratory pathogens . Newer quinolones are also active against these pathogens, but overuse is very likely to result in rapid development of resistance, and their use should be reserved for patients with treatment failure or significant drug allergies. Fed Regist, 1999 Feb 23, 64(35), 9042 - 8 New vaccine information materials for hepatitis B, Haemophilus influenzae type b (Hib), and varicella (chickenpox) vaccines, and revised vaccine information materials for measles, mumps, rubella (MMR) vaccines . Centers for Disease Control and Prevention (CDC), Department of Health and Human Services . Notice; Functional classes in the three domains of life; EMBL-EBI, Wellcome Trust Genome Campus, Cambridge, UKThe evolutionary divergence among the three major domains of life can now be addressed through the first set of complete genomes from representative species . These model species from the three domains of life, Haemophilus influenzae for Bacteria, Saccharomyces cerevisiae for Eukarya, and Methanococcus jannaschii for Archaea, provide the basis for a universal functional classification and analysis . We have chosen 13 functional classes and three superclasses (ENERGY, COMMUNICATION and INFORMATION) as global descriptors of protein function . Compositional comparison of the three complete genomes reveals that functional classes are ubiquitous yet diverse in the three domains of life . Proteins related with ENERGY processes are generally represented in all three domains, while those related with COMMUNICATION represent the most distinctive functional feature of each single domain . Finally, functions related with INFORMATION processing (translation, transcription, and replication) show a complex behaviour . In Archaea, proteins in this superclass are related with proteins in either Eukarya or Bacteria, as recognized previously . The distribution of functional classes in the three domains accurately reflects the principal characteristics of cellular life forms. J Biol Inorg Chem, 1999 Oct, 4(5), 588 - 92 Extended X-ray absorption fine structure studies on periplasmic and intracellular molybdenum-binding proteins; Duhme AK et al.; We have studied the molybdenum K-edge X-ray absorption spectra of Mo bound in the Mo-binding proteins Mop from Haemophilus influenzae, ModG from Azotobacter vinelandii and the Escherichia coli ModE transcriptional regulatory protein, and compared them with the absorption spectra of A . vinelandii ModA and monomeric molybdate . Pre-edge and extended fine structure data indicate that the Mo-binding proteins with molbindin-like domains bind tetrahedral molybdate with a Mo-O distance of 1.76 A . The molbindin subunits or sub-domains represent a novel protein fold that is used by proteins with distinct functions to bind molybdate in the cytoplasm . The high specificity of the proteins for molybdenum does not depend on a change of coordination number or geometry. Otolaryngol Head Neck Surg, 1999 Nov, 121(5), 616 - 21 Effects of influenza A virus on lectin-binding patterns in murine nasopharyngeal mucosa and on bacterial colonization; Hirano T et al.; To clarify the role of viral infection in otitis media, we intranasally inoculated mice with influenza A virus and examined histologic changes in the nasopharyngeal mucosa using a battery of lectins . Additionally, live Haemophilus influenzae or Streptococcus pneumoniae was injected into the nasopharynx after virus inoculation, and the clearance of bacteria from the nasopharynx was examined . Staining of the mucous blanket and epithelial cell surfaces in the nasopharynx with peanut agglutinin, succinyl wheat-germ agglutinin, and Bandeiraea simplicifolia agglutinin was significantly enhanced with intranasal virus inoculation when compared with that in control animals . The nasopharynx was moderately stained with Maachia amurensis agglutinin and wheat-germ agglutinin in control animals, and the staining was enhanced after virus inoculation . These findings were most remarkable 5 and 9 days after virus inoculation . The numbers of bacteria cultured from the nasopharynx were significantly increased when bacteria were injected 5 days after virus inoculation . These results suggest that an alteration in the glycoconjugate structure lining the nasopharyngeal mucosa caused by the influenza virus might be associated with the reduction in bacterial clearance. J Mol Biol, 1999 Nov 12, 293(5), 1055 - 65 The HaeIV restriction modification system of Haemophilus aegyptius is encoded by a single polypeptide; Piekarowicz A et al.; The HaeIV restriction endonuclease (ENase) belongs to a distinct class of ENases, characterized by its ability to cleave double-stranded DNA on both sides of its recognition sequence, excising a short DNA fragment that includes the recognition sequence . The gene encoding the HaeIV ENase was cloned from Haemophilus aegyptius into pUC19 using a previously described system that does not need the knowledge that a particular ENase is produced by a bacterial strain . DNA sequence analysis of the insert contained on this plasmid identified a single open reading frame (ORF), with the predicted protein having an apparent molecular mass of approximately 110 kDa . The protein encoded by this ORF was purified to homogeneity from Escherichia coli strain ER1944 carrying the haeIVRM gene on a recombinant plasmid under the control of the inducible ara promoter . The protein possessed both ENase and methyltransferase (MTase) activities . Amino acid sequence analysis was able to identify several conserved motifs found in DNA MTases, located in the middle of the protein . The enzyme recognizes the interrupted palindromic sequence 5' GAPyNNNNNPuTC 3', cleaving double-stranded DNA on both strands upstream and downstream of the recognition sequence, releasing an approximately 33 bp fragment . The ENase possessed an absolute requirement only for Mg(+2) . ATP had no influence on ENase or MTase activities . The ENase made the first strand cleavage randomly on either side of the recognition sequence, but the second cleavage occurred more slowly . The MTase activity modified symmetrically located adenine residues on both strands within the recognition sequence yielding N6-methyl adenine . Furthermore, the MTase was active as a dimer . Monaldi Arch Chest Dis, 1999 Aug, 54(4), 337 - 44 Community-acquired pneumonia . The ORIONE Board; Pozzi E; Acute infections of the deep lung contracted outside a hospital environment are termed community-acquired or home-based pneumonia . This disease is still the sixth commonest cause of death in industrialized countries . The clinical picture varies from mild involvement to cases requiring prompt therapeutic interventions and hospitalization . Identification of the aetiological agent is often impossible, and in > 50% of patients antibacterial treatment is chosen on empirical grounds alone . Streptococcus pneumoniae, Haemophilus influenzae and Mycoplasma pneumoniae are still the most commonly occurring pathogens, but aetiology varies according to age group . Presenting symptoms may differ, and distinction between typical and atypical pneumonia is complex . The degree of severity of disease must be assessed in order to determine whether outpatient treatment is sufficient or whether hospitalization is required . Invasive and noninvasive techniques may be employed to reach an aetiological diagnosis . Most cases of community-acquired pneumonia may be effectively treated at home . However, it is important to recognize high-risk conditions that require immediate hospitalization . Choice of treatment is generally based on empirical criteria, adapting antibiotic selection to the clinical characteristics of the disease and individual patient conditions. J Bacteriol, 1999 Nov, 181(21), 6797 - 805 Outer membrane lipoprotein e (P4) of Haemophilus influenzae is a novel phosphomonoesterase; Reilly TJ et al.; Haemophilus influenzae exists as a commensal of the upper respiratory tract of humans but also causes infections of contiguous structures . We describe the identification, localization, purification, and characterization of a novel, surface-localized phosphomonoesterase from a nontypeable H . influenzae strain, R2866 . Sequences obtained from two CNBr-derived fragments of this protein matched lipoprotein e (P4) within the H . influenzae sequence database . Escherichia coli DH5alpha transformed with plasmids containing the H . influenzae hel gene, which encodes lipoprotein e (P4), produced high levels of a membrane-associated phosphomonoesterase . The isolated approximately 28-kDa enzyme was tartrate resistant and displayed narrow substrate specificity with the highest activity for arylphosphates, excluding 5-bromo-4-chloro-3-indolylphosphate . Optimum enzymatic activity was observed at pH 5.0 and only in the presence of divalent copper . The enzyme was inhibited by vanadate, molybdate, and EDTA but was resistant to inorganic phosphate . The association of phosphomonoesterase activity with a protein that has also been recognized as a heme transporter suggests a unique role for this unusual phosphohydrolase. Biofizika, 1999 Jul-Aug, 44(4), 601 - 10 {Computer analysis of regulatory signals in complete bacterial genomes . Translation initiation of ribosomal protein operons}; Vitreshchak A et al.; Signals of translation initiation of operons of Haemophilus influenzae ribosomal proteins were predicted . This process is regulated by the formation of secondary RNA structures to which one of the proteins encoded in a particular operon binds . In some cases, these structures imitate the region of protein binding to rRNA . Predictions are made by comparing with homologous operons of Escherichia coli and analogous regions of rRNA and by estimating the energy of secondary structure formation . It is shown that this regulatory mechanism occurs: in operons L11, S10, S15, spc, and alpha of H.influenzae and, probably, in operon S15 of Helicobacter pylori, Bacillus subtilis, and Mycoplasma genitalium. Antimicrob Agents Chemother, 1999 Nov, 43(11), 2612 - 23 Survey of susceptibilities of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis isolates to 26 antimicrobial agents: a prospective U.S . study; Thornsberry C et al.; An antimicrobial susceptibility surveillance study of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis isolates was performed during the winter of 1996-1997 in order to determine their susceptibilities to 5 fluoroquinolones and 21 other antimicrobial agents . Broth microdilution MICs were determined for 2, 752 isolates from 51 U.S . medical centers . Of the 1,276 S . pneumoniae isolates, 64% were susceptible, 17% were intermediate, and 19% were highly resistant to penicillin . On the basis of the MICs at which 90% of isolates are inhibited and modal MICs, the hierarchy of the five fluoroquinolones from most to least active was grepafloxacin > sparfloxacin > levofloxacin = ciprofloxacin > ofloxacin . For S . pneumoniae isolates for which penicillin MICs were elevated, the MICs of the cephalosporins, macrolides, clindamycin, tetracycline, and trimethoprim-sulfamethoxazole were also elevated, but the MICs of the fluoroquinolones, vancomycin, and rifampin were not . The prevalence of penicillin-susceptible pneumococci varied by U.S . Bureau of the Census region (range, 44% in the East South Central region to 75% in the Pacific region) . In addition, S . pneumoniae isolates from blood were significantly more susceptible to penicillin than those from respiratory, ear, or eye specimens, and pneumococci from patients </=2 years old were significantly more resistant to penicillin than those from older patients (by chi-square analysis, P < 0.05) . beta-Lactamase was produced by 35% of H . influenzae isolates and 93% of M . catarrhalis isolates, resulting in increased MICs of amoxicillin and certain cephalosporins . We noted that the antimicrobial resistance patterns of S . pneumoniae isolates, which correlate with the penicillin susceptibility phenotype, vary by site of infection, age group of the patient, and geographic source of the isolate. Eur J Epidemiol, 1999 Aug, 15(7), 685 - 6 National Surveillance System and Hib meningitis incidence in Italy; Squarcione S et al.; In 1994, the Italian Ministry of Health implemented a National Surveillance System to obtain data on the incidence of bacterial meningitis and its causative agents, including Haemophilus influenzae type b (Hib) . As a consequence, case reporting of Hib meningitis is increasing year by year; in 1996, there were 126 notifications, of which 73% were in children under 2 years of age . Although underreporting still exists, parallel prospective or retrospective epidemiological surveys conducted in some Italian Regions allowed for partial correction of the incidence of Hib meningitis (up to 18.5/100,000 population in 1994). Clin Otolaryngol, 1999 Sep, 24(5), 447 - 8 Paediatric epiglottitis: the influence of the Haemophilus influenzae b vaccine, a ten-year review in the Sheffield region; Midwinter KI et al.; Cases of acute epiglottitis in children have become very uncommon in recent years . This study set out to find whether the introduction of the Haemophilus influenzae b (Hib) vaccine in the UK in October 1992 has influenced the incidence of acute epiglottitis in the Sheffield region, and whether the pathogenesis has altered . A 10-year retrospective case note review was undertaken . A total of 30 children were treated for acute epiglottitis in Sheffield Children's Hospital over that time period . A sharp decline in the number of cases was found after the vaccine was introduced . Most children presenting with the disease after October 1992 had not been vaccinated . In addition, the pathogens isolated in those children who had received the vaccine were all Streptococci . This is the first study in the UK to examine the influence of the Hib vaccine on acute paediatric epiglottitis. FEMS Immunol Med Microbiol, 1999 Nov, 26(2), 159 - 66 Purification and characterization of outer membrane protein P6, a vaccine antigen of non-typeable Haemophilus influenzae; Karalus RJ et al.; Outer membrane protein P6 is a promising vaccine antigen with potential to prevent infections caused by non-typeable Haemophilus influenzae . A convenient and reliable method for the purification of P6 and an assessment of the purity, yield, protein structure, antigenicity and immunogenicity of the purified protein are described . The method begins with intact H . influenzae and utilizes a series of incubations and centrifugations using a single buffer to remove all cell components with the exception of the peptidoglycan to which the P6 is associated . P6 is dissociated from the complex with heat and the insoluble peptidoglycan is removed by centrifugation . The procedure yields highly purified P6 . Contamination with lipooligosaccharide is less than 0.025 endotoxin U per microgr P6 . The yield of P6 is approximately 2 mg of P6 per l H . influenzae culture . The purified P6 retains both the secondary and tertiary structure as measured by circular dichroism and analysis with monoclonal antibodies . The purified P6 is immunogenic in animals . A convenient method for purifying P6 which retains antigenicity and immunogenicity will be an important tool for future studies of the vaccine potential of P6. Nucleic Acids Res, 1999 Nov 15, 27(22), 4491 - 500 Long W tracts are over-represented in the Escherichia coli and Haemophilus influenzae genomes; Shomer B et al.; The occurrence of DNA tracts of the three binary base combinations: R.Y, K.M and W;S has been mapped in the complete genomes of Haemophilus influenzae and Escherichia coli . A highly significant over-representation of W tracts is observed in both bacteria . The excess of W tracts is particularly striking in the 10% intercoding regions . Subdivision of intercoding regions into divergent (promoting), convergent (terminating) and sequential subregions shows that the excess of W tracts is most concentrated in the promoter regions . A particularly high excess of W tracts is observed in the first 200 bases 5' upstream of coding start sites . The data suggest that W tracts have a role in promoter function . A function as unwinding centers, analogous to the role of R.Y tracts in eukaryotes, is proposed . R.Y and K.M tracts are only modestly over-represented in the two bacteria. Am J Respir Cell Mol Biol, 1999 Nov, 21(5), 576 - 85 Surfactant protein D stimulates phagocytosis of Pseudomonas aeruginosa by alveolar macrophages; Restrepo CI et al.; Surfactant protein (SP)-D is an oligomeric glycoprotein belonging to the family of collagen-like lectins known as collectins, which have previously been shown to stimulate phagocytosis and other immune cell functions . The hypothesis investigated in this study was that SP-D would stimulate the phagocytosis of an important pulmonary pathogen, Pseudomonas aeruginosa . SP-D, isolated from the lavage fluid of silica-treated rats, significantly enhanced the uptake of three of six strains of P . aeruginosa by rat alveolar macrophages as analyzed by both fluorescence and electron microscopy . SP-D had only minimal effects on phagocytosis of Haemophilus influenzae . SP-D bound to live P . aeruginosa, and binding was inhibited by chelation of calcium and by a competing saccharide, inositol . In vitro killing assays demonstrated that macrophage-mediated killing of one of the mucoid strains of P . aeruginosa was modestly enhanced by SP-D . P . aeruginosa was not measurably aggregated by SP-D either macroscopically or microscopically . Further, SP-D does not appear to act as an activation ligand because adherence of macrophages to SP-D- coated slides did not stimulate the uptake of P . aeruginosa . These findings suggest that SP-D may be important in controlling the pathogenesis of P . aeruginosa in the lung. Pediatr Pulmonol, 1999 Nov, 28(5), 321 - 8 Diagnostic accuracy of oropharyngeal cultures in infants and young children with cystic fibrosis; Rosenfeld M et al.; The objective of this study was to assess the diagnostic accuracy of oropharyngeal (OP) cultures relative to simultaneous bronchoalveolar lavage (BAL) cultures in very young children with CF, and to examine the effects of bacterial density, age, and study cohort on diagnostic accuracy . Respiratory culture data were analyzed from three independent, prospective studies involving simultaneous collection of 286 OP and BAL cultures from 141 children with CF <5 years of age . For predicting any growth of Pseudomonas aeruginosa (Pa) from the lower airway in subjects </=18 months of age (mean age, 8 +/- 5 months), OP cultures had a sensitivity of 44% (95% CI 14%, 79%), specificity of 95% (90%, 99%), positive predictive value of 44% (14%, 79%), and negative predictive value of 95% (90%, 99%) . Diagnostic accuracy was similar for Haemophilus influenzae (Hi) . Specificity was significantly lower for Staphylococcus aureus (Sa) . Sensitivity for all organisms improved if a positive lower airway culture was defined as >/=10(3) or >/=10(5) cfu/mL . Specificity for Pa declined significantly with increasing age . In children with CF <5 years of age, the specificity and negative predictive value of OP cultures for Pa are high, while the sensitivity and positive predictive value are poor . Thus, in this age range, a negative throat culture is helpful in "ruling out" lower airway infection with Pa . However, a positive culture does not reliably "rule in" the presence of Pa in the lower respiratory tract . These findings may have implications for study design and interpretation as well as clinical management of young children with CF . Vet Immunol Immunopathol, 1999 Oct 18, 71(2), 143 - 9 Binding of bovine IgG2a and IgG2b allotypes to protein A, protein G, and Haemophilus somnus IgBPs; Bastida-Corcuera FD et al.; Immunoglobulin binding proteins (IgBPs) are thought to be virulence factors which enable pathogens to evade the host's immune response . Since bovine IgG2 is important in protection against pyogenic infections, the binding characteristics of Staphylococcus aureus protein A (PrA), streptococcal protein G (PrG), or Haemophilus somnus high molecular weight IgBPs to the two bovine IgG2 allotypes were examined . For PrA or PrG binding of IgG2, guinea pig red blood cells coated with specific IgG2a or IgG2b antibodies were used in a competitive binding inhibition assay with unlabeled and horseradish peroxidase-labeled PrA or PrG . To determine which sizes of H . somnus . IgBPs bind to the two IgG2 allotypes, immunoblots with H . somnus culture supernatant were probed with anti-DNP IgG2a and IgG2b . This detects only Fc binding because anti-DNP does not cross-react with H . somnus antigens . Both IgG2 allotypes bound equally well to PrA and PrG . However, IgG2b but not IgG2a bound to H . somnus high molecular weight IgBPs . The lack of differential binding of bovine IgG2 allotypes to PrA and PrG means that these IgBPs can be considered to be unbiased reagents in assays for detection of bovine IgG2 or for immunoaffinity purification . The differential binding of H . somnus IgBPs to the IgG2 allotypes indicates that animals having one allotype may be more resistant to H . somnus infection than animals having the other allotype. J Biol Chem, 1999 Oct 29, 274(44), 31391 - 400 A Haemophilus influenzae gene that encodes a membrane bound 3-deoxy-D-manno-octulosonic acid (Kdo) kinase . Possible involvement of kdo phosphorylation in bacterial virulence; White KA et al.; The lipopolysaccharide of Haemophilus influenzae contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) residue derivatized with either a phosphate or an ethanolamine pyrophosphate moiety at the 4-OH position . In previous studies, we identified a kinase unique to H . influenzae extracts that phosphorylates Kdo-lipid IV(A), a key precursor of lipopolysaccharide in this organism . We have now identified the gene encoding the Kdo kinase by using an expression cloning approach . A cosmid library containing random DNA fragments from H . influenzae strain Rd was constructed in Escherichia coli . Extracts of 472 colonies containing individual hybrid cosmids were assayed for Kdo kinase activity . A single hybrid cosmid directing expression of the kinase was found . The kinase gene was identified by activity assays, sub-cloning, and DNA sequencing . When the putative kinase gene was expressed in E . coli behind a T7 promoter, massive overproduction of kinase activity was achieved ( approximately 8000-fold higher than in H . influenzae membranes) . The catalytic properties and the product generated by the overexpressed kinase, assayed with Kdo-lipid IV(A) as the substrate, were the same as observed with H . influenzae membranes . Unexpectedly, the kinase gene was identical to a previously characterized open reading frame (orfZ), which had been shown to be important for establishing bacteremia in an infant rat model (Hood, D . W., Deadman, M . E., Allen, T., Masoud, H., Martin, A., Brisson, J . R., Fleischmann, R., Venter, J . C., Richards, J . C., and Moxon, E . R . (1996) Mol . Microbiol . 22, 951-965) . However, based solely on the genome sequence of H . influenzae Rd, no biochemical function had been assigned to the product of orfZ, which we now designate kdkA ("Kdo kinase A") . Although Kdo phosphorylation may be critical for bacterial virulence of H . influenzae, it does not appear to be required for growth. Infect Immun, 1999 Nov, 67(11), 6217 - 20 Identification of genes coding for exported proteins of Actinobacillus actinomycetemcomitans; Mintz KP et al.; Random fusions of genomic DNA fragments to a partial gene encoding a signal sequence-deficient bacterial alkaline phosphatase were utilized to screen for exported proteins of Actinobacillus actinomycetemcomitans in Escherichia coli . Twenty-four PhoA(+) clones were isolated and sequenced . Membrane localization signals in the form of signal sequences were deduced from most of these sequences . Several of the deduced amino acid sequences were found to be homologous to known exported or membrane-associated proteins . The complete genes corresponding to two of these sequences were isolated from an A . actinomycetemcomitans lambda phage library . One gene was found to be homologous to the outer membrane lipoprotein LolB . The second gene product had homology with a Haemophilus influenzae protein and was localized to the inner membrane of A . actinomycetemcomitans. Infect Immun, 1999 Nov, 67(11), 5547 - 51 Carrier properties of a protein derived from outer membrane protein A of Klebsiella pneumoniae; Rauly I et al.; We have recently cloned a new protein, recombinant P40 (rP40) . When tested in vivo after conjugation to a B-cell epitope, rP40 induces an important antibody response without the need for adjuvant . To characterize its potency, this carrier protein was coupled to a peptide derived from respiratory syncytial virus attachment G protein (G1') . After immunization of mice with the rP40-G1' conjugate, strong antipeptide antibodies were detected, whereas peptide alone was not immunogenic . To emphasize the carrier properties of rP40, a polysaccharide derived from Haemophilus influenzae type b (Hib) was coupled to it . Immunoglobulin G responses against the Hib polysaccharide were observed after coupling to rP40 . Interestingly, an antipeptide antibody response was observed despite preexisting anti-rP40 antibodies generated by preimmunization with rP40 . In addition, rP40 compares well with the reference carrier protein, tetanus toxoid (TT), since antibody responses of equal intensity were observed when a peptide or a polysaccharide was coupled to TT and rP40 . Moreover, rP40 had advantages compared to TT; e.g., it induced a mixed Th1/Th2 response, whereas TT induced only a Th2 profile . Together, the results indicate that rP40 is a novel carrier protein with potential for use as an alternative carrier for human vaccination. Chest, 1999 Oct, 116(4), 974 - 83 A multicenter study of grepafloxacin and clarithromycin in the treatment of patients with community-acquired pneumonia; Moola S et al.; STUDY OBJECTIVES: To compare the efficacies of 10-day regimens of grepafloxacin (GFX) (Raxar or Vaxar; Glaxo Wellcome; Greenford, UK), 600 qd, and clarithromycin (CLA) (Klacid, Biaxin, or Klaracid; Abbott Laboratories; Chicago, IL), 500 mg bid, in patients with community-acquired pneumonia (CAP), on the basis of clinical response, including radiographic evidence, and bacteriologic efficacy . DESIGN: Phase IIIb, double-blind, double-dummy, randomized, prospective, parallel-group, comparative study conducted at 58 centers in 11 countries . PATIENTS AND SETTING: Adult patients with signs and symptoms of CAP that was confirmed by radiographic evidence and who did not require parenteral therapy were included in the study . ASSESSMENTS: Patients were assessed before treatment, during treatment, after treatment, and at follow-up (28 to 35 days after treatment completion) . Clinical response was evaluated . Blood and sputum samples were cultured for bacterial pathogens, and serology testing was performed to detect atypical pneumonia . RESULTS: A total of 504 patients were enrolled into the trial: 251 were randomly assigned to receive GFX and 253 to receive CLA . In patients able to be clinically evaluated, clinical success rates at follow-up were 147 of 163 patients (90%) in the GFX group and 148 of 167 patients (89%) in the CLA group (95% confidence interval, -6% to 9%) . Pretreatment pathogens were confirmed in 131 of 504 patients (26%), either by culture or serology testing, the primary pathogens being Streptococcus pneumoniae (22%), Haemophilus influenzae (17%), Mycoplasma pneumoniae (25%), and Chlamydia pneumoniae (11%) . For patients able to be evaluated who had typical pathogens, bacteriologic success was achieved in 92% of the patients in each treatment group . For patients able to be evaluated who had atypical pathogens, 18 of 18 patients (100%) in the GFX and 23 of 26 patients (88%) in the CLA group had a successful clinical outcome . There were similar rates of adverse events in each group, resulting in </= 7% withdrawal from treatment; gastrointestinal events were the most common . CONCLUSIONS: GFX, 600 mg qd, was equivalent to CLA, 500 mg bid, in treating adult patients with CAP . Both treatments were well tolerated. Pediatr Infect Dis J, 1999 Oct, 18(10), 860 - 5 High incidence of Alloiococcus otitis in otitis media with effusion; Hendolin PH et al.; BACKGROUND: The etiology of otitis media with effusion (OME) is unclear . Although the majority of effusions show inflammation, culture methods yield positive results for bacteria in only 20 to 30% of cases . METHODS: The polymerase chain reaction was used for detection of three upper respiratory tract pathogens, Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae, and a fairly recently described bacterium, Alloiococcus otitis (A . otitidis), that is solely found in OME . The study included 67 middle ear effusions that were collected from 48 pediatric OME patients during ventilation tube placement . RESULTS: PCR tested positive for 57 (85.1%) of the middle ear effusions . Thirty-one (46.3%) A . otitis-, 12 (17.9%) H . influenzae-, 25 (37.3%) M . catarrhalis- and 14 (20.9%) S . pneumoniae-positive effusions were obtained . All four study organisms showed similar distribution in effusions of various duration (P = 0.72) and in different effusion types (P = 0.59) . Only the proportion of M . catarrhalis-positive effusions was lowered by recent antimicrobial therapy (P < 0.05) . Although the study organisms had equal distributions among singly and multiply positive specimens (P = 0.90), A . otitis was detected significantly more often with one of the three other species (15 of 19, 78.9%) than the other species with each other (4 of 19, 21.1%, P < 0.001) . CONCLUSIONS: The findings suggest a bacterial etiology for OME . Association of A . otitis with the three other species implies that this organism might have the capability of augmenting bacterial colonization in the middle ear. Pediatr Infect Dis J, 1999 Oct, 18(10), 854 - 9 Short course therapy with cefuroxime axetil for acute otitis media: results of a randomized multicenter comparison with amoxicillin/clavulanate; Pessey JJ et al.; BACKGROUND: Otitis media is a common infection of childhood . Increasing antibiotic resistance rates among the principal causative pathogens, Streptococcus pneumoniae and Haemophilus influenzae, are associated with failure of first line agents . OBJECTIVE: This open, randomized, multicenter study compared the clinical efficacy of a short 5-day course of cefuroxime axetil (CAE) suspension with that of amoxicillin/clavulanate (A/CA) suspension for 8 or 10 days . METHODS: Children age 6 to 36 months with acute otitis media with effusion, diagnosed by tympanocentesis and microbiologic culture, were randomized to receive CAE (30 mg/kg/day in two divided doses for 5 days) or A/CA 40 mg/kg/day in three divided doses for 10 days (A/CA-10) . In French centers A/CA was given at 80 mg/kg/day in three divided doses for 8 days (A/CA-8) . Patients were assessed 1 to 4 days after completing the course (posttreatment) and followed up at 21 to 28 days after completing the course . RESULTS: Of the 716 patients randomized, 252 were treated with CAE, 255 with A/CA-10 and 209 with A/CA-8 . In the clinically evaluable population, the proportions of patients with clinical cure at posttreatment were 175 of 203 (86%), 181 of 205 (88%) and 145 of 164 (88%) in the CAE, A/CA-10 and A/CA-8 groups, respectively, demonstrating equivalence among the three treatments . For patients <18 months old, clinical cures were 111 of 134 (83%), 116 of 131 (89%) and 83 of 99 (84%) in the CAE, A/CA-10 and A/CA-8 groups, respectively; equivalence was also demonstrated . At follow-up, 130 of 175 (74%) CAE, 121 of 172 (70%) A/CA-10, and 112 of 142 (79%) A/CA-8 had maintained cure . A total of 837 pretreatment pathogens were isolated from middle ear fluid in 73% (522 of 716) patients, the majority of isolates were S . pneumoniae (30%) and H . influenzae (27%) . The most common adverse events were gastrointestinal, the incidence of drug-related diarrhea being higher in the A/CA-10 group (18%) than in either the CAE or A/CA-8 groups (10%) . CONCLUSIONS: A 5-day course of CAE, given twice daily, was shown to be equivalent to the two regimens of A/CA for treatment of acute otitis media with effusion in children. Diagn Microbiol Infect Dis, 1999 Sep, 35(1), 37 - 44 In vitro activity of the novel ketolide HMR 3647 and comparative oral antibiotics against Canadian respiratory tract isolates of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis; Hoban DJ et al.; The in vitro activities of HMR 3647, erythromycin A, clarithromycin, azithromycin, roxithromycin, penicillin G, ampicillin, cefuroxime, trimethoprim/sulfamethoxazole, tetracycline, ciprofloxacin, and levofloxacin were determined for 1179 Streptococcus pneumoniae, 1438 Haemophilus influenzae, and 428 Moraxella catarrhalis isolated from respiratory tract specimens by 18 medical centers across Canada during 1997-1998 . On a per weight basis, HMR 3647 was the most active agent tested against S . pneumoniae with MIC90s of < or = 0.12 microgram/mL for both penicillin susceptible and penicillin intermediate isolates and 0.25 microgram/mL for penicillin-resistant isolates . HMR 3647 was also highly active against M . catarrhalis (MIC90, < or = 0.12 microgram/mL), but less active against H . influenzae (MIC90, 4 micrograms/mL). Biochem Biophys Res Commun, 1999 Oct 14, 264(1), 191 - 5 Identification of two penicillin-binding multienzyme complexes in Haemophilus influenzae; Alaedini A et al.; Dansyl-labeled penicillin, reversed-phase chromatography, and peptide mapping have been used to detect, separate, and study penicillin-binding proteins (PBPs) and PBP multienzyme complexes of H . influenzae . The cross-linking of proteins in the multienzyme complex was accomplished with the aid of cyanogen, a salt-bridge specific cross-linking agent . The chromatographic profile of the PBPs clearly showed a dramatic change in the number and identity of peaks after treatment of the bacterial cells with cyanogen . The disappearance of all seven peaks corresponding to the PBPs was accompanied by the emergence of two new peaks with molecular weights between 400 kDa and 600 kDa . The results hint at the existence of two penicillin-binding multienzyme complexes, each containing subunits that interact via salt-bridges . Chromatographic active site peptide mapping of PBPs and PBP complexes was used to determine the identity of PBPs involved in each complex . It is postulated that one multienzyme complex containing PBP 2 may be involved in cell elongation while the other complex containing PBP 3 may be responsible for cell division . Diabetes Care, 1999 Oct, 22(10), 1694 - 7 Lack of association between early childhood immunizations and beta-cell autoimmunity; Graves PM et al.; OBJECTIVE: To determine whether early childhood immunization history affects the risk of developing the beta-cell autoimmunity that precedes type 1 diabetes . RESEARCH DESIGN AND METHODS: This article describes a case-control study whose participants were 317 children aged < or = 12 years who have a first-degree relative with type 1 diabetes . The children were enrolled in a prospective cohort study of the etiology of beta-cell autoimmunity, the Diabetes Autoimmunity Study in the Young, in Denver, Colorado . The main outcome measure was beta-cell autoimmunity as determined by persistent autoantibodies against insulin, GAD, or islet cell antibody (IA-2) 512 . The number of cases with beta-cell autoimmunity was 25, and the number of control subjects (the remainder of the cohort) was 292 . RESULTS: There was no difference between cases and control subjects in the proportion receiving hepatitis B (HBV), Haemophilus influenzae b (Hib), polio, or diphtheria tetanus pertussis (DTP) vaccines before 9 months of age; in the proportion receiving HBV at birth rather than later; or in the median age at first HBV, Hib, polio, or DTP vaccination . CONCLUSIONS: The results suggest that changing the early childhood immunization schedule would not affect the risk of developing beta-cell autoimmunity or type 1 diabetes. Arch Microbiol, 1999 Oct, 172(4), 219 - 26 The dcuD (former yhcL) gene product of escherichia coli as a member of the DcuC family of C4-dicarboxylate carriers: lack of evident expression Janausch IG, Unden G. The dcuD gene (formerly yhcL) of Escherichia coli shows significant sequence similarity only to the dcuC gene of E . coli, which encodes a C4-dicarboxylate carrier (DcuC) that functions during anaerobic growth . Inactivation of dcuD had no effect on the growth of E . coli under a large number of conditions and led to no detectable changes in phenotype . Translational dcuD'-'lacZ gene fusions were not significantly expressed in the presence of dicarboxylates or monocarboxylates under oxic or anoxic conditions . Other potential substrates such as amino sugar derivatives, amino acids, and alpha-aspartyl dipeptides also did not lead to expression of dcuD . Changes in medium composition, pH, ionic strength, and temperature had no significant effects on dcuD expression . A dcuD gene amplified from a natural isolate of E . coli was not expressed in wild-type and E . coli K-12 backgrounds . Cloning of dcuD behind an inducible promoter resulted in the synthesis of a protein of the expected size (49 kDa), which, however, did not complement for the loss of DcuC or other C4-dicarboxylate carriers . It is suggested that dcuD encodes a protein of the DcuC family of anaerobic C4-dicarboxylate carriers and that dcuD is not significantly expressed or is expressed only under conditions not related to carboxylate metabolism . When two adjacent open reading frames (y0585 and y0586) from Haemophilus influenzae are fused, the resulting hypothetical protein has sequence similarity to DcuC and DcuD. Arch Otolaryngol Head Neck Surg, 1999 Oct, 125(10), 1134 - 40 Evolution of the bacteriologic features of persistent acute otitis media compared with acute otitis media: a 15-year study; Loundon N et al.; OBJECTIVES: To define the epidemiologic features of persistent acute otitis media (PAOM) and modifications of these features during the past 15 years and to investigate for possible differences in bacterial resistance between acute otitis media (AOM) and PAOM . DESIGN: Retrospective patient series . SETTING: Academic tertiary care center . PATIENTS AND METHODS: Persistent acute otitis media was defined as AOM lasting longer than 3 weeks despite 1 or several courses of antibiotic therapy, with the persistence of clinical and otoscopic signs of AOM . From 1982 to 1997, 475 children with PAOM were seen in our department . Every patient had 1 or several specimens of aspirations or swabs of spontaneous otorrhea (or both) . Microbiologic characteristics of the isolated strains (including antibiotic susceptibility) were analyzed . Four successive series of specimens were analyzed-group 1: from October 1, 1982, to June 30, 1986 (136 patients); group 2: from January 1, 1987, to December 31, 1989 (165 patients); group 3: from January 1, 1992, to April 30, 1993 (73 patients); and group 4: from January 1, 1994, to January 31, 1997 (101 patients) . During the same study periods, the bacteriologic results of patients with AOM in the same geographic region were recorded . MAIN OUTCOME MEASURES: A longitudinal comparison between the groups of patients with PAOM and a cross-comparison within each group between patients with PAOM and those with AOM . RESULTS: Obtaining repeated and multiple specimens from patients with PAOM led to a progressive decrease in the rate of sterile specimens, from 35.3% (group 1, 48 patients) to 14.9% (group 4, 15 patients) (P<.01) . During this period, the prevalence of Streptococcus pneumoniae increased in patients with positive culture results, from 18.2% (group 1, 16 of 88 patients) to 44.2% (group 4, 38 of 86 patients) (P<.001) . These strains rapidly and dramatically became resistant to penicillin (amoxicillin) (0% through 1989, 76.2% {16 of 21 patients} in 1993, and 97.4% {37 of 38 patients} in 1996) (P = .01) . The overall prevalence of Haemophilus influenzae remained stable (between 31.4% {27 of 86 patients} and 45.4% {40 of 88 patients}), but the proportion of beta-lactamase-producing strains increased from 30.0% (group 1, 12 patients) to 55.6% (group 4, 15 patients) (P=.04) . The prevalences of Pseudomonas aeruginosa and Staphylococcus aureus did not vary significantly (from 23.1% {group 2, 30 patients} to 10.7% {group 3, 6 patients} and from 10.2% {group 1, 9 patients} to 4.6% {group 4, 4 patients}, respectively) . Comparing data from patients with PAOM with those with AOM revealed that the increased resistance of H influenzae and, in particular, of S pneumoniae was more rapid and more marked in patients with PAOM than in those with AOM (highest rate of resistance in AOM: 36.0% {271 of 753 specimens} and 50.6% {398 of 787 specimens} for H influenzae and S pneumoniae, respectively; P<.001 for S pneumoniae) . CONCLUSIONS: The increase in bacterial resistance frequently encountered during otitis media is even more marked in patients with PAOM . The identification of the organism is essential when the otitis does not resolve, especially in patients with PAOM . Obtaining repeated specimens helps to decrease the rate of sterile cultures. Arch Otolaryngol Head Neck Surg, 1999 Oct, 125(10), 1117 - 20 The microbiology of recurrent rhinosinusitis after endoscopic sinus surgery; Bhattacharyya N et al.; OBJECTIVE: To determine the microbiology of recurrent sinus infections occurring in patients after endoscopic sinus surgery (ESS) . DESIGN: Retrospective review of sinus cultures obtained over a 4-year period from a consecutive series of patients who underwent ESS . SETTING: An academic general otolaryngology practice . RESULTS: A total of 290 cultures were performed in 125 patients after ESS . The female-male ratio of cultures was 2.5:1 with an average patient age of 47.3 years . This group of patients represents 14.5% of 860 patients who underwent ESS during the same period . A total of 65 patients had 1 culture performed, and 60 patients had multiple cultures . Of the 290 culture specimens, 87 (30.0%) demonstrated no growth . Gram-positive cocci predominated, accounting for 37.9% of culture results . Gram-negative rods constituted 14.8% of the isolates . Of the cultures yielding gram-negative rods, 90.7% occurred in patients who had multiple cultures (P = .03) . Fungal forms were cultured in 1.7% of the specimens . None of the Streptococcus pneumoniae isolates demonstrated penicillin-based resistance . The percentages of beta-lactamase-producing strains for Haemophilus influenzae and Branhamella (Moraxella) catarrhalis were 45.4% and 81.8%, respectively . Staphylococcal species also exhibited significant antibiotic resistance patterns, but no statistical association with multiple cultures was noted (P = .23) . CONCLUSIONS: A wide range of bacteria may be present in the infected post-ESS sinus cavity, with a considerable population of gram-negative organisms, including Pseudomonas species . Beta-Lactamase-producing organisms continue to be prevalent in postoperative sinus infections . Culture and sensitivity analyses of pathologic secretions may identify drug-resistant organisms or organisms related to difficult-to-treat infections in exacerbations of chronic rhinosinusitis in the postoperative setting. J Microbiol Methods, 1999 Oct, 38(1-2), 17 - 23 Quantitation of bacterial adherence by image analysis; Barthelson R et al.; In studies of the adherence of pathogenic bacteria to host eukaryotic cells in vitro, the counting of the bacteria is often challenging, especially if many experiments are involved . We developed a method to use digital imaging and computer-aided recognition for the quantitation of bacteria attached to cultured cells . We employed an immunocytochemical method to stain the bacteria and leave the hosts cells relatively unstained . We describe this method for use with five species of bacteria, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, and Chlamydia pneumoniae . To demonstrate an application of this method, we studied the attachment of H . influenzae and S . pneumoniae to target epithelial cell lines derived from the respiratory tract. Vaccine, 1999 Oct 14, 18(5-6), 468 - 78 Studies on a Hib-tetanus toxoid conjugate vaccine: effects of co-administered tetanus toxoid vaccine, of administration route and of combined administration with an inactivated polio vaccine; Carlsson RM et al.; A freeze-dried tetanus toxoid (T) conjugated Haemophilus influenzae type b (Hib) vaccine, was reconstituted in either diphtheria toxoid (D), DT or a combined DT and inactivated polio vaccine (IPV), and administered in an open randomized trial either intramuscularly (i.m . ) or subcutaneously (s.c.) to 287 Swedish infants at three, five and 12 months of age . When not included in the mixture, IPV was administered s.c . at a separate site . The geometric mean concentrations of Hib antibodies after primary and booster vaccinations were 1.0 and 11.6 microg/ml, respectively, with no differences related to co-administration of the carrier protein T . Antibodies against T were induced by the T conjugated Hib vaccine (Hib-T) alone in 69/95 infants aged six months, and in 87/93 children aged 13 months, although infants receiving both Hib-T and T had significantly higher concentrations . Antibody responses to Hib, D, T or polio were not negatively influenced by administration route or by mixing with IPV, except that the mixed vaccine DT-IPV induced lower anti-polio GM titers after primary vaccination than did separate IPV . More local reactions were induced by the s.c . than by the i.m . route (P-values from 0.001 to 0.01) . Slight increases in rates of local reactions and febrile events (>/=38 degrees C) occurred by order of dose . The low Hib antibody concentrations after the first two doses in this and other Swedish studies are unlikely to be of clinical relevance . The tetanus antibody response from T as a carrier protein alone was not sufficient for basic tetanus immunization, but should be considered in future use of additional T conjugated vaccines. Eur J Biochem, 1999 Nov, 265(3), 1067 - 74 Structural analysis of the lipopolysaccharide oligosaccharide epitopes expressed by Haemophilus influenzae strain RM.118-26; Risberg A et al.; The structure of the lipopolysaccharide of Haemophilus influenzae mutant strain, RM.118-26, was investigated . Electrospray ionization-mass spectrometry on intact lipopolysaccharide, O-deacylated lipopolysaccharide and core oligosaccharides obtained from lipopolysaccharide after mild acid hydrolysis provided information on the composition and relative abundance of the glycoforms . Oligosaccharide samples were studied in detail using high-field NMR techniques . The structure of the major glycoform containing phosphocholine is identical to the Hex2 glycoform described for H . influenzae RM.118-28 {Risberg, A., Schweda, E.K.H . & Jansson, P.-E . (1997) Eur . J . Biochem . 243, 701-707} . A second major glycoform, containing three hexose residues (Hex3), in which a lactose unit, beta-D-Galp-(1-->4)-beta-D-Glcp, is attached at the O-2 position of the terminal heptose of the inner core element, L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)-{beta-D-Glcp-( 1-->4)-}- L-alpha-D-Hepp-(1-->5)-alpha-Kdo, carries no phosphocholine . Instead this lipopolysaccharide glycoform is partly (40%) substituted by an O-acetyl group linked to the 6-position of the glucose residue in the lactose unit and has the following structure: Neurol Clin, 1999 Nov, 17(4), 711 - 35 Bacterial meningitis; Spach DH et al.; In recent years, investigators have made significant advances in understanding the pathogenesis of bacterial meningitis, particularly with regard to understanding the cascade of biologic events that cause excessive inflammation within the central nervous system (CNS) . Nevertheless, the most important event in the field of bacterial meningitis in the past decade is the dramatic decline in the incidence of Haemophilus influenzae meningitis in children as a result of the widespread use of the conjugated H . influenzae type b vaccine . Currently, the most important clinical challenge in this field is the emergence of the drug-resistant Streptococcus pneumoniae . This problem has significantly complicated initial management of patients with suspected bacterial meningitis . Preliminary data show promise with new conjugated S . pneumoniae vaccines. Clin Microbiol Rev, 1999 Oct, 12(4), 627 - 32 Infectious coryza: overview of the disease and new diagnostic options; Blackall PJ; Infectious coryza is a well-recognized and commonly encountered upper respiratory tract disease of chickens that is caused by the bacterium Haemophilus paragallinarum . The occurrence of recent outbreaks in North America has emphasized that the disease can be significant in meat chickens as well as layer chickens . In developing countries, coryza is commonly complicated by the presence of a range of other infections, resulting in severe disease and significant economic losses . Unusual forms of the disease, involving arthritis and septicemia, again associated with the presence of other pathogens, have been found in South America . Newly recognized bacteria such as Ornithobacterium rhinotracheale and phenotypic variant forms of both H . paragallinarum and close relatives (variant in that they no longer require V-factor for growth in vitro) have increased the difficulty associated with diagnosing the disease . There have been suggestions in both South America and South Africa that new serovars or serovar variants, associated with unusual clinical manifestations and causing vaccine failures, are emerging . Definitive evidence to confirm or deny the role of these "variants" in vaccine failures is currently not available . A new DNA-based diagnostic technique, involving PCR, has been recently described and will greatly assist in the diagnosis of infectious coryza. APMIS, 1999 Aug, 107(8), 737 - 46 Moraxella catarrhalis-induced purulent otitis media in the rat middle ear . Structure, protection, and serum antibodies; Westman E et al.; To study the effects of viable and heat-killed Moraxella catarrhalis bacteria on the middle ear mucosa and to evaluate the protection after whole-cell immunizations, Sprague-Dawley rats were challenged and rechallenged with four different M . catarrhalis strains . The animals were monitored by clinical observations, bacterial and histological samples from middle ears, and serum IgG levels . Only viable bacteria at a high concentration induced purulent otitis media, which was culture positive in 58% of the cases on day 4 . The infection was characterized by a mild acute reaction lasting otomicroscopically about 8 days, together with quantitative and qualitative changes of the goblet cells . Structurally the mucosal effects of the heat-killed bacteria were less pronounced in the early phase compared to the viable bacteria, but similar at the end of the experiment at 6 months . The intrabullar and subcutaneous immunizations evoked an IgG antibody response in all animals, and the protection rate after immunization was 50% or more . The induced protection was not strain-specific . The study showed the rat to be a possible alternative for the study of different aspects of M . catarrhalis otitis media, an infection that is clinically and structurally different from that elicited by Streptococcus pneumoniae and Haemophilus influenzae in the rat. Antibiot Khimioter, 1999, 44(9), 13 - 8 {Antibiotics in bacterial acute respiratory tract infection in children}; Tatochenko VK et al.; The data on changes in the susceptibility of the most frequent respiratory tract pathogens i.e . Pneumococcus spp . and Haemophilus influenzae within the last 15 years and Streptococcus spp., Staphylococcus spp . and Moraxella spp . at the present time as well as recommendations based on the original and some literature data on the choice of the antibacterial drugs for the initial treatment of bacterial complications of acute respiratory tract viral infections such as otitis, sinusitis and pharyngitis are presented . The necessity of decreasing the unjustified use of antibiotics in cases of uncomplicated acute respiratory tract viral infections is indicated. Rinsho Byori, 1999 Aug, 47(8), 730 - 6 {Haemophilus parainfluenzae and IgA nephropathy}; Yamamoto C et al.; IgA nephropathy (IgAN) was first reported by Berger in 1968, and characterized by diffuse IgA deposition in the mesangium . Patients with IgAN have usually episodic macroscopic hematuria accompanied with pharyngitis, gastroenteritis, bronchitis, or sinusitis . These findings suggest that IgAN is an immune-complex disease resulting from a poorly controlled mucosal immune response to environmental antigens to which the patient was chronically exposed . We reported the glomerular deposition of the outer membrane of Haemophilus parainfluenzae (OMHP) antigens and the presence of IgA antibody against OMHP in the sera of patients with IgAN . These suggest that Haemophilus parainfluenzae plays a role in the aetiology of this disease . This study was conducted to determine whether OMHP antigens induced immunohistologically evident glomerular deposition of IgA and C3 in C3H/HeN mice . Female C3H/HeN mice (4 weeks old) received intraperitoneal injection (HP-IP group), and oral administration (HP-PO group) of OMHP antigens . The control group similarly received intraperitoneal injection of PBS, and oral intake of ordinary water . The mice were sacrificed at 10, 20, 30, 40, 50 weeks after the start of the experiment . The HP-IP group showed glomerular deposition of IgA, C3 and OMHP antigens, glomerular changes (Mesangial hypercellularity and increase in mesangial matrix) after 20 weeks . The HP-PO group showed only mild deposition of IgA, and mild increase in mesangial matrix . These results suggest that OMHP antigens play a role in the glomerular deposition of IgA and C3 in C3H/HeN mice . This is the first use of OMHP antigens to establish an active model of IgAN. Lancet, 1999 Sep 25, 354(9184), 1091 - 2 Haemophilus influenzae type b meningitis in The Gambia after introduction of a conjugate vaccine; Adegbola RA et al.; PIP: This paper reports on the impact of Haemophilus influenzae type b (Hib) conjugate vaccine on the incidence of Hib meningitis in Gambia . The incidence of Hib meningitis among infants younger than 12 months of age in Gambia is greater, and the children affected are younger, compared to children in more developed countries . Between March 1993 and December 1995, children who were administered diphtheria-tetanus-pertussis (DTP) vaccination were randomly assigned a course of Hib conjugate vaccine mixed with DTP or DTP only . In Gambia, DTP vaccinations are recommended at 2, 3, and 4 months of age . The coverage of these vaccinations has been over 85% since 1990 . Hib disease has not disappeared from Gambia in the last 2 years since the national immunization program was introduced . However, the incidence rate has declined rapidly, and a longer period of vaccination may be needed to achieve a sufficient herd effect to protect unimmunized children . J Med Microbiol, 1999 Apr, 48(4), 389 - 94 An investigation into the influences of species and biotype on the type of IgA1 protease produced by isolates of Haemophilus; Senior BW et al.; A total of 59 isolates of different Haemophilus spp., mostly from clinical specimens, was characterised, biotyped and examined for production of type 1 or type 2 IgA1 protease . IgA1 protease activity was not found in any isolate of a species with no or low virulence for man including H . parainfluenzae, H . haemolyticus, H . aphrophilus, H . paraphrophilus, H . segnis, H . paraphrohaemolyticus and H . haemoglobinophilus . IgA1 protease was produced by all isolates of H . influenzae and H . aegyptius and by some isolates of H . parahaemolyticus . The type of IgA1 protease appeared to be independent of the biotype of the isolate in H . influenzae . For the first time some isolates of H . aegyptius were found that produced type 2 IgA1 protease . IgA1 protease production in H . parahaemolyticus may be associated with the virulence of the isolate. J Med Microbiol, 1999 Apr, 48(4), 383 - 8 The pathogenic role of fimbriae of Haemophilus influenzae type b in murine bacteraemia and meningitis; Miyazaki S et al.; Complement activation and development of murine bacteraemia and meningitis following intranasal instillation of cell-bound fimbriated or non-fimbriated organisms were compared to clarify the role of fimbriae in the pathogenesis of illnesses caused by Haemophilus influenza type b (Hib) . In-vitro resistance of non-fimbriate bacteria to the bactericidal effects of normal human serum was at least 400 times greater than that of fimbriate bacteria . The amount of C3 bound to fimbriate Hib was more than that to non-fimbriate Hib . When mice were infected with fimbriate bacteria, 11.5% died . When mice were infected with non-fimbriate bacteria, the mean number of viable organisms gradually increased or was constant up to day 7; 38.5% of these mice died . These in-vivo results were coincident with the in-vitro data . However, the content of polyribosyl ribitol phosphate (PRP) in fimbriate organisms was the same as in non-fimbriate organisms . These results indicate that fimbriate Hib may be less likely to produce bacteraemia and meningitis, correlating with the greater susceptibility to complement-mediated bacteriolysis and the lower mortality seen with this type of organism, although fimbriae increase adherence to epithelial cells (mucosal surface). Neth J Med, 1999 Sep, 55(3), 151 - 4 Non-surgical treatment of purulent pericarditis, due to non-encapsulated Haemophilus influenzae, in an immunocompromised patient; Ligtenberg JJ et al.; A 59-year-old woman suffering from rheumatoid arthritis was admitted with pleural empyema and pericarditis due to non-encapsulated H . influenzae, and developed signs of cardiac tamponade . Purulent pericarditis resolved after ultrasound-guided percutaneous aspiration and systemic antimicrobial therapy . Serial echocardiographic examinations showed a slowly vanishing effusion . Long term follow-up revealed no evidence of pericardial constriction . This case illustrates that life-threatening purulent pericarditis in an immunocompromised patient may respond well to non-surgical treatment. Antimicrob Agents Chemother, 1999 Oct, 43(10), 2534 - 7 Potent bacteriolytic activity of ritipenem associated with a characteristic profile of affinities for penicillin-binding proteins of Haemophilus influenzae; Inui T et al.; Ritipenem is highly bacteriolytic against Haemophilus influenzae . Bacterial lysis was shown after treatments with ritipenem and cefsulodin at their MICs and after treatments with fropenem and cefdinir at four times their MICs, indicated by decreases in the culture turbidities and by morphological changes of the destroyed cells . These beta-lactams were preferentially bound to penicillin-binding protein (PBP) 1b . Ritipenem, fropenem, and cefsulodin exhibited poor affinities to PBPs 3a and 3b, but cefdinir showed high affinities to these PBPs . Microscopic examinations revealed that selective PBP 3 inhibitors, such as aztreonam and cefotaxime, inhibited lysis induced by ritipenem . These results suggest that the preferential inactivation of PBP 1b could be essential to induce the lysis of H . influenzae cells and that binding to PBPs 3a and 3b may interfere with lysis. Vaccine, 1999 Oct 1, 17 Suppl 2, S28 - 36 Glycoprotein conjugate vaccines; Lindberg AA; The polysaccharide capsule which surrounds bacterial species like Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, Salmonella typhi, is a potent virulence factor . It protects the bacterium from phagocytosis, but capsule specific antibodies plus complement binding to the capsule opsonise the organism for phagocytosis and elimination . Purified capsules elicit T-independent antibody responses without a memory function, and are often poorly immunogenic in infants where much of invasive H . influenzae type b (Hib) and pneumococcal infection is seen . Covalent linkage of the polysaccharide, or fractions thereof, to immunogenic carrier proteins creates glycoconjugates which are T-dependent antigens and which prime for boosting either with the glycoconjugate or the capsular polysaccharide; During the 1990s, four Hib glycoconjugate vaccines have been introduced and in countries that have vaccinated the majority of children, the success has been stunning . In countries with very high immunization coverage the disease has been virtually eliminated and, to a decline of over 95% in countries with slightly lower vaccine rates . Worldwide use of Hib glycoconjugate vaccines offers the possibility of elimination of invasive Hib disease . Pneumococcal (11 serotypes with coverage of approximately 85% of invasive disease) and meningococcal (A, C, W 135, Y but not B) glycoconjugates are in pre-registration phases and offer the prospect of being as successful as the Hib glycoconjugates. Pediatrics, 1999 Oct, 104(4 Pt 1), 942 - 50 Lessons learned from a review of the development of selected vaccines . National Vaccine Advisory Committee; Peter G et al.; BACKGROUND: Although the vaccine research and development network in the United States remains vibrant, its continued success requires maintaining harmonious interaction among its many components . Changing one component is likely to affect the system overall . An examination of case studies of the development of selected vaccines would allow an examination of the network as a whole . This article presents conclusions drawn from the case study review undertaken . OBJECTIVE: Successful development of vaccines is a time-intensive process requiring years of commitment from a network of scientists and a continuum of regulatory and manufacturing entities . We undertook this work to shed light on how well the vaccine development system in the United States performs . METHOD: The National Vaccine Advisory Committee examined the research and development pathways of several vaccines that reached licensure expeditiously (hepatitis B vaccine, Haemophilus influenzae type b conjugate vaccines); some that became licensed only after considerable delay (oral typhoid Ty21a vaccine, varicella vaccine); some that are at the point of imminent or recent licensure (reassortant Rhesus rotavirus vaccine, which was licensed by the Food and Drug Administration on August 30, 1998) or near submission for licensure (intranasal cold adapted influenza vaccine); and one for which clinical development is slow because of hurdles that must be overcome (respiratory syncytial virus vaccines) . RESULTS: Some common themes emerged from the reviews of these vaccine "case histories": the expediting influence of a strong scientific base and rationale; the need for firm quantitation of disease burden and clear identification of target populations; the critical role played by individuals or teams who act as "champions" to overcome the inevitable obstacles; availability of relevant animal models, high-quality reagents and standardized assays to measure immune response; the absolute requirement for well designed, meticulously executed clinical trials of vaccine safety, immunogenicity, and efficacy; postlicensure measurements of the public health impact of the vaccine and a track record of the vaccine's safety and acceptance with large-scale use; and the critical need for international collaborations to evaluate vaccines against diseases of global importance that are rare in the United States (eg, typhoid fever) . It was clear that the critical step-up from bench scale to pilot lots and then to large-scale production, which depends on a small group of highly trained individuals, is often a particularly vulnerable point in the development process . CONCLUSIONS: One fundamental lesson learned is that within the varied and comprehensive US vaccine development infrastructure, multiple and rather distinct paths can be followed to reach vaccine licensure . The National Vaccine Advisory Committee review process should be conducted periodically in the future to ascertain that the US vaccine development network, which has been enormously productive heretofore and has played a leadership role globally, is adapting appropriately to ensure that new, safe, and efficacious vaccines become available in a timely manner. J Pharm Pharmacol, 1999 Aug, 51(8), 971 - 4 On the in-vitro antimicrobial activity of oleuropein and hydroxytyrosol; Bisignano G et al.; Secoiridoides (oleuropein and derivatives), one of the major classes of polyphenol contained in olives and olive oil, have recently been shown to inhibit or delay the rate of growth of a range of bacteria and microfungi but there are no data in the literature concerning the possible employment of these secoiridoides as antimicrobial agents against pathogenic bacteria in man . In this study five ATCC standard bacterial strains (Haemophilus influenzae ATCC 9006, Moraxella catarrhalis ATCC 8176, Salmonella typhi ATCC 6539, Vibrio parahaemolyticus ATCC 17802 and Staphylococcus aureus ATCC 25923) and 44 fresh clinical isolates (Haemophilus influenzae, eight strains, Moraxella catarrhalis, six strains, Salmonella species, 15 strains, Vibrio cholerae, one strain, Vibrio alginolyticus, two strains, Vibrio parahaemolyticus, one strain, Staphylococcus aureus, five penicillin-susceptible strains and six penicillin-resistant strains), causal agents of intestinal or respiratory tract infections in man, were tested for in-vitro susceptibility to two olive (Olea europaea) secoiridoides, oleuropein (the bitter principle of olives) and hydroxytyrosol (derived from oleuropein by enzymatic hydrolysis and responsible for the high stability of olive oil) . The minimum inhibitory concentrations (MICs) calculated in our study are evidence of the broad antimicrobial activity of hydroxytyrosol against these bacterial strains (MIC values between 0.24 and 7.85 microg mL(-1) for ATCC strains and between 0.97 and 31.25 microg mL(-1) for clinically isolated strains) . Furthermore oleuropein also inhibited (although to a much lesser extent) the growth of several bacterial strains (MIC values between 62.5 and 500 microg mL(-1) for ATCC strains and between 31.25 and 250 microg mL(-1) for clinical isolates); oleuropein was ineffective against Haemophilus influenzae and Moraxella catarrhalis . These data indicate that in addition to the potential employment of its active principles as food additives or in integrated pest-management programs, Olea europaea can be considered a potential source of promising antimicrobial agents for treatment of intestinal or respiratory tract infections in man. Vaccine, 1999 Aug 20, 18(1-2), 29 - 37 Immunization of mice with P6 of nontypeable Haemophilus influenzae: kinetics of the antibody response and IgG subclasses; Badr WH et al.; The kinetics of the anti-P6 antibody response was characterized in three strains of mice of different haplotypes (Balb/c; H-2d, C3H/H; H-2k, SJL/J; H-2s) . Anti-P6 antibodies were measured on a weekly basis by enzyme-linked immunosorbent assay (ELISA) . The primary response peaked 2 or 3 weeks after the initial injection with 40 microg of purified P6 . The response remained at a plateau for 8-10 weeks . A maximum titer of 1:1,638,400 was attained and then steadily declined . To study the ability of P6 to generate a recall response, we opted to boost the vaccinated mice with a known subimmunogenic dose of live nontypeable Haemophilus influenzae (NTHI) bacteria . After the anti-P6 antibody titers in the primed animals had stayed at baseline levels for 2 weeks, the mice were injected intraperitonealy with 10(8) cfu of NTHI in sterile saline . This challenge with live NTHI bacteria induced a very rapid and strong secondary antibody response in all mice . Finally, we demonstrated that these murine anti-P6 sera were 100% bactericidal against three strains of NTHI when tested in a complement dependant bactericidal assay. Infect Immun, 1999 Oct, 67(10), 5508 - 13 Outer membrane proteins as a carrier for detoxified lipooligosaccharide conjugate vaccines for nontypeable Haemophilus influenzae; Wu TH et al.; Nontypeable Haemophilus influenzae (NTHi) is a common cause of otitis media and respiratory tract infections . Outer membrane proteins (OMP) and lipooligosaccharide (LOS) are major surface antigens of NTHi and potential vaccine candidates . De-O-acylated LOS (dLOS) or oligosaccharide (OS) was coupled to total OMP to form dLOS-OMP and OS-OMP conjugates, while a dLOS-tetanus toxoid (TT) was synthesized for comparison . These conjugates were evaluated in mice and rabbits for immunogenicity . dLOS-OMP elicited a better boostable antibody response against LOS than did dLOS-TT, while OS-OMP was not immunogenic . Formulation of the conjugates with Ribi adjuvant significantly enhanced the immunogenicity of dLOS-OMP and dLOS-TT but not that of OS-OMP . In addition, rabbit antisera elicited by dLOS-OMP but not dLOS-TT (or OMP alone) demonstrated bactericidal activity against 40% of the NTHi strains tested . These results indicate that dLOS is a better derivative of LOS than OS and that OMP is a good carrier for NTHi LOS-based conjugate vaccines. Infect Immun, 1999 Oct, 67(10), 5352 - 60 Examination of early interactions between Haemophilus ducreyi and host cells by using cocultured HaCaT keratinocytes and foreskin fibroblasts; Zaretzky FR et al.; Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease . Keratinocytes are likely the first cell type encountered by H . ducreyi upon infection of human skin; thus, the interaction between H . ducreyi and keratinocytes is probably important for the ability of H . ducreyi to establish infection . We have used the HaCaT keratinocyte cell line grown in monolayers and in cocultures with HS27 fibroblasts to investigate H . ducreyi interactions with keratinocytes and the host-cell response to H . ducreyi infection . Using quantitative adherence and gentamicin protection assays, we determined that approximately 13% of H . ducreyi adhered to HaCaT cell monolayers, while only a small proportion (0.0052%) was intracellular . By transmission electron microscopy, we observed numerous H . ducreyi organisms adherent to but rarely within HaCaT cells cocultured with fibroblasts . Both live H . ducreyi and purified H . ducreyi lipooligosaccharide (LOS) induced significant interleukin 8 (IL-8) expression from HaCaT cell-HS27 cell cocultures . However, the level of IL-8 expression in response to LOS alone was not as pronounced . H . ducreyi LOS was a more potent inducer of IL-8 from cocultures than Escherichia coli lipopolysaccharide (LPS) at the same concentration, suggesting a unique effect of H . ducreyi LOS on cocultures . Neither live H . ducreyi nor purified H . ducreyi LOS or E . coli LPS induced tumor necrosis factor alpha expression from cocultures . H . ducreyi induced drastically different cytokine profiles from cocultures than from HS27 or HaCaT cells cultured separately . IL-8 expression by skin cells in response to H . ducreyi infection in vivo may be responsible for the massive influx of polymorphonuclear leukocytes and other inflammatory cells to the site of infection . This influx of inflammatory cells may be partly responsible for the tissue destruction characteristic of chancroid. Infect Immun, 1999 Oct, 67(10), 5345 - 51 Neutropenia restores virulence to an attenuated Cu,Zn superoxide dismutase-deficient Haemophilus ducreyi strain in the swine model of chancroid; San Mateo LR et al.; Haemophilus ducreyi causes chancroid, a sexually transmitted cutaneous genital ulcer disease associated with increased heterosexual transmission of human immunodeficiency virus . H . ducreyi expresses a periplasmic copper-zinc superoxide dismutase (Cu, Zn SOD) that protects the bacterium from killing by exogenous superoxide in vitro . We hypothesized that the Cu,Zn SOD would protect H . ducreyi from immune cell killing, enhance survival, and affect ulcer development in vivo . In order to test this hypothesis and study the role of the Cu,Zn SOD in H . ducreyi pathogenesis, we compared a Cu,Zn SOD-deficient H . ducreyi strain to its isogenic wild-type parent with respect to survival and ulcer development in immunocompetent and immunosuppressed pigs . The Cu,Zn SOD-deficient strain was recovered from significantly fewer inoculated sites and in significantly lower numbers than the wild-type parent strain or a merodiploid (sodC+ sodC) strain after infection of immunocompetent pigs . In contrast, survival of the wild-type and Cu,Zn SOD-deficient strains was not significantly different in pigs that were rendered neutropenic by treatment with cyclophosphamide . Ulcer severity in pigs was not significantly different between sites inoculated with wild type and sites inoculated with Cu,Zn SOD-deficient H . ducreyi . Our data suggest that the periplasmic Cu,Zn SOD is an important virulence determinant in H . ducreyi, protecting the bacterium from host immune cell killing and contributing to survival and persistence in the host. Infect Immun, 1999 Oct, 67(10), 5060 - 8 Identification of the znuA-encoded periplasmic zinc transport protein of Haemophilus ducreyi; Lewis DA et al.; The znuA gene of Haemophilus ducreyi encodes a 32-kDa (mature) protein that has homology to both the ZnuA protein of Escherichia coli and the Pzp1 protein of H . influenzae; both of these latter proteins are members of a growing family of prokaryotic zinc transporters . Inactivation of the H . ducreyi 35000 znuA gene by insertional mutagenesis resulted in a mutant that grew more slowly than the wild-type parent strain in vitro unless ZnCl(2) was provided at a final concentration of 100 microM . Other cations tested did not restore growth of this H . ducreyi mutant to wild-type levels . The H . ducreyi ZnuA protein was localized to the periplasm, where it is believed to function as the binding component of a zinc transport system . Complementation of the znuA mutation with the wild-type H . ducreyi znuA gene provided in trans restored the ability of this H . ducreyi mutant to grow normally in the absence of exogenously added ZnCl2 . The wild-type H . ducreyi znuA gene was also able to complement a H . influenzae pzp1 mutation . The H . ducreyi znuA isogenic mutant exhibited significantly decreased virulence (P = 0.0001) when tested in the temperature-dependent rabbit model for experimental chancroid . This decreased virulence was not observed when the znuA mutant was complemented with the wild-type H . ducreyi znuA gene provided in trans. J Microbiol Immunol Infect, 1998 Sep, 31(3), 180 - 6 Immunogenicity and safety of Haemophilus influenzae type b conjugate vaccine (HibTITER) and a combination vaccine of diphtheria, tetanus, pertussis and HibTITER (TETRAMUNE) in two-month-old infants; Huang LM et al.; A study was undertaken to evaluate the safety and immunogenicity of a combination vaccine (TETRAMUNE) of conjugate Haemophilus influenzae type b (Hib) vaccine (HibTITER) and DTP (Diphtheria, Tetanus and Pertussis) vaccine . A total of 93 healthy children were randomized to receive either TETRAMUNE (combined group), or DTP and HibTITER administered concurrently (separate group) in separate syringes at approximately 2, 4 and 6 months of age in Taiwan . Serologic responses were largely comparable between the two vaccine groups; almost all subjects were seropositive to Hib PRP (polyribosylribitol phosphate) and were protected against diphtheria and tetanus after 2 doses of vaccine and mounted prominent responses to the components of Bordetella pertussis . Subjects in the combined group did not experience more adverse reactions compared with those in the separate group . We concluded that HibTITER was highly immunogenic and safe when administered concurrently with DTP vaccine to Taiwanese children . TETRAMUNE was also safe and immunogenic and might reduce the number of injections to achieve the same protection. Curr Med Res Opin, 1999, 15(2), 105 - 12 Kinetics of antibody response to Haemophilus influenzae type b vaccines . Pennridge Pediatric Associates; Madore DV et al.; Serum antibodies to the capsular polysaccharide of Haemophilus influenzae type b (Hib) are effective in preventing or ameliorating invasive disease caused by this human pathogen . Polysaccharide and conjugate (saccharide covalently linked to protein carrier) vaccines have been developed which stimulate the production of such antibodies . The polysaccharide-specific antibody concentrations in the sera of vaccine-naive adults and toddlers on days 1, 3, 7, 14 and 28 following immunisation with one dose of the Hib polysaccharide vaccine (PRP, polyribosylribitol phosphate) or an oligosaccharide-CRM197 conjugate vaccine (HbOC, HibTITER) were determined . Antibody responses occurred within 7 days of immunisation with the maximum response usually occurring 14 days post-immunisation, irrespective of vaccine or subject age . In this small study, a significant transient decline in pre-existing antibodies was observed only in the groups receiving the polysaccharide vaccine and not in the groups receiving HbOC vaccine . Because of the small magnitude of antigen-specific antibody decline and its transient nature, it is unlikely that this observation has clinical significance. Pediatr Infect Dis J, 1999 Sep, 18(9), 816 - 22 Fifteen years of experience with bacterial meningitis; Dawson KG et al.; BACKGROUND: Introduction of Haemophilus influenzae type b (Hib) vaccines has dramatically altered the epidemiology of bacterial meningitis in children . The goal of this study was to describe these changes in a pediatric teaching hospital . METHODS: Patient charts at Children's Hospital and Regional Medical Center, Seattle, were identified by diagnosis codes and reviewed retrospectively . The 1981 to 1995 time period was chosen to incorporate three distinct 5-year periods: before the use of unconjugated Hib vaccine; between the unconjugated and conjugate vaccines; and after the conjugate vaccines were available for routine immunization of infants . RESULTS: Bacterial meningitis was identified in 806 cases . In 13 premature infant cases Escherichia coli was most frequently isolated (6 cases) . Group B Streptococcus, E . coli and Listeria monocytogenes were the most common pathogens in 87 neonatal cases . The most common pathogens in 706 cases of childhood meningitis were H . influenzae, Streptococcus pneumoniae and Neisseria meningitidis . H . influenzae was the most common pathogen in the first two time periods (73 and 69% of childhood cases, respectively), but not so in the third period (16%) . CONCLUSIONS: A changing pattern in childhood meningitis was observed during the study period . H . influenzae cases dramatically declined, altering the relative proportions of other pathogens, S . pneumoniae and N . meningitidis . However, the number of cases caused by these latter pathogens remained steady. Pediatr Infect Dis J, 1999 Sep, 18(9), 783 - 8 Immunization status of children enrolled in a hospital-based medicaid managed care practice: the importance of the timing of vaccine administration; Vivier PM et al.; OBJECTIVES: To evaluate the immunization status of children enrolled in a hospital-based Medicaid managed care practice and to assess the impact of the timing of vaccine administration on measured immunization rates . DESIGN AND METHODS: The medical records of all children between the ages of 19 and 35 months who were continuously enrolled in the Medicaid managed care practice for the last 6 months of 1996 were reviewed . Immunization status was determined for the following vaccines: diphtheria-tetanus-pertussis/diphtheria-tetanus-acellular pertussis (4 doses); Haemophilus influenzae type b (3 doses); poliovirus (3 doses); hepatitis B (3 doses); measles-mumps-rubella (1 dose); and overall for the basic series . Two assessment methods were used to determine the immunization status of the study children: (1) a count of all documented vaccines ("count"); and (2) only including vaccines that met minimal age and spacing intervals based on American Academy of Pediatrics and CDC recommendations ("interval assessment") . RESULTS: With the count method vaccine-specific immunization rates ranged from 88 to 95%, with overall coverage of 80% for the basic series . With the interval assessment method vaccine-specific immunization rates ranged from 74 to 92%, with overall coverage of 53% for the basic series . CONCLUSIONS: When all documented vaccines were included in the assessment, vaccine-specific immunization rates approached national goals, although overall coverage remained below 90% in this Medicaid managed care practice . The substantially lower immunization rates obtained by the interval assessment method demonstrate the importance of considering the issue of vaccine timing when interpreting immunization rates and the need for policies for revaccinating children who were immunized at less than recommended intervals . The results also have implications for provider education regarding the early administration of vaccines. Pediatr Infect Dis J, 1999 Sep, 18(9), 757 - 63 Safety and immunogenicity of heptavalent