Direct Streptococcus pneumoniae real-time PCR serotyping from pediatric parapneumonic effusions.

BACKGROUND: To determine the serotypes of Streptococcus pneumoniae responsible for pneumonia complicated by parapneumonic effusion in children, we performed real-time PCR based pneumococcal "serotyping" directly on parapneumonic fluid samples. METHODS: Specimens were collected at two children's hospitals in Ontario, Canada from 2009 to 2011. Samples in which S. pneumoniae was detected by PCR were tested with serotype-specific 5'exonuclease PCR assays for the 13 serotypes contained in the 13-serotype pneumococcal vaccine. RESULTS: Thirty-five S. pneumoniae PCR-positive pleural samples were studied. Pneumococcal serotyping PCR assays were positive for 34 of 35 (97%). Serotype 3 was detected most frequently, in 19/35 (54%), followed by serotype 19A in 9/35 (26%), serotype 7 F/A in 4/35 (11%), serotype 1 in 1/35 (3%), and serotype 6A also in 1/35 (3%). CONCLUSIONS: PCR testing demonstrated that the vast majority (97%) of S. pneumoniae parapneumonic effusions were caused by serotypes present in the 13-serotype vaccine that were not present in the original 7 serotype vaccine. This suggests that use of the 13-serotype vaccine could potentially prevent many S. pneumoniae pneumonias complicated by parapneumonic effusion in our region, provided serotype replacement does not occur.

Pulse probiotic administration induces repeated small intestinal Muc3 expression in rats.

Upon ingestion, probiotics may act to protect the host through a number of protective mechanisms including modulation of genes involved in intestinal innate mucosal defense such as epithelial cell-derived mucin glycoproteins and inhibitor of apoptosis proteins. To determine the specificity of effect and sustainability of response in vivo, Lactobacillus plantarum 299v (Lp299v), Lactobacillus rhamnosus R0011 (LrR0011), and Bifidobacterium bifidum R0071 (BbR0071) were added repeatedly or intermittently to the drinking water of Sprague-Dawley rats. After killing the rats via CO2 suffocation, Muc2, Muc3, neuronal apoptosis inhibitor protein (NAIP), human inhibitor of apoptosis protein 1/cellular inhibitor of apoptosis 2 (HIAP1/cIAP2), and human inhibitor of apoptosis protein 2/cellular inhibitor of apoptosis 1 (HIAP2/cIAP1) mRNA and protein levels were analyzed via RT-PCR and immunohistochemistry. Live Lp299v, BbR0071, and LrR0011 increased Muc3 protein and mRNA expression in jejunum and ileum. Heat-killed and a nonadherent derivative of Lp299v failed to induce Muc3 expression. Lp299v did induce expression of HIAP2/cIAP1 and NAIP expression. Muc3 mucin expression was elevated for 5 d after oral administration of Lp299v; however, this effect was not sustained despite ongoing daily ingestion of a probiotic. Intermittent pulse ingestion of probiotics, however, was found to repeatedly increase Muc3 expression. We conclude that selected probiotics can induce protective genes of mucosal intestinal epithelial cells, an effect that is reproducible with pulse probiotic administration.

Rapid detection of Bordetella pertussis and Bordetella parapertussis in clinical and molecular proficiency panel specimens with a novel intercalating dye-based PCR assay.

A novel rapid internally-controlled duplex polymerase chain reaction (PCR) assay for Bordetella pertussis and Bordetella parapertussis that uses a new intercalating dye, LCGreen, for amplicon detection was designed and evaluated using clinical specimens and the 2009 Quality Control for Molecular Diagnostics (QCMD) molecular proficiency testing panel. The sensitivity and specificity of the new PCR assay were equal to that of reference standard uniplex probe-based assays. The assay can be performed in 1 h including DNA extraction. The new assay thus offers a simple and rapid alternative for the detection of B. pertussis and B. parapertussis.

Acute effects of viral respiratory tract infections on sputum bacterial density during CF pulmonary exacerbations.

BACKGROUND: Airway proliferation of Pseudomonas aeruginosa bacteria is thought to trigger CF exacerbations and may be affected by the presence of viral infections. METHODS: A 2-year prospective study was conducted on 35 adults with CF. P. aeruginosa sputum density was analyzed during stable, exacerbation and post exacerbation assessments. Upon exacerbation, samples were sent for PCR detection of respiratory viruses and the sputum density of P. aeruginosa in patients with a viral infection versus those without was compared. RESULTS: Twenty-two patients experienced 30 exacerbations during the study period; 50% were associated with a viral infection. There was no change in sputum density of P. aeruginosa from the stable to exacerbation state when measured by quantitative culture or by PCR. Virus-associated exacerbations did not result in significant increases in P. aeruginosa sputum density compared to non-viral exacerbations. CONCLUSION: Sputum density of P. aeruginosa was not increased at the time of CF exacerbation and was not influenced by the presence of viral infection.

Lactobacillus plantarum 299v Prevents Caspase-Dependent Apoptosis In Vitro.

Selective microbes used as probiotics can enhance epithelial cell protection. We have previously shown that a Lactobacillus plantarum strain 299v (Lp299v) has the ability to induce mucin genes. In the current study, we utilized a cytokine model of inflammation in cell culture to study the modulation of apoptosis by this probiotic. HT-29 cells were pre-incubated with the Lp299v or L. plantarum strain adh- (Lpadh-), a non-adherent derivative of Lp299v. Cells were challenged with a mixture of cytokines (TNF-α, IFN-γ, and IL-1a) to imitate conditions of inflammation. To assess for cell death, we evaluated TUNEL, multi-caspase, and caspase-3 and caspase-7 activity assays. There was a marked decrease in apoptosis as measured by TUNEL(+) cells in samples pre-treated with Lp299v (18.7 ± 4.1%, p < 0.01) and Lpadh- (16.6 ± 3.2%, p < 0.05) prior to cytokine exposure when compared to cells (43.6 ± 6.2%) exposed to the cytokine mixture. Lp299v pre-incubation with HT-29 cells reduced caspase(+) cells in the multi-caspase activity assay (3.6 ± 0.6%, p < 0.05) compared to cells exposed to cytokines (68.9 ± 5.1%) whereas Lpadh- did not (46.8 ± 17.5%, p > 0.05). Similarly, caspase-3, caspase-7 activity was also reduced by Lp299v. Selected probiotics may confer an exogenous protective effect at the mucosal-luminal interface for intestinal epithelial cells via alteration of caspase-dependent apoptotic pathways.