The accurate diagnosis of pneumococcal disease has frequently been hampered not only by the difficulties in obtaining isolates of the organism from patient specimens but also by the misidentification of pneumococcuslike viridans group streptococci (P-LVS) as Streptococcus pneumoniae. This is especially critical when the specimen comes from the respiratory tract. In this study, three novel real-time PCR assays designed for the detection of specific sequence regions of the lytA, ply, and psaA genes were developed (lytA-CDC, ply-CDC, and psaA, respectively). These assays showed high sensitivity (<10 copies for lytA-CDC and ply-CDC and an approximately twofold less sensitivity for psaA). Two additional real-time PCR assays for lytA and ply described previously for pneumococcal DNA detection were also evaluated. A panel of isolates consisting of 67 S. pneumoniae isolates (44 different serotypes and 3 nonencapsulated S. pneumoniae isolates from conjunctivitis outbreaks) and 104 nonpneumococcal isolates was used. The 67 S. pneumoniae isolates were reactive in all five assays. The new real-time detection assays targeting the lytA and psaA genes were the most specific for the detection of isolates confirmed to be S. pneumoniae, with lytA-CDC showing the greatest specificity. Both ply PCRs were positive for all isolates of S. pseudopneumoniae, along with 13 other isolates of other P-LVS isolates confirmed to be non-S. pneumoniae by DNA-DNA reassociation. Thus, the use of the ply gene for the detection of pneumococci can lead to false-positive reactions in the presence of P-LVS. The five assays were applied to 15 culture-positive cerebrospinal fluid specimens with 100% sensitivity; and serum and ear fluid specimens were also evaluated. Both the lytA-CDC and psaA assays, particularly the lytA-CDC assay, have improved specificities compared with those of currently available assays and should therefore be considered the assays of choice for the detection of pneumococcal DNA, particularly when upper respiratory P-LVS might be present in the clinical specimen.

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We have identified an unusual group of viridans group streptococci that resemble Streptococcus pneumoniae. DNA-DNA homology studies suggested that a subset of these isolates represent a novel species that may be included in the S. oralis-S. mitis group of viridans group streptococci. We suggest that this novel species be termed Streptococcus pseudopneumoniae. A combination of phenotypic and genetic reactions allows its identification. S. pseudopneumoniae strains do not have pneumococcal capsules, are resistant to optochin (inhibition zones, less than 14 mm) when they are incubated under an atmosphere of increased CO 2 but are susceptible to optochin (inhibition zones, >14 mm) when they are incubated in ambient atmospheres, are not soluble in bile, and are positive by the GenProbe AccuProbe Pneumococcus test. The bile solubility test is more specific than the optochin test for identification of S. pneumoniae. Genetic tests for pneumolysin (ply) and manganesedependent superoxide dismutase (sodA) and identification tests with a commercial probe, AccuProbe Pneumococcus, do not discriminate between the new species and S. pneumoniae.Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is also associated with bacteremia, meningitis, otitis media, and sinusitis, accounting for approximately 3,000 cases of meningitis, 50,000 cases of bacteremia, 500,000 cases of pneumonia, and 7 million cases of otitis media each year in the United States (1). Clinical laboratories must be able to accurately differentiate S. pneumoniae from other viridans group streptococci commonly found in clinical samples. An inability to correctly identify S. pneumoniae may result in inappropriate antimicrobial therapy.S. pneumoniae is a member of the Streptococcus mitis-Streptococcus oralis group (the Smit group) of viridans group streptococci, which includes S. mitis, S. oralis, Streptococcus cristatus, Streptococcus infantis, and Streptococcus peroris. Differentiation of S. pneumoniae from other viridans group streptococci, including members of the Smit group, has conventionally been based on phenotypic characteristics, most commonly by demonstrating optochin (OPT) susceptibility and/or solubility in bile (sodium deoxycholate) (18). Identification by these two tests is satisfactory when isolates from sterile sites are tested (16). However, identification is often problematic when samples from nonsterile sites, such as respiratory samples, are tested. The results of phenotypic testing can result in ambiguous OPT susceptibility and bile solubility (BS) test results (18).Although they are uncommon, OPT resistance has been reported in S. pneumoniae and OPT-resistant subpopulations have also been reported, both of which may lead to problems in identification, especially in association with atypical colony morphologies (4,13,36,42,45,46,55,60). Gardam and Miller (21) reported that variances in OPT zone sizes are dependent on the test medium and incubation environment used, as some media and environments may result in f...

Deoxyribonucleic acid hybridization (hydroxyapatite method, 55 and 70°C) was used to characterize 38 serovars from 22 named serogroups of Leptospira interrogans and Leptospira bijlexa, 6 serovars from 4 new unnamed serogroups of Leptospira interrogans, and single serovars of the proposed species Leptospira parva and Leptonema illini. Deoxyribonucleic acid relatedness confirmed the validity of Leptospira parva and Leptonema illini. The well-accepted species Leptospira interrogans and Leptospira bijlexa, as currently defined, were extremely heterogeneous. Relatedness results revealed at least five new species among the parasitic serovars formerly included in Leptospira interrogans and two new species among the saprophytic serovars formerly included in Leptospira bijlexa. Serogrouping did not equate with species identification, as serovars from several different subserogroups belonged to different species. The new species named in this paper are Leptospira, noguchii, Leptospira weilii, Leptospira santarosai, Leptospira borgpetersenii, Leptospira meyeri, Leptospira wolbachii, and Leptospira inadai.Leptospiraceae is a family in the Spirochaetales whose type genus is Leptospira (7, 12, 14). The three Leptospira species are Leptospira interrogans, which contains a large number of serogroups whose strains are parasitic or pathogenic for humans and animals (14); Leptospira bijlexa, which contains a large number of serogroups whose strains are primarily found in fresh surface waters and moist soil and are rarely isolated from humans or animals (14); and Leptospira parva, which was isolated from tap water, is not pathogenic for hamsters, and is biochemically intermediate between L . interrogans and L . bijlexa (13). Leptonema, with the single species Leptonema illini, was proposed as a second genus in the Leptospiraceae (12). Leptonema illini deoxyribonucleic acid (DNA) has a guanine plus cytosine (G+C) content of 51 to 53 mol% (12), compared with 47.4 mol% for Leptospira parva (13), 36 to 39 mol% for Leptospira bijlexa (14), and 35 to 41 mol% for Leptospira interrogans (14). Leptonema possesses cytoplasmic tubules which are absent in Leptospira, and the structure of the basal complex on its flagella resembles that of gram-positive bacteria, whereas this structure in Leptospira resembles that of gram-negative bacteria (12). Leptonema strains were isolated from the urine of a healthy bull, a turtle, and water. They are not pathogenic for animals and are biochemically similar to Leptospira bijexa except for their ability to grow in Trypticase soy broth (1, 12). In addition to pathogenicity, Leptospira interrogans differs from Leptospira bijlexa by its inability to grow in the presence of 8-azaguanine and its inability to grow at 13°C (14-17). Two biogroups within Leptospira interrogans differ in their ability to grow in the presence of 2,6-diaminopurine (14). Leptospira parva does not grow in the presence of either of the above chemicals and does grow at 13°C (13). In general, the phenotypic characteristics that reportedly d...