classified 97 consecutive clinical isolates of streptococci, including 34 isolates of VGS, with biochemical methods as the reference method (26). Ninety-one percent of the streptococcal isolates showed agreement between the Phoenix and the reference method. Of the 12 S. mitis group isolates tested, the Phoenix system correctly identified seven isolates, with two discordant identifications, and there were three isolates for which the Phoenix system did not produce any identification. Of 22 S. anginosus group isolates, 18 were correctly identified and four were discordant. A second study evaluated the Phoenix SMIC/ID-2 panel using the API 20 Strep system as a comparator method (resolving discrepant results via 16S rRNA gene sequencing and amplification and sequencing of housekeeping genes) (5). For the VGS, 31 isolates were assayed, with only 53% concordance between the Phoenix and reference methods for S. mitis group organisms, 100% concordance for the S. anginosus group, and 75% concordance for S. sanguinis group organisms (5). Sequence-based identification Historically, DNA–DNA hybridization studies have been used to confirm species level identifications for the VGS. However, these procedures are not practical for clinical laboratories to use for identification of these organisms. Other sequence-based identification systems have subsequently been introduced for VGS species level identification. In general, 16S rRNA gene sequencing results in poor resolution to species level in the VGS. This is due to the high degree of 16S rRNA gene homology in this group of organisms; S. mitis, S. oralis, S. pseudopneumoniae, and S. pneumoniae almost always have >99% sequence homology in this gene. In light of the high degree of 16S rRNA gene sequence similarity, sequencing of alternative gene targets for reliable identification to the species level has been explored. One promising target, rnpB, was explored by Innings et al., who analyzed two variable regions of rnpB by pyrosequencing (28). Of the 43 species analyzed, all were identified to species level, except for two isolates: S. anginosus/S. constellatus and S. infantis/S. peroris. rnpB can be used for identification of VGS with high-level resolution. One other successful approach is sequence analysis of the manganese-dependent superoxide dismutase gene, described by Poyart et al. This technique was used to accurately differentiate over 29 streptococcal species, including 16 VGS species, with clear differentiation of S. mitis, S. oralis, and S. pneumoniae (44, 45). Other techniques that have been used, with various degrees of success, are sequence analysis of the 16S-23S intergenic spacer region, d-alanine-d-alanine ligase