Applicability of geometrical optics to in-plane liquid-crystal configurations.

We study the applicability of geometrical optics to inhomogeneous dielectric nongyrotropic optically anisotropic media typically found in in-plane liquid-crystal configurations with refractive indices n(o)=1.5 and n(e)=1.7. To this end, we compare the results of advanced ray- and wave-optics simulations of the propagation of an incident plane wave to a special anisotropic configuration. Based on the results, we conclude that for a good agreement between ray and wave optics, a maximum change in optical properties should occur over a distance of at least 20 wavelengths.

Simulations of a liquid-crystal-based electro-optical switch.

We present simulations of a novel liquid-crystal-based electro-optical device that enables a switching effect owing to a backreflection phenomenon. In the simulations, we exploit the optical properties of a liquid-crystal layer with a Freédericksz alignment in an unconventional way. The resulting switching effect of the proposed optical design can be controlled by means of an external electric field. Possible applications of the liquid-crystal device can be found in, but are not restricted to, optical communication systems and lighting applications.

Dynamics of pneumococcal colonization in healthy Dutch children.

A recent study of pneumococcal colonization in 3198 healthy children of 1-19 years of age in The Netherlands showed pneumococcal colonization in 19 % of the children, with a peak incidence of 55 % at the age of 2 years; an age-related serotype distribution was also found. In the present study, the genetic background and resistance profiles of 578 pneumococcal isolates from the latter study were characterized by means of chromosomal genotyping and susceptibility testing. In contrast to the age-related serotype distribution observed previously, the genetic background of the strains was not age related. Few strains were found showing close homology (>95 %) with the international clones Spain(9V)-3 (ten isolates showed homology), England(14)-9 (four isolates), Tennessee(23F)-4 (two isolates), CSR(14)-10 (one isolate) and Sweden(15A)-25 (one isolate). In total, 19 % of strains showed resistance to one or more antibiotics. Resistance to cotrimoxazole, tetracycline, erythromycin and penicillin was found in 12.9, 5.6, 5.0 and 2.7 % of isolates, respectively. Multidrug resistance was found in 1.9 % of strains. In conclusion, pneumococcal colonization isolates from healthy Dutch children represent a heterogeneous, mostly antibiotic susceptible, genetic population.

Epidemiology of nasopharyngeal carriage of Neisseria meningitidis in healthy Dutch children.

We investigated the prevalence and determinants of nasopharyngeal carriage of Neisseria meningitidis in 3200 healthy children aged 1-19 years. The incidence of meningococcal carriage was, on average, 1.5%. Peak incidences were seen at age 1 year and after age 15 years. The independent determinants of meningococcal carriage included age, regular visits to youth clubs (odds ratio [OR], 2.2) and discotheques (OR, 4.3), and pneumococcal carriage (OR, 4.1).

Molecular epidemiology of pneumococcal colonization in response to pneumococcal conjugate vaccination in children with recurrent acute otitis media.

A randomized double-blind trial with a 7-valent pneumococcal conjugate vaccine was conducted in The Netherlands among 383 children, aged 1 to 7 years, with a history of recurrent acute otitis media. No effect of vaccination on the pneumococcal colonization rate was found. However, a shift in serotype distribution was clearly observed (R. Veenhoven et al., Lancet 361:2189-2195, 2003). We investigated the molecular epidemiology of 921 pneumococcal isolates retrieved from both the pneumococcal vaccine (PV) and control vaccine (CV) groups during the vaccination study. Within individuals a high turnover rate of pneumococcal restriction fragment end labeling genotypes, which was unaffected by vaccination, was observed. Comparison of the genetic structures before and after completion of the vaccination scheme revealed that, despite a shift in serotypes, there was clustering of 70% of the pneumococcal populations. The remaining isolates (30%) were equally observed in the PV and CV groups. In addition, the degree of genetic clustering was unaffected by vaccination. However, within the population genetic structure, nonvaccine serotype clusters with the serotypes 11, 15, and 23B became predominant over vaccine-type clusters after vaccination. Finally, overall pneumococcal resistance was low (14%), and, albeit not significant, a reduction in pneumococcal resistance as a result of pneumococcal vaccination was observed. Molecular surveillance of colonization in Dutch children shows no effect of pneumococcal conjugate vaccination on the degree of genetic clustering and the genetic structure of the pneumococcal population. However, within the genetic pneumococcal population structure, a clear shift toward nonvaccine serotype clusters was observed.

Multiplex opsonophagocytosis assay (MOPA): a useful tool for the monitoring of the 7-valent pneumococcal conjugate vaccine.

Pneumococcal conjugate vaccination is highly efficacious against invasive diseases in young children. Since host protection is mainly mediated by opsonin-dependent phagocytosis, the in vitro measurement of opsonophagocytic activity of the anti-capsular antibodies is assumed to be a reliable correlate of protection to monitor vaccine efficacy. Unfortunately, the methods used so far are all tedious to perform and material-consuming. Therefore, we modified the multi-specificity opsonophagocytosis killing assay (MSOPKA) into a high-throughput method, which simultaneously measures the opsonophagocytosis against the seven serotypes covered by the current conjugate vaccine in a single assay. In the so-called multiplex opsonophagocytosis assay (MOPA), a mixture containing equal numbers of colony forming units (CFUs) of chloramphenicol-resistant serotype 4, spectinomycin-resistant serotype 6B, streptomycin-resistant serotype 9V, erythromycin-resistant serotype 14, rifampicin-resistant serotype 18C, tetracycline-resistant serotype 19F, and trimethoprim-resistant serotype 23F pneumococci was used as a target mixture and incubated with serial dilutions of test serum. After opsonophagocytosis by differentiated HL-60 cells in the presence of complement, the samples were spotted onto different blood agar plates containing the seven selective antibiotics, respectively. Opsonophagocytosis was calculated as the highest serum dilution resulting in 90% or more reduction in CFUs. The data obtained by this assay correlated well with the data obtained by the MSOPKA. In conclusion, the MOPA simultaneously measures opsonophagocytosis capacity of serum against the capsular serotypes included in the 7-valent pneumococcal conjugate vaccine in a high-throughput fashion, requiring low volumes of patient sera.

Colony blot assay: a useful method to detect multiple pneumococcal serotypes within clinical specimens.

The efficacy of pneumococal conjugate vaccines in young children may be complicated by serotype replacement. We developed a colony blot assay which enables the identification of re-colonization with novel serotypes (replacement), overgrowth by minor co-colonizing serotypes or suppression of previously predominant vaccine serotype strains as a result of vaccination. This method allows the identification of multiple serotypes in a single specimen in a ratio of 1:1000. In order to demonstrate the potential of our method, we investigated the consecutive nasopharyngeal samples of 26 children who had shown a shift in pneumococcal colonization after conjugate vaccination. Mixed colonization was found once in 15 pre-vaccination samples and four times in 26 post-vaccination samples. In the remaining children 'true replacement' had presumably occurred. Hence, we conclude that the colony blot assay is an easy to apply method, which allows the identification of different pneumococcal serotypes within single clinical specimens.

Colonisation by Streptococcus pneumoniae and Staphylococcus aureus in healthy children.

A trial with a 7-valent pneumococcal-conjugate vaccine in children with recurrent acute otitis media showed a shift in pneumococcal colonisation towards non-vaccine serotypes and an increase in Staphylococcus aureus-related acute otitis media after vaccination. We investigated prevalence and determinants of nasopharyngeal carriage of Streptococcus pneumoniae and S aureus in 3198 healthy children aged 1-19 years. Nasopharyngeal carriage of S pneumoniae was detected in 598 (19%) children, and was affected by age (peak incidence at 3 years) and day-care attendance (odds ratio [OR] 2.14, 95% CI 1.44-3.18). S aureus carriage was affected by age (peak incidence at 10 years) and male sex (OR 1.46, 1.25-1.70). Serotyping showed 42% vaccine type pneumococci. We noted a negative correlation for co-colonisation of S aureus and vaccine-type pneumococci (OR 0.68, 0.48-0.94), but not for S aureus and non-vaccine serotypes. These findings suggest a natural competition between colonisation with vaccine-type pneumococci and S aureus, which might explain the increase in S aureus-related otitis media after vaccination.

Molecular epidemiology of penicillin-susceptible non-beta-lactam-resistant Streptococcus pneumoniae isolates from Greek children.

A total of 128 Streptococcus pneumoniae isolates that were susceptible to penicillin but resistant to non-beta-lactam agents were isolated from young carriers in Greece and analyzed by antibiotic susceptibility testing, serotyping, restriction fragment end labeling (RFEL), and antibiotic resistance genotyping. The serotypes 6A/B (49%), 14 (14%), 19A/F (11%), 11A (9%), 23A/F (4%), 15B/C (2%), and 21 (2%) were most prevalent in this collection. Of the isolates, 65% were erythromycin resistant, while the remaining isolates were tetracycline and/or trimethoprim-sulfamethoxazole resistant. Fifty-nine distinct RFEL types were identified. Twenty different RFEL clusters, harboring 2 to 19 strains each, accounted for 76% of all strains. Confirmatory multilocus sequence typing analysis of the genetic clusters showed the presence of three international clones (Tennessee(23F)-4, England(14)-9, and Greece(6B)-22) representing 30% of the isolates. The erm(B) gene was present in 70% of the erythromycin-resistant isolates, whereas 18 and 8% contained the mef(A) and mef(E) genes, respectively. The pneumococci representing erm(B), erm(A), and mef genes belonged to distinct genetic clusters. In total, 45% of all isolates were tetracycline resistant. Ninety-six percent of these isolates contained the tet(M) gene. In conclusion, penicillin-susceptible pneumococci resistant to non-beta-lactams are a genetically heterogeneous group displaying a variety of genotypes, resistance markers, and serotypes. This suggests that multiple genetic events lead to non-beta-lactam-resistant pneumococci in Greece. Importantly, most of these genotypes are capable of disseminating within the community.

Molecular epidemiology of pneumococcal carriage among children with upper respiratory tract infections in Hanoi, Vietnam.

To investigate the molecular epidemiology of pneumococcal nasopharyngeal carriage in Hanoi, Vietnam, we studied 84 pneumococcal strains retrieved from children with upper respiratory tract infections. Serotypes 23F (32%), 19F (21%), 6B (13%), and 14 (10%) were found most often. A significant number of strains were antibiotic resistant. Fifty-two percent of the strains were (intermediate) resistant to penicillin, 87% were (intermediate) resistant to co-trimoxazole, 76% were resistant to tetracycline, 73% were resistant to erythromycin, and 39% were (intermediate) resistant to cefotaxime. Seventy-five percent were resistant to three or more classes of antibiotics. A high degree of genetic heterogeneity among the penicillin resistance genes was observed. In addition, the tetracycline resistance gene tet(M) and the erythromycin resistance gene erm(B) were predominantly observed among the isolates. Molecular analysis of the 84 isolates by restriction fragment end labeling (RFEL) revealed 35 distinct genotypes. Twelve of these genotypes represented a total of eight genetic clusters with 61 isolates (73%). The two largest clusters contained 24 and 12 isolates, and the isolates in those clusters were identical to the two internationally spreading multidrug-resistant clones Spain 23F-1 and Taiwan 19F-14, respectively. The remaining RFEL types were Vietnam specific, as they did not match the types in our reference collection of 193 distinct RFEL types from 16 countries. Furthermore, 57 of the 61 horizontally spreading isolates (93%) in the eight genetic clusters were covered by the seven-valent conjugate vaccine, whereas this vaccine covered only 43% of the isolates with unique genotypes. According to the serotype distribution of the nasopharyngeal pneumococcal isolates, this study suggests a high potential benefit of the seven-valent pneumococcal conjugate vaccine for children in Hanoi.

Technical aspects of radiofrequency.

Radiofrequency (RF) is an alternating electric field with an oscillating frequency of 500,000 Hz. If the resulting current flows through a percutaneously introduced electrode, heat will be produced around the electrode because the body tissue acts as a resistor. RF can, therefore, be used to ablate nervous tissue in the treatment of chronic pain. This method has gained acceptance for percutaneous cordotomy and for the treatment of trigeminal neuralgia. For spinal pain, the method had little success initially, but since the introduction of small diameter instrumentation, the results have markedly improved. he mechanism of action of RF has not been challenged until recently even though there was awareness that some observations were not consistent with the heat concept. The formation of heat is not the only occurrence during RF treatment, however. The tissue surrounding the electrode is also exposed to the RF electric field. This exposure has a biological effect as has been demonstrated both in cells in a cell culture and in the exposure to RF of dorsal root ganglia, resulting in transsynaptal induction of early gene expression in the dorsal horn. The mode of action of RF is, therefore, uncertain at the moment. The method of pulsed RF is based on the concept that the production of heat has been a by-product of RF treatment and that the clinical effect is due to exposure to the electric field. In pulsed RF, the generator output is interrupted to allow for the elimination of heat in the silent period. The early results have been encouraging, but the results of controlled, prospective studies are not yet available. Since there are now 2 almost diametrically opposed views on the mode of action of RF, it is difficult to give recommendations for treatment. The decision is easy for indications for which heat RF has traditionally been contraindicated such as the treatment of peripheral nerves and trigger points. When the application of heat carries a potential risk, for instance if the dorsal root ganglion is the target structure, the use of pulsed RF is also recommended. As for the medial branch the situation is controversial. Since there are controlled studies available showing the effect of heat lesions, it is recommended that the technique should not be changed until further studies have been completed. Finally, the equipment for RF treatment is described and safety issues are discussed.

Emergence of rifampin-resistant Streptococcus pneumoniae as a result of antimicrobial therapy for penicillin-resistant strains.

A multidrug-resistant strain of Streptococcus pneumoniae was isolated in The Netherlands during a nosocomial outbreak among 36 patients who mainly had chronic obstructive pulmonary disease. After the commencement of barrier nursing and short-term ceftriaxone-rifampin eradication therapy, the epidemic ceased. However, eradication therapy failed in 3 patients, and follow-up investigation of these patients showed the emergence of rifampin-resistant isolates.

Plasminogen-activator-inhibitor-1 4G/5G promoter polymorphism and prognosis of severely injured patients.

A single base pair insertion/deletion (4G/SG) promoter polymorphism in the plasminogen-activator-inhibitor-1 (PAI-1) gene is thought to play a part in prognosis after severe trauma. We investigated the relation between outcome of severe trauma, PAI-1 concentrations, and PAP-1 genotype in 61 patients who had been severely injured. 11 (58%) of 19 patients with genotype 4G/4G did not survive, whereas only eight (28%) of 29 patients with heterozygous genotype 4G/SG, and two (15%) of 13 patients with genotype 5G/5G died. The PAI-1 4G allele is associated with high concentrations of PAI-1 in plasma and a poor survival rate after severe trauma.

Molecular characteristics of penicillin-binding protein genes of penicillin-nonsusceptible Streptococcus pneumoniae isolated in the Netherlands.

Recently, a nation-wide molecular epidemiologic survey of penicillin-nonsusceptible Streptococcus pneumoniae has been performed in the Netherlands. In the current study, we analyzed the genes pbp1a, pbp2b, and pbp2x from these clinical isolates at the molecular level, and identified the genetic composition of the penicillin-binding domains. The pneumococcal strains were selected on the basis of differences in restriction fragment length polymorphism (RFLP) patterns of the genes pbp1a, pbp2b, and pbp2x, and represented 8, 7, and 10 distinct patterns, respectively. The genetic heterogeneity observed by sequence analysis of the pbp gene parts was comparable with the heterogeneity of the entire pbp genes as deduced from RFLP analysis. Furthermore, the mutations in the pbp sequences of the Dutch isolates invariably matched with the mutations described in pbp sequences of penicillin-nonsusceptible pneumococci isolated in other countries. Finally, novel mosaic structures were identified indicating horizontal exchange of pbp gene parts among penicillin-nonsusceptible pneumococci.

Restriction Fragment End Labeling Analysis: High-Resolution Genomic Typing of Streptococcus pneumoniae Isolates.

Streptococcus pneumoniae (pneumococcus) is one of the leading bacterial pathogens causing illness and death among young children, the elderly, and persons with certain underlying medical conditions (1). Pneumococci are often part of the normal nasopharyngeal flora, especially in young children. Pneumococcal colonization is an important risk factor: children colonized with S. pneumoniae more often develop acute otitis media than children who are not colonized (2-5).

Genetic relatedness within serotypes of penicillin-susceptible Streptococcus pneumoniae isolates.

The molecular epidemiological characteristics of all Streptococcus pneumoniae strains isolated in a nationwide manner from patients with meningitis in The Netherlands in 1994 were investigated. Restriction fragment end labeling analysis demonstrated 52% genetic clustering among these penicillin-susceptible strains, a value substantially lower than the percentage of clustering among Dutch penicillin-nonsusceptible strains. Different serotypes were found within 8 of the 28 genetic clusters, suggesting that horizontal transfer of capsular genes is common among penicillin-susceptible strains. The degree of genetic clustering was much higher among serotype 3, 7F, 9V, and 14 isolates than among isolates of other serotypes, i.e., 6A, 6B, 18C, 19F, and 23F. We further studied the molecular epidemiological characteristics of pneumococci of serotype 3, which is considered the most virulent serotype and which is commonly associated with invasive disease in adults. Fifty epidemiologically unrelated penicillin-susceptible serotype 3 invasive isolates originating from the United States (n = 27), Thailand (n = 9), The Netherlands (n = 8), and Denmark (n = 6) were analyzed. The vast majority of the serotype 3 isolates (74%) belonged to two genetically distinct clades that were observed in the United States, Denmark, and The Netherlands. These data indicate that two serotype 3 clones have been independently disseminated in an international manner. Seven serotype 3 isolates were less than 85% genetically related to the other serotype 3 isolates. Our observations suggest that the latter isolates originated from horizontal transfer of the capsular type 3 gene locus to other pneumococcal genotypes. In conclusion, epidemiologically unrelated serotype 3 isolates were genetically more related than those of other serotypes. This observation suggests that serotype 3 has evolved only recently or has remained unchanged over long periods.

Immune-protective antibodies against capsular polysaccharides do not affect natural competence of Streptococcus pneumoniae: implications for current conjugate vaccination strategies?

We studied the effect of opsonization of Streptococcus pneumoniae with capsular antibodies on horizontal transfer of DNA. Opsonization did not inhibit DNA uptake. This suggests that horizontal transfer of capsular genes, which is an important escape mechanism of the pathogen, remains a potential threat for the efficacy of conjugate vaccination.

Evaluation of the Vitek 2 system for susceptibility testing of Streptococcus pneumoniae isolates.

Vitek 2 (bioMérieux, France) is a new commercial system that allows rapid identification and rapid determination of the minimum inhibitory concentration (MIC) of Streptococcus pneumoniae by monitoring the growth kinetics of the organisms in microwells. The accuracy of the Vitek 2 system in susceptibility testing was evaluated by determining the MICs of 50 penicillin-susceptible and 150 intermediate or penicillin-resistant Streptococcus pneumoniae isolates and comparing the results with those obtained using the agar dilution method. The essential agreement between the Vitek 2 system and the reference method was 91% for penicillin, 93% for cefotaxime and ceftriaxone, and more than 94% for amoxicillin, erythromycin, ofloxacin, co-trimoxazole, tetracycline, and imipenem. One very major error (1.1%) and one major error (0.9%) were obtained for tetracycline. The minor error rate for penicillin of 19.3% was mainly due to intermediate category isolates (n = 29) being identified as resistant and susceptible isolates (n = 6) being identified as intermediate by the commercial system. The minor error rates for amoxicillin, cefotaxime, ceftriaxone, imipenem, and ofloxacin were 25.4%, 25.4%, 29.4%, 19.2%, and 31.5%, respectively. Vancomycin, tetracycline, co-trimoxazole, and erythromycin showed minor error rates of 0-6.1%. In conclusion, Vitek 2 shows good agreement with the reference method, as demonstrated by the low numbers of major errors, but it has a tendency to overestimate MICs, resulting in minor errors.

The role of radiofrequency in failed back surgery patients.

There are a multitude of potential causes for both residual leg pain and residual back pain following surgery. In a minority of patients there may be pathology from an adjoining segment or recurrence of the preoperatively existent anatomic abnormality. In many others surgery fails because the damage was already irreparable by the time the anatomic abnormality was corrected. In residual leg pain this is due to radiculopathy, with or without centralization into the dorsal horn. In residual back pain inflammation or scar formation in the anterior epidural space plays a major role. The role of radiofrequency (RF) has been limited for two reasons. First, because RF is destructive in nature, it is contraindicated in the treatment of neuropathic pain. Second, the anterior epidural space is innervated by the sinuvertebral nerve, which runs too close to the main segmental nerve to apply RF safely. Recently the concept of heat as being the primary active factor in RF lesions has come under discussion and has led to the development of pulsed RF (PRF), which is a nondestructive method of exposing tissue to RF electric fields. Because PRF is nondestructive, it is potentially suitable for the treatment of neuropathic pain and it can also be applied at the origin of the sinuvertebral nerve. The initial clinical results have been promising, but controlled studies are still lacking.

The putative proteinase maturation protein A of Streptococcus pneumoniae is a conserved surface protein with potential to elicit protective immune responses.

Surface-exposed proteins often play an important role in the interaction between pathogenic bacteria and their host. We isolated a pool of hydrophobic, surface-associated proteins of Streptococcus pneumoniae. The opsonophagocytic activity of hyperimmune serum raised against this protein fraction was high and species specific. Moreover, the opsonophagocytic activity was independent of the capsular type and chromosomal genotype of the pneumococcus. Since the opsonophagocytic activity is presumed to correlate with in vivo protection, these data indicate that the protein fraction has the potential to elicit species-specific immune protection with cross-protection against various pneumococcal strains. Individual proteins in the extract were purified by two-dimensional gel electrophoresis. Antibodies raised against three distinct proteins contributed to the opsonophagocytic activity of the serum. The proteins were identified by mass spectrometry and N-terminal amino acid sequencing. Two proteins were the previously characterized pneumococcal surface protein A and oligopeptide-binding lipoprotein AmiA. The third protein was the recently identified putative proteinase maturation protein A (PpmA), which showed homology to members of the family of peptidyl-prolyl cis/trans isomerases. Immunoelectron microscopy demonstrated that PpmA was associated with the pneumococcal surface. In addition, PpmA was shown to elicit species-specific opsonophagocytic antibodies that were cross-reactive with various pneumococcal strains. This antibody cross-reactivity was in line with the limited sequence variation of ppmA. The importance of PpmA in pneumococcal pathogenesis was demonstrated in a mouse pneumonia model. Pneumococcal ppmA-deficient mutants showed reduced virulence. The properties of PpmA reported here indicate its potential for inclusion in multicomponent protein vaccines.