Ralf René Reinert

Epidemiology of Severe Streptococcus pyogenes Disease in Europe

ABSTRACT The past 2 decades have brought worrying increases in severe Streptococcus pyogenes diseases globally. To investigate and compare the epidemiological patterns of these diseases within Europe, data were collected through a European Union FP-5-funded program (Strep-EURO). Prospective population-based surveillance of severe S. pyogenes infection diagnosed during 2003 and 2004 was undertaken in 11 countries across Europe (Cyprus, the Czech Republic, Denmark, Finland, France, Germany, Greece, Italy, Romania, Sweden, and the United Kingdom) using a standardized case definition. A total of 5,522 cases were identified across the 11 countries during this period. Rates of reported infection varied, reaching 3/100,000 population in the northern European countries. Seasonal patterns of infection showed remarkable congruence between countries. The risk of infection was highest among the elderly, and rates were higher in males than in females in most countries. Skin lesions/wounds were the most common predisposing factor, reported in 25% of cases; 21% had no predisposing factors reported. Skin and soft tissue were the most common foci of infection, with 32% of patients having cellulitis and 8% necrotizing fasciitis. The overall 7-day case fatality rate was 19%; it was 44% among patients who developed streptococcal toxic shock syndrome. The findings from Strep-EURO confirm a high incidence of severe S. pyogenes disease in Europe. Furthermore, these results have identified targets for public health intervention, as well as raising awareness of severe S. pyogenes disease across Europe.

Antimicrobial Susceptibility of Streptococcus pneumoniae in Eight European Countries from 2001 to 2003

ABSTRACT Susceptibility testing results for Streptococcus pneumoniae isolates (n = 2,279) from eight European countries, examined in the PneumoWorld Study from 2001 to 2003, are presented. Overall, 24.6% of S. pneumoniae isolates were nonsusceptible to penicillin G and 28.0% were resistant to macrolides. The prevalence of resistance varied widely between European countries, with the highest rates of penicillin G and macrolide resistance reported from Spain and France. Serotype 14 was the leading serotype among penicillin G- and macrolide-resistant S. pneumoniae isolates. One strain (PW 158) showed a combination of an efflux type of resistance with a 23S rRNA mutation (A2061G, pneumococcal numbering; A2059G, Escherichia coli numbering). Six strains which showed negative results for mef(A) and erm(B) in repeated PCR assays had mutations in 23S rRNA or alterations in the L4 ribosomal protein (two strains). Fluoroquinolone resistance rates (levofloxacin MIC ≥ 4 μg/ml) were low (Austria, 0%; Belgium, 0.7%; France, 0.9%; Germany, 0.4%; Italy, 1.3%; Portugal, 1.2%; Spain, 1.0%; and Switzerland, 0%). Analysis of quinolone resistance-determining regions showed eight strains with a Ser81 alteration in gyrA; 13 of 18 strains showed a Ser79 alteration in parC. The clonal profile, as analyzed by multilocus sequence typing (MLST), showed that the 18 fluoroquinolone-resistant strains were genetically heterogeneous. Seven of the 18 strains belonged to new sequence types not hitherto described in the MLST database. Europe-wide surveillance for monitoring of the further spread of these antibiotic-resistant S. pneumoniae clones is warranted.

Reduction in the incidence of invasive pneumococcal disease after general vaccination with 7-valent pneumococcal conjugate vaccine in Germany

General vaccination with the 7-valent pneumococcal conjugate vaccine was recommended in Germany in July 2006 for all children <2 years. The proportion of reported invasive pneumococcal disease (IPD) caused by vaccine serotypes before vaccine introduction was considerably lower than in the US. We report data from nationwide surveillance of IPD in children with two reporting sources, pediatric hospitals and microbiological laboratories in Germany. Incidence rates with regard to age groups and pneumococcal serotypes are based on capture recapture estimates combining the two reporting sources. Between July 1, 1997 and June 30, 2003, 2680 cases (an average 447 yearly cases) of IPD were observed in children <16 years in Germany compared to 223 cases between July 1, 2007 and June 30, 2008. A significant reduction in overall incidence (4/100,000-3.2/100,000) was attributed to significant reductions in children younger than 2 years (20.0/100,000-11.0/100,000). While the incidence of all serotypes included in the vaccine was reduced in the age group <2 years, the incidence of non-vaccine serotypes remained stable. These data show a first success of the pneumococcal vaccination program in Germany. Further changes in incidence and serotype distribution of IPD are subject to future surveillance.

Macrolide-Resistant Streptococcus pneumoniae and Streptococcus pyogenes in the Pediatric Population in Germany during 2000-2001

ABSTRACT In a nationwide study in Germany covering 13 clinical microbiology laboratories, a total of 307 Streptococcus pyogenes (mainly pharyngitis) and 333 Streptococcus pneumoniae (respiratory tract infections) strains were collected from outpatients less than 16 years of age. The MICs of penicillin G, amoxicillin, cefotaxime, erythromycin A, clindamycin, levofloxacin, and telithromycin were determined by the microdilution method. In S. pyogenes isolates, resistance rates were as follows: penicillin, 0%; erythromycin A, 13.7%; and levofloxacin, 0%. Telithromycin showed good activity against S. pyogenes isolates (MIC90 = 0.25 μg/ml; MIC range, 0.016 to 16 μg/ml). Three strains were found to be telithromycin-resistant (MIC ≥ 4 μg/ml). Erythromycin-resistant strains were characterized for the underlying resistance genotype, with 40.5% having the efflux type mef(A), 38.1% having the erm(A), and 9.5% having the erm(B) genotypes. emm typing of macrolide-resistant S. pyogenes isolates showed emm types 4 (45.2%), 77 (26.2%), and 12 (11.9%) to be predominant. In S. pneumoniae, resistance rates were as follows: penicillin intermediate, 7.5%; penicillin resistant, 0%; erythromycin A, 17.4%; and levofloxacin, 0%. Telithromycin was highly active against pneumococcal isolates (MIC90 ≤ 0.016 μg/ml; range, 0.016 to 0.5 μg/ml). The overall resistance profile of streptococcal respiratory tract isolates is still favorable, but macrolide resistance is of growing concern in Germany.

Increased titers of antibodies against streptococcal M12 and M19 proteins in patients with Tourette's syndrome

It has been suggested that a post-streptococcal autoimmune process may be involved in the pathogenesis of a subgroup of children with tics and obsessive-compulsive symptoms (PANDAS). Elevated antibody titers against streptococcal antigens have also been described in adult patients suffering from Tourettes syndrome (TS). In order to characterise further streptococcal antigens, we focussed on M proteins. M proteins are a major virulence factor of group A streptococci and known to evoke an immunologic cross-reaction with diverse epitopes of human tissue including brain tissue. Therefore, antibodies against M proteins may play a role in the pathophysiology of at least a subgroup of TS patients. Antibodies against M proteins were studied in 25 adult patients suffering from TS and 25 healthy controls after careful medical examination. The antibody titers against the peptides M1, M4, M6, M12 and M19 were estimated by ELISA. Our results show increased titers of antibodies against the streptococcal M12 and M19 proteins in TS patients as compared with controls, while antibody titers against M1, M4 and M6 did not differ between the TS and control groups. Elevated serum titers of antibodies against M12 and M19 proteins support the view that a streptococcus-induced autoimmune process may be involved in TS. The finding of a possible autoimmune origin of TS has implications for both pathophysiology and future therapeutic strategies.

Molecular Epidemiology of Macrolide-Resistant Streptococcus pneumoniae Isolates in Europe

ABSTRACT In many European countries, the level of pneumococcal resistance to macrolides has now passed the level of resistance to penicillin G. A total of 82 erythromycin A-resistant isolates of Streptococcus pneumoniae were collected by 11 laboratories in seven European countries. All of the isolates were tested for antimicrobial susceptibility, analyzed for clonal relatedness by multilocus sequence typing, and characterized for macrolide resistance genotypes. The prevalence of the macrolide resistance genotypes varied substantially between countries. In France (87.5% of all strains), Spain (77.3%), Switzerland (80%), and Poland (100%), strains were predominantly erm(B) positive, whereas higher levels of mef(A)-positive strains were reported from Greece (100%) and Germany (33.3%). Macrolide resistance was caused by the oligoclonal spread of some multilocus sequence types, but significant differences in clonal distribution were noted between France and Spain, countries from which high levels of macrolide resistance have been reported. Overall, sequence type 81 (Spain23F-1 clone) was by far the most widespread. The mainly erm(B)-positive serotype 14 clone (sequence type 143), first reported in Poland in the mid-1990s, is now widespread in France.

Resistance to Erythromycin and Telithromycin in Streptococcus pyogenes Isolates Obtained between 1999 and 2002 from Greek Children with Tonsillopharyngitis: Phenotypic and Genotypic Analysis

ABSTRACT Since the late 1990s, the prevalence of erythromycin-resistant Streptococcus pyogenes has significantly increased in several European countries. Between January 1999 and December 2002, 1,577 isolates of S. pyogenes were recovered from children with tonsillopharyngitis living in various areas of Western Greece. Erythromycin resistance was observed in 379 (24%) of the 1,577 isolates. All erythromycin-resistant strains along with 153 randomly selected erythromycin-susceptible S. pyogenes isolates were tested for their antimicrobial susceptibility, resistance phenotypes, and genotypes. Representative isolates underwent emm gene sequence typing. Isolates with reduced susceptibility to telithromycin (MIC, ≥2 μg/ml) were studied for multilocus sequence type, L22, L4, and 23S rRNA mutations. Of the total 379 erythromycin-resistant isolates, 193 (50.9%) harbored the mef(A) gene, 163 (43%) erm(A), 1 (0.3%) mef(A) plus erm(A), and 22 (5.8%) the erm(B) gene. Among the erythromycin-susceptible isolates, emm 1 (25%), emm 2 (12.5%), and emm 77 (12.5%) predominated. Furthermore, among the erythromycin-resistant isolates, emm 4 (30.6%), emm 28 (22.2%), and emm 77 (12.5%) prevailed. Resistance to telithromycin was observed in 22 (5.8%) of the erythromycin-resistant isolates. Sixteen (72.7%) of the 22 isolates appeared to be clonally related, since all of them belonged to emm type 28 and multilocus sequence type 52. One of the well-known mutations (T2166C) in 23S rRNA, as well as a new one (T2136C), was detected in erythromycin- and telithromycin-resistant isolates. High incidence of macrolide resistance and clonal spread of telithromycin resistance were the characteristics of the Greek S. pyogenes isolates obtained from 1999 to 2002.

Ribosomal Mutations Conferring Resistance to Macrolides in Streptococcus pneumoniae Clinical Strains Isolated in Germany

ABSTRACT Among a collection of 4,281 pneumococcal isolates, 7 strains isolated in Germany had an unusual macrolide resistance phenotype. The isolates were found to have multiple mutations in the 23S rRNA and alterations in the L4 ribosomal protein. One strain had an amino acid alteration in the L22 ribosomal protein.

Antimicrobial resistance of Streptococcus pneumoniae recovered from outpatients with respiratory tract infections in Germany from 1998 to 1999: results of a national surveillance study.

ABSTRACT Clinically significant pneumococcal isolates were prospectively collected from outpatients with respiratory tract infections by 19 different clinical microbiology laboratories in Germany. Resistance rates in a total of 961 isolates were as follows: penicillin, 6.6%; clarithromycin, 10.6%; tetracycline, 13.9%; and levofloxacin, 0.1%. Among 324 isolates from children, pneumococcal serotypes 19F (17.0%), 23F (13.0%), and 6B (11.7%) were the predominant types.

Susceptibility to tuberculosis is associated with TLR1 polymorphisms resulting in a lack of TLR1 cell surface expression

Human TLR1 plays an important role in host defense against Mycobacterium tuberculosis. Our aim was to analyze the association of the loss of TLR1 surface expression and TLR1 SNPs with susceptibility to TB. TLR1neg and TLR1pos cells from healthy individuals were identified by flow cytometry and compared by sequencing. TLR1 expression was measured using quantitative real‐time PCR and immunoblotting. TLR1 SNP analyses of healthy individuals and TB patients from EU‐C and Ghana were performed, and association of the TLR1 genotypes with increased risk of developing TB was statistically evaluated. Lack of TLR1 surface expression accompanied by impaired function was strongly associated with TLR1 SNP G743A. Genotyping of EU‐C controls and TB patients revealed an association of TLR1 743A/1805G alleles [OR 2.37 (95% CI 1.13, 4.93), P=0.0219; OR 2.74 (95% CI 1.26, 6.05), P=0.0059] as well as TLR1neg 743AA/1805GG versus TLR1pos genotypes 743AG/1805TG [OR 4.98 (95% CI 1.64, 15.15), P=0.0034; OR 5.70 (95% CI 1.69, 20.35), P=0.0015] and 743AG + GG/1805TG + TT [OR 3.54 (95% CI 1.29, 9.90), P=0.0086; OR 4.17 (95% CI 1.52, 11.67), P=0.0025] with increased susceptibility to TB. No association of G743A with TB was found in Ghana as a result of a low frequency of genotype 743AA. Our data gain new insights in the role of TLR1 in M. tuberculosis defense and provide the first evidence that TLR1 variants are associated with susceptibility to TB in a low‐incidence country.