Pathogenesis of coronavirus SD in mice. I. Prominent demyelination in the absence of infectious virus production.

Following intracerebral inoculation of 3- to 4-week-old C57 B16/J mice with coronavirus SD, 23% exhibited neurologic signs within the first week. However, only 6% died. Within the first week after inoculation (AI), we noted a panencephalitis. Prominent demyelination detected in the spinal cord on day 6 continued through day 29 AI. Demyelinated lesions in the spinal cord were either subpial with few inflammatory cells except for macrophages or perivascular with prominent accumulation of lymphocytes, plasma cells, and macrophages. Beginning on day 6 AI, IgG was detected in the lesions. Although an infectious virus was detectable in the CNS only through day 12 AI, viral antigen expression continued through day 24. We concluded that coronavirus SD persists in a nonrecoverable form throughout the initial phase of demyelination, day 6 to day 24 AI.

Comparison of cefpodoxime proxetil and cefixime in the treatment of acute otitis media in infants and children. Otitis Study Group.

OBJECTIVE: To compare the use of once-a-day cefpodoxime proxetil to once-a-day cefixime in the treatment of acute suppurative otitis media. DESIGN: Randomized, multicenter, investigator-blinded. SETTING: Outpatient. PATIENTS: A total of 368 patients (age 2 months to 17 years) were randomized to receive either cefpodoxime or cefixime in a 2:1 ratio (245 cefpodoxime, 123 cefixime); 236 patients (155 cefpodoxime, 81 cefixime) were evaluable for drug efficacy. INTERVENTIONS: Patients received either cefpodoxime proxetil oral suspension (10 mg/kg/day, once daily for 10 days) or cefixime oral suspension (8 mg/kg/day, once daily for 10 days). MAIN OUTCOME MEASURES: Clinical evaluations were performed before treatment (study day 1), at an interim visit (study day 3 through 6), at the end of therapy (study day 12 through 15), and at final follow-up (study day 25 through 38). Microbiologic evaluations were performed at enrollment and whenever appropriate thereafter. RESULTS: End-of-therapy clinical cure rates in evaluable patients were 56% for the cefpodoxime group and 54% for the cefixime group. Clinical improvement rates were 27% for both groups. Clinical response rates were not significantly different between treatment groups (P = .541; 95% confidence interval = -8.1%, 15.2%). At long-term follow-up, 17% of patients in the cefpodoxime group and 20% in the cefixime group had a recurrence of infection. Drug-related adverse events (eg, diarrhea, diaper rash, vomiting, rash) occurred in 23.3% of cefpodoxime-treated patients and 17.9% of cefixime-treated patients (P = .282). CONCLUSIONS: These findings suggest that cefpodoxime proxetil administered once daily is as effective and safe as cefixime given once daily in the treatment of acute suppurative otitis media in pediatric patients.

Diagnostic usefulness of mycobacterial skin test antigens in childhood lymphadenitis.

Nontuberculous mycobacterial (NTM) infections are a frequent cause of chronic lymphadenitis in children. Previous studies of NTM antigen skin testing were inconclusive as a result of problems with study design and antigen formulation. The present study was undertaken with the Centers for Disease Control to determine whether newly formulated NTM skin test antigens applied in a double blind manner with a standard purified protein derivative could accurately distinguish NTM infections from those caused by Mycobacterium tuberculosis. Among the 11 children enrolled at our institution the NTM antigens correctly identified the 5 children with culture-proved NTM infections, as well as 2 other children with clinical or histopathologic data consistent with NTM lymphadenitis (P = 0.003, Fisher test). None of the 11 children cross-reacted with the Centers for Disease Control-supplied purified protein derivative. The NTM antigens appear to be useful in the diagnostic evaluation of lymphadenitis and perhaps in the evaluation of children with positive purified protein derivative skin tests.

PBP profiles of Haemophilus influenzae, H. aegyptius, and the H. influenzae biogroup aegyptius associated with Brazilian Purpuric Fever.

We questioned if PBP analysis could differentiate strains of Haemophilus influenzae, H. aegyptius, and H. influenzae biogroup aegyptius associated with Brazilian Purpuric Fever. A relatively homogeneous PBP pattern was observed for all strains. The amount of penicillin bound to PBP 5 appeared to separate H. influenzae and H. aegyptius isolates, whereas PBP 5 of those strains associated with Brazilian Purpuric Fever bound an intermediate amount. We conclude that based on PBP profiles, the strains tested appear to be difficult to separate taxonomically and may represent a common species.

Antimicrobial susceptibility of clinical isolates of Haemophilus influenzae to ampicillin-sulbactam.

A total of 1092 clinical isolates of Haemophilus influenzae (306 type b; 786 non-type-b), from five medical centers were obtained during 1987 and 1988. Disk diffusion antimicrobial susceptibilities were obtained for all isolates, and broth microdilution susceptibilities were obtained for 502 isolates. Beta-lactamase was produced by 34.3% of type-b and 22.1% of non-type-b isolates, with some geographic variations. Using disk diffusion antimicrobial susceptibility testing, all isolates were susceptible to ampicillin-sulbactam, ceftriaxone, cefuroxime, and rifampin; two isolates were resistant to chloramphenicol. Whether tested using a fixed ratio of ampicillin to sulbactam of 2:1 or a fixed concentration of sulbactam, the ampicillin-sulbactam combination demonstrated good activity against clinical isolates of H. influenzae. Only 8 of the 1092 isolates did not produce beta-lactamase but demonstrated MICs of greater than or equal to 2 micrograms/ml for ampicillin.

The in vitro antibacterial activity of ceftriaxone against Streptococcus pyogenes is unrelated to penicillin-binding protein 4.

The in vitro activities of penicillin and ceftriaxone were compared against 29 strains of Streptococcus pyogenes with the result that ceftriaxone showed greater activity than penicillin. The morphological changes induced by 1/2 and 1x MIC concentrations of penicillin and ceftriaxone, respectively, were very similar using scanning electron microscopy. Competitive binding studies using 'cold' penicillin or ceftriaxone as inhibitors of radiolabeled penicillin binding demonstrated that ceftriaxone had a very low affinity for penicillin binding protein (PBP) 4 compared to that of penicillin. Since ceftriaxone had greater antibacterial activity, this suggests that PBP 4 may not be important to the in vitro activity of ceftriaxone. In contrast, the IC50 for ceftriaxone was much lower (> 200 fold) for PBPs 2 and 3 compared to PBP 4, suggesting greater avidity of these high molecular mass PBPs for ceftriaxone. These data may at least in part explain the superior in vitro activity of ceftriaxone compared to penicillin against S. pyogenes. These data, together with the observation that PBP 1 was saturated at a lower concentration of penicillin than any of the other PBPs, suggest that the inhibition of PBPs 1, 2, and 3 mediates the bactericidal activity of beta-lactam antibiotics against group A streptococci.

Virulence of non-beta-lactamase-mediated ampicillin-resistant Haemophilus influenzae.

We questioned whether strains of ampicillin-resistant, non-beta-lactamase-producing (AmpR NBLP) Haemophilus influenzae with lower affinity penicillin-binding proteins (PBPs) might have altered virulence. The virulence of resistant transformant strains and the susceptible recipient was compared using infant rats. Following intraperitoneal inoculation, there was a significantly lower mortality rate and incidence and magnitude of bacteremia with two of three transformants compared to the recipient strain. Reduced virulence was not associated with greater bactericidal activity of serum or human neutrophils or faster clearance of the transformant following intravenous injection. Heated rat or human plasma supported exponential growth of the recipient, but not the transformant, suggesting deficient in vivo multiplication. We conclude that H. influenzae with altered PBPs are less virulent in an infant rat model which may be related to differences in in vivo growth.

The penicillin binding proteins of the genus Haemophilus.

We questioned whether the penicillin binding protein (PBP) profiles of representative strains from the 19 species varied within the genus Haemophilus and whether these profiles would be of taxonomic value. Seventeen of the 19 representative strains studied had distinct PBP profiles; only those of H. avium and H. paragallinarum were identical. The data support the inclusion of H. aegyptius in the genus as a species related to but separate from H. influenzae and could not exclude H. somnus, H. agni, and H. equigenitalis from the genus. Comparative PBP analysis within the genus Haemophilus may therefore be useful taxonomically.

Autoantibody production in Kawasaki syndrome.

Although a variety of autoantibodies are produced in patients with Kawasaki syndrome (KS), their specificities in many instances are controversial and their role in disease pathogenesis is undetermined. Autoantibody production was studied in 14 patients with Kawasaki syndrome (KS). Antibodies to myeloperoxidase (MPO), the dominant antigen responsible for perinuclear antineutrophil cytoplasmic antibody (pANCA) reactivity, were detected by ELISA in 73% of acute phase and 89% of convalescent phase KS specimens, in contrast to 4% of normal adult control subjects (p < 0.002 and p < 0.001, respectively). MPO and cytoplasmic antineutrophil antibody (cANCA) levels measured by ELISA were significantly elevated above levels for adult normal control subjects (p < 0.005 and p < 0.01, respectively), but not above recently ill childhood controls. Among patients who developed a positive ANCA, antibody titers tended to rise in serial specimens despite clinical improvement. Antibodies to myocardial muscle, cardiac perimysial connective tissue, nuclear antigens (ANA), and smooth muscle were also detected in some KS patients, but titers did not differ significantly from control patients. Autoantibody results were not predictive of patients with echocardiographic abnormalities.

Safety and immunogenicity of Haemophilus influenzae type b conjugate vaccine (PedvaxHIB) in Papua New Guinean children.

BACKGROUND: In view of high mortality and morbidity from Haemophilus influenzae type b (Hib) in young Papua New Guinean children, the incorporation of a Hib conjugate vaccine into a nationwide immunization program would be of major public health benefit. The choice of the Hib conjugate vaccine will be based on the evaluation of several Hib conjugate vaccines, after consideration of such factors as the ease of incorporation into the current vaccination schedule, cost, kinetics of antibody responses and safety. METHODS: This study evaluated the safety and immunogenicity of Hib polysaccharide-Neisseria meningitidis outer membrane protein complex conjugate vaccine (PRP-OMPC) in Papua New Guinea. 95 children were recruited at Goroka Base Hospital, Eastern Highlands Province, and enrolled in the study. PRP-OMPC was administered at ages 2, 4 and 12 to 15 months. Blood was collected before each dose, one month after the second and booster doses, and at ages 18 and 24 months. Antibody to PRP (anti-PRP) was measured by radioimmunoassay. RESULTS: PRP-OMPC was generally well tolerated. At successive sampling times from the prevaccination bleed through the 1-month post-booster bleed, geometric mean titres were 0.18, 1.45, 2.54, 1.03 and 8.05 micrograms/ml, respectively (n = 60). The proportions of subjects with anti-PRP titres > or = 1.0 microgram/ml were 2%, 62%, 73%, 47% and 93%, respectively (n = 60). Persistence of anti-PRP was ascertained in 41 subjects. The GMTs at 18 and 24 months were 3.42 and 2.0 micrograms/ml, respectively. CONCLUSIONS: PRP-OMPC was found to be immunogenic after the first dose and to elicit a robust booster response. Antibody titres persisted until age 24 months, at which time 100% of subjects had anti-PRP > or = 0.15 microgram/ml. These results are consistent with previous studies in US Native American infants and in Gambian infants.

Mutation frequency of Haemophilus influenzae to rifampin resistance.

Twenty-two susceptible strains of Haemophilus influenzae were examined for mutation to rifampin resistance (minimal inhibitory concentration, greater than or equal to 10 micrograms/ml). All strains had detectable apparent mutation frequencies with a median minimal inhibitory concentration 2,000-fold greater than that of their wild-type parents. Of the type b strain mutants, 80% (8 of 10) expressed high-level resistance (minimal inhibitory concentration, 750 micrograms/ml) that was 75-fold greater than readily achievable serum concentrations.

Statistical comparison of ligand-binding kinetics.

We describe the application of generalized linear model methodology to the problem of testing differences among ligand-receptor interactions, and show that the method is analogous to weighted least squares regression methodology and F tests familiar to many investigators. The method accommodates incomplete block designs so that one can obtain kinetic parameter estimates directly comparable among samples analysed on incompletely overlapping sets of experimental runs. We demonstrate the method with data that compare saturation kinetics for a single penicillin-binding protein in isogenic ampicillin susceptible and resistant bacteria.

Penicillin-binding proteins and ampicillin resistance in Haemophilus influenzae.

Ampicillin-resistant, non-beta-lactamase-producing isolates of Haemophilus influenzae contain a variety of penicillin-binding protein (PBP) patterns that differ from the single pattern of eight PBPs characteristic of susceptible strains. During genetic transformation of resistance, only some of the anomalies in PBP pattern were transformed, specifically those relating to the penicillin-binding capacities of PBPs 4 (Mr of 62,000) and 5 (Mr of 59,000) and, in some transformations, PBP 3 (Mr of 71,000). Comparison of the binding of penicillin by PBPs 4 and 5 of three resistant transformants (derived with DNA from different donors) revealed a decrease in the rate of PBP acylation and no appreciable change in the rate of deacylation as compared to the susceptible recipient. Thus, rapid turnover of these PBPs does not play a role. Retransformation studies confirm that altered PBPs 3, 4, and 5 are associated with resistance and suggest that these PBPs are major targets for the beta-lactam antibiotics in H. influenzae.

Safety, efficacy and effectiveness of the influenza virus vaccine, trivalent, types A and B, live, cold-adapted (CAIV-T) in healthy children and healthy adults.

Influenza is a major cause of illness. We have assessed the safety, efficacy, and effectiveness of CAIV-T vaccine. A two year, multicenter, double-blind, placebo-controlled, efficacy field trial in pre-school aged children was conducted; 1602 enrolled in Year One and 1358 (85%) returned in Year Two. In both study years combined, the overall vaccine efficacy against culture-confirmed influenza was 92% (95% CI: 88, 94). The vaccine efficacy was 95% (95% CI: 62, 99) against lower respiratory illness, 94% (95% CI: 90, 96) against febrile illness and 96% (95% CI: 88, 99) against otitis media associated with culture-confirmed influenza. A multicenter, double-blind, placebo-controlled, effectiveness field trial was conducted in 4561 working adults aged 18 to 64 years. Episodes and days of febrile illness (FI), severe febrile illness (SFI), febrile upper respiratory illness (FURI), work loss, and health care use were assessed. Vaccination significantly reduced the numbers of SFI, 18.8% reduction (95% CI: 7, 29), and FURI, 26.3% reduction (95% CI: 13, 33); and led to fewer days of illness (22.9% reduction for FI, 27.3% reduction for SFI), fewer days of work lost (17.9% reduction for SFI, 28.4% for FURI), and fewer days of health care provider visits (24.8% for SFI, 40.9% for FURI). Prescription antibiotics and over-the-counter medications were also reduced. The vaccine was generally safe and well tolerated with no vaccine related serious adverse events. LAIV represents an additional important option for the control of influenza.

A permeability barrier as a mechanism of chloramphenicol resistance in Haemophilus influenzae.

Chloramphenicol resistance in Haemophilus influenzae occurs most frequently via plasmid-mediated chloramphenicol acetyltransferase production. We studied four strains with high-level chloramphenicol resistance (MIC greater than 20 micrograms/ml) which did not have detectable chloramphenicol acetyltransferase activity. The chloramphenicol resistance determinant was transformed into a chloramphenicol-susceptible laboratory H. influenzae strain from each of the four wild-type strains, enabling isogenic comparisons. By thin-layer chromatography and a bioassay, there was no evidence of non-chloramphenicol acetyltransferase modification of chloramphenicol. In vitro protein synthesis in the presence of chloramphenicol was equivalently inhibited in the chloramphenicol-resistant transformants and in the susceptible recipient. Chloramphenicol uptake by these strains during logarithmic growth was compared by high-pressure liquid chromatographic quantitation; at chloramphenicol concentrations of 5, 10, and 20 micrograms/ml the four transformants showed a decreased rate of uptake of chloramphenicol compared with the isogenic chloramphenicol-susceptible recipient. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of outer membrane proteins revealed a markedly diminished 40-kilodalton protein in the resistant transformants. We propose that the mechanism of chloramphenicol resistance in these strains is a relative permeability barrier due to the loss of an outer membrane protein.

Cloning and expression in Escherichia coli of a gene encoding nonenzymatic chloramphenicol resistance from Pseudomonas aeruginosa.

High-level chloramphenicol resistance in Pseudomonas aeruginosa may be due to enzymatic inactivation, ribosomal mutation, or a permeability barrier. We investigated the nonenzymatic resistance mechanism encoded by Tn1696, a transposon found in P. aeruginosa. A 1-megadalton DNA fragment from Tn1696 was cloned which mediated expression of chloramphenicol resistance in Escherichia coli. Comparison of the effects of chloramphenicol on in vitro translation revealed no difference between the susceptible recipient strain and the resistant transformant containing the cloned gene. The rate of chloramphenicol uptake was slower in the resistant strain, suggesting a permeability barrier to the antibiotic. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membranes demonstrated the absence of a 50,000-dalton protein in the resistant strain. DNA homology was evident between Tn1696 and chloramphenicol-resistant isolates of Haemophilus influenzae possessing altered outer membrane permeability. We conclude that chloramphenicol resistance encoded by Tn1696 is due to a permeability barrier and hypothesize that the gene from P. aeruginosa may share a common ancestral origin with these genes from other gram-negative organisms.

Electrodiagnosis reliability in the diagnosis of infant botulism.

Infant botulism is confirmed by isolation of Clostridium botulinum from stool culture or by toxin assay. Although electrodiagnosis has been described as a diagnostic tool in infant botulism, our 11-year review of toxin-confirmed cases suggests that electrodiagnosis is not a reliable tool. In the case report presented, results of electrodiagnosis were negative but enema effluent contained adequate concentrations of organism and toxin to confirm the diagnosis.