A comparison of different pre-lysis methods and extraction kits for recovery of Stretococcus agalacticae (Lancefield group B Streptococcus) DNA from whole blood

Sub-optimal recovery of bacterial DNA from whole blood samples can limit the sensitivity of molecular assays to detect pathogenic bacteria. We compared 3 different pre-lysis protocols (none, mechanical pre-lysis and achromopeptidasepre-lysis) and 5 commercially available DNA extraction platforms for direct detection of Group B Streptococcus (GBS) in spiked whole blood samples, without enrichment culture. DNA was extracted using the QIAamp Blood Mini kit (Qiagen), UCP Pathogen Mini kit (Qiagen), QuickGene DNA Whole Blood kit S (Fuji), Speed Xtract Nucleic Acid Kit 200 (Qiagen) and MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics Corp). Mechanical pre-lysis increased yields of bacterial genomic DNA by 51.3 fold (95% confidence interval; 31.6–85.1, p < 0.001) and pre-lysis with achromopeptidase by 6.1 fold (95% CI; 4.2–8.9, p < 0.001), compared with no pre-lysis. Differences in yield dueto pre-lysis were 2–3 fold larger than differences in yield between extraction methods. Including a pre-lysis step can improve the limits of detection of GBS using PCR or other molecular methods without need for culture.

The common vaginal commensal bacterium Ureaplasma parvum is associated with chorioamnionitis in extreme preterm labour

OBJECTIVE: To assess the association of vaginal commensal and low grade pathogenic bacteria including Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Group B Streptococcus (GBS), and Gardnerella vaginalis, in women who delivered preterm at less than 37 weeks gestation in the presence or absence of inflammation of the chorioamnionitic membranes.

METHODS: A case control study involving women who delivered before 37 weeks gestation with and without inflammation of chorioamnionitic membranes. A total of 57 placental samples were histologically examined for polymorphonuclear leukocyte infiltration of placental tissue for evidence of chorioamnionitis, and by type-specific nucleic acid amplification for evidence of infection with one or more of the target bacteria. Demographic data was collected for each mother.

RESULTS: Amongst the 57 placental samples, 42.1% had chorioamnionitis and 24.6% delivered in the second trimester of pregnancy; U. parvum, U. urealyticum, G. vaginalis and GBS were all detected in the study with respective prevalence of 19.3%, 3.5%, 17.5% and 15.8%; M.genitalium and M. hominis were not detected. U. parvum was significantly associated with chorioamnionitis (p value = 0.02; OR 5.0; (95% CI 1.2-21.5) and was more common in women who delivered in the second (35.7%) compared to the third trimester of pregnancy (13.9%). None of the other bacteria were associated with chorioamnionitis or earlier delivery and all G.vaginalis positive women delivered in the third trimester of pregnancy (p value 0.04).

CONCLUSIONS: The detection of U. parvum in placental tissue was significantly associated with acute chorioamnionitis in women presenting in extreme preterm labour.

Population structure and characterization of viridans group streptococci (VGS) including Streptococcus pneumoniae isolated from adult patients with cystic fibrosis (CF)

A study was undertaken to examine the population structure of viridans group streptococci (VGS) in the sputum of adult patients with cystic fibrosis (CF). Freshly expectorated sputa (n=58) from 45 adult CF patients were examined by selective conventional culture on Mitis-Salivarius agar and yielded 190 isolates of VGS. Sequence analyses of the rpnB and 16-23S rRNA ITS genes identified these isolates to belong to 12 species of VGS and included S. anginosus, S. australis, S. cristatus, S. gordonii, S. infantis, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. pneumoniae, S. salivarius and S. sanguinis. The most frequently VGS organism isolated was S. salivarius (47/190; 24.7%), followed by S. mitts (36/190; 19%), S. sanguinis (25/190; 13.2%), S. oralis (20/190; 11.0%), S. pneumoniae (19/190; 10.0%), S. parasanguinis (16/190; 8.4%), S. infantis (11/190; 5.8%), S. gordonii (7/190; 3.7%), S. anginosus (4/190; 2.1%), S. cristatus (2/190; 1.1%), S. australis (1/190; 0.5%), S. mutans (1/190; 0.5%) and S. agalactiae (1/190; 0.5%). All, but four, patients harboured at least one VGS species, which ranged from one to five streptococcal species, with a mean of 2.85 species per patient. There was no clonality at the subspecies level employing ERIC RAPD PCR. Antibiotic susceptibility was determined by Minimum Inhibitory Concentration (MIC) testing against penicillin, erythromycin and ciprofloxacin. Overall, resistance to penicillin with all VGS was 73/190 (38.4%) and 167/190 (87.9%) for erythromycin. With regard to ciprofloxacin, 27/190 (14.2%) were fully resistant, whilst a further 21/190 (11.1%) showed intermediate resistance, which equated to approximately three quarters (74.7%) of isolates being fully sensitive to this agent. In addition, as a comparator control population, we examined antibiotic susceptibility, as above, in a non-CF population comprising 12 individuals (50 VGS isolates), who were not receiving chronic antibiotics. In comparison, 8% and 38% of VGS isolates from non-CF individuals were resistant by disk susceptibility testing to penicillin and erythromycin, respectively. None of the non-CF VGS organisms were resistant to ciprofloxacin, but 42% showed intermediate resistance. (C) 2010 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

An experiment in group learning technology: evaluating critical thinking in face-to-face and computer-supported seminars

Can learning quality be maintained in the face of increasing class size by the use of Computer Supported Co-operative Learning (CSCL) technologies? In particular, can Computer-Mediated Communication promote critical thinking in addition to surface information transfer? We compared face-to-face seminars with asynchronous computer conferencing in the same Information Management class. From Garrison's theory of critical thinking and Henri's critical reasoning skills, we developed two ways of evaluating critical thinking: a student questionnaire and a content analysis technique. We found evidence for critical thinking in both situations, with some subtle differences in learning style. This paper provides an overview of this work.

A content analysis method to measure critical thinking in face-to-face and computer supported group learning

This paper gives a detailed account of the content analysis method developed at Queen's University Belfast to measure critical thinking during group learning, as used in our controlled comparisons between learning in face-to-face and computer conference seminars. From Garrison's 5 stages of critical thinking, and Henri's cognitive skills needed in CMC, we have developed two research instruments: a student questionnaire and this content analysis method. The content analysis relies on identifying, within transcripts, examples of indicators of obviously critical and obviously uncritical thinking, from which several critical thinking ratios can be calculated.