Neonatal invasive disease caused by Streptococcus agalactiae in Europe: the DEVANI multi-center study.

PURPOSE: Group B streptococcus (GBS) remains a leading cause of invasive disease, mainly sepsis and meningitis, in infants < 3 months of age and of mortality among neonates. This study, a major component of the European DEVANI project (Design of a Vaccine Against Neonatal Infections) describes clinical and important microbiological characteristics of neonatal GBS diseases. It quantifies the rate of antenatal screening and intrapartum antibiotic prophylaxis among cases and identifies risk factors associated with an adverse outcome. METHODS: Clinical and microbiological data from 153 invasive neonatal cases (82 early-onset [EOD], 71 late-onset disease [LOD] cases) were collected in eight European countries from mid-2008 to end-2010. RESULTS: Respiratory distress was the most frequent clinical sign at onset of EOD, while meningitis is found in > 30% of LOD. The study revealed that 59% of mothers of EOD cases had not received antenatal screening, whilst GBS was detected in 48.5% of screened cases. Meningitis was associated with an adverse outcome in LOD cases, while prematurity and the presence of cardiocirculatory symptoms were associated with an adverse outcome in EOD cases. Capsular-polysaccharide type III was the most frequent in both EOD and LOD cases with regional differences in the clonal complex distribution. CONCLUSIONS: Standardizing recommendations related to neonatal GBS disease and increasing compliance might improve clinical care and the prevention of GBS EOD. But even full adherence to antenatal screening would miss a relevant number of EOD cases, thus, the most promising prophylactic approach against GBS EOD and LOD would be a vaccine for maternal immunization.

Publisher Correction: Mice repeatedly exposed to Group-A ß-Haemolytic Streptococcus show perseverative behaviors, impaired sensorimotor gating, and immune activation in rostral diencephalon.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

European Multicentre Tics in Children Studies (EMTICS): protocol for two cohort studies to assess risk factors for tic onset and exacerbation in children and adolescents.

Genetic predisposition, autoimmunity and environmental factors [e.g. pre- and perinatal difficulties, Group A Streptococcal (GAS) and other infections, stress-inducing events] might interact to create a neurobiological vulnerability to the development of tics and associated behaviours. However, the existing evidence for this relies primarily on small prospective or larger retrospective population-based studies, and is therefore still inconclusive. This article describes the design and methodology of the EMTICS study, a longitudinal observational European multicentre study involving 16 clinical centres, with the following objectives: (1) to investigate the association of environmental factors (GAS exposure and psychosocial stress, primarily) with the onset and course of tics and/or obsessive-compulsive symptoms through the prospective observation of at-risk individuals (ONSET cohort: 260 children aged 3-10 years who are tic-free at study entry and have a first-degree relative with a chronic tic disorder) and affected individuals (COURSE cohort: 715 youth aged 3-16 years with a tic disorder); (2) to characterise the immune response to microbial antigens and the host's immune response regulation in association with onset and exacerbations of tics; (3) to increase knowledge of the human gene pathways influencing the pathogenesis of tic disorders; and (4) to develop prediction models for the risk of onset and exacerbations of tic disorders. The EMTICS study is, to our knowledge, the largest prospective cohort assessment of the contribution of different genetic and environmental factors to the risk of developing tics in putatively predisposed individuals and to the risk of exacerbating tics in young individuals with chronic tic disorders.

Mice repeatedly exposed to Group-A ß-Haemolytic Streptococcus show perseverative behaviors, impaired sensorimotor gating, and immune activation in rostral diencephalon.

Repeated exposure to Group-A ß-Haemolytic Streptococcus (GAS) may constitute a vulnerability factor in the onset and course of pediatric motor disturbances. GAS infections/colonization can stimulate the production of antibodies, which may cross the blood brain barrier, target selected brain areas (e.g. basal ganglia), and exacerbate motor alterations. Here, we exposed developing SJL male mice to four injections with a GAS homogenate and evaluated the following domains: motor coordination; general locomotion; repetitive behaviors; perseverative responses; and sensorimotor gating (pre-pulse inhibition, PPI). To demonstrate that behavioral changes were associated with immune-mediated brain alterations, we analyzed, in selected brain areas, the presence of infiltrates and microglial activation (immunohistochemistry), monoamines (HPLC), and brain metabolites (in vivo Magnetic Resonance Spectroscopy). GAS-exposed mice showed increased repetitive and perseverative behaviors, impaired PPI, and reduced concentrations of serotonin in prefrontal cortex, a brain area linked to the behavioral domains investigated, wherein they also showed remarkable elevations in lactate. Active inflammatory processes were substantiated by the observation of infiltrates and microglial activation in the white matter of the anterior diencephalon. These data support the hypothesis that repeated GAS exposure may elicit inflammatory responses in brain areas involved in motor control and perseverative behavior, and result in phenotypic abnormalities.

Significant Differences Characterise the Correlation Coefficients between Biocide and Antibiotic Susceptibility Profiles in Staphylococcus aureus.

There is a growing concern by regulatory authorities for the selection of antibiotic resistance caused by the use of biocidal products. We aimed to complete the detailed information on large surveys by investigating the relationship between biocide and antibiotic susceptibility profiles of a large number of Staphylococcus aureus isolates using four biocides and antibiotics commonly used in clinical practice. The minimal inhibitory concentration (MIC) for most clinically-relevant antibiotics was determined according to the standardized methodology for over 1600 clinical S. aureus isolates and compared to susceptibility profiles of benzalkonium chloride, chlorhexidine, triclosan, and sodium hypochlorite. The relationship between antibiotic and biocide susceptibility profiles was evaluated using non-linear correlations. The main outcome evidenced was an absence of any strong or moderate statistically significant correlation when susceptibilities of either triclosan or sodium hypochlorite were compared for any of the tested antibiotics. On the other hand, correlation coefficients for MICs of benzalkonium chloride and chlorhexidine were calculated above 0.4 for susceptibility to quinolones, beta-lactams, and also macrolides. Our data do not support any selective pressure for association between biocides and antibiotics resistance and furthermore do not allow for a defined risk evaluation for some of the compounds. Importantly, our data clearly indicate that there does not involve any risk of selection for antibiotic resistance for the compounds triclosan and sodium hypochlorite. These data hence infer that biocide selection for antibiotic resistance has had so far a less significant impact than feared.

Evaluation of reduced susceptibility to quaternary ammonium compounds and bisbiguanides in clinical isolates and laboratory-generated mutants of Staphylococcus aureus.

The MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates of Staphylococcus aureus. Both compounds showed unimodal MIC and MBC distributions (2 and 4 or 8 mg/liter, respectively) with no apparent subpopulation with reduced susceptibility. To investigate further, all isolates were screened for qac genes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence of qacA, qacB, qacC, and qacG genes increased the mode MIC, but not MBC, to benzalkonium chloride, while only qacA and qacB increased the chlorhexidine mode MIC. Isolates with a wild-type norA promoter or mutations in the norA promoter had similar biocide MIC distributions; notably, not all clinical isolates with norA mutations were resistant to fluoroquinolones. In vitro efflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region of norA, but these were distinct from this region of the clinical isolates. Still, none of the in vitro mutants displayed fitness defects in a killing assay in Galleria mellonella larvae. In conclusion, our data provide an in-depth comparative overview on efflux in S. aureus mutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, current in vitro tests appear not to be suitable for predicting levels of resistance that are clinically relevant.

A novel resistance mechanism to triclosan that suggests horizontal gene transfer and demonstrates a potential selective pressure for reduced biocide susceptibility in clinical strains of Staphylococcus aureus.

The widely used biocide triclosan selectively targets FabI, the NADH-dependent trans-2-enoyl-acyl carrier protein reductase, which is an important target for narrow-spectrum antimicrobial drug development. In relation to the growing concern about biocide resistance, we compared in vitro mutants and clinical isolates of Staphylococcus aureus with reduced triclosan susceptibility. Clinical isolates of S. aureus as well as laboratory-generated mutants were assayed for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) phenotypes and genotypes related to reduced triclosan susceptibility. A potential epidemiological cut-off (ECOFF) MBC of >4 mg/L was observed for triclosan in clinical isolates of S. aureus. These showed significantly lower MICs and higher MBCs than laboratory mutants. These groups of strains also had few similarities in the triclosan resistance mechanism. Molecular analysis identified novel resistance mechanisms linked to the presence of an additional sh-fabI allele derived from Staphylococcus haemolyticus. The lack of predictive value of in-vitro-selected mutations for clinical isolates indicates that laboratory tests in the present form appear to be of limited value. More importantly, detection of sh-fabI as a novel resistance mechanism with high potential for horizontal gene transfer demonstrates for the first time that a biocide could exert a selective pressure able to drive the spread of a resistance determinant in a human pathogen.

Treatment of tuberculosis in a region with high drug resistance: outcomes, drug resistance amplification and re-infection.

INTRODUCTION: Emerging antituberculosis drug resistance is a serious threat for tuberculosis (TB) control, especially in Eastern European countries. METHODS: We combined drug susceptibility results and molecular strain typing data with treatment outcome reports to assess the influence of drug resistance on TB treatment outcomes in a prospective cohort of patients from Abkhazia (Georgia). Patients received individualized treatment regimens based on drug susceptibility testing (DST) results. Definitions for antituberculosis drug resistance and treatment outcomes were in line with current WHO recommendations. First and second line DST, and molecular typing were performed in a supranational laboratory for Mycobacterium tuberculosis (MTB) strains from consecutive sputum smear-positive TB patients at baseline and during treatment. RESULTS: At baseline, MTB strains were fully drug-susceptible in 189/326 (58.0%) of patients. Resistance to at least H or R (PDR-TB) and multidrug-resistance (MDR-TB) were found in 69/326 (21.2%) and 68/326 (20.9%) of strains, respectively. Three MDR-TB strains were also extensively resistant (XDR-TB). During treatment, 3/189 (1.6%) fully susceptible patients at baseline were re-infected with a MDR-TB strain and 2/58 (3.4%) PDR-TB patients became MDR-TB due to resistance amplification. 5/47 (10.6%) MDR- patients became XDR-TB during treatment. Treatment success was observed in 161/189 (85.2%), 54/69 (78.3%) and 22/68 (32.3%) of patients with fully drug susceptible, PDR- and MDR-TB, respectively. Development of ofloxacin resistance was significantly associated with a negative treatment outcome. CONCLUSION: In Abkhazia, a region with high prevalence of drug resistant TB, the use of individualized MDR-TB treatment regimens resulted in poor treatment outcomes and XDR-TB amplification. Nosocomial transmission of MDR-TB emphasizes the importance of infection control in hospitals.

The relationship between group A streptococcal infections and Tourette syndrome: a study on a large service-based cohort.

AIM: To evaluate the relationship between diagnosis and clinical course of Tourette syndrome and group A Streptococcus (GAS). METHOD: GAS infections, anti-streptococcal, and anti-basal ganglia antibodies (ABGA) were compared between 168 patients (136 males, 32 females) with Tourette syndrome; (median [range] age [25th-75th centile] 10y [8-11y]); median Tourette syndrome duration (25th-75th centile), 3y (1y 3mo-5y 9mo) and a comparison group of 177 patients (117 males, 60 females) with epileptic or sleep disorders median age [25th-75th centile], 10y [8y-1y 6mo]). One hundred and forty-four patients with Tourette syndrome were followed up at 3-month intervals; exacerbations of tics, obsessive-compulsive symptoms, and other psychiatric comorbidities were defined by a bootstrap procedure. The effect of new GAS infections and identification of new ABGA upon risk of exacerbation was assessed using logistic regression analysis. RESULTS: Cross-sectionally, patients with Tourette syndrome exhibited a higher frequency of GAS infection (8% vs 2%; p=0.009), higher anti-streptolysin O (ASO) titres (246 [108-432] vs 125 [53-269]; p<0.001), and higher ABGA frequency (25% vs 8%; p<0.001) than the comparison group. On prospective analysis, ASO titres were persistently elevated in 57% of patients with Tourette syndrome; however, new infections or newly identified ABGA did not predict clinical exacerbations (all p>0.05). INTERPRETATION: Patients with Tourette syndrome might be more prone to GAS infections and develop stronger antibody responses to GAS, probably as a result of underlying immune dysregulation. New GAS infections are unlikely to exert, years after their onset, a major effect upon the severity of neuropsychiatric symptoms.

International external quality assurance for laboratory identification and typing of Streptococcus agalactiae (Group B streptococci).

We report the results from the first international multicenter external quality assessment (EQA) studies for molecular and serological typing of group B streptococcus (GBS) strains as part of DEVANI (Design of a Vaccine against Neonatal Infections), a pan-European program. A questionnaire-based surveillance was undertaken among eight laboratories participating in DEVANI and six laboratories not participating in DEVANI from 13 countries in order to assess their current microbiological procedures for GBS screening, diagnosis, and typing. GBS strains from three EQA distributions were characterized using molecular and serological methods based on GBS capsular polysaccharide typing. Participants were asked to test the first distribution using their current serotyping and genotyping methods. The Strep-B-Latex agglutination method was the most widely used method, with a typeability value of >90%. A multiplex PCR assay for GBS capsular gene typing was also used by 2 of 14 centers, which achieved a typeability value of 93%; this assay detected only 9 of 10 GBS capsular polysaccharide genes. From the second and third EQA studies, standardized protocols were prepared for serological and molecular typing of GBS strains based on the Strep-B-Latex agglutination method and a novel multiplex PCR assay that detected all 10 GBS capsular types (Ia to IX). These standardized protocols are being used by many European laboratories, and as the use of these methods increases, it is imperative to continuously improve and assess laboratory performance and offer training to any laboratories that have technical difficulties.

A multiplex PCR assay for the direct identification of the capsular type (Ia to IX) of Streptococcus agalactiae.

A multiplex PCR assay for the identification of serotypes Ia to IX of Streptococcus agalactiae was developed. By using a single PCR reaction containing a mix of 19 primers the assay identified each serotype by the analysis of the unique two or three bands pattern on agarose gel.

Protein array profiling of tic patient sera reveals a broad range and enhanced immune response against Group A Streptococcus antigens.

The human pathogen Group A Streptococcus (Streptococcus pyogenes, GAS) is widely recognized as a major cause of common pharyngitis as well as of severe invasive diseases and non-suppurative sequelae associated with the existence of GAS antigens eliciting host autoantibodies. It has been proposed that a subset of paediatric disorders characterized by tics and obsessive-compulsive symptoms would exacerbate in association with relapses of GAS-associated pharyngitis. This hypothesis is however still controversial. In the attempt to shed light on the contribution of GAS infections to the onset of neuropsychiatric or behavioral disorders affecting as many as 3% of children and adolescents, we tested the antibody response of tic patient sera to a representative panel of GAS antigens. In particular, 102 recombinant proteins were spotted on nitrocellulose-coated glass slides and probed against 61 sera collected from young patients with typical tic neuropsychiatric symptoms but with no overt GAS infection. Sera from 35 children with neither tic disorder nor overt GAS infection were also analyzed. The protein recognition patterns of these two sera groups were compared with those obtained using 239 sera from children with GAS-associated pharyngitis. This comparative analysis identified 25 antigens recognized by sera of the three patient groups and 21 antigens recognized by tic and pharyngitis sera, but poorly or not recognized by sera from children without tic. Interestingly, these antigens appeared to be, in quantitative terms, more immunogenic in tic than in pharyngitis patients. Additionally, a third group of antigens appeared to be preferentially and specifically recognized by tic sera. These findings provide the first evidence that tic patient sera exhibit immunological profiles typical of individuals who elicited a broad, specific and strong immune response against GAS. This may be relevant in the context of one of the hypothesis proposing that GAS antigen-dependent induction of autoantibodies in susceptible individuals may be involved the occurrence of tic disorders.

Characteristics of drug-resistant tuberculosis in Abkhazia (Georgia), a high-prevalence area in Eastern Europe.

Although multidrug-resistant (MDR) tuberculosis (TB) is a major public health problem in Eastern Europe, the factors contributing to emergence, spread and containment of MDR-TB are not well defined. Here, we analysed the characteristics of drug-resistant TB in a cross-sectional study in Abkhazia (Georgia) between 2003 and 2005, where standard short-course chemotherapy is supplemented with individualized drug-resistance therapy. Drug susceptibility testing (DST) and molecular typing were carried out for Mycobacterium tuberculosis complex strains from consecutive smear-positive TB patients. Out of 366 patients, 60.4% were resistant to any first-line drugs and 21% had MDR-TB. Overall, 25% of all strains belong to the Beijing genotype, which was found to be strongly associated with the risk of MDR-TB (OR 25.9, 95% CI 10.2-66.0) and transmission (OR 2.8, 95% CI 1.6-5.0). One dominant MDR Beijing clone represents 23% of all MDR-TB cases. The level of MDR-TB did not decline during the study period, coinciding with increasing levels of MDR Beijing strains among previously treated cases. Standard chemotherapy plus individualized drug-resistance therapy, guided by conventional DST, might be not sufficient to control MDR-TB in Eastern Europe in light of the spread of "highly transmissible" MDR Beijing strains circulating in the community.

Sequence variation in group A Streptococcus pili and association of pilus backbone types with lancefield T serotypes.

BACKGROUND: We previously reported that group A Streptococcus (GAS) pili are the T antigens described by Rebecca Lancefield. We also showed that these pili, constituted by backbone, ancillary 1, and ancillary 2 proteins, confer protection against GAS challenge in a mouse model. METHODS: We evaluated pilus distribution and conservation by sequencing the subunits of 39 new GAS isolates and used immunoblot analysis and agglutination assays to define the specificity of T sera to pilus subunits. RESULTS: GAS pili are encoded by 9 different islands within which backbone protein, ancillary protein 1, and ancillary protein 2 cluster in 15, 16, and 5 variants, respectively. Immunoblot and agglutination assays revealed that T type is determined by the backbone variant. This observation enabled us to set up a simple polymerase chain reaction assay to define the T type of GAS isolates. CONCLUSIONS: We propose the use of a tee gene sequence typing, analogous to the emm gene typing, as a valuable molecular tool that could substitute for the serological T classification of GAS strains. From our sequence analysis and from recent epidemiological data, we estimate that a vaccine comprising a combination of 12 backbone variants would protect against > 90% of currently circulating strains.

Molecular epidemiology and distribution of serotypes, surface proteins, and antibiotic resistance among group B streptococci in Italy.

Group B streptococci (GBS) comprising three different sets of isolates (31 invasive, 36 noninvasive, and 24 colonizing isolates) were collected in Italy during the years 2002 to 2005. Clonal groups were established by pulsed-field gel electrophoresis (PFGE), and selected isolates were studied by multilocus sequence typing (MLST). GBS isolates were also characterized by classical and molecular techniques for serotyping and protein gene and antibiotic resistance profiling. Some serotypes were significantly associated with a particular isolate population: serotype Ia more frequently corresponded to invasive strains than other strains, serotype V was more frequently encountered among noninvasive strains, and nontypeable strains were more common among isolates from carriers. Four major clonal groups accounted for 52.7% of all isolates: PFGE type 1/clonal complex 1 (CC1) comprised mainly serotype V isolates carrying the alp3 gene, PFGE type 2/CC23 encompassed serotype Ia isolates with the alp1 or alpha gene, PFGE type 3/CC17 comprised serotype III isolates carrying the rib gene, and PFGE type 4/CC19 consisted mainly of serotype II isolates possessing the rib gene. The same serotypes were shared by isolates of different clonal groups, and conversely, isolates belonging to the same clonal groups were found to be of different serotypes, presumably due to capsular switching by the horizontal transfer of capsular genes. Erythromycin resistance (prevalence, 16.5%; 15 resistant isolates of 91) was restricted to strains isolated from patients with noninvasive infections and carriers, while tetracycline resistance was evenly distributed (prevalence, 68.1%; 62 resistant isolates of 91). Most erythromycin-resistant GBS strains were of serotype V, were erm(B) positive, and belonged to the PFGE type 1/CC1 group, suggesting that macrolide resistance may have arisen both by clonal dissemination and by the horizontal transfer of resistance genes.

emm Types, virulence factors, and antibiotic resistance of invasive Streptococcus pyogenes isolates from Italy: What has changed in 11 years?

To investigate the epidemiology and characteristics of invasive group A streptococcal (GAS) disease over 11 years in Italy, this study compared the emm types and the superantigen toxin genes speA and speC as well as the erythromycin, clindamycin, and tetracycline susceptibilities of 207 invasive GAS strains collected during two national enhanced surveillance periods (1994 to 1996 and 2003 to 2005) and the time between each set of surveillance periods. The present study demonstrated that emm1 strains were consistently responsible for about 20% of invasive GAS infections, while variations in the frequencies of the other types were noted, although the causes of most cases of invasive infections were restricted to emm1, emm3, emm4, emm6, emm12, and emm18. During the 1994 to 1996 surveillance period, an emm89 epidemic clone spread across the northern part of Italy. A restricted macrolide resistance phenotype-type distribution of the bacteriophage-encoded speA toxin as well as of macrolide resistance genes was noted over time. Indeed, the recent acquisition of macrolide resistance in previously susceptible emm types was observed.

A post-streptococcal pathogenesis in children with tic disorders is suggested by a color Doppler echocardiographic study.

BACKGROUND: A post-streptococcal autoimmune mechanism, similar to that of rheumatic fever or Sydenham's chorea, has been hypothesized in some cases of neuropsychiatric disorder (tics and/or obsessive-compulsive disorders). A few studies on the involvement of other organs, outside the central nervous system, have been performed in these patients. AIM: To evaluate a possible post-streptococcal pathogenesis in the children affected by tic disorders and showing sign of streptococcal exposure. METHODS: A case-control study was performed at the Outpatient Division of the Child Neurology and Psychiatry, and Paediatrics Departments of the University "La Sapienza" of Rome, from September 1, 2000, to February 28, 2005. Forty-eight subjects affected by tic disorder, aged 4-16 years, with signs of a recent or intercurrent exposure to streptococcal antigens, and 18 age-matched patients affected by tic disorder but without evidence of streptococcal exposure were examined by Color doppler echocardiography. RESULTS: The rate of echocardiographic abnormalities was significantly higher (p<0.001) in the patients with sign of streptococcal exposure. In 28 out of 48 patients (58.3%), the color Doppler echocardiography showed abnormalities: 26 patients (54,3%) had a mitral regurgitation, 1 (2%) a mitral valve prolapse and finally 1 (2%) showed a kinking of the anterior mitral valve leaflet. In the control group, four children (22.2%) showed a mitral regurgitation. All of these abnormalities were not hemodynamically significant, and in many cases decreased with time. CONCLUSIONS: The higher rate of echocardiographic abnormalities observed in patients with tic disorder and exposure to group A beta-haemolytic streptococcal antigens, together with their decrease with time, suggest a post-streptococcal pathogenesis.

Detection of genes encoding internalization-associated proteins in Streptococcus pyogenes isolates from patients with invasive diseases and asymptomatic carriers.

A total of 161 Streptococcus pyogenes isolates from patients with invasive infections or from asymptomatic carriers were examined for genes (prtF1, prtF2, and fba) coding for fibronectin-binding proteins to evaluate their involvement in the pathogenesis of different streptococcal manifestations. We found no significant differences in the presence of these three genes between the two groups. Overall, the prtF2 gene was present in similar percentages among strains from both sources (61% versus 63%). Strains carrying the gene fba were slightly more common among those isolated from asymptomatic carriers (72.6% versus 65%). Also, the prtF1 gene was present in a higher, but not significant, percentage among strains from throat swabs than among isolates from invasive infections (75% versus 64.9%). However, this more detailed characterization of the genes encoding fibronectin-binding proteins allowed us to identify a strong association of genes of the erm class, coding for macrolide resistance, with prtF1 and prtF2 rather than with prtF1 alone. Since macrolide resistance was significantly associated with throat swab isolates, it may be hypothesized that proteins coded by prtF1 and prtF2 genes may be synergic in providing support for cell invasion and/or colonizing or persistence efficiency.