Streptococcus pneumoniae serogroup 6 clones over two decades.

The major evolutionary stresses on Streptococcus pneumoniae are thought to be the widespread use of antibiotics and the deployment of effective vaccines against the capsular polysaccharides. Our current knowledge of genetic lineages among pneumococcal isolates comes largely from investigations just before and after the introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) introduced in 2000. We examined 66 serogroup 6 isolates from the 1970s, long before the introduction of PCV7 and before widespread penicillin resistance was common in Birmingham, Alabama, to look for ancestors of the clones that came into play around the introduction of the PCV7 vaccine. The hypothesis was that some clonal complexes, if not individual clones, would be stable enough to persist over this period of time. We compared the 1970s isolates with 122 isolates from the 1990s in US and worldwide collections. Genotyping with pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) revealed that while some clones were probably localized to our area, others have persisted within groups that have expanded or diminished over the years.

Reduction of serum cholesterol in Watanabe rabbits by xenogeneic hepatocellular transplantation.

Transplantation of xenogeneic hepatocytes would provide a novel therapy for liver disease and would help to solve the problem of an insufficient supply of donor organs. We have tested whether xenogeneic cells infused into the liver could correct the metabolic defect in the Watanable heritable hyperlipidemic (WHHL) rabbit, an animal model for homozygous familial hypercholesterolemia, and we have investigated whether the infused cells traverse the lining of the portal vasculature. We find that porcine hepatocytes are localized in the hepatic sinusoids after surgery and subsequently migrate out of the vessels and integrate into the hepatic parenchyma. The integrated porcine hepatocytes provide functional LDL receptors that lower serum cholesterol in the WHHL rabbit by 30-60% for at least 100 days.

Ulcerative typhlocolitis associated with Helicobacter mastomyrinus in telomerase-deficient mice.

Telomerase deficiency induces early senescence and defects in proliferating cell populations, but in mice it has not been associated with inflammatory bowel disease. Genetically engineered mice lacking either telomerase reverse transcriptase (TERT) or telomerase RNA were examined for chronic diarrhea and wasting. Affected mice had pasty stools, thickened nondistensible colon walls, and contracted ceca. Histologically, the cecal mucosa was largely replaced by inflammatory infiltrate consisting of plasma cells, neutrophils, lymphocytes, and macrophages with marked widespread fibrosis and ulceration. Remaining epithelium was disorganized and hyperplastic, with multifocal dysplasia. Colonic mucosa was markedly hyperplastic with similar inflammation and epithelial dysplasia. Multifocal adenomatous hyperplasia, but no inflammation, was present in the small intestine. Microaerophilic spiral bacteria with 16S rRNA gene sequences identical to Helicobacter mastomyrinus were isolated from the colon and cecum. Severe granulomatous typhlocolitis without epithelial dysplasia developed in germ-free recombination-activating gene (RAG) knockout (KO) recipients of CD4+ T cells and inoculated with cecal contents from affected TERT KO mice and in specific pathogen-free recipient RAG KO mice and interleukin-10 KO mice inoculated with H mastomyrinus. Typhlocolitis in mice given H mastomyrinus was more severe than in mice given Helicobacter hepaticus. Telomerase-deficient mice are susceptible to helicobacter-associated typhlocolitis. H mastomyrinus causes severe disease in susceptible mouse strains.

Protection of mice from experimental infection with type III group B Streptococcus using monoclonal antibodies.

Mouse hybridoma antibodies of several major classes against group B streptococcus type III have been produced. Mice were immunized with either whole heat-killed or acid-treated organisms to obtain antibodies against both the complete (sialated) or incomplete (nonsialated) forms of the type III polysaccharide. Resulting monoclonal antibodies showed exclusive specificity for either the complete or incomplete antigen. The ability of these antibodies to protect mice from a lethal challenge of live type III organisms was tested with a mucin model that permitted use of very small inocula given intraperitoneally with antibody and mucin. Antibodies specific for the nonsialated antigen were not protective, whether of IgM, IgG2a, or IgG3 isotypes. Antibodies specific for the complete antigen were, however, highly protective, including monoclonals of IgM, IgG2a, and IgA isotypes. These mouse monoclonal antibodies against group B streptococci that are directed against either complete or incomplete antigenic determinants, and include isotypes other than IgM, should be particularly useful for studying the mechanism of protection against experimental infection.

ELISA methodology for polysaccharide antigens: protein coupling of polysaccharides for adsorption to plastic tubes.

A method is described which permits the adaption of ELISA techniques for measurement of antibody against bacterial polysaccharides. First, the polysaccharides antigen is covalently bound to poly-L-lysine, using cyanuric chloride as the coupling agent. The poly-L-lysine then adsorbs to the walls of plastic tubes, thus immobilizing the polysaccharide coupled to the poly-L-lysine. The method is simple, rapid, and utilizes small amounts of polysaccharide antigen.

Interaction of group B streptococcal type-specific polysaccharides with wheat germ agglutinin and other lectins.

The group B streptococci (GBS) are known to have type-specific polysaccharides rich in N-acetylneuraminic acid end groups, which are thought to be important immunological determinants. Wheat germ agglutinin (WGA) has affinity for N-acetylneuraminic acid as well as N-acetylglucosamine, and readily precipitates the type but not the group polysaccharide. A WGA-Sepharose affinity column was used to isolate complete type polysaccharides of representative strains of the 4 major GBS types. WGA, other lectins, and rabbit antisera were then used to characterize the products of various extraction procedures and chemical degradations, including mild acid hydrolysis and treatment with neuraminidase. Results of lectin binding studies were consistent with proposed chemical structures of types Ia, Ib and II. Differences were noted, however, between the cross-reactive antigens of pneumococcus type 14 and the desialated GBS type III polysaccharide. Although structurally similar, indirect evidence from lectin binding studies suggest that these antigens may not be identical.

Opsonophagocidal activity in sera from infants and children immunized with Haemophilus influenzae type b conjugate vaccine (meningococcal protein conjugate).

Immunization with Haemophilus influenzae type b capsular polysaccharide (polyribosylribitol-phosphate [PRP]) has resulted in limited and variable antibody radioimmunoassay in infants younger than 2 years of age. Although an H influenzae type B vaccine has been in use for several years, it is not used now for the age group at greatest risk for disease. In an effort to enhance immunogenicity, PRP has been coupled to various carrier proteins and to an outer membrane protein complex (OMPC) from Neisseria meningitidis group B. The latter approach has yielded a vaccine that elicits a good antibody response after a single 15-micrograms dose of vaccine in infants as young as 2 months of age, as measured by radioimmunoassay and immune bacteriolysis. In this report we describe the results of a pilot study using this H influenzae type B conjugate vaccine, PedvaxHIB, in children from 2 months to 4 years of age. Three different vaccine lots were examined for consistency of response. Sera were measured for antibody levels by radioimmunoassay and for functional activity using an opsonophagocytic assay using human adult neutrophils. These assays correlated well and demonstrated the excellent immune response and biologic activity of sera from infants vaccinated with this unique H influenzae type B conjugate vaccine.

Lack of correlation of in vitro adherence of Haemophilus influenzae to epithelial cells with frequent occurrence of otitis media.

The adherence to human epithelial cells, biotype and capsular type of 175 Haemophilus influenzae cultured from the upper respiratory tract were studied in a prospective study of children with recurrent otitis media. Forty-three children who had greater than 2 episodes of acute otitis media (AOM) during the first year of life were followed for at least 1 year. Cultures of the oropharynx were done periodically, and the middle ear fluid (MEF) was cultured at the time of AOM. H. influenzae was recovered from MEF in 44% of the 136 AOM episodes recorded. Thirty-one children had at least one episode of AOM caused by H. influenzae; the remaining 12 children, designated as "controls," had no otitis or had AOM caused by other organisms. The possible differences between carriage and infection strains were evaluated by comparison of MEF and oropharyngeal isolates, by pairwise comparison of MEF and oropharyngeal isolates and by pairwise comparison of multiple isolates from each host recovered at the time of AOM and during infection-free intervals. No significant differences in patterns of adherence, capsular type or biotype were found. The lack of correlation between these characteristics and infection suggests either that H. influenzae organisms have determinants of virulence yet to be defined or that variations in host susceptibility permit infection by the strain colonizing the upper respiratory tract. Adherence per se may be less important in the development of infection than in establishing and maintaining colonization within the host.

Role of adherence of Streptococcus pneumoniae in acute otitis media.

The adherence of Streptococcus pneumoniae to human nasopharyngeal epithelial cells was studied as a possible determinant in the development of acute otitis media (AOM). Pneumococcal isolates were obtained from the nasopharynx (NP) and middle ear fluid of infants followed from birth in a prospective study of pneumococcal carriage and infection. The adherence of 33 middle ear fluid isolates from 19 infants with AOM was compared with 143 strains recovered from NP cultures taken from each child both at the time of their acute infections and on other occasions. We studied 171 NP isolates from 29 "carrier" infants, who had no pneumococcal infections, for comparison. Adherence properties were not associated with any particular pneumococcal capsular types, nor were adherent strains more frequent among infants with AOM. There was no evidence to support the hypothesis that pneumococci associated with AOM have a special propensity for adherence. Adherence was a frequent characteristic of pneumococci recovered from the NP, especially in connection with upper respiratory tract infection, and may be required for the establishment of colonization but was not a property that discriminated between carriage strains and those causing AOM.

In vitro activities of cefepime versus cefotaxime and ceftriaxone against penicillin-resistant Streptococcus pneumoniae determined by Etest.

The Etest was used for determining in vitro susceptibilities of 144 unique clinical isolates of penicillin-intermediate and resistant Streptococcus pneumoniae to cefepime, cefotaxime, and ceftriaxone. MIC ranges were 0.12-8 mug/ml for cefepime and 0.06-16 mug/ml for cefotaxime and ceftriaxone. MICs for 50% of the isolates for the three agents were equivalent at 1 mug/ml, whereas MICs for 90% of the isolates were 2 mug/ml for cefotaxime and ceftriaxone, versus 4 mug/ml for cefepime. The Etest is a practical means for determining susceptibilities of S. pneumoniae to cefepime and other cephalosporins in diagnostic laboratories.

In vitro activity of ABT-773, telithromycin and eight other antimicrobials against erythromycin-resistant Streptococcus pneumoniae respiratory isolates of children.

The activity of the ketolide ABT-773 against 180 erythromycin-resistant Streptococcus pneumoniae obtained from children was compared with telithromycin, azithromycin, clarithromyin, roxithromycin, clindamycin, penicillin, levofloxacin and gatifloxacin. Ketolide MICs were all < or =1 mg/l, with ABT-773 being the most potent of all drugs tested. MIC(90)s for macrolides and azithromycin in mefE+ isolates were 16-32 compared with >128 mg/l for ermB+ isolates. ABT-773 and telithromycin MIC(90)s for mefE+ isolates were 0.125 and 0.5, compared with 0.032 and 0.016 mg/l for ermB+ isolates and 0.5 and 1 mg/l, respectively, for isolates containing both genes. Clindamycin was active against mefE+ but not ermB+ isolates. 155 isolates were resistant to penicillin. All fluoroquinolone MICs were < 1 mg/l. Further studies of ketolides for treatment of paediatric S. pneumoniae infections are warranted.

Case report: Rhodococcus equi pneumonia in a patient infected by the human immunodeficiency virus.

Rhodococcus equi, a facultative intracellular bacterium, is a common cause of pulmonary infection in farm animals, especially foals. Pulmonary and disseminated infection caused by this organism is occasionally seen in humans, especially in patients whose cell-mediated immunity has been altered by glucocorticoids or cytotoxic chemotherapy. Not surprisingly, the organism may cause serious disease in human immunodeficiency virus (HIV)-infected humans whose T cell-dependent immune system has been profoundly suppressed. This report describes an HIV infected patient with Rhodococcus equi pneumonia and reviews nine additional HIV-infected patients. Treatment in humans is not standardized. Studies in foals indicate that erythromycin and rifampin together are the treatment of choice. The patient in this report responded to this treatment briefly before relapsing and dying of the infection.

A clinically applicable technique to study cytoskeletal dynamics in normal and abnormal polymorphonuclear leukocytes isolated from small volume blood samples.

Shape change and motility of polymorphonuclear leukocytes (PMNs) are essential for host defense and require dynamic reorganizations of microfilamentous cytoskeleton by reversible polymerization of G-actin into filaments (F-actin). Although clinical disorders of actin polymerization are rare, recently described simple methodologies for assaying actin dynamics in PMNs make the technique readily applicable to clinical studies. To develop a clinically useful F-actin assay, the authors investigated the optimal preparation conditions for PMN isolation that resulted in the least in vitro cytoskeletal activation and evaluated the variability in actin dynamics in acutely and chronically infected patients. Basal and chemotactic factor-activated PMN F-actin content was measured by a previously described flow cytometric technique in fixed, permeabilized, NBDphallacidin-stained PMNs isolated by centrifugation in Percoll or Ficoll-Hypaque density gradients or by countercurrent elutriation. F-actin content is expressed as mean fluorescent channel or relative fluorescence intensity. Basal F-actin in PMNs prepared from countercurrent elutriation (mean fluorescent channel = 79.0 +/- 4.5, n = 6) or by Ficoll Hypaque (82.0 +/- 3.5, n = 4) was significantly higher than endotoxin free, Percoll purified PMNs, whether purified in bulk (56.1 +/- 7.9, n = 8) or by the small volume modification applicable to clinical studies (53.3 +/- 8.7, n = 15). Basal Ficoll Hypaque purified PMNs have evidence of shape change, whereas endotoxin free, Percoll purified PMNs are smooth and round and represent the most basal cell equivalent in F-actin content to a circulating PMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Does managed care mean more hassle for physicians?

Using the results of a 1995 nationally representative survey of physicians, this paper examines the relationship between exposure to managed care and resources expended by physicians on administrative and insurance matters. Our measures of managed care exposure are the degree to which a physician experiences a variety of managed care techniques (i.e., utilization review, capitation payment, restricted panels, gatekeepers, discounted fees, compensation links to utilization measures, profiling, protocols, and salary payment). Physicians report expending, on average, three hours per week on insurance-related matters and 4.8 hours per week on administration. Although managed care techniques affect administrative and insurance-related burdens, the direction of that effect varies according to the form that managed care exposure takes. With the exception of being salaried, none of our variables has an economically significant effect on physicians' administrative/insurance burdens, even at the outer-most edge of the 95% confidence interval. Overall, our findings contradict the widely held notion that managed care dramatically raises the administrative and insurance burden of physicians.