Bacteriological follow-up of pulmonary tuberculosis treatment: a study with a simple colorimetric assay

The viability of Mycobacterium tuberculosis (MTB) in serial sputum specimens from persistently smear positive patients was evaluated. The assay was based on oxidation-reduction of Alamar Blue and Malachite Green dyes that change their color in response to MTB growth. A total of 280 sputum specimens from 40 persistently smear positive TB patients and 40 sputa from non-tuberculosis patients were digested, decontaminated and examined microscopically. To check the MTB viability, the sediments from decontaminated samples were inoculated into three culture media: Lowenstein-Jensen (LJ) slants, Alamar Blue and Malachite Green culture tubes. We found that out of 280 smear positive specimens, the LJ culture was positive in 124 (44%). The numbers of correctly identified S+/C+ cases by Alamar Blue and Malachite Green were 118 (95%) and 116 (93%), respectively. The mean time required for reporting the positive signal in Alamar Blue culture tubes was 9 versus 11 days by Malachite Green culture tubes. In the standard LJ culture media the average detection time was 27 days (P < 0.05). The sensitivity of LJ was 99%, Alamar Blue 95% and Malachite Green 93%. The specificity was 100%, 92% and 93%, respectively. The oxidation-reduction method is rapid, sensitive and inexpensive in monitoring the treatment response of patients with pulmonary TB. Thus, using this method can be of paramount importance, particularly in resource-constrained areas.     

Drug-Eluting versus Bare-Metal Stent for Treatment of Saphenous Vein Grafts: A Meta-Analysis

Background

Saphenous vein grafts develop an aggressive atherosclerotic process and the efficacy of drug eluting stents (DES) in treating saphenous vein graft (SVG) lesions has not been convincingly demonstrated. The aim of this study was to review and analyze the current literature for controlled studies comparing DES versus bare metal stents (BMS) for treatment of SVG stenoses.

Methodology/Principal Findings

We searched several scientific databases and conference proceedings up to March 15, 2010 for controlled studies comparing target vessel revascularization (TVR) between DES and BMS. Summary odds ratios (OR) for the primary endpoint TVR and secondary endpoints infarction, stent thrombosis and death were calculated using random-effect models. A total of 29 studies (3 randomized controlled trials RCT) involving 7549 (202 in RCT) patients were included. The need for target vessel revascularization in the DES group tended to be lower compared to BMS for the 3 RCT (OR 0.50 [0.24–1.00]; p = 0.051) and for observational studies (0.62 [0.49–0.79]; p<0.001). There was no significant difference in the risk for myocardial infarction in the RCT (OR 1.25 [0.22–6.99]; p = 0.250) but a lower risk for DES based on the observational studies 0.68 [0.49–0.95]; p = 0.023. The risk for stent thrombosis was found to be non-different in the RCT (OR 0.78 [0.03–21.73], p = 0.885) while it was in favor of DES in the observational studies (0.58 [0.38 – 0.84]; p<0.001). The mortality was not significantly different between DES and BMS in the RCT''s (OR 2.22 [0.17 – 29.50]; p = 0.546) while the observation studies showed a decreased mortality in the DES group (0.69 [0.55–0.85]; p<0.001).

Conclusion

DES may decrease TVR rate in treatment of SVG stenoses. No differences in reinfarction rate, stent thrombosis or mortality was found between the DES and BMS groups in the RCT''s while the observational data showed lower risk for myocardial infarction, stent thrombosis and death in the DES group. This may be a result of patient selection bias in the observational studies or represent a true finding that was not the detected in the RCT analysis due to limited statistical power.     

A critique of widely used normalization software tools and an alternative method to identify reliable reference genes in red clover (Trifolium pratense L.)

Determination of appropriate reference genes is crucial to normalization of gene expression data and prevention of biased results in qRT-PCR studies. This study is the first attempt to systematically compare potential reference genes to detect the most constitutively expressed reference genes for accurate normalization in red clover tissues including leaves, stems and roots. To identify the best-suited reference gene(s) for normalization, several statistical algorithms such as geNorm, BestKeeper and NormFinder have been developed. All these algorithms are based on the key assumption that none of the investigated candidate reference genes show systematic variation in their expression profile across the samples being considered. However, this assumption is likely to be violated in practice. The authors therefore suggest a simple and novel stability index based on the analysis of variance model which is free from the assumption made by the algorithms. We assessed the expression stability of eight candidate reference genes including actin (ACT), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), elongation factor-1alpha (EF-1α), translation initiation factor (EIF-4a), ubiquitin-conjugating enzyme E2 (UBC2), polyubiquitin (UBQ10), sand family protein (SAND) and yellow-leaf-specific protein 8 (YLS8). Our results indicated that UBC2 and UBQ10 ranked as the two most stably expressed genes in leaf tissue. UBC2 and YLS8 were defined as optimal control genes for stem tissue. EIF-4a and UBC2 were found to be the most stable reference gene for root tissue. GAPDH and SAND showed relatively low stability in expression study of red clover. When all tested tissues were considered, we observed that YLS8 and UBC2 showed remarkable stability in their expression level across tissues.     

Genetic susceptibility to S. aureus mastitis in sheep: differential expression of mammary epithelial cells in response to live bacteria or supernatant

Staphylococcus aureus is a prevalent pathogen for mastitis in dairy ruminants and is responsible for both clinical and subclinical mastitis. Mammary epithelial cells (MEC) represent not only a physical barrier against bacterial invasion but are also active players of the innate immune response permitting infection clearance. To decipher their functions in general and in animals showing different levels of genetic predisposition to Staphylococcus in particular, MEC from ewes undergoing a divergent selection on milk somatic cell count were stimulated by S. aureus. MEC response was also studied according to the stimulation condition with live bacteria or culture supernatant. The early MEC response was studied during a 5 h time course by microarray to identify differentially expressed genes with regard to the host genetic background and as a function of the conditions of stimulation. In both conditions of stimulation, metabolic processes were altered, the apoptosis-associated pathways were considerably modified, and inflammatory and immune responses were enhanced with the upregulation of il1a, il1b, and tnfa and several chemokines known to enhance neutrophil (cxcl8) or mononuclear leukocyte (ccl20) recruitment. Genes associated with oxidative stress were increased after live bacteria stimulation, whereas immune response-related genes were higher after supernatant stimulation in the early phase. Only 20 genes were differentially expressed between Staphylococcus spp-mastitis resistant and susceptible animals without any clearly defined role on the control of infection. To conclude, this suggests that MEC may not represent the cell type at the origin of the difference of mastitis susceptibility, at least as demonstrated in our genetic model. Supernatant or heat-killed S. aureus produce biological effects that are essentially different from those induced by live bacteria.     

Glycosaminoglycan polymerization may enable osmotically inactive Na+ storage in the skin

Osmotically inactive skin Na(+) storage is characterized by Na(+) accumulation without water accumulation in the skin. Negatively charged glycosaminoglycans (GAGs) may be important in skin Na(+) storage. We investigated changes in skin GAG content and key enzymes of GAG chain polymerization during osmotically inactive skin Na(+) storage. Female Sprague-Dawley rats were fed a 0.1% or 8% NaCl diet for 8 wk. Skin GAG content was measured by Western blot analysis. mRNA content of key dermatan sulfate polymerization enzymes was measured by real-time PCR. The Na(+) concentration in skin was determined by dry ashing. Skin Na(+) concentration during osmotically inactive Na(+) storage was 180-190 mmol/l. Increasing skin Na(+) coincided with increasing GAG content in cartilage and skin. Dietary NaCl loading coincided with increased chondroitin synthase mRNA content in the skin, whereas xylosyl transferase, biglycan, and decorin content were unchanged. We conclude that osmotically inactive skin Na(+) storage is an active process characterized by an increased GAG content in the reservoir tissue. Inhibition or disinhibition of GAG chain polymerization may regulate osmotically inactive Na(+) storage.     

Platelet sparing effect of COX II inhibition used with pegylated interferon alfa-2a for the treatment of chronic hepatitis C: a short term pilot study

The role of a cyclooxygenase (COX) II inhibitor in reducing microvascular inflammation and the platelet count associated with interferon (IFN) plus ribavirin therapy of chronic hepatitis C (HCV) was assessed. Three plasma mediators (biomarkers) associated with platelet activation, inflammation and fibrosis were measured. Eighteen IFN na?ve patients were studied. Nine were treated with pegylated IFN alfa-2a (PEG-IFN alpha-2a) plus ribavirin and rofecoxib; nine were treated with PEG-IFN alpha-2a plus ribavirin. A complete blood count, liver panel and HCV-RNA were assayed weekly. Human soluble P-selectin (hs-P-selectin), human interleukin-8 (IL-8), human interleukin-13 (IL-13) and human thrombopoietin (TPO) were assayed at 4 week intervals. The COX II inhibitor reduced the platelet reduction experienced with PEG-IFN alpha-2a treatment of HCV despite a reduction in the plasma TPO level. Hs-P-selectin was increased in both groups. In contrast, human IL-8 levels declined to undetectable levels in virologic responders. Similarly, human IL-13 levels declined with therapy (P < 0.001). These data suggest that: (1) a COX II inhibition is associated with an increase in the platelet count despite a reduction in the TPO level; (2) human IL-8 and human IL-13 but not hs-P-selectin levels decline in those who experience an early virologic response.     

Median-of-subsets normalization of intensities for cDNA array data

cDNA arrays allow quantitative measurement of expression levels for thousands of genes simultaneously. The measurements are affected by many sources of variation, and substantial improvements in the precision of estimated effects accompany adjustments for these effects. Two generic nuisance variations, one associated with the magnitude of expression and the other associated with array location, are common in data from filter arrays. Procedures, like normalization using lowess regression, are effective at reducing variation associated with magnitude, and they have been widely adopted. However, variation associated with location has received less attention. Here, a simple, but effective method based on localized median is expounded for dealing with these nuisance effects, and its properties are discussed. The proposed methodology handles location-dependent variation ("splotches") and magnitude-dependent variation (background and/or saturation) effectively. The procedure is related to lowess when implemented to adjust magnitude-dependent variation, and it performs similarly. The proposed methodology is illustrated with data from the National Center for Toxicological Research (NCTR), where treatment differences in levels of mRNA from rat hepatocytes were assessed using 33P-labeled samples hybridized to cDNA spotted arrays. Normalizing intensities by the median-of-subsets removes systematic variation associated with the location of a gene on the array and/or the level of its expression. This procedure is easy to implement using iteratively reweighted least-squares algorithms. Although less sophisticated than lowess, this procedure works nearly as well for normalizing intensities based upon their magnitude. Unlike lowess, it can adjust for location-dependent effects.     

Vascular endothelial growth factor inhibits programmed cell death of endothelial cells induced by clinorotation

The principal aim of this study was to investigate short- and long-term effects of clinorotation on human endothelial cells (EA hy 926 cell line) using a three-dimensional random positioning machine. Moreover, the impact of vascular endothelial growth factor (VEGF) was addressed. Immediately, within one hour and after four and twenty-four hours an increase of apoptotic cells was detected. VEGF significantly inhibited the amount of apoptotic endothelial cells (EC). VEGF reduced the amount of fas-positive EC. Moreover, after 24 hours, proliferating EC grew in form of three-dimensional multicellular spheroids and also as monolayers. The initially formed spheroids (maximum diameter 3 mm) remained stable up to the 15th day of clinorotation. Some spheroids revealed tubular structures. In addition, a clear increase of extracellular matrix proteins such as osteopontin and fibronectin was measured. The three-dimensional clinostat represents an important tool for cell biological experiments. VEGF significantly attenuated the changes of endothelial cells induced by simulated weightlessness in a cell protective manner.     

Phenotypic characteristics and population genetics of Enterococcus faecalis cultured from patients in Tehran during 2000-2001

Conventional bacteriology techniques were used to identify enterococci isolates cultured from patients at different hospitals in Tehran during 2000-2001. The identification was confirmed using species-specific PCR targeting the D-alanyl-D-alanine ligase gene. A total of 59 isolates of Enterococcus faecalis were identified. The rates of resistance to different antibiotics were in the following order: penicillin 84%, ciprofloxacin 42%, high-level gentamicin 30%, nitrofurantoin 14%, imipenem 4%, and chloramphenicol 2%. Resistance to ampicillin was found to be rare among the Iranian isolates of E. faecalis. Multi-locus enzyme electrophoresis was then used to analyze the strains. Forty-five electrophoretic types were obtained when 10 enzyme loci were screened. Although the collection of bacterial isolates was limited in time and location, considerable heterogeneity was found. Analysis of strains for linkage disequilibrium demonstrated that the studied population is not clonal, since the index of association was not significantly different from zero (Ia = 0.0296). Enterococcus faecalis isolates recovered from patients in Tehran were genetically diverse and seemed to possess a high potential for genetic recombinations, though none were resistant to vancomycin.