Effect of antimicrobial use on pneumococcal diagnostic tests in elderly patients with community-acquired pneumonia.

Antimicrobial treatment decreases bacterial culture yields. We assessed the impact of antimicrobial treatment on pneumococcal assays in a prospective study of community-acquired pneumonia (CAP) in the elderly. We enrolled 323 cases aged ≥65 years with radiologically confirmed CAP and collected detailed data on antimicrobial exposure and pneumococcal assays on various samples. Complete antimicrobial use data were available for 303 (94%) cases; 61% had no antimicrobial exposure, 19% had received antibiotics at the acute visit only, and 20% within 2 weeks before the acute visit (15% ongoing and 5 % completed treatment). Ongoing use before the visit reduced pneumococcal detection by culture (nasopharyngeal swab 2 vs. 16% in the unexposed; high-quality sputum 0 vs. 25%) and sputum lytA polymerase chain reaction (PCR) (0 vs. 25%). Urine antigen test and serology were not affected. Among those who had received antibiotics only at the acute visit before study sampling, serology (29 vs. 15%), urine antigen (19 vs. 8%), and blood culture (9 vs. 2%) were more often positive than among the unexposed. Antimicrobial exposure before the visit reduced both culture and PCR-based detection. Patients given antibiotics at the visit had higher proportions of positive blood culture, serology, and urine antigen tests, suggesting higher pneumococcal CAP prevalence.

[Acute otitis media in ambulatory practice: epidemiological and clinical characteristics after 7 valent pneumococcal conjugate vaccine (PCV7) implementation]. / Otite moyenne aiguë en pédiatrie ambulatoire : caractéristiques épidémiologiques et cliniques après l'introduction du vaccin antipneumococcique conjugué 7 valent (PCV7).

BACKGROUND: In US, pneumococcal conjugate vaccine (PCV7) had reduced the burden of AOM and changed the profile of the disease. Prior to PCV7 implementation in France, AOM represented 8% of pediatricians visits and failure rate was 12%. The aim of this study is to describe the epidemiologic characteristics of AOM after PCV7 implementation. METHODS: From 2007 to 2008, 30 pediatricians enrolled 3141 patients 3 to 36 months old with AOM. Standardized history and physical examination findings were recorded. Factors related to AOM failures were identified by multivariate logistic regression. RESULTS: AOM accounted for 5.8% of the 43 433 visits or 6.2 cases/week per pediatrician. Among 3141 evaluable AOM cases (mean age 16.7±8 months, peak incidence at 10 months), 99% had been vaccinated with PCV7 and 42.1% attended day care (DCC). Recurrent AOM comprised 24.5% of cases and 51% of children had received ATB in the last 3 months. At the time of diagnosis, 47.1% had fever≥38,5°C, 74.5% otalgia and 4.7% otorrhea. Febrile and painful AOM accounted for 29.5% of cases and cunjunctivitis-otitis syndrome for 18.2%. ATB was prescribed in 98.7% of cases (cefpodoxime proxetil, 59% and amoxicillin/clavulanate, 37%). The failure rate was 6.4% and failure risk was greater in children in DCC (OR=1.50, [1.10;2.05]), young age<18 months (OR=1.47, [1.06;2.04]) and history of recurrent AOM (OR=1.45, [1.02;2.06]). CONCLUSION: Despite PCV7 implementation, AOM remains a very frequent childhood infection and a major reason for ATB prescriptions.

Timing of serotype 1 pneumococcal disease suggests the need for evaluation of a booster dose.

Protection against serotype 1 could not be demonstrated in two randomized trials of 9 valent pneumococcal conjugate vaccines. An analysis of the timing of type 1 cases among vaccinees and controls shows that the vaccine failures occurred among cases occurring after the first year of life. Vaccination was given as three doses in infancy with no booster dose. These data suggest that a booster dose given at 9 months of age, or early in the second year of life, should be evaluated for protection against type 1 pneumococcal disease.

Estimating the direct impact of new conjugate vaccines against invasive pneumococcal disease.

New pneumococcal conjugate vaccines (PCVs) are now becoming available. These formulations differ from the heptavalent diphtheria toxin variant conjugate vaccine (7vCRM, Prevenar/Prevnar) both in the number of serotypes and in serotype-specific immunogenicity. This review proposes an algorithm that attempts to predict the overall impact of these differences in vaccine formulation and immunogenicity on invasive pneumococcal disease (IPD) effectiveness. It builds on the principles underlying WHO licensure criteria for new PCVs, that serotype-specific anti-polysaccharide immunogenicity is potentially predictive of effectiveness. The algorithm used three sources of information: serotype-specific effectiveness data for 7vCRM, serotype-specific head-to-head immunogenicity data with 7vCRM and a recently licensed 10-valent pneumococcal non-typeable H. influenzae protein D-conjugate vaccine (PHiD-CV, Synflorix), and epidemiological information regarding the serotypes causing IPD in young children. Based on this algorithm, PHiD-CV and 7vCRM are predicted to prevent approximately 60-80% and 45-80%, respectively of IPD in young children worldwide, with significant variability by country and region.

Identification of pneumococcal serotypes from culture-negative clinical specimens by novel real-time PCR.

Pneumococcal parapneumonic empyema is an increasingly common complication in children. Conventional microbiological cultures indicate bacterial causes in as few as 8% of cases; therefore, there is a vital need for new molecular methods of detection and diagnosis. The development and clinical evaluation of real-time PCR-based assays to detect the pneumococcal capsular wzg gene of all serotypes tested are reported here, and 24 of them have been identified in clinical specimens. Using real-time PCR assays with highly specific TaqMan MGB probes that target DNA sequences within the capsular polysaccharide gene cluster, it was possible to differentiate serotypes 1, 3, 5, 4, 6A, 6B, 7F/A, 8, 9V/A/N/L, 14, 15B/C, 18C/B, 19A, 19F/B/C, 23F and 23A. These assays showed high sensitivity (five to ten pneumococcal DNA equivalents) and they were validated with 175 clinical isolates of known serotypes. The clinical value of this approach was demonstrated by analysis of 88 culture-negative pleural fluids from children diagnosed with parapneumonic empyema in three Spanish hospitals. Pneumococcal DNA was detected in 87.5% of pleural fluids, and serotypes 1, 7F and 3 were responsible for 34.3%, 16.4% and 11.9%, respectively, of cases of parapneumonic empyema in children. Such molecular methods are critical for the diagnosis of invasive pneumococcal disease and continued epidemiological surveillance in order to monitor serotype vaccine effectiveness.

Antibiotic susceptibility and molecular epidemiology of nasopharyngeal pneumococci from Spanish children.

Nasopharyngeal pneumococci were collected from 635 Spanish children aged 6 months to 6 years attending four primary healthcare centres (n = 276) or two hospital emergency rooms (n = 359); 36% of the children had received >/=1 dose of pneumococcal conjugate vaccine (PCV7). Overall, the carriage rate of Streptococcus pneumoniae was 31%, with no significant differences in carriage rates according to setting. Colonization with vaccine serotypes was significantly associated with the absence of PCV7 immunization (29.4% vs. 5.9%, p <0.001). Forty-seven per cent of all isolates were penicillin- and/or erythromycin-non-susceptible; 13 international antibiotic-resistant clones were represented among non-susceptible pneumococci and were similarly distributed among vaccine and non-vaccine serotypes.

Pneumococcal conjugate vaccine serotypes of Streptococcus pneumoniae isolates and the antimicrobial susceptibility of such isolates in children with otitis media.

The ability of the recently licensed 7-valent pneumococcal conjugate vaccine to cover isolates that cause otitis media, especially drug-resistant ones, was assessed using 500 recently obtained US isolates. Of these isolates, 418 (84%) belonged to vaccine-related serogroups, whereas 82 (16%) belonged to non-vaccine-related serogroups. Serotype 3 accounted for 48 (59%) of the non-vaccine-related serogroups. In addition, 93% of the isolates from patients < or =3 years of age belonged to serotypes that were included in or related to the heptavalent vaccine, compared with 49% of the isolates from older patients (P=.001). Most of the isolates (98%-100%) that were resistant to the antimicrobial agents tested were covered by the heptavalent vaccine, including 95.1% of the isolates from patients <2 years of age. The 7-valent pneumococcal conjugate vaccine could therefore potentially provide protection against all but 1 (type 3) of the common otitis media-associated pneumococcal serogroups identified in this study as well as against 98% of antibiotic-resistant isolates.

Geographical differences in invasive pneumococcal disease rates and serotype frequency in young children.

The development of glycoconjugate vaccines for Streptococcus pneumoniae that are effective in very young children has renewed interest in identification of which among the more than 90 pneumococcal serotypes are most likely to cause invasive pneumococcal disease (IPD). Serotype distribution is thought to vary geographically, even between regions as socioeconomically similar as western Europe and North America. To explain these variations, we note the considerable variation that exists between reported rates of IPD in young children in the USA and west European countries. We postulate that this variation is attributable to different blood-culture rates and practices, and that mild IPD is probably underdiagnosed and under-reported in western Europe. On the basis of a comparison of serotype distributions between the two regions, we also postulate that those serotypes found at similar frequencies in both regions are virulent and rarely cause mild disease. As a result, reported distributions of IPD serotypes, especially when expressed as percentages, might be strongly skewed by the distribution of clinical presentations in a particular study population.

Which pneumococcal serogroups cause the most invasive disease: implications for conjugate vaccine formulation and use, part I.

We analyzed >70 recent data sets to compare the serogroups causing invasive pneumococcal disease (IPD) with those represented in conjugate vaccine formulations. Five to 8 and 10-11 serogroups comprise at least 75% of pneumococcal isolates from young children and older children/adults, respectively, in each geographic region. Serogroups in the 7-valent formulation (4, 6, 9, 14, 18, 19, and 23) cause 70%-88% of IPD in young children in the United States and Canada, Oceania, Africa, and Europe, and <65% in Latin America and Asia. Serogroups in the 9-valent formulation (7-valent+1, 5) cause 80%-90% of IPD in each region except Asia (66%). Serogroup 1 accounts for >6% of IPD in each region, including Europe, except the United States and Canada and Oceania. In contrast, several serogroups not found in 7-, 9-, and 11-valent conjugate formulations are significant causes of disease in older children/adults. Nevertheless, each conjugate formulation could prevent a substantial IPD burden in each region and age group.

The contribution of specific pneumococcal serogroups to different disease manifestations: implications for conjugate vaccine formulation and use, part II.

To assess whether certain serogroups of Streptococcus pneumoniae are preferentially associated with specific disease manifestations, we analyzed all recent pneumococcal disease studies and assessed the relative frequency of isolation of each serogroup by clinical site (as a proxy for different disease states). In all age groups, serogroups 1 and 14 were more often isolated from blood, and serogroups 6, 10, and 23 were more often isolated from cerebrospinal fluid (CSF); in young children, serogroups 3, 19, and 23 were more often isolated from middle ear fluid (MEF). Serogroups represented in conjugate vaccines were isolated slightly less frequently from CSF than from blood or MEF. Nonetheless, serogroups in the 9-valent conjugate vaccine formulation still comprised approximately 75% of pneumococcal isolates from the CSF of young children in Europe and in the United States and Canada. These analyses indicate that pneumococcal conjugate vaccines could potentially prevent a substantial proportion of episodes of bacteremic disease, pneumonia, meningitis, and otitis media, especially in young children.

Reappraisal of the Peruvian and Brazilian lower titer tetravalent rhesus-human reassortant rotavirus vaccine efficacy trials: analysis by severity of diarrhea.

OBJECTIVES AND METHODS: With the purpose of better understanding the efficacy of the lower titer [4 x 10(4) plaque-forming units (pfu)] tetravalent rhesus-human reassortant rotavirus vaccine (RRV-TV) against diarrheal episodes of different severities, the Peruvian and Brazilian efficacy data were reanalyzed with a 20-point scoring system. Mild, moderate/severe and very severe rotavirus diarrhea were scored as 0 to 8, 9 to 14 and >14, respectively. RESULTS: In the Peruvian study one dose of vaccine yielded 64% (P = 0.04) protection against pure cases of rotavirus disease (i.e. those in which no other enteropathogen was found) with clinical scores ranging from 9 to 14. Protective efficacy against very severe rotavirus gastroenteritis could not be assessed because of the small number of cases. In Brazil there was a trend in preventing "all" and "pure" cases of rotavirus diarrhea scored 9 to 14 (44%, P = 0.06, and 45%, P = 0.08, respectively) and the vaccine was 75% (P = 0.02) protective against pure rotavirus diarrhea scored >14. No protection was observed for mild rotavirus diarrhea (scores <9). These data were compared with those from trials in Venezuela (4 x 10(5) pfu/dose), US (4 x 10(4) pfu/dose and 4 x 10(5) pfu/dose) and Finland (4 x 10(5) pfu/dose). Combining the Peruvian (one dose, pure cases) and Brazilian studies together, the levels of protection against 9- to 14-scored rotavirus diarrhea are comparable with those from the Venezuelan (47%) and American (57, 57 and 65%) efficacy trials. In Brazil the level of protection (75%) against pure, >14-scored rotavirus diarrhea is similar to the efficacy rates yielded in the three US trials (82, 80 and 69%) and the Finnish trial (100%) for episodes of the same severity. CONCLUSIONS: Our reanalysis provides evidence that, at least against moderate/severe rotavirus gastroenteritis, RRV-TV, 4 x 10(4) pfu/dose is potentially as efficacious as RRV-TV, 4 x 10(5) pfu/dose, even in settings with very high rotavirus disease burden. The reanalysis of the Peruvian data suggests that one and three vaccine doses may yield similar efficacy rates. It is also suggested that vaccine efficacy against most severe episodes in Peru and Brazil was not evident because of the trial design used in those studies (i.e. prospective, active home surveillance rather than a catchment trial), resulting in too few cases of severe disease even in the placebo group. To confirm these findings, future trials with this vaccine are necessary in developing countries with high diarrhea morbidity rates. These trials should use catchment designs and focus on the evaluation of the efficacy of one or three doses of RRV-TV against moderate to severe/very severe rotavirus diarrhea.

Prospects for the use of new vaccines in developing countries: cost is not the only impediment.

Global immunization programs represent a great public health success story. Evidence from every region documents substantial reductions in morbidity and mortality following widespread use of vaccines developed years ago. Development and introduction of new vaccines and vaccine combinations aimed at the industrialized world market are occurring at a fast pace. A number of political and economic factors will influence the rate at which developing country immunization programs incorporate those new vaccines that could have a major public health impact. Perhaps the greatest determinant of this rate is the extent to which international and bilateral agencies and national governments appreciate the potential value of new vaccines.

Beta-adrenergic receptor sequestration. A potential mechanism of receptor resensitization.

Continuous exposure of cells to hormonal agonists often causes a rapid waning of the stimulated response. This desensitization effect has been extensively studied in the beta-adrenergic receptor system, and attributed largely to the rapid phosphorylation of the receptor by two kinases. Over a similar time frame (seconds to minutes), agonists also trigger a selective loss in the capacity of receptors to bind hydrophilic but not hydrophobic ligands, a phenomenon termed sequestration. There is some evidence suggesting that sequestration represents the rapid internalization of receptors, but the functional significance of sequestration has remained unclear. Upon the removal of agonist, both desensitization and sequestration are readily reversed with similar kinetics (t1/2 approximately 3 min for both). To investigate the possibility that receptor sequestration is involved in this resensitization of the adenylyl cyclase response, we applied two distinct approaches to block receptor sequestration: by pretreating cells with sucrose and by creating a sequestration-defective beta 2-adrenergic receptor by site-specific mutagenesis. Both approaches effectively disabled receptor sequestration, with little effect on adenylyl cyclase stimulation or on desensitization. However, in both cases, no recovery from desensitization was apparent even 20 min after the removal of agonist. Similarly, pretreating cells with concanavalin A almost completely blocked receptor sequestration and resensitization but only partially inhibited other receptor functions. Our results therefore suggest that sequestration of beta 2-adrenergic receptors is a mechanism involved in reactivating and recycling desensitized receptors.

Overexpression of pp60c-src is associated with altered regulation of adenylyl cyclase.

The ability of activators of the beta-adrenergic receptor to elevate intracellular cAMP levels in murine fibroblasts is enhanced upon overexpression of avian c-src [Bushman et al. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 7462-7466]. To investigate the molecular basis for this effect, we prepared particulate fractions from control and pp60c-src overexpressing C3H10T1/2 fibroblasts and assessed the relative abilities of several activators of the beta-adrenergic receptor-Gs-adenylyl cyclase (AC) signal transduction pathway to stimulate the enzymatic response. Two- to three-fold increases in both the sensitivity and maximum responsiveness of AC to the beta-adrenergic agonist isoproterenol were consistently observed in fractions prepared from the c-src overexpressing cells. Interestingly, the AC response to two agents believed to act directly at the level of the G protein were either enhanced (NaF) or unaffected (GTP gamma S) by c-src overexpression. Finally, overexpression of c-src was associated with a reduced ability of both Mn2+ and forskolin to activate AC directly. These results suggest that overexpression of wild type c-src may affect two distinct steps in the regulation of AC exerting a positive effect at the level of Gs activation and a negative effect on AC itself. As no differences in the relative number or affinity of beta-adrenergic receptors, or in the level of AC, Gs alpha or G beta, were detected between control cells and those overexpressing c-src, we propose that pp60c-src overexpression results in a modification of one or more components in this signal transduction pathway.

Tyrosine phosphorylation of G protein alpha subunits by pp60c-src.

A number of lines of evidence suggest that cross-talk exists between the cellular signal transduction pathways involving tyrosine phosphorylation catalyzed by members of the pp60c-src kinase family and those mediated by guanine nucleotide regulatory proteins (G proteins). In this study, we explore the possibility that direct interactions between pp60c-src and G proteins may occur with functional consequences. Preparations of pp60c-src isolated by immunoprecipitation phosphorylate on tyrosine residues the purified G-protein alpha subunits (G alpha) of several heterotrimeric G proteins. Phosphorylation is highly dependent on G-protein conformation, and G alpha(GDP) uncomplexed by beta gamma subunits appears to be the preferred substrate. In functional studies, phosphorylation of stimulatory G alpha (G alpha s) modestly increases the rate of binding of guanosine 5'-[gamma-[35S]thio]triphosphate to Gs as well as the receptor-stimulated steady-state rate of GTP hydrolysis by Gs. Heterotrimeric G proteins may represent a previously unappreciated class of potential substrates for pp60c-src.

A small region of the beta-adrenergic receptor is selectively involved in its rapid regulation.

Plasma membrane receptors that couple to guanine nucleotide-binding regulatory proteins (G proteins) undergo a variety of rapid (minutes) and longer term (hours) regulatory processes induced by ligands. For the beta 2-adrenergic receptor (beta 2AR), the rapid processes include functional desensitization, mediated by phosphorylation of the receptor by the cAMP-dependent protein kinase and the beta-adrenergic receptor kinase, as well as a loss of hydrophilic ligand binding proposed to represent sequestration of receptors into a cellular compartment distinct from the plasma membrane. The slower processes include beta 2AR down-regulation (i.e., a decrease in the total cellular complement of receptors). It is not yet known whether beta 2AR phosphorylation and/or sequestration are prerequisites for down-regulation of the receptor. Like other G protein-coupled receptors, the beta 2AR molecule spans the plasma membrane seven times, and the cytoplasmic carboxyl-terminal domain has been proposed to contain molecular determinants for each of these regulatory processes. We replaced four serine and threonine residues located within a 10-amino acid segment of this domain of beta 2AR and thereby prevented agonist-promoted phosphorylation, sequestration, and rapid desensitization of the adenylyl cyclase response. In contrast, these mutations did not affect functional coupling to the stimulatory G protein Gs or long-term down-regulation. These findings thus define a small, hitherto unappreciated region of the receptor molecule that may selectively subserve its rapid regulation. In addition, with the demonstration that beta 2AR does not have to be phosphorylated or sequestered in order to enter the down-regulation pathway, the results suggest that the classical receptor endocytosis model may not be appropriate for beta 2AR regulation.

Mutations of the human beta 2-adrenergic receptor that impair coupling to Gs interfere with receptor down-regulation but not sequestration.

The integrity of coupling of the beta 2-adrenergic receptor (beta 2AR) to its guanine nucleotide-binding protein, Gs, and phosphorylation events on the receptor molecule have been proposed to be important determinants in the processes of receptor sequestration and down-regulation. However, little is known about the molecular mechanisms underlying these processes, and the regions of the receptor molecule that specifically subserve sequestration and down-regulation have yet to be delineated. To address these questions, we stably transfected eight mutant beta 2AR genes into Chinese hamster fibroblasts and evaluated the coupling, sequestration, and down-regulation properties of the mutated receptors. These mutant receptors have been previously demonstrated either to exhibit abnormal coupling to Gs or to lack functionally important phosphorylation sites for either the cAMP-dependent protein kinase or the agonist-dependent beta-adrenergic receptor kinase. All eight mutants exhibited receptor sequestration equivalent in extent to that of the beta 2AR, regardless of their coupling or phosphorylation status. However, four mutants that exhibited various degrees of impairment in coupling to Gs showed blunted receptor down-regulation patterns. Simultaneous treatment with isoproterenol and dibutyryl-cAMP did not improve the abilities of the mutant receptors to undergo down-regulation. These findings demonstrate that a variety of mutant beta 2AR with impaired coupling to Gs are, nevertheless, able to be sequestered normally. In contrast, agonist-induced down-regulation appears to require coupling of the beta 2AR to Gs but is largely independent of the generation of cAMP. Our results also suggest that molecular determinants of the beta 2AR involved in receptor sequestration are distinct from those participating in the down-regulation process.

Turning off the signal: desensitization of beta-adrenergic receptor function.

Cellular responses to many hormones and neurotransmitters wane rapidly despite continuous exposure of cells to these stimuli. This phenomenon, termed desensitization, has been particularly well studied for the stimulation of cAMP levels by plasma membrane beta-adrenergic receptors (beta AR). The molecular mechanisms underlying rapid beta AR desensitization do not appear to require internalization of the receptors, but rather an alteration in the functioning of beta AR themselves that uncouples the receptors from the stimulatory G protein Gs. This uncoupling phenomenon involves phosphorylation of beta AR by at least two kinases, PKA and the beta AR kinase (beta ARK), which are activated under different desensitizing conditions. Receptor phosphorylation by the two kinases leads to desensitization of the receptor response via distinct biochemical mechanisms, and additional cytosolic factors appear to be involved in the case of beta ARK. Numerous experimental approaches have been used recently to elucidate the molecular details of this ubiquitous biological process.

Role of phosphorylation in desensitization of the beta-adrenoceptor.

Regulation of receptors allows their responses to be modified rapidly and appropriately according to the needs of the environment. Multiple mechanisms are involved in the loss of sensitivity that follows exposure to agonists. Receptor sequestration, a rapid and transient event, and receptor downregulation, which requires more prolonged agonist exposure, contribute to this effect. However, in this article Bob Lefkowitz and colleagues focus primarily on the recent developments in understanding mechanisms of rapid desensitization involving receptor phosphorylation. Various molecular biological techniques have been used to demonstrate the important roles of two particular kinases--beta ARK and protein kinase A--in this regard.