Amplification and sequence analysis of the 16S rRNA gene can be applied to detect and identify bacteria in clinical samples. We examined 75 clinical samples (17 culture-positive, 58 culturenegative) prospectively by two different PCR protocols, amplifying either a single fragment (1343 bp) or two fragments (762/598 bp) of the 16S rRNA gene. The 1343 bp PCR and 762/ 598 bp PCRs detected and identified the bacterial 16S rRNA gene in 23 (31 %) and 38 (51 %) of the 75 samples, respectively. The 1343 bp PCR identified 19 of 23 (83 %) PCR-positive samples to species level while the 762/598 bp PCR identified 14 of 38 (37 %) bacterial 16S rRNA gene fragments to species level and 24 to the genus level only. Amplification of shorter fragments of the bacterial 16S rRNA gene (762 and 598 bp) resulted in a more sensitive assay; however, analysis of a large fragment (1343 bp) improved species discrimination. Although not statistically significant, the 762/598 bp PCR detected the bacterial 16S rRNA gene in more samples than the 1343 bp PCR, making it more likely to be a more suitable method for the primary detection of the bacterial 16S rRNA gene in the clinical setting. The 1343 bp PCR may be used in combination with the 762/598 bp PCR when identification of the bacterial rRNA gene to species level is required.

Phylogenetic analysis excluded a common source of infection in this apparent outbreak. Two of 3 patients on the same ward had closely related viruses, raising the possibility of cross-infection despite protective isolation. Analysis of deep sequencing data enabled us to establish the likely direction of nosocomial transmission.

A prospective observational study was conducted to examine whether asymptomatic VZV reactivation occurs in immunocompetent children hospitalized in an ICU and its impact on clinical outcome. A secondary aim was to test the hypothesis that vaccinated children have a lower risk of reactivation than naturally infected children. Forty immunocompetent paediatric ICU patients and healthy controls were enrolled. Patients were prospectively followed for 28 days. Clinical data were collected and varicella exposure was recorded. Admission serum levels of TNF-a, cortisol and VZV-IgG were measured. Blood and saliva samples were collected for VZV-DNA detection via real-time PCR. As a comparison, the detection of HSV-DNA was also examined. Healthy children matched for age and varicella exposure type (infection or vaccination) were also included. VZV reactivation was observed in 17% (7/39) of children. Children with VZV reactivation had extended duration of fever (OR = 1.17; 95% CI, 1.02-1.34). None of the varicella-vaccinated children or healthy controls had detectable VZV-DNA in any blood or saliva samples examined. HSV-DNA was detected in saliva from 33% of ICU children and 2.6% of healthy controls. Among children with viral reactivation, typing revealed wild-type VZV and HSV-1. In conclusion, VZV reactivation occurs in immunocompetent children under severe stress and is associated with prolonged duration of fever.

bThe recent identification of Streptococcus pseudopneumoniae (pseudopneumococcus) has complicated classification schemes within members of the "mitis" streptococcal group. Accurate differentiation of this species is necessary for understanding its disease potential and identification in clinical settings. This work described the use of the competence-stimulatory peptide ComC sequence for identification of S. pseudopneumoniae. ComC sequences from clinical sources were determined for 17 strains of S. pseudopneumoniae, Streptococcus pneumoniae, and Streptococcus oralis. An additional 58 ComC sequences from a range of sources were included to understand the diversity and suitability of this protein as a diagnostic marker for species identification. We identified three pherotypes for this species, delineated CSP6.1 (10/14, 79%), CSP6.3 (3/14, 21%), and SK674 (1/14, 7%). Pseudopneumococcal ComC sequences formed a discrete cluster within those of other oral streptococci. This suggests that the comC sequence could be used to identify S. pseudopneumoniae, thus simplifying the study of the pathogenic potential of this organism. To avoid confusion between pneumococcal and pseudopneumococcal pherotypes, we have renamed the competence pherotype CSP6.1, formerly reported as an "atypical" pneumococcus, CSPps1 to reflect its occurrence in S. pseudopneumoniae.T he mitis group of streptococci includes nasopharyngeal colonizers such as Streptococcus mitis, Streptococcus oralis, Streptococcus pneumoniae, and the recently classified Streptococcus pseudopneumoniae (2). Of these, S. pneumoniae (pneumococcus) is responsible for more than a million deaths annually and is responsible for diseases such as otitis media, pneumonia, septicemia, and meningitis. However, invasive diseases caused by other related viridans group streptococci had been documented (16,26,31).Some strains of S. pseudopneumoniae, along with S. mitis and S. oralis, have often been classified previously as "atypical pneumococci," because of their similarity to S. pneumoniae. These organisms share Ն99% identity in 16S rRNA gene sequences (2,21,43). Optochin sensitivity and bile solubility, the two standard pneumococcal phenotypic identification tests, have proven to be inconclusive for differentiating pneumococci from these atypical strains (3,4,9,10,17,19,20,22,27,29,32,34,38,39,49). Virulence factors that were once thought to be exclusive to the pneumococcus, such as pneumolysin (encoded by ply) and autolysin A (encoded by lytA), have been detected in commensal streptococcal species (18,35,49), compromising their specificity as species identification markers. The pathogenic potential of S. pseudopneumoniae (the pseudopneumococcus) has been demonstrated in a murine model (12) as well as in humans (2,18,23,24,28,40). Rapid, correct identification of this organism in the clinical setting is essential for diagnosis and for understanding its disease potential. A simple, unequivocal method to identify S. pseudopneumoniae would be valuable.Streptococci are competent for gene...

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