Prospective evaluation of thin-layer agar colour test in routine diagnosis of multidrug-resistant TB.

This study evaluated the diagnostic performance of the thin-layer agar MDR/XDR-TB Colour Test (CT), a Mycobacterium tuberculosis complex (MTBC) detection and direct drug susceptibility testing (DST) method with routine sputum, bronchoalveolar lavage and pleural fluid specimen.In a prospective study, the time and rate of MTBC detection were compared between CT, Löwenstein-Jensen, and MGIT media. Times until DST result, sensitivities, and specificities were evaluated between CT and MGIT 960 indirect DST.The cultivation of 177 pulmonary specimens resulted in 83 MTBC-positive cultures. The sensitivity of CT for MTBC detection was 81.3% with a median time of 20 days compared to 13 days and 93.5% for MGIT. The sensitivity of CT for DST results was 100% for isoniazid and levofloxacin and 94.7% for rifampicin. The specificities for isoniazid and rifampicin DST were 97.3% and 98.0% for levofloxacin. The median time until a DST result was significantly shorter with CT than the BACTEC MGIT 960 system, 20 and 27 days, respectively, independent of the specimen type used.The CT is a highly accurate and fast initial diagnostic test for high-incidence settings and could also be used as a first culture and direct DST in peripheral settings..

A comprehensive evaluation and impact of normalization of generalized tracer kinetic model parameters to characterize blood-brain-barrier permeability in normal-appearing and tumor tissue regions of patients with glioma.

RATIONALE AND OBJECTIVES: To comprehensively evaluate robustness and variations of DCE-MRI derived generalized-tracer-kinetic-model (GTKM) parameters in healthy and tumor tissues and impact of normalization in mitigating these variations on application to glioma. MATERIALS (PATIENTS) AND METHODS: A retrospective study included pre-operative 31 high-grade-glioma(HGG), 22 low-grade-glioma(LGG) and 33 follow-up data from 10 patients a prospective study with 4 HGG subjects. Voxel-wise GTKM was fitted to DCE-MRI data to estimate Ktrans, ve, vb. Simulations were used to evaluate noise sensitivity. Variation of parameters with-respect-to arterial-input-function (AIF) variation and data length were studied. Normalization of parameters with-respect-to mean values in gray-matter (GM) and white-matter (WM) regions (GM-Type-2, WM-Type-2) and mean curves (GM-Type-1, WM-Type-1) were also evaluated. Co-efficient-of-variation(CoV), relative-percentage-error (RPE), Box-Whisker plots, bar graphs and t-test were used for comparison. RESULTS: GTKM was fitted well in all tissue regions. Ktrans and ve in contrast-enhancing (CE) has shown improved noise sensitivity in longer data. vb was reliable in all tissues. Mean AIF and C(t) peaks showed ~38% and ~35% variations. During simulation, normalizations have mitigated variations due to changes in AIF amplitude in Ktrans and vb.. ve was less sensitive to normalizations. CoV of Ktrans and vb has reduced ~70% after GM-Type-1 normalization and ~80% after GM-Type-2 normalization, respectively. GM-Type-1 (p = 0.003) and GM-Type-2 (p = 0.006) normalizations have significantly improved differentiation of HGG and LGG using Ktrans. CONCLUSION: Ktrans and vb can be reliably estimated in normal-appearing brain tissues and can be used for normalization of corresponding parameters in tumor tissues for mitigating inter-subject variability due to errors in AIF. Normalized Ktrans and vb provided improved differentiation of HGG and LGG.

Hallmarks of cancer-the new testament.

Diagnosis and treatment of disease demand a sound understanding of the underlying mechanisms, determining any Achilles' heel that can be targeted in effective therapies. Throughout history, this endeavour to decipher the origin and mechanism of transformation of a normal cell into cancer has led to various theories-from cancer as a curse to an understanding at the level of single-cell heterogeneity, meaning even among a single sub-type of cancer there are myriad molecular challenges to overcome. With increasing insight into cancer genetics and biology, the disease has become ever more complex to understand. The complexity of cancer as a disease was distilled into key traits by Hanahan and Weinberg in their seminal 'Hallmarks of Cancer' reviews. This lucid conceptualization of complex cancer biology is widely accepted and has helped advance cancer therapeutics by targeting the various hallmarks but, with the advancement in technologies, there is greater granularity in how we view cancer as a disease, and the additional understanding over the past decade requires us to revisit the hallmarks of cancer. Based on extensive study of the cancer research literature, we propose four novel hallmarks of cancer, namely, the ability of cells to regress from a specific specialized functional state, epigenetic changes that can affect gene expression, the role of microorganisms and neuronal signalling, to be included in the hallmark conceptualization along with evidence of various means to exploit them therapeutically.

Evaluating feasibility of high resolution T1-perfusion MRI with whole brain coverage using compressed SENSE: Application to glioma grading.

PURPOSE: To evaluate the efficacy of optimized T1-Perfusion MRI protocol (protocol-2) with whole brain coverage and improved spatial resolution using Compressed-SENSE (CSENSE) to differentiate high-grade-glioma (HGG) and low-grade-glioma (LGG) and to compare it with the conventional protocol (protocol-1) with partial brain coverage used in our center. METHODS: This study included MRI data from 5 healthy volunteers, a phantom and 126 brain tumor patients. Current study had two parts: To analyze the effect of CSENSE on 3D-T1-weighted (W) fast-field-echo (FFE) images, T1-W, dual-PDT2-W turbo-spin-echo images and T1 maps, and to evaluate the performance of high resolution T1-Perfusion MRI protocol with whole brain coverage optimized using CSENSE. Coefficient-of-Variation (COV), Relative-Percentage-Error (RPE), Normalized-Mean-Squared-Error (NMSE) and qualitative scoring were used for the former study. Tracer-kinetic (Ktrans,ve,vp) and hemodynamic (rCBV,rCBF) parameters computed from both protocols were used to differentiate LGG and HGG. RESULTS: The image quality of all structural images was found to be of diagnostic quality till R = 4. NMSE in healthy T1-W-FFE images and COV in phantom images increased with-respect-to R and images provided optimum quality till R = 4. Structural images and maps exhibited artefacts from R = 6. All parameters in tumor tissue and hemodynamic parameters in healthy gray matter tissue computed from both protocols were not significantly different. Parameters computed from protocol-2 performed better in terms of glioma grading. For both protocols, rCBF performed least (AUC = 0.759 and 0.851) and combination of all parameters performed best (AUC = 0.890 and 0.964). CONCLUSION: CSENSE (R = 4) can be used to improve the resolution and brain coverage for T1-Perfusion analysis used to differentiate gliomas.

Decolonisation of MRSA and its effect on surgical site infections--a study in a tertiary care institute.

BACKGROUND: Two-thirds of surgical site infections (SSI) because of Staphylococcus aureus are caused by Methicillin resistant Staphylococcus aureus (MRSA). This study was done to assess the efficacy of topical 2% mupirocin with 2% chlorhexidine gluconate body wash in decolonizing MRSA and its impact in preventing SSI because of MRSA. The various risk factors associated with MRSA carriers and SSI were also studied because of paucity of data in the developing world. METHODS: We did a non-randomised interventional trial in 602 patients undergoing elective general surgical operations. All patients in case (297) group were screened for MRSA and those positive were decolonised with topical 2% mupirocin calcium ointment and daily baths with 2% chlorhexidine antiseptic solution for 5 days. Control (305) group patients underwent surgery without decolonisation. Postoperatively, all patients were followed up for SSI for 30 days. RESULTS: Prevalence of MRSA carriers was 7.5% with decolonisation rate of 95.2%. The SSI incidence was 21.3%. The significant risk factors for SSI were type of anaesthesia (p = 0.002), duration of surgery (p = 0.001) and preoperative hospital stay (p = 0.001). There was a significant association between MRSA carrier positivity at the time of surgery and SSI (p = 0.041). CONCLUSIONS: There was no reduction in rate of SSI or other nosocomial infections in patients undergoing elective general surgical operations following preoperative MRSA decolonisation with 2% mupirocin and 2% chlorhexidine gluconate in MRSA carriers. MRSA carrier status was a significant risk factor for SSI but not for other nosocomial infections.

Breaking the 'harmony' of TNF-α signaling for cancer treatment.

Tumor necrosis factor-alpha (TNF-α) binds to two distinct receptors, TNFR1/p55 and TNFR2/p75. TNF-α is implicated in the processes of tumor growth, survival, differentiation, invasion, metastases, secretion of cytokines and pro-angiogenic factors. We have shown that TNFR2/p75 signaling promotes ischemia-induced angiogenesis via modulation of several angiogenic growth factors. We hypothesized that TNFR2/p75 may promote tumor growth and angiogenesis. Growth of mouse Lewis lung carcinoma (LLC1) and/or mouse melanoma B16 cell was evaluated in wild type (WT), p75 knockout (KO) and double p55KO/p75KO mouse tumor xenograft models. Compared with WT and p55KO/p75KO mice, growth of tumors in p75KO mice was significantly decreased (twofold) in both LLC and B16 tumors. Tumor growth inhibition was correlated with decreases in vascular endothelial growth factor (VEGF) expression and capillary density, as well as bone marrow-derived endothelial progenitor cells incorporation into the functional capillary network, and an increase in apoptotic cells in LLC xenografts. Gene array analysis of tumor tissues showed a decrease in gene expression in pathways that promote tumor angiogenesis and cell survival. Blocking p75 by short-hairpin RNA in cultured LLCs led to increases in TNF-mediated apoptosis, as well as decreases in the constitutive and TNF-mediated expression of angiogenic growth factors (VEGF, HGF, PLGF), and SDF-1α receptor CXCR4. In summary, p75 is essential for tumor angiogenesis and survival in highly vascularized murine lung tumor xenografts. Blocking p75 expression may lead to tumor regression. This may represent new and effective therapy against lung neoplasms and potentially tumors of other origin.

Exposure of mice to arsenic and/or benzo[a]pyrene does not increase the frequency of Aprt-deficient cells recovered from explanted skin of Aprt heterozygous mice.

Exposure to inorganic arsenic in drinking water is linked to cancer in humans, but the mechanism of arsenic-induced cancer is not clear. Arsenic is not a powerful point mutagen, but can cause chromosome malsegregation and mitotic recombination, two events that can cause loss of tumor suppressor alleles and thereby contribute to the evolution of cancerous cells. To determine whether arsenic increases the frequency of allele loss due to either malsegregation or mitotic recombination in vivo, Aprt(+/-) hybrid mice were exposed to sodium arsenite (10 mg/L) in their drinking water for 10 weeks. To determine whether arsenic enhances the action of a known mutagen, half of the arsenic-treated mice were exposed to benzo[a]pyrene (BaP) for 8 weeks by skin painting (500 nmoles/week). Cells were taken from painted dorsal skin and cultured in the presence of 2,6-diaminopurine (DAP), to select colonies lacking adenosine phosphoribosyl transferase (Aprt) activity. The frequency of DAP-resistant (DAP(r)) colonies varied substantially within the treatment groups, but there was no significant difference between the groups. Analysis of DNA from DAP(r) colonies suggested that mitotic recombination contributed to the loss of wild-type Aprt allele. Whether arsenic or BaP enhanced or diminished the frequency of this process could not be deduced from these data.

Co-mutagenic activity of arsenic and benzo[a]pyrene in mouse skin.

Exposure to inorganic arsenic in drinking water is linked to skin, lung and bladder cancer in humans. The mechanism of arsenic-induced cancer is not clear, but exposure to arsenic and polycyclic arylhydrocarbons (PAH) is more carcinogenic than exposure to either type of carcinogen alone. Arsenic can also generate reactive oxygen species, suggesting that oxidation of DNA may play a role in carcinogenesis. Oxidization of guanosines in polyG tracts is known to cause frameshift mutations, and such events can be detected in situ using the G11 placental alkaline phosphatase (PLAP) transgenic mouse model, which reports frameshift mutations in a run of 11 G:C basepairs by generating cells containing heat-resistant alkaline phosphatase activity. PAH can also induce frameshift mutations. In the study described here, FVB/N mice carrying the G11 PLAP transgene were crossed to C57Bl/6 mice. Half of the hybrid mice were given drinking water with sodium arsenite (10 mg/L) for 10 weeks. Half of the arsenic treated mice were also exposed to benzo[a]pyrene (BaP) by skin painting (500 nmol/week) for 8 weeks. Another group of mice was exposed to BaP but not arsenic. The effect on frameshift mutation was assessed by staining sections of skin tissue to detect cells with PLAP activity. Arsenic alone had no significant effect. On average, mice given BaP alone had approximately three times more PLAP-positive (PLAP+) cells. By contrast, mice exposed to both arsenic and BaP exhibited 10-fold more PLAP+ cells in the skin, and these cells were often arranged in large clusters, suggesting derivation from stem cells. Whereas combined treatment produced more PLAP+ cells, stable BaP adduct levels and arsenic burdens were not higher in mice exposed to both agents compared to mice exposed to either one agent or the other.

The potential of inductively coupled plasma-mass spectrometric detection for capillary electrophoretic analysis of pesticides.

In this work, the potential of inductively coupled plasma-mass spectrometry (ICP-MS) coupled to capillary electrophoresis (CE) to determine organophosphorus pesticides (OPPs) is demonstrated. Element specific detection of (31)P with ICP-MS is performed for the detection of OPPs. Three common OPPs, including glyphosate, glufosinate, and aminomethylphosphonic acid (AMPA), were analyzed by CE-ICP-MS to demonstrate its applicability for the analysis of OPPs. The advantages of using ICP-MS with respect to other common detectors, such as flame photometric detection (FPD), for CE analysis of OPPs are shown. Additionally, different CE separation conditions were studied to achieve complete baseline separation of the pesticide compounds in short migration times. Two CE buffer systems were evaluated for the separation of OPPs using ICP-MS detection. A buffer solution containing 40 mmol.L(-1) ammonium acetate at pH 9.0 and an applied voltage of +20 kV were finally selected leading to a separation time of 10.0 min. Both migration time and area relative standard deviations (%RSD) were evaluated and their respective values were in the intervals of 1.1-3.3% and 2.7-5.3%. Detection limits obtained with the CE-ICP-MS system were in the range of 0.11-0.19 mg.L(-1) (as compound) yielding an enhancement of 130- to 230-fold with respect to FPD. The proposed methodology was finally applied for the determination of the OPPs mentioned above in natural river water samples.

Studying the distribution pattern of selenium in nut proteins with information obtained from SEC-UV-ICP-MS and CE-ICP-MS.

In this work, size exclusion chromatography (SEC) with UV and inductively coupled plasma mass spectrometry (ICP-MS) detection was used to study the association of selenium to proteins present in Brazil nuts (Bertholletia excelsa) under five different extraction conditions. As expected, better solubilization of proteins was observed using 0.05molL(-1) sodium hydroxide and 1% sodium dodecylsulfate (SDS) in Tris/HCl buffer (0.05molL(-1), pH 8) as compared to 0.05molL(-1) HCl, 0.05molL(-1) Tris/HCl or hot water (60 degrees C). Due to non-destructive character of Tris-SDS treatment, this was applied for studying molecular weight (MW) distribution patterns of selenium-containing nut proteins. Three different SEC columns were used for obtaining complete MW distribution of selenium: Superdex 75, Superdex Peptide, and Superdex 200 were tested with 50mmolL(-1) Tris buffer (pH 8), 150mmolL(-1) ammonium bicarbonate buffer (pH 7.8), phosphate (pH 7.5), and CAPS (pH 10.0) mobile phases. Using Superdex 200 column, the elution of at least three MW fractions was observed with UV detection (200-10kDa) and ICP-MS chromatogram showed the co-elution of selenium with the two earlier fractions. The apparent MWs of these selenium-containing fractions were respectively about 107 and 50kDa, as evaluated from the column calibration. For further characterization of individual selenium species, the defatted nuts were hydrolyzed with proteinase K and analyzed by capillary electrophoresis (CE) with ICP-MS detection. The suitability of CE for the separation of selenite, selenate, selenocystine and selenomethionine in the presence of the nut sample matrix is demonstrated. Complete separation of the above mentioned selenium species was obtained within a migration time of 7min. In the analysis of nut extracts with CE-ICP-MS, selenium was found to be present mainly as selenomethionine.

Hydride generation interface for speciation analysis coupling capillary electrophoresis to inductively coupled plasma mass spectrometry.

A novel hydride generation (HG) interface for coupling capillary electrophoresis (CE) with inductively coupled plasma mass spectrometry (ICPMS) is presented in this work. The CE-HG-ICPMS interface was applied to the separation and quantitation of common arsenic species. Lack of a commercially available HG interface for CE-ICPMS led to a three concentric tube design allowing alleviation of back pressure commonly observed in CE-HG-ICPMS. Due to the high sensitivity and element-specific detection of ICPMS, quantitative analysis of As(III), As(V), monomethylarsonic acid, and dimethylarsinic acid was achieved. Optimization of CE separation conditions resulted in the use of 20 mmol L(-1) sodium borate with 2% osmotic flow modifier (pH 9.0) and -20 kV applied potential for baseline resolution of each arsenic species in the shortest time. Hydride generation conditions were optimized through multiple electrophoretic separation analyses with 5% HCl and 3% NaBH(4) (in 0.2% NaOH) determined to be the optimum conditions. After completion of system optimization, detection limits obtained for the arsenic species were less than 40 ng L(-1) with electromigration time precision less than 1% within a total analysis time of 9.0 min. Finally, the interface was used for speciation analysis of arsenic in river and tap water samples.

Studies of various elements of nutritional and toxicological interest associated with different molecular weight fractions in Brazil nuts.

On-line hyphenation of size exclusion chromatography (SEC), UV, and inductively coupled plasma mass spectrometry (ICP-MS) was used to study the molecular weight distribution patterns of several elements in Brazil nuts (Bertholletia excelsa). This technique was used for the elemental speciation of different elements of nutritional and toxicological interests such as Mg, Fe, Co, Mo, Ag, Hg, and Pb. Elemental fractionation in Brazil nuts was studied using a Superdex peptide column with resolving capacity in the range of 14 to 0.18 kDa. Three different mobile phases, Tris buffer solution (pH 8.0), phosphate buffer (pH 7.5), and CAPS buffer solution (pH 10.0), were tried for the SEC fractionation. Size exclusion fractionation of all the extracted solutions was performed using a 50 mmol L(-)(1) Tris buffer (pH 8) as the mobile phase at a flow rate of 0.6 mL min(-)(1). Three different extractions, 0.05 mol L(-)(1) NaOH, 0.05 mol L(-)(1) HCl, and hot water at 60 degrees C, were performed, and the association of elements with various molecular weight fractions was evaluated. Total elemental concentrations in the extracted samples were determined and compared with the values obtained after total digestion to calculate the recovery values. Generally, high extraction efficiency was obtained with the NaOH solution as compared with HCl and hot water except in the case of magnesium, for which HCl was found to be a good extractant. Chromatographic elution profiles for these extractions were quite distinct from each other in most cases. Most of the elemental species were found to be associated with high molecular weight fractions. To study the differences obtained during the sample-processing step, the results obtained for nuts with shell were treated differently from those obtained for nuts purchased without shell and were compared.

Fast speciation analysis of iodophenol compounds in river waters by capillary electrophoresis-inductively coupled plasma-mass spectrometry with off-line solid-phase microextraction.

An analytical methodology for the fast separation and determination of iodophenol species in natural water samples was developed using capillary electrophoresis (CE) coupled to inductively coupled plasma-mass spectrometry (ICP-MS). Based on the element-specific and highly sensitive detection provided by ICP-MS, the methodology has been applied to the analysis of 2-iodophenol, 4-iodophenol, and 2,4,6-triiodophenol. The use of solid-phase microextraction (SPME), after proper optimization, improved the signal by a factor of 100 leading to detection limits in the sub microg.L(-1). Different desorption conditions of iodophenol compounds from the SPME microfiber were studied to achieve the optimum preconcentration factor and best analytical performance. Different CE conditions were studied to achieve complete baseline separation of iodophenols in short migration times. Three different CE buffer systems were evaluated using ICP-MS detection. A buffer solution containing 20 mmol.L(-1) 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) and an applied potential of +22 kV were finally selected leading to a maximum separation time of 6.6 min. A relative standard deviation (%RSD) of about 5.0% for ten consecutive determinations was obtained. Finally, the speciation methodology developed was utilized for the determination of iodophenol compounds in natural water samples.

Speciation of arsenic in different types of nuts by ion chromatography-inductively coupled plasma mass spectrometry.

In this work the quantitative determination and analytical speciation of arsenic were undertaken in different types of nuts, randomly purchased from local markets. The hardness of the whole nuts and high lipid content made the preparation of this material difficult for analysis. The lack of sample homogeneity caused irreproducible results. To improve the precision of analysis, arsenic was determined separately in nut oil and in the defatted sample. The lipids were extracted from the ground sample with the two portions of a mixture of chloroform and methanol (2:1). The defatted material was dried and ground again, yielding a fine powder. The nut oil was obtained by combining the two organic extracts and by evaporating the solvents. The two nut fractions were microwave digested, and total arsenic was determined by inductively coupled plasma mass spectrometry (ICP-MS). The results obtained for oils from different types of nuts showed element concentration in the range 2.9-16.9 ng g(-)(1). Lower levels of arsenic were found in defatted material (<0.1 ng g(-)(1) with the exception of Brazil nuts purchased with and without shells, 3.0 and 2.8 ng g(-)(1) respectively). For speciation analysis of arsenic in nut oils, elemental species were extracted from 2 g of oil with 12 mL of chloroform/methanol (2:1) and 8 mL of deionized water. The aqueous layer, containing polar arsenic species, was evaporated and the residue dissolved and analyzed by ion chromatography-ICP-MS. The anion exchange chromatography enabled separation of As(III), dimethylarsinic acid (DMAs(V)), monomethylarsonic acid (MMAs(V)), and As(V) within 8 min. Several types of nuts were analyzed, including walnuts, Brazil nuts, almonds, cashews, pine nuts, peanuts, pistachio nuts, and sunflower seeds. The recovery for the speciation procedure was in the range 72.7-90.6%. The primary species found in the oil extracts were As(III) and As(V). The arsenic concentration levels in these two species were 0.7-12.7 and 0.5-4.3 ng g(-)(1), respectively. The contribution of As in DMAs(V) ranged from 0.1 +/- 0.1 ng g(-)(1) in walnuts to 1.3 +/- 0.3 ng g(-)(1) in pine nuts. MMAs(V) was not detected in almonds, peanuts, pine nuts, sunflower seeds, or walnuts, and the highest concentration was found in pistachio nuts (0.5 +/- 0.2 ng g(-)(1)).