Pneumolysin polymerase chain reaction for diagnosis of pneumococcal pneumonia and empyema in children

Streptococcus pneumoniae is the most important cause of childhood pneumonia and empyema, yet the diagnosis of pneumococcal infections by conventional methods is challenging. In this study, the clinical value of the pneumolysin-targeted real-time polymerase chain reaction (PCR) method for the diagnosis of pneumococcal pneumonia and empyema was evaluated with 33 whole blood samples and 12 pleural fluid samples. The analytical sensitivity of the PCR assay was 4 fg of pneumococcal DNA, corresponding to two genome equivalents of pneumococcal DNA per reaction. The PCR assay correctly detected all clinical isolates of S. pneumoniae tested, whereas all nonpneumococcal bacterial organisms tested were negative by PCR. In a clinical trial, S. pneumoniae was detected by PCR in the pleural fluid of 75% of children with empyema, increasing the detection rate of pneumococcus almost tenfold that of pleural fluid culture. However, in whole blood samples, PCR detected S. pneumoniae in only one child with pneumonia and one child with pneumococcal empyema and failed to detect S. pneumoniae in three children with blood cultures positive for S. pneumoniae. The present data indicate that pneumolysin-targeted real-time PCR of pleural fluid is a valuable method for the etiologic diagnosis of pneumococcal empyema in children. The ease and rapidity of the LightCycler technology (Roche Diagnostics, Mannheim, Germany) make real-time PCR an applicable tool for routine diagnostics. In the evaluation of blood samples, blood culture remains the superior method for the diagnosis of bacteremic pneumococcal disease.     

Diagnostic value of procalcitonin in pleural effusions

This study was to determine the diagnostic value of procalcitonin (PCT) in the differentiation of infectious and non-infectious causes of pleural effusion. From January 2005 to April 2005, we measured the PCT levels of pleural effusion from 76 patients using an immunoluminometric assay. The types of pleural infusions studied were para-pneumonic effusion (n = 26), empyema (n = 7), tuberculous pleurisy (n = 8), malignant pleural effusion (n = 25) and transudative pleural effusion (n = 8). The PCT levels were low in transudative pleural effusions (0.188 ± 0.077 ng/mL) and tuberculous pleurisy (0.130 ± 0.069 ng/mL), but high in empyema (5.147 ± 3.056 ng/mL), para-pneumonic effusion (1.091 ± 0.355 ng/mL), and malignant pleural effusion (0.241 ± 0.071 ng/mL). The receiver-operating characteristic curve analysis for an optimal discrimination between empyema and para-pneumonic effusion from non-para-pneumonic effusion could be performed at a cut-off point of 0.18 ng/mL with area under the curve of 0.776 (sensitivity: 69.7%, specificity: 72.1%). The correlation was found between pleural effusion PCT and serum PCT levels in 16 patients (r ² = 0.967, p < 0.001). In conclusion, a high pleural effusion PCT level suggests the presence of empyema and para-pneumonic effusion.     

Blastocystis hominis and Dientamoeba fragilis in patients fulfilling irritable bowel syndrome criteria

Studies have suggested a possible role for Blastocystis hominis and Dientamoeba fragilis in the etiology of irritable bowel syndrome (IBS). We studied the prevalence of B. hominis and D. fragilis in patients with IBS-diarrhea (IBS-D). Three hundred and thirty patients were enrolled, 171 (52%) with IBS-D and 159 (48%) were controls, respectively. Stool microscopy, culture, and polymerase chain reaction (PCR) for B. hominis and D. fragilis were done. B. hominis was positive by stool microscopy in 49% (83/171) of IBS compared to 24% (27/159) in control (p < 0.001). B. hominis culture was positive in 53% (90/171) in IBS compared to 16% (25/159) in control (p < 0.001). B. hominis PCR was positive in 44% (75/171) in IBS compared to 21% (33/159) in control (p < 0.001). D. fragilis microscopy was positive in 3.5% (6/171) in IBS-D compared to 0.6% (1/159) in control (p = 0.123). D. fragilis culture was positive in 4% (7/171) in IBS compared to 1.3% (2/159) in control (p = 0.176). D. fragilis PCR was positive in 4% (6/171) in IBS-D compared to 0% (0/159) in control (p = 0.030). B. hominis is common, while D. fragilis was less prevalent in our patients with IBS-D. B. hominis and D. fragilis culture had a better yield compared to stool microscopy and PCR.     

Relationship Between Eosinophilia and Levels of Chemokines (CCL5 and CCL11) and IL-5 in Bronchoalveolar Lavage Fluid of Patients With Mustard Gas-Induced Pulmonary Fibrosis

Objective Therefore, this study was designed to analyze the bronchoalveolar lavage (BAL) fluid concentrations of IL-5, RANTES (CCL5) and eotaxin (CCL11) and also to examine the relationship between the percentage and absolute number of the BAL eosinophils and these measured chemokines in patients with sulfur mustard (SM) gas-induced pulmonary fibrosis (PF). Patients Fifteen veterans with mustard gas-induced PF and 14 normal veterans as control group. Intervention Pulmonary function tests, tests for D_LCO, computed tomography scans of the chest, analyses of BAL fluids for RANTES (CCL5), eotaxin (CCL11), and IL-5 were performed in all cases. Results Eosinophilic alveolitis was the predominant feature (p < 0.0001). There were significant differences in CCL5, CCL11, and IL-5 levels of BAL fluid between patients with PF and controls (p < 0.0001, p < 0.0001, and p = 0.001, respectively). The concentrations of CCL5 and CCL11 showed positive correlations with percentage (r = 0.57 and p = 0.03; r = 0.52 and p = 0.04, respectively) and absolute counts (r = 0.54 and p = 0.04, r = 0.53 and p = 0.04, respectively) of BAL eosinophils. There were significant positive correlations between the concentrations of IL-5 and the proportion and total cell number of eosinophils in BAL (r = 0.67 and p = 0.01; r = 0.59 and p = 0.02, respectively) too. Conclusion A significant correlation between BAL CCL5, CCL11, and IL-5 levels and eosinophils in patients with pulmonary fibrosis due to SM gas inhalation has been demonstrated, suggesting that these C–C chemokines and IL-5 contribute to the recruitment of eosinophils cells in the lung in these victims.     

Pulmonary cryptococcosis in patients without HIV infection: factors associated with disseminated disease

Cryptococcus neoformans is an uncommonly recognized cause of pneumonia in HIV-negative patients. Because of its propensity to disseminate to the meninges and other sites, a lumbar puncture is recommended for patients with pulmonary cryptococcosis, regardless of other risk factors. This study explored clinical and laboratory features to help predict which patients had pulmonary disease alone versus those who had pulmonary plus extrapulmonary disease. A retrospective chart review at 15 medical centers was performed from 1990 to 2000 of all HIV-negative patients who had pulmonary cryptococcosis. Demographic, clinical, radiographic, and laboratory features were evaluated to determine factors that differentiated those patients who had extrapulmonary disease. Among 166 patients who had pulmonary cryptococcosis, 122 had pulmonary infection only and 44 had pulmonary plus extrapulmonary (disseminated) disease. A negative serum cryptococcal antigen titer was more common in patients with pulmonary disease alone (p < 0.01). Multivariate analysis demonstrated that patients who had disseminated disease were more likely than those who only had pulmonary disease to have cirrhosis (p = 0.049), headache (p < 0.001), weight loss (p = 0.003), fever (p = 0.035), altered mental status (p < 0.001), and to be receiving high-dose corticosteroids (p = 0.008). In this large cohort of HIV-negative patients with pulmonary cryptococcosis, there were easily distinguished clinical and laboratory features among patients with pulmonary disease alone versus those with pulmonary plus extrapulmonary disease. These findings may be helpful in the evaluation of HIV-negative patients with pulmonary cryptococcosis with regard to the need for lumbar puncture or to search for disseminated disease.     

LYVE-1 upregulation and lymphatic invasion correlate with adverse prognostic factors and lymph node metastasis in neuroblastoma

Neuroblastoma (NB) accounts for 15% of all childhood cancer deaths. The majority of patients have widespread lymphatic and/or haematogenous metastases at diagnosis, but lymphangiogenesis has not been well documented. Sixty-seven NBs were immunostained for the lymphatic endothelial marker, LYVE-1, and the lymphatic density (LD) and lymphatic invasion (LI), were counted in LYVE-1-expressing lymphatics. LYVE-1-stained lymphatic vessels and LI were present in 26/67 (39%) and 14/67 (21%) of the NBs, respectively. Central LD (CLD) and LI were higher in NBs from stage 4 (p = 0.012, p = 0.004, respectively), high-risk group (p = 0.030, p = 0.002), NBs with high mitosis karyorrhexis index (MKI) (p = 0.011, p = 0.005), unfavourable histology group (p = 0.040, p = 0.017) and distant lymph node metastasis (LNM) (p < 0.001 for each). Marginal LD (MLD) was higher in patients with LNM (p < 0.001). CLD and MLD correlated with LI (p < 0.001 each). Total LYVE-1 protein levels, quantified by a sensitive enzyme-linked immunosorbent assay (n = 55), were also higher in NBs from patients with stage 4 disease (p = 0.046), high-risk group (p = 0.028), MYCN-amplified NBs (p = 0.034) and LNM (p = 0.038). Kaplan–Meier analysis showed that the presence of CLD was associated with both worse OS at 5 years (77% [95% CI: 62–87%] versus 60% [95% CI: 32–80%], p = 0.062) and EFS (74% [95% CI: 58–85%] versus 43% [95% CI: 15–69%], p = 0.070) and LI with OS (71% [95% CI: 57–81%] versus 56% [95% CI: 26–78%], p = 0.055). Significant upregulation of LYVE-1 and the presence of LI in patients with stage 4 and high-risk disease, MYCN-amplification and LNM suggests that LYVE-1 may have value as predictors of outcome.     

High JC virus load in tongue carcinomas may be a risk factor for tongue tumorigenesis

The John Cunningham virus (JCV) asymptomatically infects a large proportion (~90%) of the population worldwide but may be activated in immunodeficient patients, resulting in progressive multifocal leukoencephalopathy. Recent reports demonstrated its oncogenic role in malignancies. In this paper, the presence of JCV-targeting T antigen was investigated in tongue carcinoma (TC, n = 39), dysplastic tongue epithelium (DTE, n = 15) and glossitis (n = 15) using real-time polymerase chain reaction (PCR) and in situ PCR and immunohistochemistry, and JCV copies were analyzed with the clinicopathological parameters of TCs. The results demonstrated that glossitis and DTEs had significantly lower copies of JCV (410.5 ± 44.3 and 658.3 ± 53.3 copies/μg DNA respectively) than TCs (981.5 ± 14.0, p  < 0.05). When they were divided into three groups with 0–200 copies/μg DNA (low), 201–1,000 (moderate) and more than 1001 (high), TCs showed 3 (7.6%) in the low group, 21 (53.8%) in the moderate group and 15 (38.4%) in the high group and glossitis showed 11 (73.3%) in the low group, 0 (0%) in the moderate group and 4 (26.6%) in the high group. The DTEs occupied an intermediate position between them (p < 0.001). In situ PCR demonstrated that the nuclei of TC and DTE cells are sporadically T-antigen positive but not in nasal turbinate epithelial cells. Immunohistochemistry for T-antigen protein revealed four positive cases only in TCs. The existence of JCV T-antigen DNA was not associated with the clinicopathological variables of TCs. In conclusion, the presence of JCV may be a risk factor of tongue carcinogenesis.     

Subacute Inflammatory Activation in Subjects with Acute Coronary Syndrome and Left Ventricular Dysfunction

Several lines of evidence indicate that increased inflammatory cytokine levels can be used for risk prediction in patients with acute coronary syndrome (ACS). This study therefore aimed to evaluate correlations between levels of soluble interleukin (IL)-2 receptor (sIL-2r), IL-6, and IL-8 and in-hospital incidence of acute heart failure (AHF) and left ventricular (LV) systolic dysfunction in the subacute phase of ACS. In 48 consecutive patients with ACS, circulating levels of sIL-2r, IL-6, and IL-8 were ascertained 72–96 h after onset of symptoms. Clinical data, LV function, and in-hospital incidence of AHF were also evaluated. IL-8 levels were significantly higher in patients with pulmonary edema (1,829 ± 2,496 vs 456 ± 624 pg/ml, p < 0.05); sIL-2r, IL-6, and IL-8 levels were increased proportionally to Killip class (r = 0.35, p < 0.05; r = 0.48, r = 0.47, p < 0.01) and in patients with LV ejection fraction (LVEF) < 30%. Levels of sIL-2r were inversely related to LVEF in subjects with acute myocardial infarction (r = −0.51, p < 0.05). Soluble IL-2r and IL-8 levels were related to mitral regurgitation severity (r = 0.34, p < 0.05; r = 0.37, p < 0.05). Levels of sIL-2 were proportional to LV end-diastolic diameter (r = 0.49, p < 0.001) and LV end-systolic diameter (r = 0.58, p < 0.001). Number of cytokines with circulating values above upper level of normal was significantly correlated with Killip class and LVEF (r = 0.40, r = −0.38, p < 0.05). sIL-2r, IL-6, and IL-8 are increased in patients with ACS and systolic dysfunction or AHF. These data suggest that inflammatory cytokine activity detectable in peripheral blood may be useful in identifying subjects with a worse clinical course.     

Does the Presence of Anti-CCP Autoantibodies and Their Serum Levels Influence the Severity and Activity in Rheumatoid Arthritis Patients?

This study aims to investigate the association of the presence and of the titer of autoantibodies against cyclic citrullinated peptides (aCCP), with clinical manifestations and disease activity in a cohort of patients with rheumatoid arthritis (RA). From January 2000 through December 2005, 135 patients were diagnosed with RA at the Rheumatology Unit of our hospital. Demographic, clinical, laboratory, and therapeutic parameters were evaluated in all patients at study entry and at every follow-up visit. Positivity in aCCP and also their levels were determined for all patients. At the end of the study, we reevaluated the above parameters, dividing patients into aCCP positive and aCCP negative. From 135 patients, 53.3% were aCCP positive. The majority of aCCP-positive patients were males (p < 0.001), positive to rheumatoid factor (p < 0.001) and current smokers (p < 0.05). At diagnosis, aCCP-positive patients presented with higher tender joint counts (p < 0.001) and swollen joint counts (p < 0.001), and exhibited more active disease, expressed by higher disease activity scores for 28 joints (DAS-28) (p < 0.001). At the end of the study, aCCP-positive patients also displayed more active disease, with higher DAS-28 (p < 0.001), and more severe disease, as this was indicated by the higher radiological Larsen score (p < 0.001). The serum levels of aCCP were not found to be associated with disease activity and severity. In early RA, the presence of aCCP is associated with increased disease activity and severity. This was found to be independent of circulating levels of aCCP.     

Association between multiple sclerosis and Candida species: evidence from a case-control study

Candida infection among multiple sclerosis (MS) patients has not been studied in depth. We determined whether there is an association between serological evidence of Candida infection and MS. Blood specimens were obtained from 80 MS patients and 240 matched controls. Immunofluorescence analysis and ELISA were used to detect Candida species antibodies and slot-blot to detect antigens. Using immunofluorescence analysis, moderate to high concentrations of serum antibodies to Candida famata were present in 30 (37.5%) MS patients vs. 30 (12.5%) controls (p < 0.001). Results for Candida albicans were 47.5% (38/80) in MS patients vs. 21.3% (51/240) in controls (p < 0.001), for Candida parapsilosis 37% (28/80) vs. 17.1% (41/240) (p < 0.001) and for Candida glabrata 46.3% (37/80) vs. 17.5% (42/240) (p < 0.001), respectively. After adjusting for age and gender, the odds ratios (95% confidence intervals) for MS, according to the presence of Candida antigens were: 2.8 (0.3–23.1, p = 0.337) for Candida famata; 1.5 (0.7–3.4, p = 0.290) for Candida albicans; 7.3 (3.2–16.6, p < 0.001) for Candida parapsilosis; and 3.0 (1.5–6.1, p = 0.002) for Candida glabrata. The results were similar after excluding ten patients on immunosuppressants. The results of this single study suggest that Candida species infection may be associated with increased odds of MS.     

<Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> pneumonia in cancer patients without traditional risk factors for infection, 1997–2004

In order to elucidate the spectrum of Stenotrophomonas maltophilia pneumonia in cancer patients without traditional risk factors, 44 cancer patients (cases) with S. maltophilia pneumonia in whom S. maltophilia pneumonia risk factors were not present were compared with two S. maltophilia pneumonia risk groups (controls) including 43 neutropenic non-intensive care unit (ICU) and 21 non-neutropenic ICU patients. The case and control patients had similar demographic and underlying clinical characteristics. Compared with case patients with S. maltophilia pneumonia, neutropenic patients had higher exposure to carbapenem antibiotics (58 vs. 41%; p < 0.03), more frequent hematologic malignancy (95 vs. 64%; p < 0.0003), and they presented with concurrent bacteremia more often (23 vs. 0%; p < 0.0005). Patients with S. maltophilia pneumonia in the ICU needed vasopressor therapy more frequently than cases (62 vs. 5%; p < 0.0001). Hospital-acquired S. maltophilia pneumonia was more common among controls than cases (98 vs. 61%; p < 0.000002). Among the cases, 15 (34%) received outpatient oral antimicrobial therapy, while 29 were hospitalized and eight (28%) were subsequently admitted to the ICU. The mean duration of ICU stay, even among these eight patients (19 ± 40 days), was comparable to that of patients with neutropenia (23 ± 26 days) and those who developed S. maltophilia pneumonia during their ICU stay (34 ± 22 days; p = 0.46). The overall infection-associated mortality in the 108 patients with S. maltophilia pneumonia was 25%. Twenty percent of patients without traditional risk factors for S. maltophilia pneumonia died due to progressive infection. In a multivariate logistic regression analysis, only admission to the ICU predicted death (odds ratio 33; 95% confidence interval, 4.51–241.2; p < 0.0006). The results of this study indicate S. maltophilia pneumonia is a serious infection even in non-neutropenic, non-ICU patients with cancer. This work was presented in part at the 15th European Congress of Clinical Microbiology and Infectious Diseases, Copenhagen, Denmark, April 2–5, 2005 (abstract no P1374) and at the 45th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington D.C., December 16–19, 2005 (abstract no K-1535).     

Preliminary Evidence for the Cross-Cultural Utility of the Type D Personality Construct in the Ukraine

Background  Type D personality is a risk indicator in cardiac patients. The validity and reliability of the Type D Scale (DS14) have been confirmed in Western Europe but not outside this context. Purpose  We examined the structural, convergent, and divergent validity and the reliability of the DS14 in the Ukrainian setting. Method  Healthy Ukrainian respondents (n = 250) completed the DS14, the Eysenck Personality Questionnaire, the State Trait Anxiety Inventory, and the Beck Depression Inventory. A subsample (n = 57) completed the DS14 again after 4 weeks. Results  The prevalence of Type D personality was 22.4%. The two-factor structure and the validity of the DS14 were confirmed. The DS14 subscales were internally consistent (Cronbach’s α = 0.86/0.71; mean inter-item correlation = 0.48/0.27) and stable over a 4-week period (r = 0.85/0.63). Type D individuals had significantly higher mean scores on anxiety (p < 0.001), depressive symptoms (p < 0.001), and negative affect (p < 0.001), and lower scores on positive affect (p < 0.001) compared to non-Type D individuals. Conclusion  Preliminary evidence suggests that the Ukrainian DS14 is a valid and reliable measure. Future studies are warranted to test the utility of the scale in cardiac patients in the Ukraine, including whether Type D also predicts adverse health outcomes beyond the boundaries of Western Europe.     

Classical and Follicular Variant Papillary Thyroid Carcinoma: Comparison of Clinical,Ultrasonographical, Cytological,and Histopathological Features in 444 Patients

Follicular variant papillary thyroid carcinoma (FVPTC) is the most common variant of papillary thyroid carcinoma (PTC) after classical PTC (CPTC). In this study, we aimed to compare functional status, ultrasonographical features, cytological results, and histopathological characteristics of patients with CPTC and FVPTC. Preoperative thyroid functions, thyroid autoantibodies, ultrasonographical features, cytology, and histopathology results of 354 (79.9%) CPTC and 90 (20.3%) FVPTC patients were reviewed retrospectively. Sex distribution, mean age, thyroid autoantibody positivity, and thyroid dysfunctions were similar in two groups. Among 320 patients with preoperative ultrasonography (US) findings, a hypoechoic halo was observed more frequently (p = 0.003), and marginal irregularity was observed less commonly (p = 0.024) in FVPTC lesions. In CPTC, rate of malignant cytology (p = 0.001), and in FVPTC, rate of suspicious cytology (p < 0.001) were significantly higher. Histopathologically, mean tumor diameter was markedly higher in FVPTC compared to CPTC (16.89 ± 13.86 vs 10.64 ± 9.70 mm, p < 0.001), while capsular invasion and extrathyroidal spread were significantly lower in patients with FVPTC (p = 0.018 and p = 0.039, respectively). FVPTC tend to have more benign features in US and less malignant results in cytology. Higher tumor size in FVPTC might be explained by the recognition of clinical importance of these lesions after reaching particular sizes due to benign US features.     

Monotherapy versus combination antibiotic therapy for patients with bacteremic <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> community-acquired pneumonia

The purpose of this study was to examine the impact of antimicrobial monotherapy vs combination therapy on length of stay and mortality for patients with Streptococcus pneumoniae pneumonia. Thirty-nine percent of patients received monotherapy, while 61% received combination therapy. Although there was no significant difference in mortality (OR 1.25, 95% CI = 0.25–6.8), there was a significant increase in length of stay for patients who received combination therapy (p = 0.02). Patients with bacteremic pneumococcal pneumonia treated with empiric combination therapy had no significant difference in mortality; however, they did have increased length of stay after adjusting for severity of illness. Randomized controlled trials are needed to determine what is the optimal empiric antimicrobial regime for patients with community-acquired pneumonia.     

Real-time PCR strategy and detection of bacterial agents of lymphadenitis

The aim of this study was to compare 16 S rRNA gene amplification and sequencing with a systematic real-time PCR assay screening strategy that includes all common known pathogens recovered from lymph node biopsy specimens. Lymph node biopsy samples sent to our laboratory from January 2007 to December 2008 were tested in the study. Lymph nodes were screened for the presence of any bacteria by PCR amplification and sequencing targeting the 16 S rRNA gene and also by a specific real-time PCR strategy that includes Bartonella henselae, mycobacteria, Francisella tularensis, and Tropheryma whipplei. By testing 491 lymph nodes, we found that the sensitivity of our specific real-time PCR assay strategy was significantly higher than 16 S rRNA PCR amplification and sequencing for the detection of Bartonella henselae (142 vs 98; p < 10⁻⁴), Francisella tularensis (16 vs 10, p < 10⁻⁴), and mycobacteria (8 versus 3, p < 10⁻⁴). None of the samples was positive for Tropheryma whipplei. Our study demonstrates the usefulness and specificity of a systematic real-time PCR strategy for molecular analysis of lymph node biopsy specimens and the higher sensitivity compared with standard 16 S rRNA gene amplification and sequencing.     

Tissue transglutaminase expression in celiac mucosa: an immunohistochemical study

Tissue transglutaminase (tTG) constitutes the main autoantigen in celiac disease (CD). The aim of the study was to clarify weather celiac disease is associated with changes in tTG expression in duodenal mucosa. Tissue transglutaminase was assessed immunohistochemically (clone CUB 7402) in duodenal biopsy specimens from 22 untreated CD patients, ten normal controls (NC) with unremarkable duodenal mucosa, and nine disease nonceliac controls (DC). In 15 CD patients duodenal biopsy specimens were repeatedly assessed after these patients had been prescribed gluten-free diet. Positive pixel count algorithm of ImageScope was used for quantitative evaluation of immunohistochemistry. Tissue transglutaminase expression in superficial epithelium differed significantly between the three groups (p < 0.001). It was increased in DC in relation to NC (p < 0.001) and in CD—in relation to NC (p < 0.001) and DC (p = 0.003). In CD and DC, cryptal epithelium was stained more intensively than in NC (p < 0.001), but there was no difference between CD and DC (p = 0.507). The same pattern was seen in lamina propria. A significant decrease in tTG expression in all the compartments was seen in repeatedly assessed samples. Untreated CD is associated with tTG overexpression, which is reversible. Tissue transglutaminase up-regulation does not seem to be specific for CD and can appear in other pathological conditions.     

Plasma 1,25 Dihydroxy Vitamin D<Subscript>3</Subscript> Level and Expression of Vitamin D Receptor and Cathelicidin in Pulmonary Tuberculosis

Introduction  Vitamin D₃, which exerts its effect through vitamin D receptor (VDR), is known for its potent immunomodulatory activities. Associations between low serum vitamin D₃ levels and increased risk of tuberculosis have been reported. Study Subjects and Methods  Plasma 1,25 dihydroxy vitamin D₃ levels (1,25(OH)₂ D₃) and ex vivo levels of VDR protein from peripheral blood mononuclear cells were studied in 65 pulmonary tuberculosis (PTB) patients and 60 normal healthy subjects (NHS) using enzyme-linked immunosorbent assay-based methods. Using real-time polymerase chain reaction (PCR), induction of VDR, cathelicidin, and CYP27B1 mRNA were studied in live Mycobacterium tuberculosis-stimulated macrophage cultures treated with or without 1,25 dihydroxy vitamin D₃. VDR and CYP27B1 (-1077 A/T) gene polymorphisms were studied using PCR-based methods. Results  1,25(OH)₂ D₃ were significantly increased (p = 0.0004), while ex vivo levels of VDR protein were significantly decreased in PTB patients (p = 0.017) as compared to NHS. 1,25(OH)₂ D₃ levels were not different between variant genotypes of CYP27B1. A trend towards decreased levels of VDR protein was observed among NHS with BsmI BB and TaqI tt genotypes compared to NHS with other genotypes. Relative quantification of mRNA using real-time PCR revealed increased VDR mRNA expression in live M. tuberculosis-stimulated culture in PTB patients (p < 0.01) than normal healthy subjects. Cathelicidin mRNA expression was significantly increased in vitamin D₃-treated cultures compared to unstimulated and M. tuberculosis-stimulated culture in both patients (p < 0.001) and NHS (p < 0.05). Conclusions  The present study suggests that PTB patients may have increased 1,25(OH)₂ D₃ levels, and this might lead to downregulation of VDR expression. Decreased VDR levels could result in defective VDR signaling. Moreover, addition of 1,25(OH)₂ D₃ might lead to increased expression of cathelicidin which could enhance the immunity against tuberculosis.     

Self-collected vaginal swabs for the quantitative real-time polymerase chain reaction assay of Atopobium vaginae and Gardnerella vaginalis and the diagnosis of bacterial vaginosis

The aim of this study was to assess the feasibility of using self-collected vaginal specimens for the quantitative real-time polymerase chain reaction (qPCR) assays of bacterial vaginosis (BV)-associated bacteria versus practitioner-collected swabs. A cross-sectional study included 190 pregnant women enrolled before 20 weeks’ gestation from September 2008 to November 2009. Self- and practitioner-collected swabs were taken during the same prenatal visit for each woman, qPCR assays performed for each, and the results compared. The quantification of the human albumin gene was used as an internal control to ensure sampling quality and accurate comparisons. The level of agreement of the qPCR assays for each microorganism was calculated with the Spearman product moment correlation coefficient and the kappa statistic. In all, 370 vaginal samples (185 self- and 185 practitioner-collected swabs) had a narrow range of values for the number of albumin gene copies and a significant correlation coefficient (Spearman’s rho = 0.532; p < 0.001). The agreement between both sampling methods was excellent (Spearman’s rho was 0.748 for Atopobium vaginae, 0.918 for Lactobacillus species, 0.940 for Gardnerella vaginalis; p < 0.001), especially for high concentrations of A. vaginae (≥10⁸ copies/mL; kappa value = 0.973; p < 0.001) and G. vaginalis (≥10⁹ copies/mL; kappa value = 0.903; p < 0.001). This study demonstrates the validity and reliability of self- versus practitioner-collected swabs for the molecular quantification of Lactobacillus species, G. vaginalis, and A. vaginae.     

Bacterial diversity of periodontal and implant-related sites detected by the DNA Checkerboard method

The aim of this study was to compare the microbial composition of the subgingival biofilm from teeth and implant sulci in relation to contents originating from internal parts of the implant, abutment and implant prosthesis. Twenty subgingival biofilm samples from the mesial and distal aspects of each tooth/implant and 29 samples from the internal parts of titanium implants, abutments and implant prostheses were evaluated for the presence of 18 bacterial species using DNA Checkerboard and the differences between samples from teeth and implants were assessed with Pearson’s correlation analysis. The periodontal and peri-implantar sulci presented significantly higher bacterial counts than the implant-related sites (p < 0.05 and p < 0.01, respectively). The highest counts were observed for Capnocytophaga gingivalis, Prevotella intermedia, P. nigrescens and P. micra. The correlation between the counts in the periodontal and peri-implantar sulci was r = 0.66 (p < 0.001). Weaker correlations between samples from the internal parts of the implants and periodontal sulcus (r = 0.49; p < 0.001) or peri-implant sulcus (r = 0.42; p < 0.001) were found. All 18 bacterial species were detected to be colonising the subgingival sulcus of teeth and implants, and implant components in the evaluated patients. Significant correlations between the microbiota were found, the strongest being between the periodontal and peri-implantar sulci.     

Improved detection of canine Angiostrongylus vasorum infection using real-time PCR and indirect ELISA

This study reports the development of a real-time PCR assay and an indirect ELISA to improve on current detection of canine Angiostrongylus vasorum infection. A highly specific fluorescent probe-based, real-time PCR assay was developed to target the A. vasorum second internal transcribed spacer region and detected DNA in EDTA blood, lung tissue, broncho-alveolar larvage fluid, endotracheal mucus, pharyngeal swabs and faecal samples. PCR was fast (∼1 h), highly efficient when using EDTA blood samples, consistently detected a single molecule of parasite DNA and did not amplify DNA from other parasitic nematodes or definitive host species. An indirect ELISA was also developed using the soluble protein fraction from adult A. vasorum worms. Some cross-reactive antigen recognition was observed when tested against sera from dogs infected with Crenosoma vulpis (n = 8), Toxocara canis (n = 5) and Dirofilaria immitis (n = 5). This was largely overcome by setting the cut-off for a positive result at an appropriately high level. Field evaluation of the real-time PCR and ELISA was conducted by testing sera and EDTA blood from dogs with suspected A. vasorum infection (n = 148) and compared with the Baermann's larval migration test in faeces. Thirty-one dogs were positive by at least one test. Of these, 20 (65%) were detected by the Baermann method, 18 (58%) by blood PCR, 24 (77%) by ELISA and 28 (90%) by blood PCR and ELISA together. Combined testing using real-time PCR and ELISA therefore improved the detection rate of A. vasorum infection and holds promise for improved clinical diagnosis and epidemiological investigation.