Genotoxicity assessment of water soluble fractions of biodiesel and its diesel blends using the Salmonella assay and the in vitro MicroFlow (R) kit (Litron) assay

The designation of biodiesel as an environmental-friendly alternative to diesel oil has improved its commercialization and use. However, most biodiesel environmental safety studies refer to air pollution and so far there have been very few literature data about its impacts upon other biotic systems, e.g. water, and exposed organisms. Spill simulations in water were carried out with neat diesel and biodiesel and their blends aiming at assessing their genotoxic potentials should there be contaminations of water systems. The water soluble fractions (WSF) from the spill simulations were submitted to solid phase extraction with C-18 cartridge and the extracts obtained were evaluated carrying out genotoxic and mutagenic bioassays [the Salmonella assay and the in vitro MicroFlow (R) kit (Litron) assay]. Mutagenic and genotoxic effects were observed, respectively, in the Salmonella/microsome preincubation assay and the in vitro MN test carried out with the biodiesel WSF. This interesting result may be related to the presence of pollutants in biodiesel derived from the raw material source used in its production chain. The data showed that care while using biodiesel should be taken to avoid harmful effects on living organisms in cases of water pollution. (C) 2011 Elsevier Ltd. All rights reserved.

PCR colorimetric dot-blot assay and clinical pretest probability for diagnosis of Pulmonary Tuberculosis in Smear-Negative patients

Abstract Background Smear-negative pulmonary tuberculosis (SNPTB) accounts for 30% of Pulmonary Tuberculosis (PTB) cases reported annually in developing nations. Polymerase chain reaction (PCR) may provide an alternative for the rapid detection of Mycobacterium tuberculosis (MTB); however little data are available regarding the clinical utility of PCR in SNPTB, in a setting with a high burden of TB/HIV co-infection. Methods To evaluate the performance of the PCR dot-blot in parallel with pretest probability (Clinical Suspicion) in patients suspected of having SNPTB, a prospective study of 213 individuals with clinical and radiological suspicion of SNPTB was carried out from May 2003 to May 2004, in a TB/HIV reference hospital. Respiratory specialists estimated the pretest probability of active disease into high, intermediate, low categories. Expectorated sputum was examined by direct microscopy (Ziehl-Neelsen staining), culture (Lowenstein Jensen) and PCR dot-blot. Gold standard was based on culture positivity combined with the clinical definition of PTB. Results In smear-negative and HIV subjects, active PTB was diagnosed in 28.4% (43/151) and 42.2% (19/45), respectively. In the high, intermediate and low pretest probability categories active PTB was diagnosed in 67.4% (31/46), 24% (6/25), 7.5% (6/80), respectively. PCR had sensitivity of 65% (CI 95%: 50%–78%) and specificity of 83% (CI 95%: 75%–89%). There was no difference in the sensitivity of PCR in relation to HIV status. PCR sensitivity and specificity among non-previously TB treated and those treated in the past were, respectively: 69%, 43%, 85% and 80%. The high pretest probability, when used as a diagnostic test, had sensitivity of 72% (CI 95%:57%–84%) and specificity of 86% (CI 95%:78%–92%). Using the PCR dot-blot in parallel with high pretest probability as a diagnostic test, sensitivity, specificity, positive and negative predictive values were: 90%, 71%, 75%, and 88%, respectively. Among non-previously TB treated and HIV subjects, this approach had sensitivity, specificity, positive and negative predictive values of 91%, 79%, 81%, 90%, and 90%, 65%, 72%, 88%, respectively. Conclusion PCR dot-blot associated with a high clinical suspicion may provide an important contribution to the diagnosis of SNPTB mainly in patients that have not been previously treated attended at a TB/HIV reference hospital.

Comparative performance evaluation of hepatitis C virus genotyping based on the 5' untranslated region versus partial sequencing of the NS5B region of brazilian patients with chronic hepatitis C

Background Genotyping of hepatitis C virus (HCV) has become an essential tool for prognosis and prediction of treatment duration. The aim of this study was to compare two HCV genotyping methods: reverse hybridization line probe assay (LiPA v.1) and partial sequencing of the NS5B region. Methods Plasma of 171 patients with chronic hepatitis C were screened using both a commercial method (LiPA HCV Versant, Siemens, Tarrytown, NY, USA) and different primers targeting the NS5B region for PCR amplification and sequencing analysis. Results Comparison of the HCV genotyping methods showed no difference in the classification at the genotype level. However, a total of 82/171 samples (47.9%) including misclassification, non-subtypable, discrepant and inconclusive results were not classified by LiPA at the subtype level but could be discriminated by NS5B sequencing. Of these samples, 34 samples of genotype 1a and 6 samples of genotype 1b were classified at the subtype level using sequencing of NS5B. Conclusions Sequence analysis of NS5B for genotyping HCV provides precise genotype and subtype identification and an accurate epidemiological representation of circulating viral strains.

Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.

Comparison of flow cytometry and indirect immunofluorescence assay in the diagnosis and cure criterion after therapy of American tegumentary leishmaniasis by anti-live Leishmania (Viannia) braziliensis immunoglobulin G

The aim of this study was to compare the techniques of indirect immunofluorescence assay (IFA) and flow cytometry to clinical and laboratorial evaluation of patients before and after clinical cure and to evaluate the applicability of flow cytometry in post-therapeutic monitoring of patients with American tegumentary leishmaniasis (ATL). Sera from 14 patients before treatment (BT), 13 patients 1 year after treatment (AT), 10 patients 2 and 5 years AT were evaluated. The results from flow cytometry were expressed as levels of IgG reactivity, based on the percentage of positive fluorescent parasites (PPFP). The 1:256 sample dilution allowed us to differentiate individuals BT and AT. Comparative analysis of IFA and flow cytometry by ROC (receiver operating characteristic curve) showed, respectively, AUC (area under curve) = 0.8 (95% CI = 0.64–0.89) and AUC = 0.90 (95% CI = 0.75–0.95), demonstrating that the flow cytometry had equivalent accuracy. Our data demonstrated that 20% was the best cut-off point identified by the ROC curve for the flow cytometry assay. This test showed a sensitivity of 86% and specificity of 77% while the IFA had a sensitivity of 78% and specificity of 85%. The after-treatment screening, through comparative analysis of the technique performance indexes, 1, 2 and 5 years AT, showed an equal performance of the flow cytometry compared with the IFA. However, flow cytometry shows to be a better diagnostic alternative when applied to the study of ATL in the cure criterion. The information obtained in this work opens perspectives to monitor cure after treatment of ATL.

Next-Generation Sequencing-Based Mutations Scanning Strategy of the BCR-ABL Kinase Domain in Patients with PhiladelPhia-Chromosome Positive Leukemias Treated with Tyrosine Kinase Inhibitors

In chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia patients resistant to tyrosine kinase inhibitors (TKIs), BCR-ABL kinase domain mutation status is an essential component of the therapeutic decision algorithm. The recent development of Ultra-Deep Sequencing approach (UDS) has opened the way to a more accurate characterization of the mutant clones surviving TKIs conjugating assay sensitivity and throughput. We decided to set-up and validated an UDS-based for BCR-ABL KD mutation screening in order to i) resolve qualitatively and quantitatively the complexity and the clonal structure of mutated populations surviving TKIs, ii) study the dynamic of expansion of mutated clones in relation to TKIs therapy, iii) assess whether UDS may allow more sensitive detection of emerging clones, harboring critical 2GTKIs-resistant mutations predicting for an impending relapse, earlier than SS. UDS was performed on a Roche GS Junior instrument, according to an amplicon sequencing design and protocol set up and validated in the framework of the IRON-II (Interlaboratory Robustness of Next-Generation Sequencing) International consortium.Samples from CML and Ph+ ALL patients who had developed resistance to one or multiple TKIs and collected at regular time-points during treatment were selected for this study. Our results indicate the technical feasibility, accuracy and robustness of our UDS-based BCR-ABL KD mutation screening approach. UDS was found to provide a more accurate picture of BCR-ABL KD mutation status, both in terms of presence/absence of mutations and in terms of clonal complexity and showed that BCR-ABL KD mutations detected by SS are only the “tip of iceberg”. In addition UDS may reliably pick 2GTKIs-resistant mutations earlier than SS in a significantly greater proportion of patients.The enhanced sensitivity as well as the possibility to identify low level mutations point the UDS-based approach as an ideal alternative to conventional sequencing for BCR-ABL KD mutation screening in TKIs-resistant Ph+ leukemia patients

Ultrasensitive chemiluminescence bioassays based on microfluidics in miniaturized analytical devices

The activity carried out during my PhD was principally addressed to the development of portable microfluidic analytical devices based on biospecific molecular recognition reactions and CL detection. In particular, the development of biosensors required the study of different materials and procedures for their construction, with particular attention to the development of suitable immobilization procedures, fluidic systems and the selection of the suitable detectors. Different methods were exploited, such as gene probe hybridization assay or immunoassay, based on different platform (functionalized glass slide or nitrocellulose membrane) trying to improve the simplicity of the assay procedure. Different CL detectors were also employed and compared with each other in the search for the best compromise between portability and sensitivity. The work was therefore aimed at miniaturization and simplification of analytical devices and the study involved all aspects of the system, from the analytical methodology to the type of detector, in order to combine high sensitivity with easiness-of-use and rapidity. The latest development involving the use of smartphone as chemiluminescent detector paves the way for a new generation of analytical devices in the clinical diagnostic field thanks to the ideal combination of sensibility a simplicity of the CL with the day-by-day increase in the performance of the new generation smartphone camera. Moreover, the connectivity and data processing offered by smartphones can be exploited to perform analysis directly at home with simple procedures. The system could eventually be used to monitor patient health and directly notify the physician of the analysis results allowing a decrease in costs and an increase in the healthcare availability and accessibility.

A pyrosequencing assay for the rapid discrimination of mitochondrial lineages in the Salmo trutta species complex

The European trout (Salmo trutta species complex) is genetically very diverse consisting of five distinct mitochondrial lineages that probably originated in the Pleistocene. Here, we describe a novel pyrosequencing protocol to generate two short sequence reads from the mitochondrial control region, which allow the unambiguous identification of all five lineages. The approach was found to be easily transferable between laboratories and should be a valuable tool for the assessment of genetic diversity in trout. Pyrosequencing-based assays for molecular species identification are expected to be generally useful whenever multiple positions in a short DNA sequence need to be assessed.

Rivaroxaban: Quantification by anti-FXa assay and influence on coagulation tests A study in 9 Swiss laboratories

INTRODUCTION: Rivaroxaban (RXA) is licensed for prophylaxis of venous thromboembolism after major orthopaedic surgery of the lower limbs. Currently, no test to quantify RXA in plasma has been validated in an inter-laboratory setting. Our study had three aims: to assess i) the feasibility of RXA quantification with a commercial anti-FXa assay, ii) its accuracy and precision in an inter-laboratory setting, and iii) the influence of 10mg of RXA on routine coagulation tests. METHODS: The same chromogenic anti-FXa assay (Hyphen BioMed) was used in all participating laboratories. RXA calibrators and sets of blinded probes (aim ii.) were prepared in vitro by spiking normal plasma. The precise RXA content was assessed by high-pressure liquid chromatography-tandem mass spectrometry. For ex-vivo studies (aim iii), plasma samples from 20 healthy volunteers taken before and 2 - 3hours after ingestion of 10mg of RXA were analyzed by participating laboratories. RESULTS: RXA can be assayed chromogenically. Among the participating laboratories, the mean accuracy and the mean coefficient of variation for precision of RXA quantification were 7.0% and 8.8%, respectively. Mean RXA concentration was 114±43?g/L .RXA significantly altered prothrombin time, activated partial thromboplastin time, factor analysis for intrinsic and extrinsic factors. Determinations of thrombin time, fibrinogen, FXIII and D-Dimer levels were not affected. CONCLUSIONS: RXA plasma levels can be quantified accurately and precisely by a chromogenic anti-FXa assay on different coagulometers in different laboratories. Ingestion of 10mg RXA results in significant alterations of both PT- and aPTT-based coagulation assays.

Oxidative damage to DNA related to survivorship and carotenoid-based sexual ornamentation in the common yellowthroat

Carotenoid-based sexual ornaments are hypothesized to be reliable signals of male quality, based on an allocation trade-off between the use of carotenoids as pigments and their use in antioxidant defence against reactive oxygen species. Carotenoids appear to be poor antioxidants in vivo, however, and it is not clear whether variation in ornament expression is correlated with measures of oxidative stress (OXS) under natural conditions. We used single-cell gel electrophoresis to assay oxidative damage to erythrocyte DNA in the common yellowthroat (Geothlypis trichas), a sexually dichromatic warbler in which sexual selection favours components of the males’ yellow ‘bib’. We found that the level of DNA damage sustained by males predicted their overwinter survivorship and was reflected in the quality of their plumage. Males with brighter yellow bibs showed lower levels of DNA damage, both during the year the plumage was sampled (such that yellow brightness signalled current OXS) and during the previous year (such that yellow brightness signalled past OXS). We suggest that carotenoid-based ornaments can convey information about OXS to prospective mates and that further work exploring the proximate mechanism(s) linking OXS to coloration is warranted.

Levels and determinants of inflammatory biomarkers in a Swiss population-based sample (CoLaus study)

Objective to assess the levels and determinants of interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and C-reactive protein (CRP) in a healthy Caucasian population. Methods population sample of 2884 men and 3201 women aged 35 to 75. IL-1β, IL-6 and TNF-α were assessed by a multiplexed particle-based flow cytometric assay and CRP by an immunometric assay. Results Spearman rank correlations between duplicate cytokine measurements (N = 80) ranged between 0.89 and 0.96; intra-class correlation coefficients ranged between 0.94 and 0.97, indicating good reproducibility. Among the 6085 participants, 2289 (37.6%), 451 (7.4%) and 43 (0.7%) had IL-1β, IL-6 and TNF-α levels below detection limits, respectively. Median (interquartile range) for participants with detectable values were 1.17 (0.48–3.90) pg/ml for IL-1β; 1.47 (0.71–3.53) pg/ml for IL-6; 2.89 (1.82–4.53) pg/ml for TNF-α and 1.3 (0.6–2.7) ng/ml for CRP. On multivariate analysis, greater age was the only factor inversely associated with IL-1β levels. Male sex, increased BMI and smoking were associated with greater IL-6 levels, while no relationship was found for age and leisure-time PA. Male sex, greater age, increased BMI and current smoking were associated with greater TNF-α levels, while no relationship was found with leisure-time PA. CRP levels were positively related to age, BMI and smoking, and inversely to male sex and physical activity. Conclusion Population-based levels of several cytokines were established. Increased age and BMI, and to a lesser degree sex and smoking, significantly and differentially impact cytokine levels, while leisure-time physical activity has little effect.

Association between circulating cytokine levels, diabetes and insulin resistance in a population-based sample (CoLaus study)

OBJECTIVE: The associations between inflammation, diabetes and insulin resistance remain controversial. Hence, we assessed the associations between diabetes, insulin resistance (using HOMA-IR) and metabolic syndrome with the inflammatory markers high sensitivity C-reactive protein (hs-CRP), interleukin-1beta (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α). DESIGN: CROSS-SECTIONAL STUDY: PARTICIPANTS: 2884 MEN AND 3201 WOMEN AGED 35 TO 75: METHODS: CRP was assessed by immunoassay and cytokines by multiplexed flow cytometric assay. In a subgroup of 532 participants an oral glucose tolerance test was performed to screen for impaired glucose tolerance (IGT). RESULTS: IL-6, TNF-α and hs-CRP were significantly and positively correlated with fasting plasma glucose, insulin and HOMA-IR. Participants with diabetes had higher IL-6, TNF-α and hs-CRP levels than participants without diabetes; this difference persisted for hs-CRP after multivariate adjustment. Participants with metabolic syndrome had increased IL-6, TNF-α and hs-CRP levels; these differences persisted after multivariate adjustment. Participants in the highest quartile of HOMA-IR had increased IL-6, TNF-α and hs-CRP levels; these differences persisted for TNF-α and hs-CRP after multivariate adjustment. No association was found between IL-1β levels and all diabetes and insulin resistance markers studied. Finally, participants with IGT had higher hs-CRP levels than participants with a normal OGTT, but this difference disappeared after controlling for body mass index (BMI). CONCLUSION: subjects with diabetes, metabolic syndrome and increased insulin resistance present with increased levels of IL6, TNF-α and hs-CRP, while no association was found with IL-1β. The increased inflammatory state of subjects with IGT is partially explained by increased BMI. © 2012 Blackwell Publishing Ltd.

qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λnDNA) and mtDNA (λmtDNA) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.

Twelve years' detection of respiratory viruses by immunofluorescence in hospitalised children: impact of the introduction of a new respiratory picornavirus assay

Background Direct immunofluorescence assays (DFA) are a rapid and inexpensive method for the detection of respiratory viruses and may therefore be used for surveillance. Few epidemiological studies have been published based solely on DFA and none included respiratory picornaviruses and human metapneumovirus (hMPV). We wished to evaluate the use of DFA for epidemiological studies with a long-term observation of respiratory viruses that includes both respiratory picornaviruses and hMPV. Methods Since 1998 all children hospitalized with respiratory illness at the University Hospital Bern have been screened with DFA for common respiratory viruses including adenovirus, respiratory syncytial virus (RSV), influenza A and B, and parainfluenza virus 1-3. In 2006 assays for respiratory picornaviruses and hMPV were added. Here we describe the epidemiological pattern for these respiratory viruses detected by DFA in 10'629 nasopharyngeal aspirates collected from 8'285 patients during a 12-year period (1998-2010). Results Addition of assays for respiratory picornaviruses and hMPV raised the proportion of positive DFA results from 35% to 58% (p < 0.0001). Respiratory picornaviruses were the most common viruses detected among patients ≥1 year old. The seasonal patterns and age distribution for the studied viruses agreed well with those reported in the literature. In 2010, an hMPV epidemic of unexpected size was observed. Conclusions DFA is a valid, rapid, flexible and inexpensive method. The addition of assays for respiratory picornaviruses and hMPV broadens its range of viral detection. DFA is, even in the "PCR era", a particularly adapted method for the long term surveillance of respiratory viruses in a pediatric population.

Development of PCR/dot blot assay for specific detection and differentiation of taeniid cestode eggs in canids

We report the development of a colourimetric PCR/dot blot assay targeting the mitochondrial gene NADH dehydrogenase subunit 1 (nad1) for differential diagnosis of taeniid eggs. Partial sequences of the cestode nad1 gene were aligned and new primers were designed based on conserved regions. Species-specific oligonucleotide probes (S-SONP) for canine taeniid cestodes were then designed manually based on the variable region between the conserved primers. Specifically, S-SONP were designed for the Taenia crassiceps, T. hydatigena, T. multiceps, T. ovis, T. taeniaeformis, Echinococcus granulosus (genotype 1), E. multilocularis and E. vogeli. Each probe showed high specificity as no cross-hybridisation with any amplified nad1 fragment was observed. We evaluated the assay using 49 taeniid egg-positive samples collected from dogs in Zambia. DNA from 5 to 10 eggs was extracted in each sample. Using the PCR/dot blot assay, the probes successfully detected PCR products from T. hydatigena in 42 samples, T. multiceps in 3 samples, and both species (mixed infection) in the remaining 4 samples. The results indicate that the PCR/dot blot assay is a reliable alternative for differential diagnosis of taeniid eggs in faecal samples.