Temporal trends of invasive Streptococcus pneumoniae serotypes and antimicrobial resistance patterns in Spain from 1979 to 2007.

Temporal trends of serotypes from invasive pneumococcal disease (IPD) in Spain from 1979 to September 2007 under antibiotic and vaccine pressure were analyzed. A significant trend in pneumococcal conjugate 7-valent vaccine (PCV7) serotypes (except serotype 4) was found, whereby the prevalence increased from the early 1980s and decreased in the 2000s for all but serotype 23F, which began decreasing in the late 1980s. Among the major non-PCV7 serotypes, a significant decrease was observed for serotypes 1, 5, and 7F in the 1980s. From the late 1990s, serotypes 1, 5, 6A, 7F, and 19A increased significantly, while serotypes 3 and 8 showed similar but nonsignificant trends over time. The incidence of IPD cases was 10.7/100,000 for the period 1996 to 2006, with reporting coverage ranging from 18% to 43%. A significant decrease in IPD incidence due to PCV7 serotypes was observed, while the incidence of non-PCV7 serotypes increased, with the consequence that there was no clear pattern in the overall incidence of IPD. Penicillin nonsusceptibility was correlated with the proportion of PCV7 serotypes. Erythromycin nonsusceptibility increased in association with long-half-life macrolide consumption and then decreased in 2004 to 2007. The increase in PCV7 serotypes and antibiotic nonsusceptibility related to antibiotic consumption in the 1980s and 1990s was reversed in the 2000s, probably as a result of PCV7 immunization. The decrease in IPD incidence due to PCV7 serotypes was mirrored by an increase in that of non-PCV7 serotypes. The impact of various preventive/therapeutic strategies on pneumococcal evolution is serotype dependent, and the dynamics remain unpredictable.

Has the licensing of respiratory quinolones for adults and the 7-valent pneumococcal conjugate vaccine (PCV-7) for children had herd effects with respect to antimicrobial non-susceptibility in invasive Streptococcus pneumoniae?

OBJECTIVES: The aim of the study was to analyse the evolution of antibiotic non-susceptibility in Spanish invasive Streptococcus pneumoniae after licensure of respiratory-quinolones for adults and 7-valent pneumococcal conjugate vaccine (PCV-7) for immunization of children. METHODS: All invasive pneumococci received in the Reference Laboratory (January 2000-August 2007; n = 12 957 isolates) were serotyped, and susceptibility to penicillin/erythromycin/levofloxacin was determined. Antibiotic consumption and PCV-7 doses/year were provided by IMS and the manufacturer, respectively. RESULTS: In 2000-07, PCV-7 distribution (doses/1000 inhabitants

Dependence of correlations between antibody titres and opsonophagocytosis on pneumococcal serotype and patient morbidity in pre- and post-pneumococcal vaccination states.

Pre- vs. post-vaccination changes in correlations between IgG concentrations (ELISA titres) and opsonophagocytic activity (OPA) against Streptococcus pneumoniae serotypes 6B, 14 and 23F induced by the 23-valent polysaccharide vaccine were studied in paired serum samples received from elderly individuals, haemodialysed patients and kidney transplant recipients by the Spanish Pneumococcal Reference Laboratory. The pre- and post-vaccination parameters considered were: ELISA and OPA titres and the percentage of subjects with post-vaccination OPA values above the cut-off levels; the correlations between OPA and ELISA (Spearman correlation coefficient, r); and the amount of IgG needed to obtain OPA (beta coefficient). Non-significant pre-vaccination correlations between OPA and ELISA were found. Vaccination increased the correlation coefficient between OPA and ELISA to a statistically significant level for serotypes 6B, 14 and 23F in samples from haemodialysed patients, for serotypes 14 and 23F in samples from elderly individuals, and for none of the serotypes in samples from transplant recipients. In all cases, except for serotype 23 in transplant recipients, vaccination increased the beta coefficient, indicating that lower amounts of IgG were needed to obtain high OPA titres. A globally lower response was obtained for serotype 23 and/or transplant recipients.

Human lymphocytotoxic monoclonal autoantibodies from a highly sensitized renal dialysis patient.

A panel of 5 human monoclonal autolymphocytotoxic antibodies (IRM-3, IRM-4, IRM-7, IRM-8, and IRM-10) of the IgM class was established from a highly sensitized renal dialysis patient (IRM), by the generation of mouse-human heterohybridomas. This panel was screened for reactivity against foreign and autoantigens by ELISA, and for reactivity against different tissue sections and HEp-2 slide preparations by indirect immunofluorescence. Cytotoxicity screening of heterohybridoma supernatants gave broad panel reactivity profiles, being cytotoxic against B cells from patient IRM and also against most B cells tested and less reactive with chronic lymphocytic leukemia B cells; T cells were the least sensitive target. Immunoblotting showed that monoclonal IRM displayed some heterogeneity in their binding profiles, although all of them recognized a cellular structure of 26 kDa. None of the heterohybridoma cell lines exhibited cytoplasmic nor surface staining with an anti-CD5 mAb. Results obtained showed that all the autolymphocytotoxic mAbs generated were also able to react against certain nuclear and cytoplasmic self-structures as well as foreign compounds. Monoclonal antibody IRM-7 and, to a lesser degree, IRM-10 exhibited multispecific properties similar to those observed for polyreactive or natural antibodies.

Lack of suppressive antibody activity in sera from patients with active-phase autoimmune diseases.

To investigate the presence of a suppressive antibody activity in sera from patients with autoimmune diseases, the IgG autoreactivity in whole serum was compared to that of the IgG fraction purified by affinity chromatography on protein G-sepharose. Competitive inhibition assays on the binding to histone, dsDNA, RNP and thyroglobulin of the purified IgG fraction by the autologous IgM present in serum without IgG and depleted of <100 kD components (named IgM fraction) were also performed. The IgG reactivity to the autoantigens tested was considerably increased in the IgG fraction than in the whole serum drawn from a healthy control and from three SLE patients in an inactive-phase of disease. Addition of the IgM fraction to the autologous purified IgG resulted in a dose-dependent inhibition of IgG binding to the autoantigens tested. However, no differences were observed between the autoreactivity of the IgG in whole serum and that of the purified IgG fraction from active-phase SLE patients, or from two patients with autoimmune thyroiditis, and the autologous IgM fractions did not inhibit significantly binding to the autoantigens under study of the purified IgG fraction. Our findings support the concept that the IgG autoreactivity in physiological conditions is regulated by idiotypic interactions between IgG and IgM, and suggest that this regulation is broken in the active phase of autoimmune diseases and that clinical remission from SLE could be associated with the restoration of this control mechanism. Additionally, qualitative differences, such as polyreactivity or change of idiotype in the autoreactive IgG fraction from active-phase disease might contribute to escape of regulation.

[Comparison of the microdilution method (PASCO), the Etest and disc-diffusion in streptococcus pneumoniae antibiotic susceptibility testing]. / Comparación del método de microdilución (PASCO) con el Etest y la difusión con disco en el estudio de la sensibilidad de Streptococcus pneumoniae a los antibióticos.

MIC values and inhibition zone diameters for penicillin, cefotaxime, and erythromycin where determined in 75 consecutive Streptococcus pneumoniae blood isolates by microdilution (PASCO), disc-diffusion and Etest. Using the Etest, 40% and 29.3% of the isolates showed low- or high-level resistance to penicillin and erythromycin respectively, whereas 9.3% were intermediate to cefotaxime. No high-level resistance to cefotaxime was found. No errors were found with the comparison of penicillin and cefotaxime results by microdilution and Etest, with just 2 (2.7%) and 3 (4%) minor errors appearing respectively. On the other hand, when comparing penicillin susceptibility results by disc-diffusion (1 mg oxacillin disc) and Etest, 6 (8%) major errors and 18 (24%) minor errors were found.

Molecular epidemiology of enterovirus 71, coxsackievirus A16 and A6 associated with hand, foot and mouth disease in Spain.

Hand, foot and mouth disease (HFMD) is a childhood illness frequently caused by genotypes belonging to the enterovirus A species, including coxsackievirus (CV)-A16 and enterovirus (EV)-71. Between 2010 and 2012, several outbreaks and sporadic cases of HFMD occurred in different regions of Spain. The objective of the present study was to describe the enterovirus epidemiology associated with HFMD in the country. A total of 80 patients with HFMD or atypical rash were included. Detection and typing of the enteroviruses were performed directly in clinical samples using molecular methods. Enteroviruses were detected in 53 of the patients (66%). CV-A6 was the most frequent genotype, followed by CV-A16 and EV-71, but other minority types were also identified. Interestingly, during almost all of 2010, CV-A16 was the only causative agent of HFMD but by the end of the year and during 2011, CV-A6 became predominant, while CV-A16 was not detected. In 2012, however, both CV-A6 and CV-A16 circulated. EV-71 was associated with HFMD symptoms only in three cases during 2012. All Spanish CV-A6 sequences segregated into one major genetic cluster together with other European and Asian strains isolated between 2008 and 2011, most forming a particular clade. Spanish EV-71 strains belonged to subgenogroup C2, as did most of the European sequences circulated. In conclusion, the recent increase of HFMD cases in Spain and other European countries has been due to a larger incidence of circulating species A enteroviruses, mainly CV-A6 and CV-A16, and the emergence of new genetic variants of these viruses.

Sustained high prevalence of pneumococcal serotype 1 in paediatric parapneumonic empyema in southern Spain from 2005 to 2009.

The epidemiology and microbiological characteristics of paediatric parapneumonic empyema (PPE) before the introduction of the new generation of conjugate pneumococcal vaccines (10-valent and 13-valent) are described. All patients <14 years old admitted to a tertiary paediatric hospital with a diagnosis of PPE were prospectively enrolled from January 2005 to December 2009. Pneumococcal serotyping of culture-negative pleural fluid samples was performed using a multiplex real-time PCR assay. Overall, 219 patients had PPE. Incidence rates for PPE remained stable during the study period with a not significant increase in 2009 compared with 2005 (p 0.13), and were temporally associated with higher circulation of pandemic influenza A H1N1 during the last quarter in our population (p 0.001). Pneumococci were detected in 72% of culture-positive and 79% of culture-negative samples. Serotypes were determined in 104 PPE cases. Serotype 1 was the most prevalent serotype identified (42%) followed by serotypes 7F (20%), 3 (16%), 19A (8%) and 5 (7%). Serotype distribution remained similar during all time periods. Pneumococcal serotype 1 remained the most common cause of PPE during the 5-year study. The new generation of pneumococcal conjugate vaccines offers potential serotype coverage of 73% (10-valent) and 99% (13-valent) in the population studied suffering from PPE. Continuous epidemiological and molecular studies are paramount to monitor the impact of pneumococcal vaccines on the epidemiology of PPE.

Influence of penicillin/amoxicillin non-susceptibility on the activity of third-generation cephalosporins against Streptococcus pneumoniae.

To study the influence of penicillin/amoxicillin non-susceptibility on the activity of third-generation cephalosporins, 430 consecutive penicillin non-susceptible Streptococcus pneumoniae 2007 isolates received in the Spanish Reference Pneumococcal Laboratory were tested. For comparative purposes, 625 penicillin-susceptible 2007 isolates were also tested. Susceptibility was determined by agar dilution using Mueller-Hinton agar supplemented with 5% sheep blood. Penicillin-susceptible strains were susceptible to amoxicillin, cefotaxime and ceftriaxone, 99.8% to cefpodoxime and 99.5% to cefdinir, and were inhibited by 0.12 microg/ml of cefditoren and 4 microg/ml of cefixime. Penicillin-intermediate strains were susceptible to cefotaxime and ceftriaxone, with <50% susceptibility to cefdinir and cefpodoxime. The MIC(50) and MIC(90) values of cefditoren were 0.25 microg/ml and 0.5 microg/ml, respectively, whereas cefixime exhibited only marginal activity (MIC(90)=16 microg/ml). Penicillin-resistant strains were resistant to cefdinir and cefpodoxime, with 74.8% and 94.1% susceptibility to cefotaxime and ceftriaxone, respectively. Cefditoren MIC(50)/MIC(90) (0.5/1 microg/ml) were lower than cefotaxime and ceftriaxone. Among amoxicillin non-susceptible strains, susceptibility to cefdinir and cefpodoxime was <10%, and susceptibility to cefotaxime decreased from 87.9% in the intermediate category to 63.0% in the resistant group. Cefditoren MIC(50)/MIC(90) (0.5/1 microg/ml) were lower than cefotaxime. In conclusion, the activity of cefixime, cefdinir and cefpodoxime was highly affected by penicillin/amoxicillin non-susceptibility, while parenteral third-generation cephalosporins exhibited higher intrinsic activity (MIC(90)=1 microg/ml for penicillin-resistant and 2 microg/ml for amoxicillin-resistant strains). Cefditoren exhibited one-dilution lower MIC(90) values for these strains, even against those of the most troublesome serotypes.

In vitro activity of oral cephalosporins against pediatric isolates of Streptococcus pneumoniae non-susceptible to penicillin, amoxicillin or erythromycin.

The aim of this study was to evaluate the effects of penicillin, amoxicillin or erythromycin resistance on the in vitro activity of oral cephalosporins against Streptococcus pneumoniae pediatric isolates. A total of 282 pediatric isolates received during 2005 in the Spanish Reference Pneumococcal Laboratory were tested by agar dilution: 104 strains were penicillin-susceptible, 72 intermediate, and 106 resistant. Serotypes 9 and 14 were the most troublesome with <10% susceptibility to oral cephalosporins. Cefditoren exhibited the highest intrinsic activity against penicillin/amoxicillin-resistant pneumococci, with MIC(90s )of 0.5 microg/ml, followed by cefotaxime (2 microg/ml), cefpodoxime (4 microg/ml), cefuroxime (16 microg/ml), and cefaclor/cefixime (>or= 32 microg/ml), with 0% susceptibility to cefaclor, cefuroxime and cefpodoxime. Cefditoren 0.5 microg/ml inhibited 95.3%, 95.5%, and 98.6% of penicillin-, amoxicillin-, and erythromycin-resistant isolates, respectively. Susceptibility to oral cephalosporins shifted from >90% in penicillin-susceptible isolates to approximately 38% for cefuroxime/cefpodoxime and approximately 7% for cefaclor in penicillin-intermediate, and to 0% in resistant isolates. Despite the different in vitro activity of oral cephalosporins, full resistance to penicillin or amoxicillin implied lack of susceptibility to all oral cephalosporins with defined CLSI breakpoints, rendering them inadequate as empirical treatment in countries with a high prevalence of penicillin resistance.

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.

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.

Human polyreactive IgM monoclonal antibodies with blocking activity against self-reactive IgG.

Natural IgM antibodies have been found to be involved in the control of IgG reactivity in normal serum. The authors investigated the blocking activity of four human IgM monoclonal antibodies (BY-2, BY-7, BY-10 and IRM-7) derived from B-cells from blood samples of three renal dialysis patients, which had shown multispecific properties similar to those observed for natural polyreactive autoantibodies. To achieve this, competitive inhibition assays were performed with these MoAbs on the binding of IgG purified from a healthy control, three patients with SLE, and two patients with autoimmune thyroiditis, to histone, dsDNA, RNP and thyroglobulin. MoAbs inhibited binding of self-reactive IgG to histone and dsDNA, but not to thyroglobulin or RNP, of natural and active or inactive phase disease-associated autoreactive IgG. The inhibitory effect of the MoAbs was mediated by V-region dependent interactions with autoreactive IgG, as shown by the ability of these MoAbs to block the binding of F(ab')2 fragments of autoreactive IgG to antigens (histone and dsDNA). The blocking of autoantibody activity was dose-dependent with maximal inhibition occurring at a specific molar ratio between the patient's IgG and a given MoAb. In contrast, MoAbs did not inhibit binding of IgG alloantibodies present in the sera of four polytransfused renal dialysis patients to target antigens on the surface of different cells. These results support the concept of a functional idiotypic network regulating autoimmune responses, and suggest that the IgM MoAbs under study may be natural polyreactive antibodies belonging to the physiological network of autoantibodies with highly connected V-regions, capable of binding and functionally neutralizing V-regions of natural and pathologic autoantibodies.

Application of cellular ELISA (CELISA) to the detection of human monoclonal autoantibodies.

This work describes the application of cellular enzyme-linked immunosorbent assay (CELISA) for the detection of both polyreactive and monospecific human monoclonal antibodies against autoantigens. The CELISA is ideally suited for the screening of a large number of hybridoma culture supernatants, being, in this way, superior to other methods commonly used for the detection of autoantibody activity, such as indirect immunofluorescence on tissue sections and slide cell preparations, in terms of speed and sensitivity. This assay demonstrated higher sensitivity than ELISA using autoantigenic extracts from rabbit thymus, human spleen, nucleoprotamine, and salmon sperm nuclei, and enzyme immunoassays using ssDNA, dsDNA, and affinity purified autoantigens as substrate. The CELISA has been also successfully applied to the detection of autolymphocytotoxic antibody activity in heterohybridoma supernatants.

Effects of amoxicillin subinhibitory concentrations on the cross-protection developed by pneumococcal antibodies in mouse sepsis caused by an amoxicillin-resistant serotype 6B Streptococcus pneumoniae strain.

A model of mouse sepsis caused by a serotype 6B Streptococcus pneumoniae strain (amoxicillin MIC of 8 microg/ml) was developed to investigate the therapeutic effect of an amoxicillin dose (3.12 mg/kg of body weight three times daily for 48 h) producing, over the whole treatment period, subinhibitory concentrations in serum (peak concentration [C(max)]: 6.1 microg/ml) in animals that prior to infection had been passively immunized with a 6B or 23F hyperimmune serum (obtained by immunization with a whole-cell heat-inactivated inoculum and diluted to produce no protective effect by itself). Mortality in nonimmunized animals treated with antibiotic (3.12 mg/kg) was 90%, and mortality in animals immunized but not treated with the antibiotic was 100%. Antibiotic treatment in immunized animals produced mortality rates

Identification of cation-independent mannose 6-phosphate receptor/insulin-like growth factor type-2 receptor as a novel target of autoantibodies.

Two human monoclonal autoantibodies, B-33 and B-24, were generated from the B cells of a patient with scleroderma. Both monoclonal antibodies (mAbs) were composed of mu and lambda chains, and recognized cytoplasmic vesicular structures by indirect immunofluorescence on Hep-2 cell line slides, although mAb B-24 showed an additional diffuse cytoplasmic staining pattern. By Western blot, mAb B-24 exhibited a polyreactive-like binding pattern, whereas mAb B-33 failed to recognize any electroblotted Hep-2 antigen. The polyreactive versus monospecific behaviour of mAbs B-24 and B-33 was further confirmed by enzyme-linked immunosorbent assay (ELISA) with a variety of foreign and autoantigens. The N-terminal sequence of a protein band isolated by affinity chromatography with mAb B-33 was identical to that of cation-independent mannose 6-phosphate receptor (CI-MPR), also known as the insulin-like growth factor type-2 receptor (IGF-2R). Immunofluorescence experiments on Hep-2 cell line slides demonstrated a striking co-localization between the staining pattern exhibited by these mAbs and the pattern obtained using a goat anti-CI-MPR serum, indicating the recognition by B-24 and B-33 of a structure located predominantly in late endosomes. Sequence analysis of the V-region gene segments of B-33 and B-24 showed both to be identical, except for the existence of a point mutation in B-33 located in the H-complementarity-determining region 3 (H-CDR3) (position 100D), which produces a non-conservative replacement of Gly by Ser. This single replacement appears to be responsible for the dramatic change in reactivity of human mAb B-33. The data shown here provide new evidence of the critical role played by the H-CDR3 region in distinguishing a polyspecific from a monospecific antibody. A population study demonstrated the existence of immunoglobulin G (IgG) reactivity against CI-MPR/IGF-2R in serum specimens from five individuals with different pathological conditions, thus indicating that this molecule is a potential target for the human autoimmune response.