The investigation of invasive diseases due to S. pyogenes (iGAS) was studied for the first time in Greece in the line of enhanced surveillance. The results of the initial three years (2003 to 2005), of this study are present here. During this period 101 cases from several geographic areas in Greece were studied, covering approximately 38% of the total Greek population (census paper 2001: 4,186,410). Epidemiological and clinical information collected included: age, gender, specimen type, date of isolation, clinical manifestations, treatment, outcome within seven days and risk factors. Antibiotic sensitivity was performed by measuring the MICs for erythromycin, clindamycin and tetracycline using the E-test method. MLSB resistance phenotypes were screened by double-disk diffusion susceptibility test. The presence of erythromycin resistance gene was detected using the polymerase chain reaction (PCR). Multiplex PCR was performed for detection of speA, speB and speC toxin genes. The M-typing was performed by sequencing part of emm gene. PFGE for gonotypic typing was performed after SmaI restriction of total DNA. Sixty eight children 68 (67.3 %) and thirty three adults (32.7%) were included in the study. Age range was between 6 months and 90 years (mean 19.8 years). The average annual incidence was 0.8 cases/100,000 inhabitants/year. The higher incidence was recorded among the age groups 5-9 years, 3-4 years (0.64, 0.45, cases/100,000 inhabitants/year, respectively). The total incidence presented seasonal distribution with high predominance in early spring and summer. iGAS strain were isolated from blood, deep abscesses, wound, synovial fluid, pleural fluid, cerebrospinal fluid and bronchoalveolar lavage in 42, 35, 8, 7, 4, 3 and 1% of the cases, respectively. Twelve isolates (11.9%) were resistant to erythromycin (EryR, MIC ? 0.5 mg/L). Six of these (5.9% of the total) expressed the M phenotype, and four (4.0% of the total) the inducible resistance, IRMLSB, phenotype. The remaining two EryR isolates (2.0% of the total) also expressed clindamycin resistance (MIC >256 mg/L) and the constitutive resistance, CR-MLSB, phenotype. Twenty six isolates (25.7%) were non-susceptible to tetracycline(TetR, MIC ? 4 mg/L). Only five isolates (5.0%) were resistant to both erythromycin and tetracycline. All four M phenotype isolates were positive only for the mefA gene, the IR-MLSB isolates were positive only for ermTR and the two CR-MLSB isolates were positive only for ermB. These resistance genes were not detected among the remaining 89 susceptible (EryS) strains. Regarding the three spe toxin genes tested –speA, speB and speC– most of the isolates possessed both speB and speC. 32 isolates only the spe4 gene (31.7%), 25 isolates (24.7%) both speB+speA, while 41 isolates (40.6%) both speB+speC. Three isolates (3.0%) possessed the genes speB+speC+speA. All but four of the 101 isolates were typed by the emm-gene sequencing protocol and assigned to a known emm-type. The most frequent types (?5%) were emm-type 1, 12, 4, 6 and 95, (26.7%, 8.9%, 5.0%, 5.0% and 5.0% respectively). All 27 emm-1 strains were fully susceptible to all antimicrobial agents tested. All but two emm-1 strains and only two of the remaining emm-types possessed speA gene. The mef gene was found in emm-types 4, 12 and 75). The emm-types 11, 19, 50, 80, 101, 108, 113, and 117 were found only in adults while the types 22, 75, 77, 78, 83 and 115 only among pediatric patients. emm-types 1, 3, 6 and 29 were more frequently found among STSS cases. XIV Most frequent clinical manifestations were cellulitis, bacteremia, necrotizing fasciitis/myositis, toxic shock (STSS), arthritis, pneumonia, pleurisy, osteomyelitis, and meningitis, in 52.5%, 45.5%, 22.0%, 11.0%, 9.0%, 7%, 4%, 4% and 3% of cases, respectively. Bacteremia was more frequently among adults (p=0,054). The most frequent risk factors were trauma/skin lesion (27.5%), Varicella virus infection (19.2%), surgical operation (10.8), immunosupression (5.8%), tonsillitis (5.8%), malignancy (5.8%), steroid use (4.2%) and drug abuse (3.3%). Varicella infection was detected as a risk factor only among children although a marked decrease was noticed during the last year of the study, possibly due to the recently established Varicella vaccination among pediatric population in Greece. Trauma/skin lesion was dominant among adults. Immunosupression and malignancy were more frequent among adults than children. Children were treated more frequently with surgery (27.3 %) (p=0.003). Only 2 cases were fatal. Both were with meningitidis ant STSS and isolated stains belonged to emm-1 type. Fatality rate among children was 2.9 %. PFGE yielded a total of 50 chromosomal fingerprints, and in general proved more discriminatory than emm-typing (which produced 28 types). Given the fact that a single PFGE type correspond to only a specific emm-type, this study confirm the place of other researchers that PFGE-typing may in fact predict emm- types among S. pyogenes isolates. In conclusion, the erythromycin rate (11.9%) reported here is lower than those reported in previous Greek studies, which concerned mostly isolates from superficial infections. The erythromycin resistance was mostly due to the presence of mef gene, responsible for efflux mechanism. Tetracycline resistance was high (26%). Half of isolates belonged to five emm-types:1, 12, 4, 6 and 95. All emm-1 strains were susceptible to erythromycin. Differences were detected between pediatric and adult cases of iGAS infections in Greece. PFGE in general proved more discriminatory than emm-typing, and subdivided (PFGE-subtypes) further within specific emm-types. Both methods provide useful information in the epidemiological study and the molecular investigation of invasive GAS infections and none of them can substitute the other.
